Overviews: Thirty-Five Years of Cell Biology (International Review of Cytology, Volume 100)

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME100

Overviews: Thirty-Five Years of Cell Biology

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY PIET BORST BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO CHARLES J . FLICKINGER OLUF GAMBORG M. NELLY GOLARZ DE BOURNE YUKIO HIRAMOTO YUKINORI HIROTA K. KUROSUMI GIUSEPPE MILLONIG ARNOLD MITTELMAN AUDREY MUGGLETON-HARRIS DONALD G. MURPHY

ROBERT G. E. MURRAY RICHARD NOVICK ANDREAS OKSCHE MURIEL J. ORD VLADIMIR R. PANTIC W. J. PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL L. EVANS ROTH JOAN SMITH-SONNEBORN WILFRED STEIN HEWSON SWIFT K. TANAKA DENNIS L. TAYLOR TADASHI UTAKOJI ROY WIDDUS ALEXANDER YUDIN

INTERNATlONAL

Review of Cytology Editor-in-Chief

G . H. BOURNE St. George's University School of Medicine St. George's, Grenada West Indies

Associate Editors

K. W . JEON

M. FRIEDLANDER

Department of Zoology University of Tennessee Knoxville, Tennessee

Jules Stein Eye Institute UCLA School of Medicine Los Angeles. California

VOLUME100

Overviews: Thirty-Five Years of Cell Biology Edited by K. W. JEON Department of Zoology University of Tennessee Knoxville, Tennessee

ACADEMIC PRESS, INC. 1987 Harcourt Brace Jovanovich, Publishers

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9 8 7 6 5 4 3 2 I

Contents PREFACE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

One Hundred Volumes of the International Review of Cytology GEOFFREYH. BOURNE

I. Introduction

.........................................

11. Early Studies in 111. A New Era in Cell Biology . . . . . . . . . . . . . . . . . . . . IV. International Review of Cytology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

13

Development of Centrifuges and Their Use in the Study of Living Cells H. W. BEAMSAND R. G. KESSEL I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. History of Centrifuge Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Effects of Centrifugal Force on Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IV. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 17 22 43 44

The Chromosome Cycle and the Centrosome Cycle in the Mitotic Cycle DANIEL MAZIA

........................ ......... .. . .... .. .. ._ . ........ . . ........ ... .. ..

I. Introduction . . . . . . . . . . . . . . . . . . . 11. Variants

49 50

entrosome Cycle Occupy the Whole IV. V. VI. VII.

........................ ........... 11 Cycle . . . . . . . . . . . . . . . . . The Chromoso The Centrosome Cycle in the Whole Cell Cycle . . . . . . . . . . . . . . . . . . Establishment of the Mitotic Apparatus: Boveri’s Rules . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51 53 63 79 88 89

vi

CONTENTS

Cell Reproduction DAVIDM. PRESCOTI

I. Introduction . . . . . . . . . . .

............................

93 95

.............. 11. Cell Growth and Reproduc 111. Regulation of G I Transit . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100

The Relationship of Go to the Cell Cycle . The GI/S Border . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear Structure and DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Events of the G2 Period . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . .......................................

118 119 120 122 123 123

IV . V. VI. VII. VIII.

The Early Days of Electron Microscopy of Nerve Tissue and Membranes J. DAVID ROBERTSON I. 11. 111. IV. V. VI. VII. VIII. IX. X.

Introduction Formative Ye Early Academic Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Military Service and Medical Practice . . Ph.D. Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Kansas Period ................................................... The London Period . . . .............. The Harvard Period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Duke Period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . .................. References . . . ... ..

129 129 130 132 133 143 152 177 185 195 196

Ionized Groups on the Cell Surface: Their Cytochemical Detection and Related Cell Function SATIMARU SENO

. . . _ . _ . . . . . _ _ . . . . . . _203 . I. Introduction I Tissues . . . . . . . . . . . . . 204 11. Ionic Groups 111. Cytochemical and Histochemical Detection of Ionized Groups by Ionic Dyes . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 IV. Reactions between Polyanionic and Polycationic Macromolecules . . . V. Ionized Groups of Acid Polysaccharides and Proteins Estimated by the ............................ ..... 210 VI. Colloidal Probes . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 VII. Effect of Aldehyde Fixation on Ionized Groups of Cells and Tissues . . . . . . . . , . 221 ..._. 225 VIII. IX . Conclusion . . .

CONTENTS

vii

Nucleocytoplasmic Interactions in Morphogenesis J . BRACHET

I . Old Reminiscences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Nucleocytoplasmic Interactions in Acetabularia . . . . . ...................... 111. Nucleate and Anucleate Fragments of Sea Urchin Eggs ..................... IV . Nucleocytoplasmic Interactions in Xenopus Oocytes and Eggs . . . . V . Cleavage of Fertilized Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . A Very Brief Overview of Later Stages of Development . . . . . . . . . . . . . . . . . VII . Cytoplasmic Determinants (Germinal Localizations) ........................ VIII . Nuclear Determinants (Genes) of Early Embryonic D ecular Embryology . . . . . IX . ............ References . . . . . . . ........................ . . . . . . . . . . . . . . . . . . . .

249 252 264 271 286 290 293 30 1 307 309

Protistan Phylogeny and Eukaryogenesis JOHN 0. CORLISS

I . Introduction and Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Reflections on the “Protist” Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Bases for Renewed Interest in “Lower” Eukaryotes ... IV . Diversity within the Protist Conglomerate . . . . . . . . . ... V . Data of Supposed Phylogenetic Significance . .......................... VI . Major “Evolutionary Lines” of Protists . . . . .......................... VII . Seemingly Isolated Groups ............................................ VIII . Progress and Prognoses. Problems and Frustrations ........................ IX . Hopes and Conclusions ............................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

319 320 323 328 330 333 345 355 351 358

Protozoological Approaches to the Cellular Basis of Mammalian Stress Repair S . H . HUTNERAND S . L . MARCUS

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Economics and Practicalities in Stress-Repair Research: The Protozoological Gambit ................................. 111. Stress Proteins ................................... . . . . . IV . Hemoflagellates (Trypanosomatids): Biopterin. Heme and Fe. Oxidative Stress. Polyamines . . . . . . . .............. V . Chrysomonads. Vitamin BIZ.Carnitine VI . Tetrahymena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Hypoxia ......... ........... VIII . Concluding Reflections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

386 394 404 408 410 415

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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371 379 382

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Preface

This year, the International Review of Cytology marks its 35th year of continuous publication and its 100th volume; it is fitting to celebrate the occasion by a commemorative volume containing special articles contributed by “elder statesmen” of cell biology. The International Review of Cytology has played a pivotal role in disseminating new findings and developments in the rapidly expanding field of cell biology to its worldwide readership, and we owe much and are grateful to the founding editors, Drs. Geoffrey H. Bourne and James F. Danielli, for their keen insight and diligent work. We sincerely regret that Dr. Danielli passed away in 1984 without seeing the 100th volume of the series. This volume is by no means a comprehensive overview of the progress of cell biology for the past 35 years but merely offers a representative sampling. In Chapter 1, Dr. Bourne describes his early involvement in the study of cells and recalls the steps he took, as one of the founding editors, to bring the International Review of Cytology to reality. Drs. Beams and Kessel trace the history of the important development of centrifuges and their use in cell studies in Chapter 2. Various aspects of cell reproduction and specific cell cycle stages are discussed in Chapters 3 and 4 by Drs. Mazia and Prescott, respectively. Dr. Robertson gives an autobiographical account of his endeavors with nerve and membrane structure in Chapter 5, and Dr. Seno’s Chapter 6 contains a survey of various ionized groups on cell surfaces. Professor Brachet shares his insight and rich experience with studies on morphogenesis in Chapter 7. In Chapter 8 , Dr. Corliss expounds on his long-standing interest in protistan phylogeny as it has enveloped him over the years, and Drs. Hutner and Marcus present an unconventional approach to the study of mammalian stress phenomena using protozoans as a model system in Chapter 9. In the hope of making this unique volume more valuable, I asked authors not only to overview scientific progress on given subjects but also to share personal experiences and anecdotes. Many authors have done so, and their articles are rich with humor and interesting aspects of biologists’ lives. For example, cell biology is termed “not a notoriously self-critical field” (Dr. Mazia), and as late as 1952 “so little was known about cell reproduction that it was difficult to find a basis from which to start” (Dr. Prescott). An accidental slit in the animal pole of a frog oocyte produced a translucent spherule (germinal vesicle) and opened new possibilities for research in 1938 (Dr. Brachet). As a general practitioner, Dr. Robertson “built [his] own examining table from pine boards, had an oldix

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PREFACE

fashioned iron pot-bellied stove for heat, and [his] water supply was a bucket hanging from the ceiling. . . .” Then, “a graduate student in microbiology and biochemistry at Cornell was parachuted onto the Physics Department at MIT in 1935 . . . to assess the desirability of million-volt X-rays for deep tumor” (Dr. Hutner). These articles teach us many wonderful lessons and remind us how far we have come in the study of cells. The 200th commemorative volume will probably tell us very different stories. Several other prominent cell and developmental biologists had been invited to contribute to this volume but could not do so because of other commitments. I regret that a few other planned chapters are not included here because the manuscripts did not reach us by press time, but they may appear in regular volumes in the future. I thank the authors for their kindness to have undertaken such colossal tasks and especially for their willingness to share their personal views and experiences of scientific endeavor with us. As usual, the staff members at Academic Press have not spared their expert and willing assistance, for which I am very grateful.

KWANGW. JEON

INTERNATIONAL REVIEW OF CYTOLOGY, VOL lo0

One Hundred Volumes of the International Review of Cytology GEOFFREYH. BOURNE St. George’s University

School of Medicine,

St.

George’s, Grenada, West Indies

I. Introduction In the 19th century, following the enunciation of the cell theory by Schleiden and Schwann in 1938, there was a good deal of interest in the chemical nature of the cell structures that could be seen under the microscope. Then with the development of aniline dyes, many of which picked out beautifully the larger cell components, the delineation of the structure of these objects took priority over their chemistry. In the late 1940s, after the second World War, the development of new physical techniques made it possible to start interpreting the physical and physicochemical activities of cell components in relation to the molecular arrangement. Noteworthy in this line of research were the studies of Danielli and Davson (1935) concerning the nature of the cell membrane; in fact, the modem conception of the unit membrane in cell structure had its origin in Danielli’s work (see later). The last few years of the 1940s saw a ferment of interest in the molecular structure of cells (soon to be exacerbated by the results coming from the newly developed electron microscope) and also in the location and mode of action of metabolically active chemical molecules such as enzymes. Back in the 1920s Bles, publishing in the Quarterly Journal of Microscopical Science, made a very important step into the modem world of cell biology by his study of the intracellular localization of peroxidases in protozoa using a solution of benzidine. His preparations picked out granules associated with bases of cilia. This was at a time when the location of important metabolic compounds such as vitamins, enzymes, and hormones within the cell was completely unknown. It was not even known whether these compounds were associated with the existing known organelles or whether they were simply distributed in the cytosol. I came into the area of cell biology, histology, etc., at the end of the 1920s. I was especially interested in the chemical nature of mitochondria and the Golgi apparatus and their function in the cell. In the account that follows I refer in some detail to some of my early work which was carried out in an attempt to understand the chemical nature, location, and possible metabolic significance of the 1 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.

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GEOFFREY H. BOURNE

substances present in the cell. This reference is made not to place significance on my own investigations which commenced at the University of Western Australia, but to show the background of the growth and the conception of the International Review of Cytology and the intellectual climate prior to and during the period when it was born.

11. Early Studies in Cell Biology A. REDUCING AGENTS In 1928 Albert Szent-Gyorgyi was working at Cambridge University, England on a very powerful reducing substance which he had found in the adrenal gland (Szent-Gyorgyi, 1928). The reducing effect of this substance was such that if a macroscopic slice of adrenal gland was dropped into a solution of silver nitrate in water the cortex of the gland darkened and became completely black in a few minutes but the medulla remained unaffected. Subsequently, Szent-Gyorgyi went back to Hungary and showed that the Hungarian red pepper contained what was an apparently identical reducing substance: Szent-Gyorgyi suggested that it was vitamin C. I had been interested in this reducing substance from the beginning and when I read Szent-Gyorgyi’s suggestions I felt that we could use this reducing property for microscopic purposes, particularly since the substance would reduce metal salts, such as silver nitrate. Frozen sections of adrenal gland dropped into solutions of different strengths of silver nitrate and also of gold chloride were quickly penetrated and cells and their contents suddenly blackened. Under the microscope, silver nitrate preparations showed various-sized black granules in the cell, whereas gold chloride tended to show scattered multiple very fine granules. I was not sure what this difference meant, but the larger black granules and globules in the cytoplasm of the cells in the silver preparations were located in a way which suggested that the vitamin might be associated with the Golgi apparatus. I also placed small pieces of adrenal gland in silver nitrate until they became black, fixed them, embedded them in wax, and sectioned them; in this way better and thinner preparations than the frozen sections were obtained, which could be subjected to more detailed microscopical examination. (Freeze-drying in cytology was of course some distance in the future.) Because of the osmotic effect of silver nitrate a variety of other solvents were tried. Enough silver nitrate was found to be taken up by chloroform that the gland would blacken with this reagent, but chloroform was very destructive to the cytoplasm of cells. Since vitamin C was a water-soluble vitamin, we wanted to fix pieces of the gland before exposing it to silver nitrate so that the localization of the black granules after reduction would more truly represent the distribution of the vitamin in the cell. Most fixatives, however, were aqueous and contained chemicals which destroyed vitamin C, so attempts were made to fix slices of

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gland in formaldehyde vapor derived by heating paraformaldehyde. Although the tissue was well washed after fixation, formaldehyde is a reducing substance itself and probably created artifacts with the silver nitrate, and we found later it also destroyed the vitamin. The results of these many studies were published in Nature in 1933 (Bourne, 1933a,t), in which it was established that all the reducing reaction of the cell was cytoplasmic and the nucleus did not appear to contain any of the vitamin. It was suggested that in the cytoplasm, vitamin C appeared to be, at least in part, in the Golgi material. Another more detailed paper was published in the Australian Journal of Experimental Biology (Bourne, 1934). While these studies were going on in my laboratory, Charles Leblond was working on the same vitamin and the same problem in the laboratory of Professor Anton Giroud in Paris. In 1934 Leblond published his doctoral thesis on the cytological localization of vitamin C. An important modification of the technique was made by Leblond and that was the acidification of the silver nitrate solution by acetic acid. This had the benefit of not only making the silver nitrate reaction more specific, but the acetic acid also had a fixative effect in the cells. Many years later, I looked at tissues treated with this reagent under the electron microscope; the reagent was found to have a very destructive effect, especially on the cytoplasm of cells. Subsequently, it was shown that concentrations of vitamin C equal to those found in the adrenal cortical cells were also to be found in the corpus luteum cells and in the Leydig cells of the testis, both of which were also steroid hormoneproducing cells; this suggested a relationship between vitamin C and steroid production. However, Zilva, using the silver nitrate reagent, demonstrated a high vitamin C concentration in cells of the anterior pituitary glands, a finding which raised many questions. During my remaining few years in Australia, I applied the silver-nitrate technique to study the reaction in most organs of the body and also in many invertebrates and protozoa. At this time also, I began to develop the histochemical localization of vitamin A using antimony trichloride, with which vitamin A gives a blue color. My preparations indicated a concentration of this vitamin in mitochondria. From Australia, I wefit to Oxford University, working first in the Anatomy Department and subsequently in the Physiology Laboratory. By this time biochemical reactions for vitamin C were well established, and in the Laboratory of Professor Sir Rudolf Peters, I began chemical titration of various organs employing different procedures and comparing the results with the silver-nitrate reactions. At this point, I was diverted into the interrelationship of the adrenal cortex with other endocrines, and when the second World War broke out I directed all my energies to the role of vitamin C in wound healing.

B. WOUNDHEALING There was experimental evidence in the literature that vitamin C was essential for proper healing of wounds, but all the experimenters had used orange or lemon

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GEOFFREY H. BOURNE

juice as a source of the vitamin and of course it contains a number of compounds other than vitamin C. However, I showed that pure synthetic vitamin C promoted the healing of wounds, that the tensile strength of the wound varied directly with the amount of vitamin C up to a plateau, and that the tensile strength was related to the amount of collagen. Since the ossein of bone is close to collagen, I also showed, by developing a technique for measuring the amount of bone formed after a standard lesion was inflicted on the bone, that the amount of ossein formed in the healing process was directly related to the amount of pure vitamin C administered and that vitamin C was not specifically related to the mineralization process. If the proper ossein framework was formed it seemed to have the innate capacity to mineralize. In the early 1940s the study of the chemical nature of cell components was given a boost by George Gomori at the University of Chicago, who published a paper which described a technique for localizing the enzyme alkaline glycerophosphatase in microscopic sections of tissues (Gomori, 1939). I pioneered this technique in Europe and made an extensive series of studies on the technique itself and the results it produced. I had a special interest in this enzyme because it was supposed to be important in the production of bone. However I found, and in a subsequent paper Danielli also showed, that there is a significant increase in phosphatase in the production of collagen in healing wounds where no mineralization was involved. Subsequently, I showed that the role of glycerophosphatase in the bone was not related to the laying down of mineral, as many people had thought, but was directly related to the production of the inorganic matrix, the ossein.

111. A New Era in Cell Biology

A.

PHYSICOCHEMICAL STUDIES

This was a period when many physical and chemical processes were beginning to make their way into cytology. Danielli’s work on membranes had a substantial impact in the area, and the work of Schulman and others showed the importance of applying physicochemical techniques and thought to the study of cells. Even though we were in the middle of the war and bombing raids, I felt that it was time to bring together what was known about the application of physicochemistry to cytology, and I conceived of an edited volume which would cover the main areas: the book was to be called Cytology and Cell Physiology (Bourne, 1942). It seemed obvious in this case that it should be written principally by people who were currently working in wartime Britain and that to produce such a book on what was then very much an academic subject in the middle of air raids, etc., was an act of defiance of all that war stood for. Many of the chapters were in fact

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written in air-raid shelters during air raids with bombs falling around, and one of the reviewers of the book, when it appeared, wrote quite harshly about the fact that I had mentioned this in the preface. However, I felt strongly that people who could write such recondite material in the middle of such appalling destruction and death should be recognized for their dedication to science as well as their bravery. Cytology and Cell Physiology was published by the Oxford University Press and subsequently went into three editions, one more by Oxford University Press and one by Academic Press of New York. It was placed by John Fulton in the museum of the History of Medicine at Yale University. The book signaled the beginning of a new era in cell biology, and Cytology and Cell Physiology was the forerunner of the International Review of Cytology. When I went on a visit to the United States in 1950 I found that many of the graduate students in biological sciences, a number of whom have since become famous, were using the book and it was very well known. I believe it had a significant impact at that time in directing the subject of cytology along the lines it needed to go.

B. ENZYMEHISTOCHEMISTRY After a spell in the armed forces in Southeast Asia I moved from Oxford to the University of London. There I participated actively in the rapidly developing field of enzyme histochemistry , being the first to apply the oxidative enzyme techniques to the study of muscles, including human muscle, and showing that oxidative enzymes were primarily in the muscle mitochondria or sarcosomes. Subsequently, with Evelyn Beckett, I made a detailed study using a range of enzyme techniques of muscle biopsies taken from people suffering from a variety of neuromuscular diseases. This study led King Engel at the National Institutes of Health Clinical Research Center to cany the work further and eventually to use it to sort out many of the neuromuscular diseases whose clinical relationships were obscure. Subsequently, extensive studies were made with Dr. M. Nelly Golarz on the histochemistry of muscular dystrophy. Other areas which gave interesting results were the identification of a high concentration of oxidative enzymes in the striated ducts of the salivary glands due of course to their localization in the abundant mitochondria in those duct cells. ATPase and other phospate-splitting enzymes were found to be concentrated in the intercalated discs of heart muscle fibers. Phosphatases and esterases were found to be exceptionally active in taste buds and in the epithelium overlying them and also in the basal layer of cells in the olfactory epithelium. A very extensive study of this phenomenon was carried out with A . F. Baradi and was used as a basis to compound a hypothesis of taste. This was published as a review in Volume 2 of the International Review of Cytology. In the late 1940s and early 1950s the availability of radioactive isotopes led to

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GEOFFREY H. BOURNE

the development of radioautography. I was one of the first to use this technique for the microscopical localizations of such isotopes in tissues and certainly the first in Europe to do so, developing a technique for mounting sections directly on the emulsion of a photographic plate.

IV. International Review of Cytology A. CONCEPTION In the late 1940s, with such a wide variety of activities going on in my own laboratories and laboratories all over the world and with the technique of electron microscopy in its early stages, it was not surprising that my mind began to turn toward the problem of keeping up with all these fast-developing techniques which were being applied to the study of cells and the information they were producing. The concept of a review journal which would bring all these developments under one cover began to form. I felt that the reviews in such a journal should not be just a recital of published papers and their results, but should be an in-depth discussion of the literature with the impact of the authors’ own views and research in the area. I felt that it would be desirable for a good deal of theoretical material to be included if it was appropriate. By 1950 the pace of development and the interest in cell biology was becoming difficult to follow, and the time seemed ripe for the production of the review I had been thinking about. 1 finally decided to go ahead with transferring all these ideas into action, but there were some important areas in which I had limited experience, and I felt that it would be important to have a coeditor who could handle them. While I was working in Oxford Jim Danielli had been working in Cambridge. He had already made a reputation in the area of cell-membrane studies and his interests were directed toward their molecular composition. Jim Danielli had been interested in cell membranes from an early stage in his career and had a very fruitful association with H. Davson and also with E. N. Harvey. Among the early workers on the nature of the surface of cells, Overton had proposed a lipid layer as the interface between the environment and the cell. This was based partly on the fact that lipids spread as a monomolecular layer on aqueous surfaces. This view was modified by the classic experiment of Gorter and Grendel in 1925 in which they measured the surface area of erythrocytes, then extracted the lipid and found that the surface area, after spreading it on an aqueous surface, was twice the surface area of the erythrocytes. The conclusion was logical that the membrane surrounding the erythrocytes was two lipid molecules thick. They would almost certainly be oriented with their polar groups directed toward the aqueous phases (the interior of the cell and the environment)

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and with the molecular tails directed toward each other in the middle of the bilayer. For some years this conception, which was expanded from erythrocytes to other cells, prevailed. Then Danielli and Harvey in 1935 showed that surfacetension studies of cells did not support the idea that the membrane was made up only of a bimolecular leaflet of lipid molecules. However the surface-tension studies could be reconciled by assuming that proteins were associated with the membrane. In the same year Danielli and Davson (1935) proposed the paucimolecular model of such a system, devised experiments for assembling such structures, and explained their role in cell activity such as pinocytosis and membrane permeability. Later the electron microscope gave a morphological basis to Danielli’s model, and now, 50 years later, the biology of membranes is still dominated by Danielli’s contributions, his model remaining essentially correct. Rosen (1985) says that this model of the cell membrane (the unit membrane) played a role “comparable in its influence, integrative power, and wealth of implications only to the Watson-Crick model of DNA. ” I got to know Danielli personally in the early 1940s. He had already received considerable acclaim for his membrane work. We kept in touch at various scientific meetings and when, in 1940, I decided to go ahead with the production of Cytology and Cell Physiology, Danielli was the obvious choice to write on cell membranes. After the second World War when I returned from Southeast Asia, Vladimir Korenchevsky, the founder of the British Society for Research in Aging, prevailed upon me to take over the Secretaryship of the Society from him. Here the paths of Jim Danielli and myself crossed again. During the 1940s he had become very interested in the biological processes involved in aging, especially at the cellular level, an interest which extended into the 1950s and 1960s. Both he and I believed that a cellular factor was significant in the aging of a multicellular organism, and he suggested that somatic mutations in cells caused metabolic errors which affected the aging process. My own studies indicated that there was a higher acid-phosphatase activity in aging tissues, and I suggested that cellular aging was related to a “leakage” of hydrolytic enzymes from “lysosomes” into the cytoplasmic matrix (Yapp and Bourne, 1957). Danielli’s experiments with transplanting amoeba nuclei into previously enucleated amoebae to produce types of cells different from anything in nature finally led to his interest in exobiology and an association with NASA. Here again our paths crossed, since at this time I too was associated with NASA and was especially interested in the changes which would take place in the cells of various organs in organisms exposed to weightlessness for long periods of time. Danielli was interested in the forms of life that might be found in space and made significant studies and contributions in this area. These NASA studies by both of us were made some time after the International Review of Cytology came to life in 1952.

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I have gone into some detail on my own research interests and those of Danielli to indicate our range of interests and their reciprocal nature. We applied these interests to the operation of the International Review of Cytology, sharing the tasks in the light of our various individual interests and experience. B. GESTATION In 1950 when I decided to activate my ideas about the founding of a review it was not surprising that Danielli’s name was one of the first to come to my mind for a coeditor. Of all the others I thought of, Danielli’s work seemed the most complementary to mine and his personality appeared to be the most compatible. I felt sure I could work with him. One day in my office at the London Hospital Medical School I decided to ring Jim up and discuss the project. He was immediately enthusiastic and wondered what we should call the review series. I had thought of the name International Review of Cytology and suggested it to him. He thought that was just right and we decided to adopt it. At the present time the term “cytology” suggests the days when cell structure was studied under the microscope, often by the use of aniline dyes, whereas the term “cell biology” is used to denote the functional, physical, chemical, metabolic, and submicroscopic and molecular studies of cells. What Jim Danielli and I were thinking of in 1950 was in fact “cell biology,” but the term was not in common usage as it is today, so we used the word that was in use, and “cytology” took its place in the title. Following my first telephone call to Jim Danielli about the International Review of Cytology we had a number of personal meetings to discuss the policy, format, etc., of the new review. The next job was to cast around for a publisher who would be interested in the subject and who would understand and sympathize with what we were trying to do. We found in Kurt Jacoby and Walter Johnson, of the relatively newly formed Academic Press, Inc., two sympathizers, and we began negotiations with them. We finally came to a mutual agreement to proceed with the review in the summer of 1950, and we stayed with them for the next 35 years. We suggested to the publishers that the title of the series be the International Review of Cytology, and they thought this was appropriate, remembering that, at the time, cytology and cell biology were synonymous terms. All the modem developments of cell biology, including molecular biology, have been and are still being reviewed in the International Review of Cytology. In 1956, after the International Review of Cytology had been founded, I was invited by Emory University in Atlanta, Georgia, to become the chairman of the Anatomy Department in the Medical School there. With larger research facilities and substantially more research money, I launched into many areas applying the various techniques of histochemistry. Dr. M. Nelly Golarz and I did an extensive

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study of the histochemistry of muscular dystrophy in man and animals and later moved on to study muscle changes in bed rest and weightlessness under a grant from the Space Agency. Dr. T. R. Shantha and I discovered the perineural epithelium-a multilayered stratified squamous epithelium which surrounds nerve bundles and terminal nerve fibers from their point of origin in the central nervous system to their termination in the appropriate end organ. We investigated its structure and histochemistry in detail. Dr. Tewari and I made an extensive study of the enzyme histochemistry of the central nervous system. Dr. S. Manocha prepared an atlas of the distribution of enzymes in the rhesus monkey brain under my direction and guidance. Dr. D. Brandes worked with me on the histochemistry of the prostate. Dr. M. Sandler and I made a detailed study of the histochemistry of the aortic wall in the development of atheroma. There were many other projects during the period of 5 years I was there. Then, in 1962, I became the director of the Yerkes Primate Research Center of Emory University and carried many of the projects over into that institution, although I was not able to carry the same personal and detailed interest into them as I had before. An exception was the study of histochemical and ultrastructural changes of muscle and other tissues and organs in the bodies of monkeys exposed to bed rest with special reference to weightlessness. This work was carried on for 16 years. These studies indicated that I maintained an active participation in research in the postnatal development period of the International Review of Cytology. There was a similar intensive personal participation in research by Jim Danielli as well. Neither of us felt that the launching of the review was the signal to sit back and “put our feet up.” C. EARLYVOLUMES

In the foreword to the first volume, Danielli and I said, “It is proposed to keep the scope of the International Review of Cytology as wide as possible-to deal with all aspects of cell biology including morphological and chemical studies of both cells and tissues. Papers presenting new theories of general interest will be welcomed.” When the International Review of Cytology commenced, it was planned that it would appear once a year and that it would be truly international. The International Society of Cell Biology was asked, and agreed, to extend its auspices over the Review at its birth. The society had been formed in 1948 and Danielli had been active in its formation. Subsequently it became the International Federation for Cell Biology. It took about a year to assemble the authors for the first volume. Seven of them were British, five American, two Dutch and Danish, and one Swiss. There were 16 articles. They covered historical aspects of the subject, techniques (freezedrying and electron-microscopic studies of tissue sections, histochemistry of

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esterases, factors controlling staining of tissue sections with acid and basic dyes), the nucleus (nuclear reproductions and nucleocytoplasmic relations), and enzymes (relation to cell nutrition, relation to cell membrane penetration, and protoplast surface enzymes). There were other articles on bacterial cytology, bacteriophage reproduction, folding of protein molecules and osmosis, structural agents in mitosis, spermatozoan behavior, and cytology of mammalian epidermis and its associated glands. Volume 1 appeared in the early part of 1952; the foreword was written by Danielli and me in January of that year. The scientific reviews of the volumes, in general, were very favorable, and interest was shown in the new series by libraries and institutions. We also received encouragement from a number of prestigious individuals in the field. In late 1953 Volume 2 appeared; the foreword was written in August of that year and emphasized our policy that the term “cytology” covered both “cytology” and “cell physiology” and reaffirmed that our reviews would be drawn from the wide field covered by those areas. We also affirmed our policy that contributions would be by invitation, but we did agree to consider reviews submitted voluntarily. Over the years, we have received remarkably few either invited or unsolicited articles that have not been printed. Two important reviews appeared in Volume 2. In the first, Hewson Swift reviewed the development of the quantitative cytology of nucleoproteins, and in the second, David Glick gave a critical review of quantitative histo- and cytochemistry, covering the analysis of large single cells, microdissected samples, and whole microtome sections. Glick discussed centrifugal separation, the use of absorption histospectroscopy, and radioautographic analysis of small and large cells. In the same volume, J. Chayen gave a review of work on the intracellular localization of vitamin C, a technique I described earlier in this chapter. A review of the enzyme histochemistry of gustatory and olfactory epithelia was provided by Baradi and myself. Other subjects covered included reviews on phosphatases, a redox pump, growth of explanted tissues, ion secretion, bacteria, electron microscopy of tissue sections, and others. Eight countries were represented among the authors. Authors from the United States were in the majority with seven articles, and other authors were from Australia, France, Ireland, Belgium, Holland, and Germany, as well as three from England. While it is invidious to pick out specific authors as being especially distinguished for their excellent contributions to the first ten volumes, we are proud to mention a few of them: Asboe Hansen, Max Alpert, Christian de Duve, Ed Dersey, Holger Hydtn, A. G. Everson Pearse, Roy G. Williams, RenC Couteaux, Alfred Marshak, Albert Coons, L. C. U. Junquiera, F. Sjostrand, Jerome Gross, Arther Pollister, Don Fawcett, Paul Weiss, Johannes Rhodin, Eduardo de Robertis, Charles Oberlin, Saul Wischnitzer, and C. M. Pomerat.

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D. PROGRESS The volumes continued to come out at the rate of one a year until 1966, when they were increased to two volumes per year. We produced three volumes per year during 1973 and 1974, and four volumes between 1975 and 1977. The rate of five to seven volumes per year has continued since 1978, and it is likely to remain at that level for some time. During the 1970s, we also introduced occasional special volumes in which all articles were related to a specific subject and were edited or written by someone who was a specialist in that area. For the first two decades of the review, that is from 1952 to 1970, we coeditors ourselves combed the literature to select authors and subjects for review, and from time to time we asked our colleagues in cell biology for suggestions. However, the increase in research and publication in cell biology made it necessary to formalize this procedure. We appointed a board of Advisory Editors whose knowledge covered an extensive area of cell biology. They were requested to send in their suggestions each year, and the job of writing to the proposed authors was shared by the two coeditors. The names of the first board of Advisory Editors were published in Volume 29, 1970. The distinguished members of this board were H. W. Beams, W. Beermann, Howard A. Bern, W. Bernhard, Gary G . Borisy, Robert W. Briggs, R. Couteaux, B. Davis, N. B. Everett, Don Fawcett, H. Holter, Winfrid Krone, K. Kurosumi, Giuseppe Millonig, Andreas Oksche, Jean-Paul Revel, Helmut Ruska, Wilfred Stein, Elton Stubblefield, Hewson Swift, J. B. Thomas, and Tadashi Utakoji. Ten of the members were from the United States, four from Germany, two each from France and Japan, and one each from Italy, Denmark, Israel, and Holland. Of these advisors, 11 are still with us after 15 years (and 100 volumes). They are Beams, Bern, Borisy, Couteaux, Kurosumi, Millonig, Oksche, Revel, Stein, Swift, and Utakoji. We owe a special thanks to these eleven for their many contributions and for the prestige their names have lent to the International Review of Cytology over the years. In 1967, Kwang Jeon, who was working at the Center for Theoretical Biology at the State University of New York with Dr. Danielli, was appointed Assistant Editor. Dr. Jeon’s name first appeared on the title page of the International Review of Cytology in Volume 22. Kwang Jeon has made a fine contribution to the series. After more than 30 years, each of us coeditors was well satisfied with the work and cooperation of the other. In all that time we had never had a confrontation or a serious disagreement. As far as I am concerned it was a perfect association, and I believe Jim Danielli felt the same. Danielli’s death in 1984 was not unexpected after some months of serious illness, but it still came as a shock. It was hard to believe that man who was himself so much a part of the history of cell biology,

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with his membrane studies, his work on senescence and on the synthesis of artificial cells, and his work in many other areas, had been taken from us. I cannot begin to indicate how sorry I am that his death stopped him from seeing the 100th volume in print and from being the joint author with me of this chapter. All of us who had worked with him felt we suffered a serious loss, and it was reflected in the discussion we had concerning the future of the International Review of Cytology.

V. The Future of the Znternational Review of Cytology: A Survey of Cell Biology After many conferences, it was decided that the new editorial format would consist of myself as Editor-in-Chief and two Associate Editors, Kwang Jeon, and Martin Friedlander, then from The Rockefeller University. This decision was made toward the end of 1984, and a meeting to discuss future plans was held early in 1985. The first meeting of the new editorial board to plan future issues of the International Review of Cytology was held in June 1985. The IRC will gain much from two Associate Editors who, between them, cover a wide area of knowledge and are very active in their special fields. Thus, the International Review of Cytology is now underway with its new editorial structure, but its course remains the same, that is, to review the fastadvancing fronts of cell biology, to “publish critical discussions of data published elsewhere and of new theoretical material. ” The majority of contributions will continue to be made by invitation. We will continue to solicit suggestions for subjects to be reviewed and authors to review them from our 35 Advisory Editors, and the Board of Editors will then discuss these suggestions and issue invitations to authors to write appropriate reviews. No publications of this type can succeed without the willing cooperation and expertise of the publishers, and we have been fortunate in our choice of Academic Press. In the beginning, we dealt directly with Kurt Jacoby, whose experience and insight proved a tower of strength to us. Later, as he became older, he delegated much of the work to his younger colleagues, who have proved most helpful and enthusiastic for the success of the series. The subject of cell biology continues to grow and, from being an academic study confined to the laboratory, it has burgeoned to a level at which it has a profound medical and social impact. The Watson and Crick DNA helix has turned into recombinant DNA with all its practical results and implications for the future. The International Review of Cytology plans to keep pace with these and other developments, and the next 100 volumes can be expected to bring up-to-date

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reviews of the important and exciting developments in the field of cell biology to its host of loyal readers.

REFERENCES Bourne, G. H. (1933a). Nature (London) 132, 859. Bourne, G. H. (1933b). Nature (London) 132, 874. Bourne, G. H. (1934). Ausr. J. Exp. Biol. 12, 123. Bourne, G. H., (1942). “Cytology and Cell Physiology,” 1st Ed. Clarendon, Oxford. Danielli, J. F., and Davson, H. (1935). J. Cell. Physiol. 4, 495. Danielli, J. F., and Harvey, E. N. (1935). J. Cell. Physiol. 4, 483. Gorter, E., and Grendel, F. (1925). J. Exp. Med. 41, 439. Rosen, R. (1985). J . SOC.Biol. Struct. 8, 1. Szent-Gyorgyi, A. (1928). Biochem. J . 22, 1387. Yapp, E. B., and Bourne, G. H., eds. (1957). “The Biology of Ageing.” Institute of Biology Symposium, No. 6.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 100

Development of Centrifuges and Their Use in the Study of Living Cells H. W. BEAMS AND R. G. KESSEL Department of Biology, University of Iowa,Iowa City, Iowa 52242

I. Introduction Gravity is among the most important environmental conditions to which living organisms are constantly exposed and to which they must conform. It acts at the earth’s surface in a given direction and at a relatively constant intensity. Early naturalists were curious about the fact that, regardless of their orientation when planted, young seedlings would generally grow with their roots directed downward (positive geotropism) and their shoots upward (negative geotropism). However, the stimulus for this behavior was unknown until the centrifuge experiments of Knight (1806), which provided evidence to show that gravity was in some way responsible for directing the orientation of the plants. Taking advantage of a small stream which traversed his garden, Knight devised a water wheel in such a way that seeds could be planted in various positions on the wheel that rotated around a vertical axis, thus developing a centrifugal force similar to, but several times greater than, gravity (Fig. 1). He observed that the plants responded in a characteristic way to the centrifugal force or accelerated gravity: the roots grew radially away from (centrifugally) and the shoots radially toward (centripetally) the axis of rotation. Thus, the centrifugal force overrode the normal pull of gravity on the young growing plants. Sachs (1882) found when growing plants were slowly rotated (one turn in about 15 to 20 minutes) about a horizontal axis, a condition which constantly changed their direction and orientation to the pull of gravity, that the roots and shoots grew in various directions relative to the gravitational field, largely overcoming the normal effects of gravity on their growth and orientation. Even today the different responses of plants to gravity, “positive for roots, negative for stems, and diageotropic for branches and leaves, are an aspect of development which is not at all well understood” (Galston, 1971), but one that is currently being actively studied (Audus, 1969; Larsen, 1971; Gray and Edwards, 1971; Iversen, 1982). Animals too, including man, have been studied relative to the effects of gravity and centrifugation on their locomotion, orientation, and various anatomical and physiological systems (Wunder and Lutherer, 1964; Wunder, 1966, 15 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any farm reserved.

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FIG. 1 . Diagram of initial centrifuge method using a wheel to study effects of centrifugal force on plants. After Knight (1806). Figure reproduced from Smith et al. (1942). FIG.2. Diagram of a low-speed ultracentrifuge developed by Svedberg and Nichols. For explanation of figure, see Svedberg and Pedersen (1940). FIG. 3. Diagram of an early air turbine ultracentrifuge developed by I. W. Beams. See J. W. Beams (1937) for additional explanation.

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1971; Halstead, 1984). In this connection, Brown (1971) has stated that “they have learned to cope with and make use of it-learned in the evolutionary as well as the ontogenetic sense. Recent advances in relatively long periods of space travel have produced a special need for further study on the biological effects of different intensities of gravity-accelerated (centrifugation), reduced, and the absence of gravity (weightlessness)-on man, plants, and other organisms. In fact, a number of studies have been made and more are anticipated on this subject and the possibility of eventually establishing an orbiting biological station in space (Taylor, 1977; Klein, 1981; Halstead, 1984). There is a large literature dealing with the use of the centrifuge, especially the ultracentrifuge, which has played an important role in biochemistry and industry. These studies also usually describe the various types of centrifuges used, the techniques involved, the mathematical analysis of the results obtained, and the basic concepts of the sedimentation theory (Svedberg and Pederson, 1940; J. W. Beams, 1937; Lewis and Weiss, 1976; Schachman, 1959; Williams, 1963; Pickels, 1944, 1952; Birnie and Rickwood, 1978; Rickwood, 1984). ”

11. History of Centrifuge Development As pointed out by Sheeler (1981), “The practical application of the centrifugal force dates back more than a thousand years to the extraction of ‘tung oil,’ a substance used in paints and varnishes. . . .” However, its use as an experimental method in biology is much more recent. Many different types of apparatus have been used to generate centrifugal force in addition to those used by Knight (1806) and Sachs (1882). These include various types of hand, water, oil, air, and electrically driven centrifuges, the flywheel of an engine (Morgan, 1903), and parts of a cream separator (Mottier, 1889; Svedberg, 1940). Needless to say, the centrifuge has proven to be a most valuable instrument for separating particles of different size, shape, and density from solutions, and it has found wide application in biochemistry, industry, and the clinical diagnostic laboratory for the separation and concentration of a variety of substances, including viruses (Lewis and Weiss, 1976). The Sharples super centrifuge with a single bearing revolved a maximum of 40,000 rpm and exerted a force of 41,000 g (E. N. Harvey, 1932a). We intend here to present a short history of the development of the ultracentrifuge and to list some of the more interesting aspects of the effects of ultracentrifugation on living cells and on their structure, physical properties, vitality, survival, and development. In this connection, Mazia (1961) has stated that “ultracentrifugation of organelles within the living cell provides a means of in vivo manipulation of living components,” which, we might add, is not readily available by other methods. Because of the breadth of this subject only a small segment of the many papers

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dealing with it can be cited here. Further references to the literature may be found in the bibliographies of the papers cited.

OILTURBINE ULTRACENTRIFUGE A. SVEDBERC An important advance in the technique of centrifuging came in 1924, when Svedberg and Rinde developed a relatively low-speed, electrically driven centrifuge constructed in part from the components of a cream separator (Fig. 2). They called this instruments a low-speed ultracentrifuge, and the displacement of the particles could be observed and photographed during the experiments (Svedberg and Nichols, 1923; Svedberg and Pedersen, 1940). Studies by Svedberg and Rinde (1924) and their associates were directed at improving the speed of rotation and the elimination of convection occurring in the rotor while in operation. These conditions were accomplished by driving the rotor by means of a small turbine operated by a stream of oil under pressure and by enclosing the rotor in an atmosphere of hydrogen. This development led to the construction of a more sophisticated ultracentrifuge capable of producing forces of several hundred thousand times gravity. By use of this instrument they were able to determine the molecular size and weight of a number of proteins, thus opening a whole new field for important biochemical investigations. It is not clear whether or not the Svedberg ultracentrifuge was ever used to study living cells. For a detailed history of the construction and development of the Svedberg oilturbine ultracentrifuge, see Nichols (1928), Svedberg and Pedersen (1940), Gray (1951), and Pedersen (1976). Svedberg (1934) recognized the need for the development of a less expensive and less complicated ultracentrifuge when he said, “Measurement in strong centrifugal fields requires a complicated and expensive machinery for handling it. ” Because the oil-turbine ultracentrifuge was one of the most reliable means for determining molecular weights of substances such as proteins, Svedberg remarked that “this circumstance may serve for a justification for wasting money and time on the construction and use of this unwieldy tool,” the use of which, it may be added, resulted in Svedberg receiving the Nobel Prize in 1926. It was clear by this time that a great need existed for the development of a less costly and less complicated apparatus to generate high centrifugal force that could be made available to a large number of investigators. In fact, as Gray (195 1) pointed out, fewer than one dozen ultracentrifuges of the Svedberg type were ever made. The answer to this problem came with the development of the air-turbine drive ultracentrifuge first described by Henriot and Hugenard ( 1925, 1927) and subsequently developed into a practical laboratory ultracentrifuge by J. W. Beams and collaborators (J. W. Beams, 1930, 1937; J. W . Beams and Weed, 1931; J. W. Beams et al., 1933). Because this type of air-turbine drive

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ultracentrifuge has been used in studies dealing with the effects of high centrifugal force on living cells, a short description of it will be included here. B . AIR-TURBINE ULTRACENTRIFUGE The type of air-turbine ultracentrifuge that has been generally used in the study of living cells is illustrated in Fig. 4. It is composed of two basic components, a stator usually constructed of duralumin or brass, and a rotor constructed of a strong metal such as alloy steel or duralumin. The rotors may vary considerably in size; we have found rotors of 15 to 40 mm in diameter the most suitable for our studies. The rotor may be machined to carry small plastic tubes (Fig. 4)or to form a small bowl closed at the top by a small screw cap (Fig. 4).When in operation, compressed air enters the stator cup and escapes through the diagonally bored holes in the stator cone, where the jets of air impinge on the flutings of the cone-shaped base of the rotor (Fig. 4).This causes the rotor to rise slightly above the stator cone, where it rests on an air column and is forced to rotate by the jets of air striking the flutings of the rotor. The rotor is stabilized on the supporting air cushion and prevented from being blown out of the stator cup because of the forces (Bernouli) produced by the air pressure and its motion between the rotor and the stator cup (J. W. Beams et al., 1933). The rotor speed is regulated by the air pressure driving it, and the temperature of the rotor varies only slightly from that of the air flow supporting and driving it (E. N. Harvey, 1932a). While this design of the air-turbine ultracentrifuge has been used to a limited extent for biochemical studies, the slight convection caused by the rotor running in air often led to an incomplete separation of the material being centrifuged. This problem was eliminated (J. W. Beams, 1937) by simply placing the rotor in a vacuum chamber and joining it by means of a flexible shaft to an air-driven, air-supported turbine (Fig. 3). Many modifications of the air-turbine ultracentrifuge have been made to fit the needs for use in special investigations. This design greatly reduced the friction, heating, and hence convection of the substance being centrifuged, a condition which has made it an important and economical instrument for the sedimentation of small particles and molecules. Likewise, it was first used by J. W. Beams to separate uranium 235 from uranium 238, the result of which has led to the development of the so-called “gas centrifuge,” which is at present widely used in the uranium enrichment programs of several countries (Olander, 1978; Russel, 1985). Relatively small and inexpensive air-driven ultracentrifuges (airfuge), capable of generating forces up to 160,000 g or more, are now produced commercially (Chervenka, 1980). The development of the electron microscope together with that of the ultracentrifuge has permitted investigations into the more highly complex structural organization of cells.

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FIG. 4. Photo illustrates the rotor (R) and stator (S) components of an air turbine ultracentrifuge. FIG.5. Photo of the microscope centrifuge. From E. B . Harvey (1937).

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It is interesting to note that more recently developed small rotors (steel balls) have been spun by means of a magnetic field at speeds of “1.5 million revolutions per second” producing at their periphery more than a billion times gravity (J. W. Beams, 1961). By use of a similarly designed instrument Katano and Shimizu (1979) have obtained centrifugal forces of over 100 million times gravity. C. THE MICROSCOPE CENTRIFUGE The microscope centrifuge for use in the study of living cells was first described by E. N. Harvey and Loomis (1930). It was subsequently modified and developed by E. N. Harvey (1931a,b, 1932a,b, 1934). The microscope centrifuge was developed to observe under relatively high magnification the changes occurring in living cells while in a centrifugal field of several thousand times gravity and to compare these observations to those seen in the same cells after removal from the centrifuge (Figs. 5-8). The optical system of the microscope centrifuge consists of a motor driving a rotating disc which is fitted with a specially arranged lens system of a compound microscope (Fig. 5). The image of cells while being centrifuged at 10,000 rpm is comparable to that observed through a microscope with a similar lens system. E. N. Harvey (1934) also adapted the air-turbine ultracentrifuge (J. W. Beams et al., 1933) rotor to perform as a centrifuge microscope. With this instrument he was able to obtain relatively clear images of cells being subjected to a centrifugal force of 84,000 g , and he suggested that the intensity of the centrifugal force generated by this instrument was limited only by the strength of the glass chamber carrying the cells; “this might be put at 200,000 X g” (E. N. Harvey, 1932a, 1934). He noted only a slight variation in temperature within the rotor during its operation. Pickels (1936) also adapted the air-turbine ultracentrifuge to function as a microscope centrifuge.

D. SUSPENSION MEma One of the important problems in the centrifugation of many types of living cells and tissues is to prevent them from being crushed by the action of the high centrifugal force. In the case of isolated cells, this condition may be largely eliminated by supporting them in an isotonic medium of similar density, such as isotonic sucrose, neutralized gum arabic, soluble starch, or fiscol. When centrifuging portions of organs or tissues, such as liver, the pieces are placed in the rotor containing a physiological solution (Beams and King, 1934b; Guyer and Claus, 1939, 1942). The cells adjacent to the centrifugal side of the rotor or bowl may be somewhat compressed or flattened while serving as a kind of buffer or cushion for those more centripetally located in the tissue.

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111. Effects of Centrifugal Force on Living Cells

A. ANIMALEGGS Early naturalists observed that the eggs of several different species of animals were composed of different visible materials suspended in a relatively clear substance, the protoplasm. They became curious as to the nature, function, and importance of these various materials in the life history of the cell. They noted that certain of the cellular materials, such as the starch grains in certain plant cells and the pigment and yolk materials in the frog’s egg, were displaced when the eggs were inverted relative to the field of gravity. This condition suggested that the various visible materials were not all of the same density. Eventually, low-speed centrifugation was used to stratify more completely the intracellular materials in an effort to further study their function. However, Morgan (1927), in whose book references and discussion of the early literature may be found, commented concerning it that “the speed of rotation was not high enough to bring about a quick separation of the materials of the animal egg” and that it was left for Lillie (1906), Lyon (1906), Morgan and Spooner (1909), Conklin (1917), Wilson (1937), and others to use sufficiently high centrifugal force to cause a sharp stratification of the materials within the eggs and to record the effects of this treatment on their survival and subsequent development. As noted by Mottier (1 899), previous to this time no extensive studies of the effects of centrifugation had been made on the individual cells of plants, but several studies dealing with its effect on the plant as a whole (geotropism) had been made. Following these initial studies, a large number of investigations were begun to determine in more detail the effects of various intensities of centrifugal force on the eggs of the frog, Ascaris, various species of marine invertebrates, somatic cells, protozoa, and certain plant cells. These studies included (1) the structure, displacement, stratification, viscosity, and relative density of the different nuclear and cytoplasmic materials, (2) the effects of this treatment on the development of the cells, especially their viability, polarity, symmetry, division, growth, and differentiation, and (3) the ultrastructure and biochemistry of the cells, especially as they relate to the organization of living matter. The importance of these studies has been emphasized by de Duve (1979, who said “I have roamed through living cells, but with the help of a centrifuge rather than of a microscope,” and in like manner Watson (1976) has stated that the centrifuge is “perhaps the most striking contribution of physical chemistry to the study of biological molecules. . . .” To this Mazia (1961) has added, “Of the various methods of studying the physical properties of bodies inside the cell, none is more free from the hazards of artifact than the use of centrifugal force.” It also provides a means of manipulating the components within the living cell.

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The results of centrifuging the eggs of many different invertebrate species, as well as those of the frog, have provided evidence that their contents may be stratified in the order of their relative specific gravities (Morgan, 1927). Of special interest in this connection is to what extent this treatment may “alter the normal course of development and thereby demonstrate the morphogenetic nature of some of the displaced substances within the egg protoplasm” (Costello, 1939). It is not possible here to list all of the many studies that have been made on this subject in a wide variety of animal species. Only a few special cases will be cited to illustrate the different types of reactions the eggs of different species have to centrifugation. Extensive studies on both fertilized and unfertilized eggs of many different species of animals have helped to determine the nature and stage in which the cytoplasmic determinants occur and become morphogenetically active. From such studies, eggs of different species have sometimes been referred to as “mosaic,” “intermediate,” or “regulative” depending upon the stage at which the differentiation capacities of the egg occur. For literature dealing with the effects of centrifugation on the eggs of a variety of species the reader is referred to Morgan (1927), Conklin (1931), Wilson (1937), Harrison (1945), Raven (1959), Davidson (1968), Reverberi (1971), Dohmen and Verdonk (1979), Whitaker (1979), Slack (1983), and Jeffery (1985). 1. Regulative Eggs (Arbacia) Although a few earlier studies had been made on the effects of centrifuging the Arbacia egg, it was Lyon (1906, 1907) who first used sufficiently high force (6400 g) to produce a displacement and sharp stratification of the materials in both the unfertilized and fertilized eggs of Arbacia. He observed that the visible contents of the egg stratified into four distinct regions, arranged from the centrifugal to the centripetal poles as follows: (1) pigment, (2) granules, (3) clear layer, and (4) a layer of a white-appearing, compact group of globules (fat?). He observed that if the stratified eggs were allowed to stand for sufficient time the materials underwent a redistribution to a more or less normal condition and, if fertilized, developed more or less normally. Centrifuged fertilized eggs showed some abnormal distribution of the pigment in the cells of the morula stage, but “no difference between the plutei from normal and,centrifugalized eggs could be noted as regards activity or longevity” (Lyon, 1907). Among the extensive studies of the effects of centrifugation on the eggs of echinoderms is that of E. N. Harvey and E. B. Harvey. When the unfertilized eggs of Arbacia punctulata are strongly centrifuged (10,000 g) in an isotonic sucrose solution of the same (graded) density, they stratify, elongate, and become dumbbell shaped and separate into two nearly equal halves: one colorless and one pigmented (E. B . Harvey, 1932, 1933a,b, 1936). Each half, in turn, when treated in a similar way, separates into clear, granular, yolk, and pig-

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H. W. BEAMS AND R. G . KESSEL

ARBAClA PUNCTULATA

(2 0 ........... .*.;. ... .......... , *.....

.

W h i t e Half

..... :: . ........ .................. ................ ..*.'....,,..:.:;. .;:;~;;~:*....:.:.:. .:*.' .::., ::,::; .,:::::

Whole Egg Centrifuged

y . . &

Red Half

FIG. 6. Diagram of centrifuged Arbuciu eggs illustrating the fragmentation produced by centrifugal force. From E. B. Harvey (1936). FIG.7. Photomicrographs of unfertilized, centrifuged Arbuciu eggs showing stages in separation similar to those shown in Fig. 6. Photomicrographs are oriented so that the direction of applied centrifugal force is toward the bottom of the figure. From E. B . Harvey (1937). FIG. 8. Photomicrographs of fertilized, centrifuged Arbuciu eggs showing more stretched eggs resulting from changed viscosity after fertilization. From E. B. Harvey (1937).

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mented quarters (Figs. 6 and 7). Each quarter, when treated with a suitable parthenogenic agent, cleaves and in some cases forms a blastula (parthenogenetic merogons) composed of as many as 500 cells. Similar results were obtained by centrifuging the eggs of several different species of sea urchins. From these results Harvey (1936) concluded that no special visible or movable granules are essential for development, and “therefore it must be the ‘ground substance’ which is the material fundamental for development-the matrix which is not moved by the centrifugal force and which in the living egg is optically empty.” Figures 7 and 8 illustrate centrifuged Arbacia eggs. Figure 7 is a photograph of a group of unfertilized eggs taken immediately upon removal from the centrifuge microscope, while Fig. 8 shows a group of fertilized eggs photographed in the centrifuge microscope while rotating at 10,000 rpm. These figures illustrate two different conditions in the centrifugation of Arbacia eggs: ( I ) some contraction occurs in the egg upon removal from the centrifuge; and (2) there is a marked stretching because of the lower viscosity in the fertilized eggs. Anderson (1970) centrifuged the eggs of Arbacia and found that they become stratified into a number of nonhomogeneous layers. With the centrifugal force used, the cortical granules were not displaced from the cortical ooplasm. Continued centrifugation of initially stratified eggs results in the separation of the eggs into nucleated and nonnucleated halves. Upon further centrifugation the halves may be separated into quarters. When activated by sperm or hypertonic seawater, none of the fractions (halves and quarters), except the nucleated half, will develop to plutei larvae. In fragments of Nereis eggs produced by centrifugation, while they are capable of fertilization, only the ones containing the germinal vesicle will cleave (Costello, 1940). Evidence from recent studies on the cortex of certain invertebrates’ eggs indicates that it is not usually displaced by centrifuging as is the endoplasm. Because of this, it has been suggested that the factors for determining polarity and symmetry probably reside in the cortex (Raven, 1959, 1970).

2 . Mosaic Eggs The “mosaic” egg, as opposed to the “regulative” egg, usually contains better-defined regions possessing specific morphogenetic or determinative substances, which, if displaced or disturbed by centrifugation or by other methods, usually give rise to malformed larvae. The annelids, nematodes, mollusks, insects, and ascidians are examples of mosaic eggs. Extensive reviews of the literature in which the centrifuge has been widely used in the study of mosaic eggs may be found in the publications of Hegner (1909), Wilson (1937), Morgan (1927), Conklin (1917, 1931), Raven (1959, 1970), Davidson (1968), Clement ( 1971) , Reverberi ( 1979), Dohmen and Verdonk ( 1979), and Jeffeq ( 1985). The results of the many studies of centrifuging a wide variety of invertebrate eggs seem to indicate that the difference between the regulative and mosaic type of eggs is fundamentally one of degree and time in which differentiation and

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segregation of the formative determining substance become active in the egg as mentioned above. Most of the evidence indicates that the various visible bodies observed in many centrifuged eggs, such as pigment granules, yolk bodies, and lipid bodies, are not the “organ-forming substance.” Of special interest is the nature of the polar lobe in certain annelids and mollusks. It has been revealed by means of centrifugation and deletion techniques to contain important and essential determinants. For a recent discussion and review of the literature relative to this subject see Dohmen (1983a,b) and Dohmen and Verdonk (1979). Davidson et al. (1965) found that removing the polar lobe from the eggs of flyanassa obsoleta depresses gene activity for many hours. This condition, coupled with the classical studies showing the presence of morphogenetic substance in the cytoplasm, suggests ‘‘that gene activation in early embryogerlesis may be mediated by cytoplasmically localized materials, thus accounting for initial nuclear differentiation” (Davidson et al., 1965). Jeffery (1983a) has reviewed the literature relative to the problem of RNA and embryonic localization. In a recent study of the Chaetopterus egg, Jeffery (1985) found that, by centrifuging the egg, the cortical organelles as well as poly(A)+ RNA, histone mRNA, and actin mRNA molecules were displaced to the centrifugal end of the cell. “ f n situ hybridization of stratified eggs extracted with NP 40 indicated that the CD [cortical cytoskeletal domain], with its associated organelles and mRNA molecules, is displaced to the centrifugal zone as a unit” (Jeffery, 1985). Further studies of Chaetopterus eggs revealed that the CD is displaced by centrifugation immediately prior to first cleavage into only one of the first two blastomeres. Embryos containing the CD in only one blastomere continued to cleave but formed defective larvae, a result suggesting that the cortical CD is necessary for normal development (Swalla et al., 1985). Frog eggs have also been subjected to varying intensities of centrifugal force for different periods of time with results ranging from little effect on the development of the embryos (Morgan, 1927), to twinning (Gerhart et al., 1983), and to the formation of dual neural tubes (Beams et al., 1934).

FIG. 9. Diagram of Ascaris rnegalocephala egg showing tetrapolar spindle resulting from blocked divisions due to centrifugation. Two masses of chromatin resulting from chromosome diminution are also shown. From King and Beams (1937a). FIGS. 10, 11, 13. Photomicrographs illustrate effects of ultracentrifugation on Ascaris megalocephala eggs at different stages of chromosome diminution. Direction of applied centrifugal force is toward bottom of figures. The ultracentrifugation has blocked cytokinesis so that mitotic chromosomes (MC) and eliminated chromatin (EC) masses resulting from chromosome diminution are present. From King and Beams (1938). FIG. 12. Photomicrograph of multinucleate Ascaris egg resulting from blocked cytokinesis during ultracentrifugation. From King and Beams (1938). FIG. 14. Photomicrograph of centrifuged Fucus egg. Rhizoid outgrowth (toward bottom of figure) occurs at the centrifugal end of a fertilized egg. Reproduced from Whitaker (1940).

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’

3. Germ-Cell Determinants Boveri (1910) and Hogue (1910) used the centrifuge method to study the germ-cell determinants in the eggs and early embryos of Ascaris. As a result, they were able to show that chromosome diminution was not inherent in the chromosomes themselves, but due to factors residing in the cytoplasm. King and Beams (1938), by ultracentrifuging the fertilized eggs of Ascaris, blocked cytokinesis while division of the chromosomes continued. Under these conditions, the cells destined to form germ plasm undergo chromosome diminution (Figs. 9-13). King and Beams (1938) suggested that a continuous formation of the diminisher substance occurs in the blocked, noncleaving cells, and when the concentration becomes critical, chromosome diminution occurs in the cells destined to form the germ line. Hegner (1909) has shown that the eggs of certain beetles, upon centrifugation, become stratified into three layers, and the pole disc containing the germ-cell determinants is displaced en masse to the heavy end of the egg. He concludes that “the cytoplasm and nuclei of centrifuged eggs are forced out of their usual positions, but often normal development takes place. ” Howland (194 1) reported that the eggs of Drosophila melanogaster, when centrifuged, stratify into five zones. The pole plasm was not markedly displaced from its normal terminal position. Abnormalities such as deletions, dislocations, and abnormal rotations of the embryo occurred. For more recent studies of the effects of centrifugation on the developing oocytes of insects refer to Bownes (1977), Brown and Schubiger (1977), and Kalthoff (1983). 4. Polarity and Symmetry Centrifugation has been frequently used to study the polarity and symmetry of many invertebrate eggs. Only a few examples dealing with these subjects will be cited here. Lillie (1909) suggested that the polarity of the Chaetopterus egg is established in the ovary, the unattached end becoming the animal pole and the attached end the vegetative pole. Upon centrifuging the eggs of this organism, the visible granules were displaced and stratified, but no evidence of mass movement of the ground substance occurred. Accordingly, he concluded that the factors for determining polarity and bilaterality must reside in the “ground substance.” In a similar study on the eggs of Arbacia, Morgan and Spooner (1909) found also that the factors which determine polarity seemed to be independent of any of the visibly displaceable material in the egg. By use of the centrifuge in a study of the polarity of the Crepidula egg, Conklin (1917) concluded that “polarity, and what is generally implied by the ‘organization of the egg’, resides in the cortical layer of cytoplasm and in the internal frame work of spongioplasm.” Raven (1970) likewise suggests that the factors for determining polarity in eggs are probably localized in the cortex.

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Pease (1939) concluded from his studies of the effects of centrifuging the egg of Dendruster that two factors are involved in fixing the dorsal-ventral axis, one which is located in the cortical layer that is not displaced by the centrifuging and one located in the endoplasm that may be displaced by the centrifuging. The method of centrifugation has proved to be an important one for the study of polarity in certain plant cells. References to the large literature dealing with the effects of various physicochemical methods, including ultracentrifugation, on the polarity of certain plant cells may be found in the review by Bloch (1943). Whitaker (1940) extensively studied the various physicochemical factors, including ultracentrifugation, on the polarity of the eggs of Fucusfurcutus. He found that when the centrifuged eggs were reared singly or in small cultures at a pH of 7.8 to 8.1 a high percentage of the rhizoids emerged at their centrifugal poles (Fig. 14). Beams (1937) had previously observed that when ultracentrifuging the eggs of Fucus serrutus and allowing them to develop in crowded cultures their polarity in a high percentage of cases was unaffected. Whitaker (1940) observed that the eggs of Fucus furcutus, when ultracentrifuged, showed a high percentage of the rhizoids forming on their centrifugal halves. He also observed that when the ultracentrifuged eggs were allowed to develop in crowded cultures the “group effect” seemed to override the centrifugation effect so that under these conditions his findings were similar to those of Beams (1937). Pollen grains of the snapdragon, when ultracentrifuged and allowed to develop, form a high percentage of the pollen tubes at their centrifugal poles. Schecter (1935) was able to alter the polarity of the alga Grzfithsiu by centrifugation. When Elodea cells are ultracentrifuged at 350,000 g for 30 minutes a marked stratification of their constituents occurs (Beams, 1949). The chloroplasts and cytoplasm are displaced to the centrifugal end of the cell just centrifugal to the vacuole. This treatment does not usually kill the cells and recovery generally follows. At first, rapid Brownian movement takes place in the cytoplasm, causing some loosening of the packed chloroplasts. The cytoplasm continues to chum and extend centripetally along the surface of the vacuole, carrying with it some of the small clumped groups of chloroplasts. Eventually, the cytoplasmic streaming is reestablished and the chloroplasts assume their normal distribution in the cytoplasm. Only about 7% of the cells show a reversed direction of cytoplasmic flow (polarity). Northern and Northern ( 1938) reported that repeated centrifugation and displacement of the chloroplasts through the cells of Spirogyru produces a marked lowering of their viscosity. Virgin (1949), using the centrifuge microscope, observed in cells of the leaves of Helodeu densu that the cytoplasm and chloroplasts were displaced to the centrifugal end of the cells. He was able to observe that the return movements of the plasma and chloroplasts start before the end of centrifugation, and he concluded, “We are therefore dealing with a motive force in the plasma that is of

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H. W. BEAMS AND R. G.KESSEL

such an order of magnitude that it is able to overcome superimposed force (the centrifugal force) in the opposite direction. ” B. VISCOSITY STUDIES It is interesting to note that protoplasm, “the physical basis of life,” may exist in a rather wide range of physical states. Its change under certain conditions from a sol to a gel and vice versa is well known and has been implicated in many different aspects of cellular activity, such as protoplasmic streaming, cell division, ameboid movement, organelle movements, and fertilization. Because of their relatively large size, the invertebrate egg, protozoa, and certain plant cells have been favorite objects for viscosity studies. Centrifugation studies on cytoplasmic streaming in plant cells have been made, and for references refer to Beams ( 1949). Seifriz (1936) stated that “protoplasm may be of any viscosity above a minimum of ten to twenty times that of water, to the practically infinite value of a firm jelly.” It is difficult to determine the absolute viscosity of cells because of the variation in density in different regions and the frequent viscosity changes occurring within them. More often the methods cited are used to determine the relative viscosity of the protoplasm of cells, that is, whether it is gel- or sol-like in consistency. Because of lack of space, we will mention only those methods commonly used to measure protoplasmic viscosity. These include (1) centrifugation, (2) the electromagnetic method, (3) microdissection, and (4) Brownian movement of granules. Discussion and literature pertaining to viscosity studies of cells may be found in Heilbrunn (1928, 1943), Seifriz (1936, 1942), and Chambers (1924). Kamiya and Kiyoko (1958) stated that the driving mechanism responsible for rotational streaming in Nitellu is located at the interface between the cortical gel and the outer edge of the endoplasmic layer. By use of the centrifuge, they observed that “the motive force, which is the shifting force generated at this interface, was determined in the internodal cell of Nitellaflexilis to be within the range of 1-2 dynes/cm2 at room temperature.” C. SOMATIC CELLS Somatic tissue cells, because of their relatively small size and high viscosity, usually require a higher centrifugal force for a longer period of time to displace and stratify thkir contents as compared to the larger, more fluid eggs previously described. This problem was largely overcome by the development of the airturbine ultracentrifuge previously described. It has permitted a study of the displacement and stratification of the organelles and other constituents in a

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variety of tissue cells (Beams, 1951; Moroz, 1984). We will mention a few ultracentrifugation studies that have been made on somatic tissue cells. 1, Organelles

During the late 1920s and early 1930s much debate occurred regarding the reality of the Golgi apparatus, first described by Camillo Golgi in 1898 (see Beams and Kessel, 1968b; Whaley, 1975, for discussion and literature). Because it could not be easily seen in the living cell, it was considered by many as an artifact, a special mitochondrion or vacuome. By aid of the ultracentrifuge Beams and King (1934a) found that uterine gland cells of the rat, when exposed to 400,000 g for 30 minutes, had the Golgi apparatus displaced to the centripetal end of the cell (compare Figs. 15 and 16). Similarly treated spermatocytes of Helix (Beams and Tahmisian, 1953) showed the Golgi apparatus to be displaced to the centripetal end of the cell and the mitochondria to the centrifugal end, thus demonstrating the sharp difference in specific gravity existing between these two organelles (Fig. 17). Ultracentrifuged root-tip cells of the bean also revealed the Golgi material (osmiophilic platelets of Bowen, 1928) to be displaced to the centripetal end of the cell (Fig. 18), thus providing strong evidence that it was a real component of the plant cell and homologous to the Golgi apparatus of animal cells (Beams and King, 1935). The evidence derived from ultracentrifugation and electron microscope studies of cells has established the Golgi apparatus as a real and important cellular organelle, and it was described in the recent words of Farquhar and Palade (1981): “The Golgi apparatus (complex)-( 1954- 1981)-from artifact to center stage.” 2. Blood a. Reticulocytes. As noted above, centrifugation may cause a displacement of the organelles in certain egg cells. Equally striking is the displacement of the organelles in the reticulocyte of the rat (Beams and Kessel, 1966b). Reticulocytes ultracentrifuged at 100,000to 400,000 g for periods of 5 to 30 minutes are illustrated in Figs. 22 and 23. The cells become greatly elongated, sometimes separated into two fragments, and their organelles are stratified from the centripetal end to the centrifugal end as follows: groups of ribosomes (polysomes), mitochondria, and a larger, more dense layer of cytoplasm. The latter contains many fine granules which may represent hemaglobin. Of special interest is that the ribosomes are not displaced from the cortex adjacent to the cell membrane (Figs. 22 and 23, arrows). The degree of stretching and the relative size of the centrifugal and centripetal portions of the cell vary with the degree of maturation and differentiation of the reticulocyte. The more mature the cell the less basophilic material is present until finally the erythrocyte stage is reached.

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b. Erythrocytes. As mentioned above, the erythrocyte has lost most, if not all, of the visible constituents present in the reticulocyte. In spite of this, ultracentrifugation of the erythrocytes clearly produces a layer at the centrifugal end of the cell that may be stained by Heidenhain’s hematoxylin (Fig. 21). If Mallory’s triple stain is used on similarly treated cells, three layers of distinct color may be clearly revealed, indicating that the rat erythrocyte contains at least two and perhaps three substances which differ not only in their relative specific gravity, but also physicochemically, as indicated by their differential staining reactions (Beams and Kessel, 1966a). The erythrocytes of the frog and Necturus, when ultracentrifuged, may be pulled into two or more fragments. The centrifugal portion contains the nucleus and the centripetal portion is nonnucleated (Beams and King, 1945). The fact that the fragments of the erythrocytes produced become rounded instead of maintaining a flattened shape indicates that a change in their viscosity and membrane properties has occurred. 3. Plant Zalokar (1960) centrifuged living hyphae of Neurospora at 50,000 g for 15 minutes whereupon they were sharply stratified. The hyphae were not killed by the centrifugation and were able to grow after the stratified layers became redistributed. A number of cytochemical tests were made on the stratified layers and the results compared to those obtained from centrifuged and homogenized hyphae. Zalokar concluded that “while the results of both methods were found to be in general agreement, some products of homogenization were considered to represent artifacts. When the mesophyll cells of the spinach leaf are centrifuged at a force of 350,000 g for periods of 15 to 20 minutes, their components become stratified in ”

FIG. 15. Photomicrograph of normal uterine gland cells of rat. Normal position of Golgi apparatus (GA) denoted. After Beams and King ( I 934a). FIG.16. Photomicrograph of ultracentrifuged rat uterine gland cells. Note that Golgi apparatus (GA) has been displaced centripetally in the cell, while the mitochondria have been displaced centrifugally in the cell. After Beams and King (1934a). FIG. 17. Photomicrograph of ultracentrifuged Helix spermatocyte. Note that Golgi apparatus (GA) has been displaced centripetally in the cell, while mitochondria (M)have been displaced centrifugally in the cell. After Beams and Tahmisian (1953). FIG. 18. Photomicrograph of ultracentrifuged root-tip cells of germinating bean. Dictyosomes (Golgi complexes) (GA) have been displaced to the centripetal end of the cells. After Beams and King (1935). FIG. 19. Photomicrograph of grasshopper spermatids. The centrifugal force has apparently caused separation of acrosomes (c) from some of the spermatid nuclei. After Beams (1948). FIG. 20. Photomicrograph of ultracentrifuged rat liver cell showing displacement of mitochondria (M) centrifugally. After Beams and King (1934b).

FIG.21. Photomicrograph of ultracentrifuged rat erythrocytes showing hematoxylin-stained material (black) displaced toward the centrifugal end of the cell. After Beams and Kessel (1966a). FIGS.22, 23. Transmission electron micrographs of ultracentrifuged rat reticulocytes showing stages in displacement of polyribosomes into centripetal end of reticulocyte. Applied centrifugal force is toward bottom of figures. Some polyribosomes in cortex (arrows) appear not to be displaced. After Beams and Kessel (1966b).

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the order of their decreasing density as follows: chloroplasts, peroxisomes, mitochondria, endoplasmic reticulum, Golgi bodies, and vacuole and lipid bodies (Beams et al., 1979). The starch grains are displaced to the centrifugal end and the plastoglobuli to the centripetal end of the chloroplast. In some extreme cases the chloroplasts are pulled into two unequal parts, the centrifugal fraction containing the starch grains, dense stroma, and stretched thylakoids and the centripetal fraction containing mainly plastoglobuli and lipid bodies. When whole duckweeds are treated in a similar manner to the spinach mesophyll cells, comparable results are obtained. The duckweeds were not killed by the ultracentrifugation and, in time, a redistribution of the stratified components, growth, and the establishment of a new colony occurred (Beams et al., 1979). The duckweed appears to be the most advanced organism in the evolutionary scale to withstand such a high centrifugal force and still survive. We have recently found that rotifers will survive a force of 200,000 for 7 days (Beams and Kessel, unpublished). Bouch (1963) centrifuged living excised roots of pea at 20,000 g for a period of 24 hours. The intracellular materials became stratified in the order of their relative densities. After centrifuging, the roots were placed in a nutrient media where they grew at a rate comparable to the controls. The stratified layers became redistributed to their normal condition in a relatively short time. 4. Cell Division (Animal and Plant) Few subjects have been more extensively studied than the mechanism of cell division. Our purpose here is to illustrate some of the effects that ultracentrifugation has on the nucleus and mitotic apparatus of certain cells of both animals and plants (Beams and King, 1936, 1938; Beams and Mueller, 1970). In all of the ultracentrifuged cells illustrated the force has been directed toward the bottom of the plate. All the diagrams in Fig. 24 are ultracentrifuged chick mesenchyme cells except the top left figure, which is a control cell which illustrates the nucleus, nucleolus, and interphase chromatin. Different effects of ultracentrifugation on these cells are illustrated in the diagrams (Fig. 24). The nucleus and cell membranes are extensively stretched. The stretching of the spindle fibers and the displacement of the chromatin are also illustrated. These results suggest that the chromatin is the densest material in the cell and is displaced centrifugally (Fig. 25). As this occurs, the spindle fibers may be greatly stretched, distorted, and sometimes detached from the chromosomes. In some cells the chromosomes are fragmented, but often portions remain attached to the spindle fibers at the kinetochores. In certain other cells the mitotic spindle may be displaced in toto (e.g., E. B. Harvey, 1935). In ultracentrifuged liver cells the chromatin is stretched in the direction of the centrifugal force, but at the centripetal end of the nucleus some of the chromatin is definitely attached to the nuclear membrane in a position between the pores (Figs. 26 and 27).

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Root-tip cells from germinating wheat seeds, like animal cells, have their mitotic apparatus markedly displaced (Fig. 28) by ultracentrifugation (Schaede, 1930; Beams and King 1938, 1939; Kostoff, 1938; Hilbe, 1941). The interphase chromosomes are often greatly stretched and displaced centrifugally, and the nucleolus is sometimes forced through the nuclear membrane (Figs. 29 and 30). Figures 32-34 depict cells in different stages of mitosis that have been subjected to ultracentrifugation. They frequently show the chromosomes torn free of the spindle fibers (Figs. 32 and 33), and those cells in anaphase or telophase often form binucleate cells (Figs. 28 and 34). Kostoff ( 1 938) observed that centrifuging the germinating seeds of several different plants gave rise to monosomic, trisomic, polysomic, and tetraploid cells. He also noted that centrifuging the seeds sometimes produced semisterile plants and various abnormal leaf structures. These results support the established view that the mitotic spindle is a relatively rigid body and the chromosomes are the most dense of the mitotic figure components. For literature relative to the effects of centrifuging plant cells, see Beams and King (1939). D. CANCERCELLS

Guyer and Claus (1939) and Cowdry and Paletta (1941) suggested that different types of neoplastic cells may react to ultracentrifuging in different ways, some showing little stratification as compared to normal cells, presumably because of their relatively high viscosity (Guyer and Claus, 1939, 1942). Other studies showed that the nuclei of cancer cells stratify readily, indicating a relatively low viscosity (Cowdry and Paletta, 1941). Beams and Kessel (1968a) reported that Ehrlich ascites tumor cells become greatly stretched when ultracentrifuged and the nuclei are displaced to the centrifugal end of the cell. The interphase chromosomes are more dense than the nucleoplasm and are stretched and displaced centrifugally. When this occurs, it was revealed that the interphase chromosomes adhere to the nuclear envelope in a way comparable to those shown in Fig. 3 1 . In general, it was observed that the

FIG.24. This series of diagrams shows effects of ultracentrifugation on interphase and mitotic chick mesenchyme cells. Normal cell is shown at top left. See Beams and King (1936) for additional deSCriptiOtl. FIG.25. Photomicrograph of ultracentrifuged dividing male germ cell of grasshopper. Displaced chromatin is located toward bottom of figure and centrifugal end of cell. Centrioles (C) are also present. From Beams (1948). FIG.26. Photomicrograph of ultracentrifuged rat liver cell showing interphase chromatin attached to nuclear envelope (arrow) at centripetal end of nucleus. After Beams and Mueller (1970). FIG.27. Transmission electron micrograph similar to Fig. 26. Nuclear pores (NP) and chromatin (Ch) are identified. After Beams and Mueller (1970).

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ascites cells may be more stretched and appear less sharply stratified than do many normal ultracentrifuged somatic cells. However, Beams and Kessel (1968a) pointed out that there is little direct evidence from their studies that the results observed are associated with the state of malignancy. Mateyko and Kopak (1964) investigated the physical properties of human gynecological tumors by both microsurgical and centrifugation methods. They observed that the cells are readily stratified from the centripetal to the centrifugal end as follows: “lipid cap, hyaline zone with vacuoles, light granules, nucleus and dense granules.” They noted a turbulent redistribution of the intracytoplasmic particles and also noted that the cells have a low cytoplasmic consistency as well as the ability to undergo cytoplasmic streaming. It is apparent that none of the ultracentrifuge studies on tumors to date seems to have established beyond a reasonable doubt any difference in viscosity between the malignant and normal cell. E. PROTOZOA

The centrifuge has been used as a tool in the study of the physical nature of a number of protozoa (McClendon, 1909; Yancy, 1931; Patten and Beams, 1936; King and Beams, 1937b; Daniels and Breyer, 1966; see Beams and King, 1941, for literature). 1. Paramecium

When Paramecium caudatum is centrifuged at 2 1,000g for 5 to 10 minutes in a solution of gum acacia of similar density, it becomes stretched and elongated. The chromatin of the macronucleus is displaced toward the centrifugal end of the organism (Fig. 35). In some instances, the chromatin is forced free of the achromatic matrix. The displaced chromatin does not usually degenerate in the cytoplasm, but reorganizes a new chromatin matrix. Animals which survive centrifuging regain their usual shape and the displaced materials return to their usual distribution (King and Beams, 1937b). Harvey (1932) reports that Jensen has used the centrifuge in a rather novel experiment to determine the motive force of a paramecium. He found that a

FIG. 28. This series of six drawings illustrates ultracentrifugation effects on root-tip cells of wheat in different stages of division. After Beams and King (1938). FIGS.29, 30. Photomicrographs showing stages in progressive displacement of nucleolus centrifugally in root-tip cells so as to become forced through the nuclear envelope. FIG.3 1. Photomicrograph of ultracentrifuged root-tip cells with centrifugally displaced chromatin. Note that some chromatin attachments persist centripetally at the nuclear membrane (arrow). FIGS.32-34. Photomicrographs of ultracentrifuged plant root-tip cells showing displacement of mitotic chromosomes from mitotic spindle.

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paramecium 0.25 mm long and weighing 0.000175 mg could lift 0.00158 mg or nine times its own weight. He calculates that it would take 600 paramecia to lift 1 mg.

2 . Amoeba Daniels and Breyer (1966) centrifuged Amoeba proteus and Pelomyxa illinoisensis at forces ranging from 70,000 to 140,000 g for varying periods of time. They were especially interested in the effects on the nucleus, which they found to be stratified into three layers: centripetal nucleoplasm, middle chromatin, and centrifugal nucleolar mass. The nucleolar mass was also separated into a centripetal electron-opaque layer and a centrifugal electron-lucent layer. They studied the structure of the nucleolar layers and their possible relationship to differences in radiosensitivity between the two species of amoeba. Heilbrunn (1943), by use of the centrifuge, determined that the viscosity of Amoeba dubia varied from 2 to 14 centipoise, depending somewhat on the temperature of the environment. Harvey and Marsland (1932) centrifuged Amoeba dubia in the centrifuge microscope and observed that the granules fall in jerks, suggesting a rather substantial organization within the cytoplasm. 3 . Euglena When Euglena sp. are centrifuged at 100,000 g for 2 to 3 minutes they are sharply stratified (Patten and Beams. 1936). The orientation of the stratified layers bears no special relation to the morphological polarity of the organism (Fig. 38); that is, the Euglena may orient itself in the centrifuge with the flagellar end pointing either centrifugally or centripetally. Paramylum grains and neutral red staining bodies are forced into the centrifugal half of the organism, the chloroplasts form a belt near the middle, and bodies identified as mitochondria collect in the centripetal half of the organism. In some cases, the components of the stigma seem to be slightly disorganized. Organisms usually recover from this

FIG.35. Photomicrograph of ultracentrifuged Paramecium showing stretched condition of the organism and the macronucleus. After King and Beams (1937b). FIG. 36. Photomicrograph of ultracentrifuged Gleocapsa with centrifugally displaced granular components. After Beams and Kessel (1977). FIG. 37. Photomicrograph of ultracentrifuged bacteria (Rhizobiurn). The bacteria have been stretched, and hematoxylin-stained material has been displaced toward the centrifugal end of the bacteria. After Beams et al. (1982). FIG.38. Diagram of ultracentrifuged Euglena showing stratified components. See Patten and Beams (1936) for additional details. FIG.39. Transmission electron micrograph of a portion of ultracentrifuged Anabaena. Note displacement of thylakoids from cortex of centripetal end (arrows). N, Nuclear material. After Beams and Kessel (1977).

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treatment and their components eventually assume a normal distribution within the Euglena.

F. BLUE-GREEN ALGAE Anabaena Beams and Kessel(l977) ultracentrifuged the cells of Anabaena and observed that they were stratified as follows: photosynthetic lamellae in the centripetal end, nucleoplasm in the central region, and dense granular cytoplasm in the centrifugal end of the cell (Fig. 39). In some cells, the photosynthetic lamellae are pulled away from the cortex, leaving bits of their structure still attached to it (Fig. 39). As the photosynthetic lamellae are displaced centrifugally a large vacuole appears in the centripetal end of the cell. The nucleoplasm does not possess a membrane and no evidence was found that it is in any way attached to the cortex or surface membrane. Figure 36 shows two ultracentrifuged Gleocapsa cells with displaced granules at the centrifugal end, stretched photosynthetic lamellae in the middle region, and nucleoplasm at the centripetal end of the cell. Many of these cells recover from this treatment.

G . BACTERIA Little evidence exists concerning the viscosity of the protoplasm of bacteria. This was reflected in the statement of Knaysi (1956) when he said, “The value of viscosity is, certainly, sufficiently high to prevent Brownian movement. When ultracentrifuged, certain bacteria such as Spirillum volutans (King and Beams, 1942) and rhizobia (Beams et al., 1982) have their intracellular contents displaced much in the same manner as eukaryotic cells. While a positive identity of the displaced components has not been made, they undoubtedly include the nucleoplasm. The rhizobia are considerably stretched by the action of the centrifugal force (Fig. 37). The fact that in certain blue-green algae and bacteria the nucleoplasm can be displaced through the cytoplasm without becoming mixed with it suggests that the nucleoplasm must possess considerable stability. An interesting question is: in the absence of a nuclear membrane or envelope, how is the integrity of the nucleoplasm maintained, and how are metabolic exchanges between the nucleoplasm and cytoplasm regulated? ”

H. GROUNDSUBSTANCE (CYTOSKELETON) Taylor (193 1) has pointed out that many nonliving colloidal systems have been successfully stratified in the ultracentrifuge as developed by Svedberg and oth-

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ers, “but to what extent the living ground substance would endure the rigors of such enormous forces (10,000-100,000 times gravity) and remain living, is, of course, exceedingly problematical. ” From the results obtained by ultracentrifuging living cells, it is evident that most of the visible inclusions may be displaced, stratified, and in some cases fragmented from the cell without killing it (Beams, 1943). Accordingly, the basic factors which endow the cell with life must reside in the nonvisible components of the ground substance (cytomatrix). A similar view was expressed by Frey-Wyssling (1948) when he stated, “For this reason the framework must either possess very coarse meshes or else it must be possible for the important groupings, whose mutual positions have been altered by centrifugation, to be restored to their original arrangements. Recent studies with the aid of the electron miscroscope, the high-voltage electron microscope, and immunofluorescence microscopy have established in the ground substance the presence of a cytoskeleton composed of microfilaments, intermediate filaments, microtubules, and microtrabeculae (Porter, 1984; Porter and Tucker, 1981; Brinkley, 1982; Jockusch, 1983, and many others). Since certain living cells will withstand a centrifugal force of 400,000 g for 10 days, it is interesting to speculate concerning the changes that may have occurred in the cytoskeleton during this treatment. That is, to what extent are the elements of the cytoskeleton disrupted, torn apart, and compressed by the rapid movement of bodies such as oil droplets, yolk bodies, mitochondria, Golgi bodies, pigment granules, chloroplasts, and starch grains through it? The answer to this question awaits further investigation. ”

IV. Concluding Remarks It seems clear that the development and application of the technique of ultracentrifugation to living cells and small organisms has proved to be a very useful cytological tool in the exploration of the nature of protoplasm. Many of the earlier studies were directed toward a better understanding of the basis for polarity, bilaterality, and differentiation, especially as studied in the female gamete. In addition, questions associated with the reality and relative viscosity of organelles were also topics of great interest to earlier biologists. Additional questions of interest concerned the upper limits in centrifugal forces that might be sustained by small organisms, cells, and various cellular constituents. Survival of cells when subjected to extreme centrifugal force and their ability to redistribute their stratified contents were also topics of interest. The fact that certain small organisms such as algae, duckweed plants, and rotifers can withstand relatively high centrifugal force for several days and then seemingly recover from this treatment represents a most unusual and interesting phenomenon.

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The fact that ultracentrifugation is an experimental technique that can be applied to living cells has no doubt enhanced its utility as a cytological tool for biological investigations. A remaining question of some interest concerns whether the tool has the potential for continued utility in the investigation of the functioning of cells and the nature of protoplasm. The answer to this question clearly seems to be affirmative. In view of the development of modem techniques in cell biology (e.g., high-voltage electron microscopy, freeze-fracture, immunocytochemistry, immunofluorescence, and other techniques), it should clearly be possible to enhance our understanding of the complex interactions characteristic of living protoplasm. For example, it should be possible by using extreme ultracentrifugal forces to better understand gravitational effects on life, including the responses of cellular macromolecules and organelles to these forces and the ability of this living material to reorient and redistribute following exposure to this extreme force. For example, the “structureless-appearing hyaloplasm” is now known to be filled with a variety of tubules, filaments, and trabeculae embedded in a cytosol, which have only relatively recently been extensively explored in terms of cellular function. The application of high centrifugal force in experimental studies should help to provide us with additional information about the nature of the cytoskeleton, its resistance to deformation and recovery, and its relationship to various important organelles and cellular compartments. The utility of this technique, to a considerable degree, should continue to reside in the fact that it is a kind of microdissection device that can be applied to living cells.

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INTERNATIONAL REVIEW OF CYTOLOGY.VOL. 100

The Chromosome Cycle and the Centrosome Cycle in the Mitotic Cycle DANIELMAZIA Hopkins Marine Station of Stanford University, Pacific Grove, California 93950

“Possible, but not interesting,” Lonnrot answered. “You‘ll reply that reality hasn’t the least obligation to be interesting. And I’ll answer you that reality may avoid that obligation but that hypotheses may not.” Jorge Luis Borges, “Death and the Compass”

I. Introduction Cell biology is not a notoriously self-critical field. We cell biologists are not reticent about announcing breakthroughs and making promises of imminent revolutions. However, one cannot summarize the history of research and thought about mitosis as a progress from primitive glimmerings to modern revelations. Nothing we have learned about mitosis since it was discovered a century ago is as dazzling as that discovery itself. The discovery was not merely a resolution of questions about how cells divide. Almost immediately, it defined the agenda for much of the cell biology of the 20th century. We have made progress during the century, despite a certain contentiousness that has always been characteristic of our field. (Disagreements about hypotheses tend to become conflicts of beliefs.) The discoverers, wherever their shades now rest, should be reasonably happy with the outcome of their ideas after a century, if understandably annoyed by the fact that few of us read their writings. Consider the state of the field around the end of the 19th century. “Indirect cell division” (meaning only that one cannot see the nucleus) calls for the “metamorphosis of the nucleus into threads.” The threads take up certain common dyes and become “chromatin.” The “chromatin” can be identified with the “nuclein” already known to biochemists, later known as DNA. The threads are called chromosomes; the chromosomes can be counted. It becomes clear that the numbers and combinations of chromosomes are displaying the mechanisms of sexual reproduction and inheritance-and we are already in trouble because the constancy of chromosomes seems incompatible with differentiation. The disposition of the chromosomes is managed by a spindle and the spindle is organized by poles and the poles are embodied in centrosomes (earlier known as ‘‘polar corpuscles”). The chromosomes are clearly double, and sister chromosomes are joined to poles by spindle fibers. The engagement of sister chromo49 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.

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somes to opposite poles is governed by strict rules. The chromosomes are moved; mitosis is “karyokinesis.” It is not hard to relate the movements to the “spindle fibers” that stand out in the microscopic preparations. Of course those old-time investigators of mitosis (all of whom are quite young at the time when they are making their discoveries) see themselves as modern. They are as eager as we now are to explain their observations in physical and chemical terms. As has been mentioned, DNA becomes the characteristic substance of chromosomes very early in the story. The formation of the mitotic apparatus is seen as the assembly of an “archoplasm”; the image is not fundamentally different from present-day views about the organization of cytoskeletal elements of the mitotic apparatus. The behavior of spindle poles tempts speculations about electromagnetic forces. Hypotheses about the role of contractile fibers in the movements of chromosomes are prominent; muscle contraction provides a seductive analogy to chromosome movement. I have ventured the above glance backward (without scholarly citations) only to show how the agenda for later and present-day work was set by the mitotic process itself as it reveals itself in dimensions accessible to the light microscope. Here, to “explain” is to account for what we apprehend with our eyes. It will be some time before an Einstein will feel at home in our business.

11. Variants

It is possible to give quite uniform account of mitosis in “higher” (read “multicellular”) organisms, more so than in the past as superficial differences such as differences between mitosis in plants and in animals become easy to explain. On the other hand, the immense increase of knowledge of mitosis in “lower” eukaryotic organisms reveals more and more variants of the mitotic design. A review of the information as of 1980 (Heath, 1980) tabulates the features of mitosis in more than 200 species. The newer facts exclude some generalizations while strengthening others. The best summaries are to be found in past volumes of the International Review of Cytology (Kubai, 1975; Fuge, 1977; Heath, 1980). The only feature of mitosis in eukaryotic cells that covers all the variants is the establishment of connections between poles (centrosomes) and kinetochores (centromeres) by microtubules. In the case of the yeast Saccharomyces cerevisiae, there is only one microtubule per chromosome (Peterson and Ris, 1972). Since it is so logical that the chromosome-to-poleconnections are the essence of accurate separation of chromosomes, it is all the more gratifying that we can draw that conclusion objectively from the descriptive comparison of a large number of organisms. Given those connections, different organisms can exploit different gadgetry to

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partition the genomes into two daughter nuclei. The comparative natural history of mitosis is extremely instructive. While the very term “mitosis” was inspired by the condensation of chromatin into compact threads, there are organisms in which the condensation is not seen (cf. Table 1 in Fuge, 1977). There are other cases in which the chromosomes appear to be condensed throughout the cycle. The best-studied case is found in Dinoflagellates (Kubai, 1975); that case has been doubly interesting in creating doubt about the universal occurrence of histones in chromosomes of eukaryotic cells. In the interesting case of Trypanosoma cruzi, the chromatin does not condense. Nevertheless, the chromosomes display kinetochores resembling those seen in higher organisms (Solari, 1980). In the course of mitosis, the kinetochores are aligned on a metaphase plate in the strictly intranuclear spindle. Older studies on “lower” eukaryotic organisms suggested that nuclear envelopes do not disperse during mitosis. Now comparative studies show a spectrum of arrangements between such a fully “closed” plan and the extreme “open” plan of classical mitosis (Fig. 1 in Heath, 1980). Spindle poles may be external to the nucleus, located at a perforation of the nuclear surface, or apposed to the inner or outer faces of the nuclear envelope. The centrosomes (under various names) may be displayed in a variety of structural forms or may not be advertised by any structure that has been seen by present-day methods. Kinetochores may be as distinct as they are in T . Cruzi (Solari, 1980) or in cells of higher organisms (Alov and Lyubiskii, 1977; Rieder, 1982)-or they may be “invisible” as they are, paradoxically, in the one case (yeast) where they have been characterized fully by their nucleotide sequences (for review see Clarke and Carbon, 1985). Kinetochores may be inserted in the nuclear envelopes in cells in which the envelopes persist while the spindle is external to the nucleus. Surveying the overall mitotic cycle, the comparative approach provides tabulations of cases where metaphase plates are formed or not formed. All of the styles of chromosome separation are found among the “lower” eukaryotic organisms: chromosome-to-pole movement, pole-pole separation, or combinations of the two. The admirable work on the variants of mitosis, even when summarized so scantily, does sustain the opinion that the essence of the problem is the accurate connection of centromeres (kinetochores) and centrosomes by microtubules, enforcing the law: sister chromosomes may not connect to the same pole.

111. The Chromosome Cycle and the Centrosome Cycle

Occupy the Whole Cell Cycle The early workers divided the reproductive cycle of the cell into a “resting” period and a period during which the processes of mitosis were carried out. It is nearly a century since 0. Hertwig, in a textbook published in 1893, included two

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phenomena in his characterization of the mitotic period: “a regular ordering of the nuclein comes into view” and “various substances . . . come into closer mutual relationship with the cytoplasm when the nuclear membrane dissolves.

”

A. THE Two CYCLES This essay will examine a modern theme that did not come to the attention of the classical workers: the theme that the events of the mitotic cycle run through the whole cell cycle. The mitotic activities that occupy the whole cell cycle are the chromosome cycle and the centrosome cycle-including , of course, the underlying processes that drive them. The chromosome cycle consists of decondensation --f replication condensation + splitting += decondensation. The centrosome cycle is being worked out only now and what I will have to say, apart from insisting that the cycle runs through the whole cell cycle, includes a certain amount of speculation. The elements of the centrosome cycle are reproduction + division-bipolarization of the cell. Later we will consider a sequence of changes of shapes of centrosomes that are analogous to, but not as well defined as, the changes seen in the typical chromosome cycle.

-

B. THE MITOTICPHASE There is no need to change our vocabulary because we have become aware of the continuity of the mitotic cycle in the cell cycle. The characteristics of the conventional mitotic phase (M phase) are still valid. In general it is the period when sister chromosomes are engaged to sister poles. In “higher” eukaryotic cells (and many “lower”) it is the period of the highest order of chromosome condensation. Finally, it is a period when longitudinally divided sister chromosomes can be revealed microscopically and when they split apart actively at the onset of mitosis. Historically, the very early observation that chromosomes split longitudinally was a point of departure toward the chromosome theory of inheritance and a defeat of theories that explained differentiation by unequal distribution of chromosome material (Mayr, 1982, p. 677). The splitting-apart of sister chromosomes that marks the beginning of anaphase does not depend on “pulling” by the poles. It is seen just as well when there is one pole or none. For example, it is seen in the “nonpolar” mitotic cycles of activated but not fertilized sea urchin eggs, described in older literature and characterized later by the writer (Mazia, 1974). It is only recently that the problem has come under experimental investigation, with a number of publications relating the splitting of chromosomes to a brief period of elevated intracellular Ca2 levels (Hepler, 1985). We know so little about the splitting-apart of sister chromosomes, even though it announces the critical transition from the end of one cell cycle to the beginning +

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of the next. Now we can hope that the “cytostatic factor” will provide a tool for the study of the splitting-apart of sister chromosomes at the metaphase/anaphase transition. This Ca-sensitive factor is responsible for metaphase arrest in meiosis I1 in amphibia and it can also arrest mitosis at metaphase in cleaving embryos (Meyerhof and Masui, 1977; Masui et al., 1984).

C. OLDVIEWS RECONSIDERED In 1961, I was able to designate certain events of interphase as “preparations for mitosis” and to incorporate them into a diagram, a “Time Map of Mitosis’’ (Mazia, 1961). The field had come a long way from the notion of a “resting” cell, but one was not yet ready to assert that strictly mitotic events, such as chromosome decondensation and condensation, run continuously through interphase. Some of the “preparations for mitosis” on the time map have not earned a place in the present-day picture. The idea of a special energy reservoir as a prerequisite to mitosis has not survived a closer analysis of the experimental evidence. A modern time map would include the bipolarization of the cell as a preparation for division but would not specify “separation of centrioles.” I certainly would not include a “trigger” between interphase and mitosis; that has not been a productive concept. There must be transitional events in the flow of mitotic time, but the study of so many cell cycles has taught us that cells rarely pause on the brink of mitotic prophase. The term “mitogenesis” has come into favor among workers on cell-population kinetics, but it does not refer to a transition from interphase to mitotic phases. (Usually, it is measured by the number of cells entering the S phase, not the number entering the M phase.) According to the following discussion, “mitogenesis” could be measured as the number of cells that begin to decondense their chromosomes after the chromosome cycle is arrested at some point beyond mitotic telophase. I f a prediction is allowed, Ipredict that the start-up of postmitotic chromosome decondensation will be recognized as the point of leverage for the regulation of the cell cycle.

IV. The Chromosome Cycle in the Cell Cycle A. CHROMOSOME REPLICATIONIN INTERPHASE: BACKGROUND It is a long time since it seemed necessary to argue that chromosomes persist from mitosis to mitosis even though they disappear from sight during interphase. By the time of publication of the final edition of Wilson’s (1928) book he could present strong arguments for the continuity of chromosomes, although he was

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hardly prophetic in remarking: “That the continued presence of chromatin . . . is essential to the genetic continuity of the chromosomes has . . . become an antiquated hypothesis. The discovery that chromosome replication (signified by the synthesis of DNA) takes place during interphase was made in the early 1950s. One could infer that the quantitative doubling of DNA measures the doubling of chromosomes. For many of us, this seemed to be sufficient evidence for DNA as genetic material. To those who were interested in the mitotic cycle, it seemed important to relate the replication of DNA in interphase to the fact that the chromosomes are decondensed during that period. At the time, many of us were picturing the nucleus as a porous bag containing a tangled spaghetti of unraveled chromosomes in a sauce called “nuclear sap.” Only quite recently have cell biologists returned to serious work on the following questions. Where are the chromosomes in the interphase nucleus? What is their structure? What is their relationship to nonchromosomal components? (Cf. Cook and Laskey, 1984, for contributions by several groups.) ”

DECONDENSATION/CONDENSATION DURING INTERPHASE B . CHROMOSOME Facts that were available in the early 1960s could be put together in the proposal that the chromosome cycle runs through interphase (Mazia, 1963). In the normal cycle, condensed chromosomes do not replicate. In many cases, the S phase does not start for some time after the preceding division. Work on DNA synthesis in the test tube shows that chromatin is a poor primer unless it is opened up in some way. Thus one can imagine that the decondensation that starts at telophase has to continue into the early part of interphase. The time required to reach the S phase would be the time required for the maximum decondensation of the chromosomes. “Open” chromosomes replicate and it can be supposed that each replicating segment condenses as soon as it replicates. This supposition would explain the fact that chromosomes cannot replicate twice in the same cycle. By the end of the S period,the chromosomes have started the condensation which will become conspicuous at prophase. C. EVIDENCE FOR

THE

CONTINUOUS CHROMOSOME CYCLE

1. The accessibility of the chromosomal DNA to various reagents changes during interphase, as predicted from a decondensation/condensation cycle. The binding of actinomycin D to DNA in synchronized HeLa cells increases during G , , is maximum around S, and decreases in G , (Pederson and Robbins, 1972). Even after chromatin is isolated from synchronized populations, the susceptibility of the DNA to attack the DNase follows the prediction that the chromatin will “open” gradually during G , and will “close” in G , (Pederson, 1972).

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Studies on Chinese hamster ovary cells provide similar evidence of chromosome decondensation followed by condensation during interphase, differing in some details from the results on HeLa cells. Binding of heparin by nuclei (Hildebrand et al., 1978) and susceptibility of nuclei to DNase I (Prentice et al., 1985) have been assayed. Work with isolated chromatin from HeLa cells in various phases of the cell cycle indicates that the structure is most open during the S period and most closed during the M period. The conclusions are drawn from studies on the binding of ethidium bromide and on circular dichroism (Nicolini and Belmont, 1982). The results of all the studies support the general proposition that the decondensation/condensation cycle runs through interphase. Viewed more closely, the differences in the results of different studies reflect differences in the cells studied and differences in what is “seen” by the various probes. 2. Microscopic study of the interphase nucleus by advanced methods generally sustains the idea that the S period coincides with a period within interphase when the chromatin is most dispersed. One finds considerable evidence in the electron microscopic (EM) literature. While the evidence is drawn from fixed and processed material, experience sustains some confidence in the images of chromosome condensation if not in finer details of chromosome structure. An especially interesting study (Setterfield et al., 1978) uses mouse L cells carrying a temperature-sensitive mutant for DNA replication. In cells going through the normal cycle, the chromatin is most dispersed during the S phase. In the mutant cells, whose cycle is arrested at the S period, the chromatin remains dispersed until the cells are restored to a temperature at which the cycle can resume. A method of flow cytophotometry by which DNA content is related to the state of aggregation of chromatin as indicated by image analysis has been applied to populations of HeLa cells, with results that demonstrate decondensation and condensation of chromatin during interphase (Nicolini and Belmont, 1982). In general, the studies of the way chromatin “looks” during the whole cell cycle support the broad thesis that the decondensation/condensation cycle runs continuously through the cell cycle. The above section will soon seem quite out of date. The current upsurge of interest in the interphase nucleus promises a real anatomy of the relationships among chromosomes, matrix, and envelope that will supersede our bowl of chromosomal spaghetti suspended in nuclear sauce.

3. Premature chromosome condensation (PCC) confirms the chromosome decondensationlcondensation cycle during interphase. The discovery of premature chromosome condensation by Robert T. Johnson and Potu N. Rao in 1972 was, in my opinion, the beginning of the future of the

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study of chromosomes in interphase. The basic phenomena are now well established and well documented (Rao, 1982, and other authors in monograph cited). a. Fusion of a cell in interphase with a cell in the M phase (usually a cell arrested by an agent such as colchicine) causes the condensation of the chromosomes of the interphase nucleus. b. The prematurely condensed chromosomes of the interphase nucleus reflect the actual condition of those chromosomes. For example, chromosomes are seen to be single before the S phase and clearly doyble after the S phase. c. The ‘‘chromosome condensing factor” in the cell in M phase is cytoplasmic, diffusible, and is not species specific. The accumulating evidence on PCC (Sperling, 1982) tolerates the hypothesis that chromosomes decondense gradually during the G, period; the length of the chromosomes increases and can be correlated with cell cycle “age.” The recondensation during G, can be measured as a shortening of the chromosomes. The fact that the replicated chromosome can be distinguished in G, as two visibly separated chromosomes is pertinent to the problem of the later splitting-apart of sister chromosomes at anaphase. One would also like to know where the kinetochore DNA is located in the G,-phase chromosomes. The work on PCC confirms the thought that the chromosomes are most expanded during the S phase. Combination of radioautography with the PCC technique makes that point. The original light-microscopic images suggested that the S-phase chromatin was not only dispersed but possibly fragmented; the descriptive term was “pulverized.” While the idea that chromosomes might be broken up during their replication would no longer be shocking, it seems more likely that the decondensation in S goes to the point where some portions of the chromosome cannot be resolved by light-microscopic methods. The fragments that are seen would be regions that have not yet decondensed fully or regions that have recondensed. Some very vivid images of chromosomes in the S phase, obtained by scanning EM (Mullinger and Johnson, 1983; Gollin et al., 1984), now show that the “pulverized” condition is explained by small aggregates of fibers, with more dispersed fibers between them; the integrity of the whole chromosomes is preserved (cf. Fig. 6 in Gollin et al., 1984). The blocks of aggregated fibers are often arranged linearly and some are paired. Combining the evidence from light microscopy and scanning EM one might venture some conclusions that are quite comfortable for some models of chromosome structure. The light microscope, seeing the GI decondensation as an increase in length, would be reporting on the extension of a packed structure, perhaps the “solenoid” favored by some models. When this structure has been fully extended, the scanning EM would be detecting the currently replicating chromatin as the more dispersed strands, while the compacted strands would be chromatin that has not replicated or has already replicated and has begun to pack up for the forthcoming karyokinetic phase.

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Examining the images of PCC, one notices immediately that the chromosomes in G, are never as strongly condensed as metaphase chromosomes. The PCC procedure does result in the dispersal of the nuclear envelope, and the term “prophasing” has been suggested (Matsui et al., 1982). From the literatureold, new, scattered, and indecisive-I would judge that most voters feel that the transition from prophase to full metaphase condensation involves the formation of major coils or solenoids by an additional factor. It is not, however, a problem that will be resolved by polls. Most of the work on PCC has been done with vertebrate cells in culture. Some studies on sea urchin eggs not only confirm the basic discoveries about the chromosome cycle, but give access to additional questions (Poccia et al., 1978; Krystal and Poccia, 1979). Here, fertilization of eggs by sperm offers a natural method of cell fusion on a large scale. Unfertilized eggs may be forced into the cell cycle by various means of artificial activitation, starting the cell cycle. The “activated” eggs may be fertilized at any time in the ongoing cell cycle (Mazia, 1974). Thus, we may examine what happens to the condensed chromosomes of the sperm cell when they “inject” themselves into the cytoplasm of eggs that are in the S period or of eggs that have advanced into mitosis. The answer is clear and predictable from the chromosome condensation cycle we have been discussing. Chromosomes “injected” as sperm nuclei during interphase decondense; those that enter the egg during the mitotic period are condensed. We can go one step further. We know how to start the underlying cell cycle in fragments of sea urchin eggs that have no nuclei (Nishioka and Mazia, 1977). Does the cycle of decondensing and condensing activity proceed even when no nucleus is present? The question is asked by fertilizing anucleate fragments after they have been activated (Krystal and Poccia, 1979). The immediate decondensation or condensation of the sperm chromosomes is observed; it does depend in a cyclical fashion on the time that has elapsed since the activation of the egg. Thus there is an underlying cytoplasmic cycle of chromosome decondensing or condensing activity that does not depend on the presence of chromosomes. In one way, this experiment is more complete than those involving the fusion of mitotic cells with cells in interphase. In the usual PCC experiments, the condensing activity contributed by the mitotic cell is generally dominant; it is not easy to demonstrate decondensing activity. In the experiments on sea urchin eggs, we see the complete underlying cycle.

D. SOMESTEPSTOWARD

A

CLOSERVIEWOF THE CHROMOSOME CYCLE

In this section, I merely sketch some trends in research that bear on the problems of the chromosome cycle. My purpose is to indicate how the methods and outlook of the molecular biology of the last decade have begun to face problems that have been under consideration for a century.

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1. Models of Chromosome Structure Originally, “indirect cell division” was characterized as “mitosis” because it was observed as “the metamorphosis of nuclei into threads” (Flemming, 1880). That problem could be defined as the emergence of condensed mitotic chromosomes from the dispersed chromatin of the interphase nuclei. It seems to me that the problem has been simplified with the discovery of PCC.We can now see that chromosomes in interphase are not so different from metaphase chromosomes when we bring them into view with condensing factors. Even chromosomes in the S phase can be regarded as very relaxed versions of those we see at metaphase (Sedat and Manuelidis, 1977). The famous special cases that we have known for a long time-chromosomes visible in interphase in dipteran salivary gland cells and in certain flagellates-may not be so very exceptional. In short, there may be less to chromosome decondensation than meets the eye-literally; a relatively small degree of opening up of structure may have a very large effect on our ability to observe chromosomes optically or electron-optically. The current ideas about the higher order of chromosome structure deal with the packing of the fundamental chromatin fiber in interphase chromosomes and mitotic chromosomes. The models fall into two classes. The conception that the fundamental fiber is DNA packed as a chain of nucleosomes does not seem to be a matter of controversy. a. Coiling. The explanation of chromosome condensation by successive orders or coiling has a long history. By the 1940s, the literature contained many images of condensed chromosomes in which coiled coil structures were clearly displayed, and controversies about finer orders of coiling aroused considerable passion. The principle of the coiled chromatin thread is still under consideration. There seems to be general agreement that the chromatin threads that we see with the EM at all stages of the chromosome cycle-threads with diameters in the 20to 30-nm range-are tightly coiled primary chromatin threads, the 10-nm nucleosome filaments. The idea that further condensation follows from the helical winding of the 20- to 30-nm strands is pursued in a model which sees the next step as the formation of “solenoids,” hollow coils with a diameter of about 400 nm. Condensation at metaphase is seen as a further coiling of the solenoids (Bak and Zeuthen, 1977; Sedat and Manuelidis, 1977). The advocates of the fully helical model of condensed chromosomes find that it accomodates itself to molecular-structural restraints but, in our present context, we would like to know how it performs in a complete decondensationlcondensationcycle. The helical model can be contrasted with another-and also persuasive-image of the chromosome. b. The Scaffold. (Lewis et al., 1984; Hadlaczky, 1985). The fundamental experiment is simple; DNA and histones are removed from condensed chromosomes; yet we still see chromosomes, reasonably faithful to the original structure. Therefore, the morphology of chromosomes is maintained by a “scaffold”

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composed of nonhistone proteins. The chromatin thread (nucleosome filaments) hangs on the scaffold, projecting as loops. Here, I cannot do justice to an impressive body of evidence, but will only try to fit the model into the questions we are asking about the whole chromosome cycle. For our immediate purposes, the model would see the chromosome condensation in the mitotic period as the structuring of the scaffold. How, then, do we explain the decondensed chromosome? The scaffold is characterized by some well-defined proteins and these proteins are found in the matrix of the interphase nucleus. It can be supposed, then, that decondensation would be expressed as a restructuring of the scaffold which would persist through interphase as a more expanded structure, so far identified only by its proteins. If this class of models prevails, we would have to learn how the nucleus doubles all the scaffolds during the S period, since the chromosomes emerge from replication as distinguishable sister pairs. Having mentioned that the coiled-coil version of the chromosome has a long history, I am tempted to mention the fact that the scaffold-except that we called it a “backbone”-was flourishing in the 1940s before it vanished into oblivion. The question then, as now, was: does the integrity of chromosomes (dipteran salivary gland chromosomes were the material) require DNA? The tools were the then-available nucleases and the reports said that the structure remained after DNA was removed. The conclusion was that the “backbone” was made of “residual proteins,” those remaining after histones as well as DNA were removed. (There is no moral in this brief remembrance of things past; then, as now, chromosomal proteins were seeking employment.)

2 . Molecules in Chromosome Condensation The chemistry of chromosome condensation has been under study for at least three decades. Experiments became possible with the improvement of methods for isolating nuclei, and reasonable hypotheses could be composed on the basis of a growing knowledge of the interactions of DNA with histones and other proteins. The most interesting agents were divalent cations and polyamines such as spermidine (Rao, 1982). The general result was that polycations did indeed produce conspicuous aggregation of the chromatin, but the chromatin thus condensed did not really resemble mitotic chromosomes. Still, it is clear from more recent work that the ionic conditions are decisive for the packing of the nucleosome filament into 20- to 30-nm chromatin fibers (Thomas, 1984). The older approaches to chromosome condensation in the mitotic cycle had a fatal defect; we were completely wrong in our image of the fundamental chromatin fiber. We came to know the length of DNA, we learned about the categories of histones and their molar ratios, and we even took pictures in which surface-spread chromatin appeared as strings of beads. We taught that chromatin was seen consistently in interphase nuclei as 20- to 30-nm fibers. Still, our thinking was muddled by an obsolete vocabulary; one could get nowhere by thinking of a chromatin fiber as a long “nucleoprotein.”

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It is scarcely more than a decade since it was established that fully extended chromatin is a chain of nucleosomes (Olins and Olins, 1974; Kornberg, 1974) and that the 20- to 30-nm chromatin fiber of the real world represents the packing of the chain of nucleosomes as a coil or solenoid. The packing, already representing a high degree of condensation, is attributed to ionic conditions and especially to the presence of the H1 histone. Despite a variety of interpretations, it seems to be agreed that the H1 histone binds the nucleosome filaments into the chromatin fiber. The molecular basis of the further condensation of chromatin is not yet clear, but it has been related to modifications of histones. One hypothesis is that the condensed state is correlated with the complete phosphorylation or “superphosphorylation” (Gurley et al., 1978) of the HI histone. Experiments on several kinds of cells (Hildebrand et al., 1978; Bradbury and Matthews, 1982) show a sharp increase of HI phosphate during the mitotic period. This may be preceded by an increase in the activity of histone kinase. The argument is strengthened by the observation that premature chromosome condensation induced in sea urchin eggs is accompanied by an increase in phosphorylated HI (Krystal and Poccia, 1981). As several authors have noted, the observation may seem paradoxical. If the effect of H1 on condensation dependend on its charge as a polycation, we would not expect a cause-effect relationship between phosphorylation and condensation. Other histones, the H2A and H2B, are modified during the cell cycle by combination with the widespread protein, ubiquitin. In a number of cases, it has been found that ubiquitin is associated with these histones during interphase but is disassociated during mitosis (Matsui et al., 1979; Mueller et al., 1985). In the case of the slime mold Physarum, where the synchrony of the cycle is very precise, the removal of ubiquitin from H2A and H2B is sharply confined to the metaphase stage; by anaphase, the histones are again ubiquitinated. These beginnings hardly add up to a definitive picture of chromosome condensation, in terms either of chromosome structure or of the mechanism of condensation. In fact, they seem rather crude when we allow ourselves to think of the fine points of condensed chromosome structure as expressed in the karyotype and even more precisely in banding, etc. However, we do confirm the original hunch that histones are molecules responsible for the packing of chromatin in eukaryotic cells.

E. FACTORS Factors are molecules that are still innocent of chemistry. We know them by what they do and trust them ultimately to expose their identities. The following brief summary is being written at a time when factors governing chromosome cycles are becoming practical tools for work on the cycles. The

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existence of cytoplasmic, diffusible chromosome-condensing factors (CCFs) could be defended a long time ago; it explained the exact synchrony of the chromosome cycles whenever several nuclei share the same cytoplasm. The exploitation of techniques of cell fusion for studies of PCC proved the existence of CCFs and demonstrated that they are not specific. The lack of specificity was displayed in recent experiments in which amphibian oocytes were used for the assay of mammalian CCF and mammalian nuclei were shown to respond to the cycling activitity of CCF in amphibian eggs. Some really new insights into the chromosome cycle have arisen from an unexpected source. Work on problems of the induction of ovulation and the maturation of oocytes in various animal groups led to the discovery of the maturation-promoting factor (MPF). The appearance of MPF leads to the breakdown of germinal vesicles and the condensation of chromosomes for meiotic divisions (reviews by Masui and Clarke, 1979; Masui, 1985; Masui et al., 1984). In our compartmentalized science, one might have considered this to be an interesting special case, relevant to the endocrinology of the production of mature ova in higher animals. It turns out that the breakdown of germinal vesicles involves the same process as the dispersion of nuclear envelopes in ordinary mitosis, leading to the formation of a meiotic (or mitotic) apparatus. When extracts of cultured cells in the mitotic phase are injected into amphibian oocytes, the result is the breakdown of the germinal vesicle and subsequent chromosome condensation; cells in the ] phase contain MPF. When nuclei from different sources are injected into the maturing oocytes, their chromosomes condense in synchrony with the condensation of the meiotic chromosomes already present. By now, there is a considerable variety of evidence that demonstrates the correlation of the rises and declines of the amount of MPF (measured by bioassay) with the increase and decrease of chromosomecondensing activity. Moreover, there is evidence for factors that inactivate MPF and can be regarded as chromosome-decondensing factors (Masui and Clarke, 1979; Gerhart et al., 1984). The basic fact is that the cytoplasm of cells in which MPF activity is low will bring about the decondensation of chromosomes. The first steps toward the characterization of MPF show that we are dealing with proteins which are inactivated, perhaps irreversibly by Ca2+ ions. Here is an enticing link between the chromosome-condensation cycle, the cycle of the synthesis and breakdown of special proteins (e.g., cyclin; Evans et al., 1983), and the fluctuations of Ca2+ levels during the cell cycle (Poenie et al., 1985; Suprynowicz and Mazia, 1985)-three types of change that are prominent in the current generation of studies on mitotis. In the newest wave of research the condensation and decondensation of chromosomes is being studied in extracts of cells (Lohka and Masui, 1983; Lohka and Maller, 1985; Masui, 1985). The very fact that parts of the chromosome cycle can be separated from the intact cell gives some assurance that the problem

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is somewhat more simple than it had seemed to be. (Or that cell biologists are less intimidated by cells than they used to be.) If demembranated sperm heads are injected into concentrated extracts of amphibian eggs, their chromatin decondenses, a nuclear envelope is formed, the DNA-replicating activity is turned on, and prophase-like nuclei develop. The observations reflect the natural history of the highly condensed sperm chromatin after it has entered an egg. We may generalize that the results represent the history of chromatin in the ordinary cell cycle, as it passes from the relatively condensed state at telophase through decondensation in G, and then the beginning of condensation as replication proceeds. The condensing system resulting in nuclear envelope breakdown and chromosome condensation can be followed in cell-free preparations to which MPF is added (Kirschner, 1985). Cycles of rise and fall of MPF activity can be followed in extracts of amphibian eggs (Masui, 1985). The pursuit of MPF as a factor is giving direction to the study of the chromosome cycle. As an emerging field of analysis, it may arrive at some wonderful new molecule that interacts directly with chromosomes or as a complex association of components in a mixture that can be squeezed out of amphibian oocytes. The first methods of extracting active MPF demand a minimal dilution of the cytoplasm and the presence of particles, probably vesicles in the extracts (Lohka and Maller, 1985). The molecular resolution of these remarkable extracts-or shall we call them isolated cytoplasms?-will be achieved in time; meanwhile they can answer many questions about the variables that drive the chromosome cycle. OF THE CHROMOSOME CYCLE F. SUMMARY

The speculation that the chromosome cycle of decondensation/condensation runs through the whole cell cycle is no longer a speculation. We can make sense of it in terms of an elementary view of the cell cycle. It is not surprising that chromosomes must be in an “open” state for the complex biochemistry of their replication, nor that the chromatin is packaged compactly for its deployment during the mitotic phases. The decondensation of chromosomes during the first part of interphase tempts a further speculation: That it is the event that decides whether the cell will proceed toward replication. In cells which do not enter the cycle (the Go condition in popular terminology) the decondensation of the chromosomes would be arrested in a posttelophase state. Just possibly, the mechanisms of chromosome decondensation are levers in the control of the cell cycle. The study of the fundamental unit of chromosome condensation, the 30-nm nucleosome filament, is far advanced. Promising models of further orders of condensation will surely lead us to an understanding of the principles of the packing of chromatin into microscopic chromosomes. The deeper problems have

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to do with the precision of structure that is more than packing: the construction of mitotic chromosomes with every genetic segment in its place in three dimensions. We will encounter it in its extreme form: the positioning on the kinetochores. The growing body of work arising from the discovery of the MPF provides the setting for a major advance in the understanding of the chromosome cycle. The nature and mode of action of the factor is a prominent problem, but the workers in the field are turning thoughts to a still deeper level. They find reason to propose that there are underlying cycles in the cell which drive the events that are expressed in the chromosome cycle and other events of the cell cycle (Newport and Kirschner, 1984; Masui, 1985; Kirschner, 1985). Whether or not the case is advanced by borrowing the term “oscillator,” we keep coming back to the thesis that one of the deep components of a living cell is “time.”

V. The Centrosome Cycle in the Whole Cell Cycle The centrosome-even its name-is enjoying a renascence. For decades, its very existence as a body was in doubt even though its functions as a mitotic pole could not be overlooked. It has not yet regained the recognition awarded to it by Boveri (1901, pp. 4-6): that it is a major, permanent reproducing organ of the cell-the real division organ of the cell. In the following text, I will pursue that conception of the centrosome, which was so prominent at the beginning of this century. We may have to wait for the next century, not so far away, before we can know whether the centrosome is truly a key to the understanding of the cellas-organism, the instrument through which a cell is constructed out of the molecules commanded by the genes. Meanwhile we can begin to cope with welldefined problems of centrosomes as mitotic poles. A. A REVISEDCONCEPTION OF THE CENTROSOME 1. Embodiment

The discovery of the centrosome a century ago came about by a wonderful conjunction of visual intelligence and the beauty of mitosis in the eggs of an unbeautiful roundworm parasite of horses, Ascaris megalocephala. The poles of the mitotic spindle revealed clearly stainable compact corpuscles. The so-called “polar corpuscles” were later designated as centrosomes. Chromosomes do move to the poles and one can see poles as particles; what could be more satisfactory? By 1901 Boveri could make a formal definition of centrosomes which made it clear that centrioles (where they existed) were contained within centrosomes. The conception of the centrosome as a permanent organ of cells that is present

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in all eukaryotic cells floundered when cytologists were unable to find the anticipated corpuscular structures in many kinds of cells. By the time Wilson summarized the status of the centrosome in 1928 even he had retreated into uncharacteristic vagueness. In my review in 1961, I managed to say a good deal about the reproduction and the functions of centrosomes while avoiding any assertions about their corporeal attributes (Mazia, 1961). The ontological legitimacy of the centrosome has improved only recently but is still compromised by our inability to reply to our students’ first and inevitable question: “What is it made of?” Meanwhile, we have been learning a great deal about centrosomes. Work on “lower” eukaryotic organisms, as discussed above, has revealed a variety of quite definite and often beautiful structures corresponding to centrosomes. The powerful methods of yeast genetics have exposed the genetics and life histories of very characteristic centrosomes, alias spindle pole bodies. In the last few years, centrosomes have been separated from cells for interesting studies of the initiation of the assembly of microtubules (MTs) in v i m (Kuriyama and Borisy, 1981) or after injection of the the isolated bodies into cells (Karsenti et al., 1984). (The citations are just samples of the literature.) In many reports, the centrosomal material that actually initiates MTs is designated as “pericentriolar material,” although the very findings lead to the conclusion that the centriole is a passenger and a dispensable one at that. The concept of the centrosome can be generalized and unified by abandoning the old definitions that called for a corpuscular body with a constant morphology. We can, instead, regard the centrosome as a flexible, ultimately linear body which can take on different shapes in different cells and will undergo changes of shape during the whole cell cycle in any cell. This conception of the centrosome will be discussed at some length but might have been inferred from observations of mitosis. If we assume that mitotic poles have any physical embodiment at all, then we cannot fail to notice that chromosomes do not converge toward a focal point (thus toward a compact centrosome) in all cases. In very many cells, the chromosomes seem to be moving through a barrel-shaped spindle toward a flat pole (hence a flat centrosome). I am aware of cases where the poles seem quite disorganized and which have led to arguments about the reality of centrosomes. The linear model that I will be proposing has no difficulty with somewhat confused poles; our problem will be to account for order, not to explain disorder. Imagination is aided by comparing a centrosome to a chromosome rather than to commonplace cytoplasmic “organelles.” It is large, it is permanent, it is present in small numbers, and it reproduces once in each cell cycle. As will be seen, it condenses in one part of the cell cycle and seems to disperse at other stages, tempting the inference that it has disappeared. History recalls that continuity of chromosomes, which are invisible in most cells most of the time, was a matter of controversy even at a date when the evidence from genetics was

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irrefutable. Evidence for the permanance of centrosomes has had to wait for methods that have emerged only recently.

2 . Identification of Centrosomes The early cytologists stained centrosomes in fixed and paraffin-sectioned material by the iron-hematoxylin method and sometimes obtained images that can be confirmed by all modem criteria. In EM cytology, centrosomes may be observed as osmiophilic regions. Osmiophilia is not, at the present state of knowledge, a specific cytochemical indicator, but osmic staining around the poles can be quite informative about changes of the shapes of centrosomes during the mitotic cycle. A great deal can be learned about centrosomes without seeing them at all, thanks to their functions as microtubule-organizing centers (MTOCs). If centrosomes are origins of MTs, then MTs are “pointers” to their origin. Images of arrangements of MTs delineate shapes of centrosomes (Paweletz et al., 1984) and can tell us when centrosomes divide and when the separating centrosomes have established the poles of a forthcoming mitosis. The introduction of anticentrosome serum has turned speculations about the existence, locations, and shapes of centrosomes into facts. The observations have the quality of facts because they enable us to see the centrosomes by fluorescence-microscopicand EM methods (Brenner and Brinkley, 1982; Collarco-Gillam et a l . , 1983). At this stage, the immunocytochemical methods in use claim no inherent cytochemical specificity; the images are validated by the other methods. In practice the methods agree quite well. The arrangements of microtubules (observed with antibodies to tubulin) do predict the locations of centrosomal antigens and vice versa. Anyone who has reflected on the history of the centrosome problem can wait patiently for the chemical characterization of the centrosome. The antigenicity is taken as the proof that the centrosome has a chemistry; therefore it exists and can have properties of shape, size, and perpetuation. So far, the available anticentrosome antibodies are autoantibodies, mostly from human patients. Fortunately, the centrosomal antigens that react with the autoantibodies are not highly specific for species; the human autoantibody reacts with centrosomes of other mammals and with those of sea urchin eggs and plants. Undoubtedly, we will soon have monoclonal antibodies to components of centrosomes; work toward that goal has already appeared (Kuriyama and Borisy, 1985). 3 . Natural History of Centrosomes What do we demand of the centrosomes? Why pursue the now-radical postulate that “the centrosome is the true division-organ of the cell,” as Boveri (1901, p. 5) put it, “Das Centrosoma ist das eigentliche Teilungsorgan der Zelle . . .”?

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The specific demands are that (1) every cell inherits a centrosome, (2) the centrosomes double in each cycle, (3) sister centrosomes separate to form poles, (4) each pole engages to one sister chromosome, (5) each of the sister chromosomes is moved to the centrosome to which it is engaged, and (6) the cell divides through a plane midway between the opposed centrosomes. These statements appear to be elementary, but it may be helpful to assert them as rules of cell reproduction, each of which defines a field of research. A brief summary of the present state of affairs follows. Of course, it expresses judgments by this author. a. Centrosomes have been found in many kinds of cells, including types which were supposed to lack them. The most striking instance is the recent demonstration of centrosomes in plant cells (Wick, 1985; Clayton et al., 1985) by the use of human anticentrosome antibodies. In a way, these new findings represent a turning point; the existence of centrosomes in higher plants has been an awkward problem, explicitly, for a century. In all of the following discussion, I will find it unnecessary to make any distinction between animals and plants. They differ categorically only in mitotic processes involving cell walls. That there are variants within each group hardly needs saying. b. The presence or absence of centrioles does not seem to be significant for the role of centrosomes as mitotic poles. That is clear in the case of higher plants. The case of the mammalian egg is especially informative because the centrosomes do not contain centrioles during early cleavages but display centrioles at later stages. The phenotypic centriole of the spermatozoon that enters the mouse egg at fertilization seems to play no part in the establishment of the mitotic apparatus, as though it has been ousted from its home in the centrosome (Schatten et al., 1985). There are enough cases where centrioles, when present, serve as useful advertisements of centrosomes; they help us to locate centrosomes and perhaps to count the number of subunits in a centrosome. I will not go further. There is too much risk in confusing Mona Lisa’s smile with the Lady herself. c. The continuity of centrosomes may appear to be broken and, in the important instance of the fusion of gametes at fertilization, may need to be broken. The century of work and controversy has not solved one of the fundamental problems of the natural history of centrosomes. In the youthful days of “cell biology,” when centrosomes were very exciting, it seemed reasonable to suppose that the centrosomes of fertilized eggs comprised contributions from both parents, and so it was claimed in Fol’s theory (1891) of the “quadrille of the centers.” The theory was refuted and replaced by the doctrine that the centrosomes of the developing egg were derived from one parent, classically the male. The problem of what happens to the centrosome contributed by the other parent was made very complicated with the discovery of artificial parthenogene-

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sis; an adequately insulted egg will regain centrosomes and will divide. Here was just the paradox that would make for a long debate that became uninteresting until the recent revival of serious work on centrosomes. The argument, whether the induction of mitotic poles in parthenogenesis was a restoration of an inactive maternal centrosome or whether centrosomes can be generated de now, was not productive. If the views to be presented below are valid, it is not difficult to imagine how a centrosome can lose its capacity to function as a mitotic pole and how it can regain that capability. In most eggs, according to the classical work, it is the sperm cell that brings in the active centrosome. In the case of the sea urchin egg the classical view has been confirmed by recent work by Sluder and Rieder (1985) and Schatten et al. (1986). In the former work, the fusion of the paternal pronucleus with the maternal is blocked. The centrosome introduced with the spermatozoon generates a bipolar spindle, engaging the paternal chromosomes. The maternal chromosomes enter what we call a “nonpolar” mitosis; the chromosomes go through the mitotic cycle in a monastral figure that cannot orient them or move them. The “nonpolar” figure says that the maternal centrosome has not been destroyed but has lost the conformation needed for it to serve as a mitotic pole. The latter study is direct: observations based on the anticentrosome antibodies prove that the centrosomes of the egg are derived from the spermatozoon in the sea urchin egg. Now there is evidence that the mammalian egg uses the maternal centrosome for cleavage (Schatten et al., 1985; Schatten et al., 1986). Recent work using anticentrosome antibody fails to detect the centrosomal antigen in the mouse spermatozoon. The centrosome for the cleavage of the egg appears to be derived from centrosomal material in the cytoplasm of the mature oocyte, initially dispersed but detectable. In either case, it appears that all the centrosomes in all the cells of an animal derived from one of the gametes. The centrosome of the other “disappears” during maturation. In the classical case, such as the sea urchin egg, in which the maternal centrosome disappears, the techniques of artificial parthenogenesis can “renature” the maternal centrosome (Mazia, 1984), restoring its ability to make mitotic poles. The proposal invokes the model of the centrosome that I will discuss below. Much of the derives from studies of the “nonpolar” mitotic apparatus in unfertilized sea urchin eggs that are activated to enter the cell cycle. The “nonpolar” mitotic cycle is similar to the normal bipolar in almost all respects; the chromosome cycle proceeds (Mazia, 1974) within a distinct apparatus whose fine structure is almost identical with that of a normal spindle (Paweletz and Mazia, 1978, 1979) but there are no poles, the chromosomes are not oriented, and the cell does not divide. The fact that the “nonpolar” mitotic apparatus is organized by the residual but “denatured” maternal centrosome is now confirmed by staining of the disarrayed centrosome with anticentrosome antibodies (H. Schatten and G . Schatten, personal communication).

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d. Centrosomes do divide into two at some point in the cell cycle. Fission has been observed by all the methods of identification of centrosomes, including staining with anticentrosome antibodies (Brenner and Brinkley , 1982). In cells in which astral rays point to the sites of centrosomes, the doubling of the asters has been a convenient symptom of their division. The duplication of centrosomes does explain the binary division of cells, yet efforts to discover a fundamental replicative event have not been successful. To put the situation more accurately, we have been unable to relate the duplication of the centrosome to known mechanisms of the replication of chromosomal DNA or viral RNA. The search for DNA or RNA in centrosomes, as of 1982, has been summarized by Wheatley (1982); his discussion refers to the centriole but it covers the literature that would be relevant more broadly to the centrosome. There is a literature on a possible role of RNA in the MT-initiating function of isolated centrosome material, but the reports are not consistent. No one has been able to prove that the duplication of centrosomes is forestalled by the inhibition of the synthesis of nucleic acids. For example, recent experiments with uphidicolin, a reliable inhibitor of the replication of chromosomal DNA, indicate that centrosomes multiply when chromosome replication is suppressed. The most extreme example is seen in some kinds of eggs (e.g., starfish) in which cleavages continue after suppression of chromosome replication (Nagano et ul., 1981). Bipolar spindles without chromosomes can be observed. The finding is most important to our thinking about the centrosome cycle. Quite a few observers, observing the doubling of centrioles, have noted that the time of visible doubling is around the S period for the reproduction of chromosomes. It would appear that the underlying cell cycle coordinates the timing of the centrosome cycle and the chromosome cycle even though the two depend on different reproductive mechanisms. (That is hardly suprising, since we know surely that the two cycles are coordinated during the mitotic phase.) The lack of evidence for a self-replicating genomic component of the centrosome has to be balanced against the powerful evidence for the doubling of the inherited centrosomes in each cell cycle. The principle of cell reproduction by binary division depends on the inheritance of one pole and the production of two poles. How shall we take account of the facts? (a) The most convenient attitude is to refuse to consider the question because it is premature. (b) A second position is that reproduction of an entity may not always involve a replicating genome in that entity. The cell may know how to make only one copy of an entity in the course of one cell cycle. (c) The replication of centrosomes may depend on unorthodox mechanisms that fall outside our hitherto-received doctrines of molecular biology. It may belong in the new “RNA world” (Gilbert, 1986), in which genomic RNAs are replicated by catalytic RNAs. Could the reproduction of centrosomes be a vestige of that world? In that case, the reproduction of centrosomes might provide the material for learning something about the postu-

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lated all-RNA system of self-replication-difficult, but more practical than a voyage back to the primeval plant. e. The centrosome cycle occupies the whole cell cycle. Admitting our ignorance of the fundamental replicative mechanism we can define the centrosome cycle in terms of the division and separation of the centrosomes to establish mitotic poles. The cycle entails continuous change in the shapes of the centrosome, as first asserted by Boveri (1901) in his chapter, “The Centrosome as a Cyclical Structure.” These views on the centrosome cycle were revived and restated in recent publications (Paweletz et al., 1984; Mazia, 1984), and I believe that they have now received very strong confirmation in the more recent literature, to be discussed below. They have not yet gained the attention that would lead to serious criticism. A closer view of the centrosome cycle will be given in a separate section, below. f. Centrosomes are MTOCs, but they are much more: They are organizers of half spindles. They dictate the planes of cytoplasmic division. In a sense, the function of centrosomes as MTOCs was emphasized even before MTs as such were discovered. When consulting the old literature, one may have considerable confidence in reading “spindle fibers” as “MTs.” The recognition of centrosomes as MTOCs began with EM observations, was confirmed by experiments on living material, and now is in a stage where isolated centrosome material is used widely for experiments on the growth of MTs in vitro. We are now seeing the first experiments in which isolated, if not pure, centrosomes are combined with isolated chromosomes in studies on the formation of pole-to-kinetochore connections (Mitchison and Kirschner, 1985). In the cell in mitosis, we see the centrosome as the focus of a halfspindle. The half spindle is anything but a region of ordinary cytoplasm threaded by MTs. In living cells we often discern it optically as a region of relative transparency, indicating that large light-scattering particles have been excluded. In some kinds of living cells, the boundaries of the mitotic apparatus are outlined by mitochondria or other structures. Methods of isolating the mitotic apparatus separate it as a discrete body. Thus we will think of the centrosome as the organizer of a distinct and coherent domain of the cytoplasm. The EM reveals membranes within the spindle, sometimes occupying most of the space through which the MTs run. Often membranes are oriented, running parallel to MTs. The striking image of the whole mitotic apparatus isolated without the use of detergents, showing it as a coherent mass of membranous vesicles containing microtubules, might well be the truest picture we have of the spindle as a whole as it exists in the cell (Silver et al., 1980). Going to a more molecular description, we now have an increasing list of interesting molecules that are concentrated sharply in half spindles. That is to say that these components remain segregated in the separating half spindles during

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anaphase. In addition to tubulin and MT-associated proteins, one finds calmodulin (Brinkley et al., 1978), Ca2+ transport enzymes (Hafner and Petzelt, 1986), and dynein (Hirokawa et al., 1985). Kinesin, the latest addition to the list of molecules involved in motility, is located in half spindles (Scholey et al., 1985). The centrosome as the organizer of the half spindle is responsible for the mobilization of many structural and molecular components of the cell in mitosis. The list is bound to increase. Surely one of the most remarkable responsibilities of centrosomes is the determination of the plane of cytoplasmic division following mitosis. So far as I know, there is no exception to the rule that the plane of division is midway between the poles and at a right angle to the axis defined by the poles. Recent observations indicate that the rule holds even in cases where the cell divides under treatment with aphidicolin without forming a metaphase plate. A compelling hypothesis, applied to animal cells and especially to eggs with asters at the poles, proposes that the poles send signals of some sort to the cell surface, dictating the assembly of a contractile ring around the equator (Rappaport, 1971; Rappaport and Rappaport, 1984). The evidence that the contractile ring is an actinomyosin system is strong. The idea of the “signal” has been demystified in the last few years with the demonstration of the transport of cytoplasmic components, such as vesicles, along MTs. Thus, a possible mechanism for communication between centrosomes and the cell surface is in hand, but we would still have to know the nature of the “signal” that is being transported and we would still have to explain how the transport is being directed to the equatorial region of the cell surface. B. THE CENTROSOME CYCLE:SEPARATION OF POLES Boveri’s (1901) firm proposal of a centrosome cycle seems to have been ignored, even though it was supported by observation and explained the observations of others. For example, Figure 58 of Wilson’s (1928) “The Cell in Development and Heredity” illustrates the centrosome cycle during mitosis of the sea urchin egg; it confirms Boveri’s images, and most of the details have now been reconfirmed by modem methods, as will be shown below. Nevertheless, the idea of “The Centrosome as a Cyclical Structure” is not mentioned explicitly in Wilson’s great book (dedicated to Boveri). One can suppose different reasons for the overlooking of a great idea by an investigator so acclaimed as Boveri; one is that he did not have time to write short papers. In my review in 1961 (Mazia, 1961, p. 122), I asked why chromosomes in anaphase do not always converge on a point pole, but move very often as though drawn to a flat pole and even diverge in some cases! The timid suggestion was to “. . . disallow the hypothesis that the centriole is necessarily a single unit. A simple alternative is that the centers are compound structures. The subunits

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might ordinarily be clustered in a compact centriole or might be arrayed in a line or plane to give nonconvergent figures.” It seems to me that the concept of an MTOC (Pickett-Heaps, 1969) demands that the forms of structures built of MTs (such as half spindles) be indicators of the shapes of structures that generate MTs (such as centrosomes). Meanwhile, the notion that centrioles in animal cells are themselves the MTOCs was put to rest some time ago (Peterson and Bems, 1980; Fuge, 1977). It survives in the jargon that refers to the MT-organizing activity as “pericentriolar. ’ ’ There is now sufficient evidence to assert that centrosomes do have various shapes and that the shapes change during the cell cycle. Boveri had based his scheme of the centrosome cycle on observations of sea urchin eggs; in that particular case, his description has been confirmed by all modem criteria: osmiophilia (Endo, 1979, 1980; Paweletz et a l . , 1984), paths of MTs (Paweletz et al., 1984), and immunocytochemistry (Schatten et al., 1986). Modem methods confirm that centrosomes are broad and flat in spindles whose ends are blunt and in which MTs run parallel to the axis rather than converging toward the poles. The evidence for the centrosome cycle has increased substantially since it was reviewed by me in 1984. Thanks to the exploitation of anticentrosome antibodies, we can compare the cycles in sea urchin eggs and in mouse eggs (Schatten et al., 1986) and cells of onions (Wick, 1985; Clayton et al., 1985). The centrosome cycle is a fact. One can generalize and look for meaning in the succession of changes of shape in the course of the cycle. Three propositions:

1. The centrosome cycle is essentially the same in all kinds of cells of “higher” eukaryotic organisms. No distinction need be made between plant and animal cells. The presence or absence of centrioles is not significant for the functions of centrosomes as mitotic poles. 2. The changes of form of the centrosomes during the cell cycle represent stages in division of centrosomes. Division is emphasized because, as has already been confessed, we do not know the mechanisms of the replication that is prerequisite to division. 3. The mechanism of division is the mechanism of separation of poles for the next mitosis. A generalized centrosome cycle is shown diagrammatically in Fig. 1. The diagram represents the common but not universal case in which the centrosomes divide during interphase, at which time they are close to the nuclear envelope. In digerent cells, the phases of the centrosome cycle may have different relationships to the phases of the chromosome cycle. For example, pole separation for the following division may be taking place before a current mitotic cycle is completed.

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FIG. 1. Scheme of the centrosome cycle. See text Section V, B for more complete interpretation. The diagrams refer to the typical case in which the division of the centrosome (black) takes place on the surface of the nucleus (gray). The centrosome is compact around prophase to metaphase (A) and spreads and becomes flattened during the mitotic period (B, C). The extent of flattening determines the shape of the spindle poles (i.e., pointed or flat). The spreading continues around the surface of the nucleus (D, E) and leads to the division of the centrosome (F). The daughter centrosomes condense@, H) and form the poles for the forthcoming mitosis. Thus the sequence of changes of shapes of centrosomes is an expression of their division, and the division brings about the formation of the poles for the next mitosis. The replication of the centrosomes, as contrasted with their division, is not represented in the figure. As is discussed in the text, the nature of the replication remains unknown.

The following paragraphs describe the centrosome cycle as represented in Fig. 1. The letters correspond to the letters in the diagram. A. The centrosome is generally compact at the time of formation of the mitotic apparatus, i.e., the transition from prophase to metaphase. In plant cells as well as in animal cells, the polar ends of the spindles are pointed around the time of metaphase; the spindles are spindle shaped. B and C. The centrosomes become thinner and flatter; the polar ends of the spindles are more or less blunt. If the poles are already flat during anaphase, the chromosomes will move in parallel and not converge; such “barrel” spindles are common in plant cells but not uncommon in animal cells. In some animal cells, the flattening of the poles will not yet have started during anaphase; the spindles will remain pointed.

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D and E. The centrosome continues to spread as the nucleus re-forms. The thin, broad centrosome is difficult to resolve. For the study of these stages, it is useful to observe types of cells that contain centrioles. The latter are indicators of the separation of future poles. By the same token, we can conclude that the centrioles are moved apart by the expansion of the centrosomes in which they lodge (cf. Fig. 8a in Paweletz et a l . , 1984). We do not yet know the extent of spreading of the centrosome around the nucleus; it might even surround the nucleus completely. Certainly, it is extensive and explains the frequent reports that MTs seem to grow out of the nuclear surface. F. The expanded centrosome splits into two. The spreading has been the mechanism of division of the centrosome AND a mechanism of separation of poles for the next division. G and H. The daughter centrosomes draw themselves into more compact forms and we now have two compact poles for the establishment of the mitotic spindle. To repeat, the description of the centrosome cycle as a sequence of changes of shape is a description of the division cycle of the centrosome. Further, the division cycle accounts f o r the bipolarization of the cell for the forthcoming division. Existing evidence tells us that such a cycle exists, although the above scheme does include interpretations and interpolations. Work on sea urchin eggs, mouse eggs, and plant cells does at least verify Boveri’s observation that compact centrosomes broaden, flatten, divide, and condense during the cell cycle. (We can still wonder that he could see what he drew but, as one reads the older literature, one can also wonder that so many other fine cytologists drew the cycle without seeing it.) If the scheme is not a fantasy, we may try to place it in the real world of cells, where mitosis looks different in different kinds of cells, and in the real world of cell biologists, where each of us likes to think of his favorite cell as the archetype of the cell. Variants of the centrosome cycle arise from a few variables, here listed. 1. The centrosome cycle is not tightly synchronous with the chromosome cycle. By convention, the phases of the cell cycle are defined by the phases of the chromosome cycle (now including the interphase as well as the mitotic periods). The seemingly large differences in the appearance of the mitotic apparatus in different cells are not profound. Pointed spindles (and convergent chromosomes) at nominal anaphase only tell us that the centrosomes have not begun to divide at the time when the chromosomes are moving to the poles. Barrel-shaped spindles (chromosomes moving on parallel paths at nominal anaphase) merely mean that the poles are progressing in their division for the next mitosis and are now flat. In experiments in which we retard the chromosome cycle relative to the centrosome cycle, we can have chromosomes moving to abnormally flat poles or even to already-divided poles. If the centrosomes have advanced into their divi-

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sion before the chromosomes advance into anaphase, the chromosomes separate to four poles and produce four nuclei (Mazia et al., 1960; Sluder, 1978; discussed in Mazia, 1984). The only requirement that is normal for all cells is that the poles be separated by the time of the formation of the mitotic apparatus. Even this rule may be violated in interesting mutants in which the centrosomes have doubled but not separated by the time of the formation of the mitotic apparatus (Wang et al., 1983). There, the doubling of the centrosornes is confirmed by counts of centrioles. The mitotic apparatus is monopolar at the restrictive temperature. Here is a case of duplication of centrosomes while the division-bipolarization cycle is incapacitated by the mutations. In experiments on the sea urchin egg (Mazia et al., 1960; worked out fully by Sluder and Rieder, 1985) it has been proved that one division-bipolarization cycle can proceed without duplication of centrosomes, demonstrating that a normal pole may carry a pair of centrosomes. 2. Centrosomes can assume a great variety of shapes, some of which may be extreme deviations from a compact corpuscle. In Section V,C I will propose that the shapes of centrosomes derive from a fundamentally linear structure, which can take on varying three-dimensional conformations. 3. Figure 1 only indicates the overall shapes of centrosomes as they go through the common division-bipolarization cycle. Our habits of seeing may exaggerate variations among different cells. For example, the division of a centrosome may look like the separation of two asters. Figure 1 says that the centrosome is thin and flat during its division, but we will see separating asters if there is a concentration of MT-initiating units at its two polar ends. We can understand why early generations of cell biologists were impressed by the distinction between spindles with asters and “anastral” forms. Nowadays, asters are being detected in cases that were regarded earlier as “anastral” (Bajer and MolC-Bajer, 1981).

C. A MODELOF THE CENTROSOME Our discussion assumes that centrosomes are integral structures. A cell normally inherits one (more likely one pair) from its mother. In the normal case, the cell has made two and only two poles by the time it divides; we assume a doubling of centrosomes somewhere in the cycle. In the case of the sea urchin egg, we have good evidence that the inherited pole contains a pair of unit centrosomes and that the doubling produces two pairs. The units are “assayed” by experiments designed some time ago (Mazia et al., 1960) in which the division of the centrosomes was permitted while their replication was prevented. (The experimental design is based on the fact that the chromosomes are arrested at a metaphase stage. The centrosomes cannot reproduce during the arrested mitosis, but they do continue into the divisionbipolarization phase of their cycle.) Two normal poles divide to make four, but

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each of the four can only make a monopolar mitotic apparatus in the next cycle; hence the normal poles are “bivalent” (Mazia et al., 1960, 1981; Sluder, 1978; Sluder and Begg, 1985). The assay has been confirmed directly by the tracking of centrioles, using high-voltage EM (Sluder and Rieder, 1985). Two poles, each containing two centrioles, divide into four poles, each of which contain one centriole and give rise to four monopolar mitotic apparatus. In this case, at least, the number of centrioles is a valuable indicator of the number of potential poles. If we may generalize, mitotic poles are “bivalent” and they may divide at least once without replication. Such evidence for the integral centrosome is based on the extreme precision of the formation and behavior of mitotic poles: a reproductive cycle that is essentially infallible. Mitosis is bipolar and we explain the few exceptions by changes (small) in the number of centrosomes. We cannot claim to have direct evidence for the physical continuity of a centrosome at all times. (The problem is analogous to the early doubt about the integral character and the physical continuity of the chromosomes.) In fact, there are some cases in which the unitary character of centrosomes needs to be defended. One is the polyarchal formation of the mitotic apparatus in meiosis in some plant cells (discussed by Wilson, 1928, p. 54) which was well known to cytologists at the turn of the century. Polyarchal mitotic spindles have now been discovered in an animal cell type, a neuroblastoma, in culture (Spiegelman et al., 1979). As the cells enter mitotis, many “poles” are seen around the nuclear region; one would guess that the cell was forming a multipolar mitotic apparatus. However, by the time of metaphase, the apparent “poles” have come into focus in a bipolar mitotic apparatus which has aligned the chromosomes in a normal metaphase plate. Recent work on mouse eggs (Maro et al., 1985; Schatten et a f . , 1986) calls attention to a considerable number of small asters in the cytoplasm at the time of maturation, suggesting the presence of numerous separated MOTCs. Here too, the small MT-nucleating sites gather together to form two correct poles at the correct time. One cannot exclude the possibility that centrosomes can be fragmented at some stages of the cycle and reassembled with great precision when the time comes to separate the chromosomes. However, we can interpret the evidence of spread-out centrosome activity as evidence for a spread-out linear centrosome. We have already examined the changes of shape in the division cycle of the centrosome. We now imagine more extreme changes, in which the centrosome appears to be greatly extended-‘‘unwound,’’ so to speak, but able to rewind to form a compact pole. The seemingly separate foci of MTOC activity would represent nodes of the expanded centrosome, not really separate from each other. The linear centrosome is a speculation, but it does offer a reasonable way of accounting for various shapes and changes of shape (reasonable to us biologists who are familiar with the creation of structure in two and three dimensions by the

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folding, bending, coiling, and looping of long threads). The linear centrosome would be very long, but our decades-long struggle with chromosome structure has accustomed us to the problems of packing molecular threads into micrometer dimensions. The minimum specifications for a centrosome call for considerable complexity. First, we are inclined to postulate the presence of a genomic component, even though the efforts to identify one have been disappointing. We can only say that the number of centrosomal units doubles at some point in the chromosome cycle. That follows from the work on “assays” of units, discussed above, in which we can prevent the doubling while allowing the existing “bivalent” units to divide into “monovalent” units. The monovalent units are expressed vividly as monopolar mitotic apparatus. We can think of a model of the centrosome as an MT-“ordering” organ even as we recognize that it has additional functions. As an MT-ordering organ it is a determiner of the origins and directions of MTs. In the model I propose that the linear unit centrosome (whatever its constitution) is a carrier of MT-initiating units. One supposes each such unit to be a group of molecules (or small assembly) responsible for the initiation and the direction of growth of one MT. The folding and twisting of the centrosomes determines where MTs will start and will point them in the direction of growth. The conformation of the linear centrosome, determining the origins and directions of MTs, embodies the information for the structural forms that are built by MTs. It is no longer necessary to argue that the MTs of the mitotic spindle are initiated by centrosomes. At least, that is my judgment of the trend of the recent literature. Some items of evidence for the newer confidence are:

1. Observations on monopolar spindles prove that single poles make half spindles; in half spindles chromosome-to-pole connections are made only by kinetochores that face centrosomes (Bajer et al., 1980; Mazia et al., 1981). 2 . Methods for displaying the molecular orientation of tubulin in MTs of the spindle indicate that all the MTs of a half spindle have the same orientation, the one expected if all the tubules grow from the centrosome (Eutenauer and McIntosh, 1981). 3 . Experiments in vitro, mixing isolated centrosome material and isolated chromosomes in an MT-assembly medium, indicate the growth of MTs from centrosomes and the capture of MTs by kinetochores (Mitchison and Kirschner, 1985). The postulate that the centrosome is a carrier of discrete MT-initiating units is still unproved. True, there is a general quantitative relation between the “concentration” of centrosome material and the number of MTs it generates. One can

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FIG. 2. Model of the flexible linear centrosome. The conformation of the linear centrosome determines the three-dimensional structures built of microtubules. The linear element carries microtubule-generating units, each of which specifies the origin and direction of one microtubule. Left, the centrosome functions as a flat pole, generating a “barrel” form of half spindle. Right, a compact centrosome generates a half spindle with a pointed pole and an aster.

deduce the relationship from various kinds of evidence. For example, fragments of centrosomes observed after irradiation of cells generate fewer MTs than do whole centrosomes (Sato et al., 1983). However, we now have direct evidence, since we can compare amounts of centrosome antigen at given sites in cells with amounts of tubulin at the same sites, using antibodies to tubulin. Of course the immunocytochemical methods are not quantitative, but the relationship is obvious in Fig. 2 of Schatten et al. (1986); the bigger and brighter the fluorescence images of the centrosomal foci, the bigger and brighter the images of the MT asters. The model, as illustrated in Fig. 2, postulates that individual MTs are generated by discrete initiators, each of which determines the starting point and the direction of growth of an MT. A considerable literature, dating back to the earliest work on the assembly of MTs in vitro, records the effort to identify such initiators or nucleating elements. At present, more and more investigations use centrosome material for studies of the initiation and growth of MTs in v i m (e.g., Mitchison and Kirschner, 1984). That strategy should lead ultimately to the identity of the suspected initiator. The model of the linear centrosome requires that the shape of a structure built of MTs be determined by the “native” folding of the linear centrosome. While isolated if impure centrosomes have been adding a good deal to our knowledge of the assembly of MTs, it is not easy to demonstrate that they can generate anything more complex than a simple aster. According to the model we are discussing, an aster is the most random structure possible; a perfectly symmetrical aster would grow out of a completely scrambled association of initiators carried on a randomly folded linear centrosome. The inference is that centrosomes are “denatured” in the present methods of isolation.

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Native centrosomes should, depending on their source, generate forms such as half spindles with pointed or flat ends and with or without asters. The model demands that some centrosomes be able to generate phenotypic centrioles, implying that they carry MT-initiating units arranged so as to give rise to nine triplet groups of MTs. (When we “see” a centriole we are seeing an arrangement of 27 MTs, generated by an underlying arrangement of MT initiators; we do not see the initiators.) Once we think of phenotypic centrioles as MT structures generated by linear centrosomes, we can account for the disappearance and reappearance of centrioles and for aberrant centriolar morphologies such as C shapes or unusual numbers of MTs. That does not solve the “central enigma” (Wheatley, 1982) posed by the centriole, but perhaps it forwards the inquiry to the correct address.

D. SUMMARY: THE CENTROSOME AS

AN

ORGANOF THE CELL

Although there are signs of life, I am reluctant to judge whether study of the centrosome is about to assume an important place in cell biology. The factual part of my discussion does, in my opinion, provide reasonable confirmation of the main theses that were set forth early in the century: the centrosome exists in all kinds of eukaryotic cells; it is permanent throughout the cell cycle; it goes through a cycle of changes of form during the cell cycle; it divides and it accounts for mitotic poles and the normal bipolarity that dictates normal binary division of cells; it seems to double once in each normal cycle; and it generates structure other than that of the mitotic apparatus, a topic I have not discussed. The speculations I have ventured to present are rather conservative in that they apply established ways of thinking to a body about which we know very little. (1) The centrosome doubles in each cell generation and therefore we look for a replicating genomic component; (2) it undergoes a consistent cycle of changes of shape and there we appeal to a linear structure, capable of changes of conformation; and (3) it generates consistent patterns of MTs in three dimensions and therefore we put the still-undiscovered MT-initiating elements into linear structure. The proposal represented in Fig. 1, which identifies the changes of shape with the division of the centrosome and identifies the division with the production of two mitotic poles, is an interpretation of facts. The broader hypothesis that the centrosome is a bearer of information about cell morphology is a speculation that extends beyond Boveri’s often-quoted assertion that the centrosome is the “kinetic center” of the cell. To summarize the speculation: The conformation of the linear element ‘ ‘determines the arrangement of (MT)-initiatingunits in a centrosome and the orientation of the individual units determines the morphology of structure built of microtubules” (Mazia, 1984). The idea appears in a recent study of the fine structure of one manifestation of a centrosome, the “cell center” that generates the microtubules in fish erythrophores, where the microtubules guide the move-

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ments of pigments. The authors (Gershon et af., 1986) used computer graphics methods to analyze images obtained by high-voltage electron microscopy. The analysis revealed stacks of flat, dense bodies in which the centrioles are embedded. The authors concluded: “From these findings and considerations, it seems that the cell center is an organelle where information about the three-dimensional structure of the cell is stored.” Speculating on the basis of facts about the centrosome cycle and of interpretations that are expressed in a model of the centrosome, I am also expressing the feeling that something truly fundamental is missing in our image of the cell and that the “something” exists at a level of complexity higher than that of molecules and perhaps more comparable to the complexity of chromosomes. So far, our doctrines about how the cell makes itself start with chromosomal genes and proceed through transcription, nuclear processing, and translation to create a wealth of macromolecules. Now, knowing so much about how cells make molecules, we can allow ourselves to look ahead into the problems of how molecules make cells. We can go some distance with the concept of self-assembly; we have to admit that viruses manage pretty well. Still, centrosomes exist and do double, divide, and construct the mitotic apparatus and manage its nearly infallible operations. Cell biology may yet add interpretation to the chain-of-command that describes the dictates of genes. If so, the centrosome will make its claims to be the organ of interpretation through which structure is managed. Thus, one makes a bet on the future of cell biology, knowing that a bet is only a guess that hopes to become a prophecy.

VI. Establishment of the Mitotic Apparatus: Boveri’s Rules By the establishment of the mitotic apparatus (MA), I mean the formation of a bipolar mitotic spindle in which sister chromosomes are connected to opposite poles. The chromosome cycle and the centrosome cycle intersect. Given the reproductive accuracy of the chromosome cycle and the centrosome cycle, the near infallibity of mitotic cell division has to be explained by the accuracy of the centrosome-to-kinetochore connections which exclude the engagement of sister kinetochores to the same poles. An excellent recent overview (Nicklas, 1985) summarizes efforts at explanation. A. BOVERI’SRULES The now-prevailing ways of looking at the problem of the establishment of the MA can be derived from the rules stated formally by Boveri in 1888 (Boveri,

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1888, p. 98). It is useful to repeat these rules because the problems they raise are demanding renewed attention. In free translation: 1. “The chromatin elements are able to make connection with the archoplasmic fibrils only on their narrow sides.” Comment: “Chromatin elements” refers to sister chromosomes or chromatids; the doubleness of the mother chromosome at this stage was recognized. “Narrow sides” anticipates kinetochores; the point was important because it explained how chromosomes divided lengthwise. The longitudinal splitting was the historic basis of the principle that mitosis distributes chromosomes equally to daughter cells (Mayr, 1982). “Archoplasm” was Boveri’s term for what we would now call cytoskeleton but with special reference to cytoplasmic structures organized explicitly by centrosomes, such as asters and spindles. 2. “If the first fibril from a sphere (pole) connects with one side of a (chromatin) thread, other fibrils from the same pole can only connect to the same side, even if the other side is still free.” Comment: “Sphere” refers to the cytoplasm organized around a centrosome; its substance was called “archoplasm” (We could, if we felt the need, revive the term “archoplasm” for the substance of the zones organized by centrosomesfree from large particles, rich in membranes and MTs.) The fact that all the fibrils (MTs to us) at the same kinetochore are connected to the same pole is the challenge we still face. Why can a kinetochore not receive MTs from two poles or (recognizing an alternative view) why can individual MTs originating at a kinetochore not connect to two poles? 3. “If one thread (sister chromosome) is already connected with one pole, the radii (fibrils, MTs) of the other pole can connect only with the other sister chromosome.” The quotations from the then 25-year-old Boveri are not intended as genuflections to the past. They are here as a completely valid statement of the basic rules of mitosis and they make the difficulties evident. Why is a centrosome unable to engage two kinetochores? Why is it impossible for two centrosomes to hook up to one kinetochore? The system is not infallible under all conditions. Aneuploidy is a common result of growing cells in culture. In yeast, gross defiance of Boveri’s rules can be studied in mutants (Broach, 1986). B. THE PUPPETSHOW Boveri and later workers, up to our time, pictured the MA as a puppet theater in which strings (spindle fibers, MTs) tied kinetochores to centrosomes. Connections were made initially according to Boveri’s second rule: the first connection of one chromosome to a pole excludes all possibility of any connection of the sister chromosome to that pole. No chromosome can connect to two poles. The

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metaphase arrangements are explained mechanically. Connection of a kinetochore to a pole sets up a virtual “pull,” orienting that kinetochore toward the pole and “pulling” it toward its pole. The sister kinetochore is quickly connected to the other pole and therefore “pulled” toward it. The metaphase plate (in cases where it occurs) represents the equilibrium of forces on a stillunseparated sister pair of chromosomes. The mechanical implication, recognized in the literature, is that the “pulling” force is proportional to the length of the kinetochore-to-pole fiber; thus the equilibrium position is midway between the poles. The term “orientation by pulling” is used in the literature. The theory of “orientation by pulling” enjoys a good deal of experimental support. For example, it predicts the finding that a chromosome that is displaced from the spindle by micromanipulation will return to the metaphase position after MT connections are reestablished (Nicklas and Kubai, 1985). Some difficulties arise when the very first-and supposedly decisive-stages of the engagement of chromosomes to poles are examined. A single kinetochore may be associated initially with MTs from both poles; that is seen very clearly in EM images of the second meiotic division of Urechis (Luykx, 1965, Figs. 1 and 2). In that case, the commitment of one kinetochore to only one pole would be a second step. Similar observations are reported for the case of the first prometaphase of Drosophilu spermatocytes (Church and Lin, 1982). The description includes a variety of unpredicted configurations: kinetochores not engaged to adjacent MTs, kinetochores connected to both poles, and kinetochores connected to poles that they do not face, etc. The observations, and others cited by the authors, do not necessarily contradict the broad hypothesis of “orientation by pulling,” but they throw very serious doubt on the most obvious interpretation: that the first linkage of a fiber to a kinetochore, depending on the direction in which the kinetochore happens to be facing at the decisive moment, decides the engagement and destination of the chromosome.

C. THEMONOPOLAR MITOTICAPPARATUS The monopolar MA challenges our suppositions about the engagement of the chromosomes to poles, since “orientation by pulling” calls for the action of two poles. Monopolar MAS were encountered by the old cytologists. Boveri (1888, Fig. 62a) figured a clear example in the Ascaris egg; two separate half spindles were seen in the same egg. The chromosomes were aligned as in a metaphase plate. Wilson (1928, p. 168) discussed the problem of “monocentric mitosis” and called attention to cases in which the chromosomes form a “. . . group which typically lies on one side of the aster,” an arrangement that “corresponds to the stage of the equatorial plate in normal mitosis.” Although the older literature includes quite a few descriptions of monopolar MA, the reader does not sense much excitement. A recent upsurge of interest is

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addressed to contemporary problems. Monopolar MAS found in cultures of newt cells and studied by time-lapse recording are providing important information about chromosome movements in relation to poles, impressing us with dynamic relationships between chromosomes and single poles (Bajer et al., 1980; Bajer and Molt-Bajer, 1981). Chromosomes, with only one of the sister kinetochores connected to the pole, move back and forth in relation to the pole. Clearly, chromosome movement does not depend on two poles. In relation to some propositions I will consider later, it is important to note that the chromosomes, when they connect to the pole, do not simply migrate to that pole, as might be predicted from the simplest version of the “orientation by pulling” image. Studies on sea urchin eggs (Mazia et al., 1981) take advantage of an experimental trick whereby large numbers of monopolar spindles may be generated. The experimental design has been discussed above (Section V,C). From the EM study, it is clear that the phenomena of the metaphase ordering of the chromosomes can be carried out with a single pole. The chromosomes form the equivalent of a metaphase plate, the kinetochores are oriented, and only the kinetochore facing the single pole makes MT connections. No MTs appear on the sister kinetochore that faces away from the pole. Monopolar MAS are produced in a temperature-sensitive mutant line of Syrian hamster cells (Wang et al., 1983). The defect is a failure of the centrosomes to separate; duplicated centrosomes remain in a single pole. A perfect half spindle forms; the chromosomes line up in a virtual metaphase and one of the sister kinetochores connects to the pole. The oldest and the newest descriptive features of the monopolar MAS are informative and disturbing to almost any hypothesis that calls for the collaboration of two poles. (1) The monopolar MA forms a half spindle; it is not merely a monaster. That is shown clearly in the oldest illustrations. (2) The monopolar apparatus is real and coherent; it can be isolated as a body by the methods used for isolating the normal MA (Mazia et al., 1981). ( 3 ) The chromosomes are aligned in the equivalent of a metaphase plate, although the plate is curved. (4) The kinetochores are oriented relative to the pole; one sister faces the one pole, the other faces, quite exactly, away from the pole. (5) The kinetochore facing the pole is connected to that pole by MTs; the one facing away is innocent of MTs. Legally, the monopolar MA provides a brilliant confirmation of the basic law of mitosis. Only one of the sister kinetochores can attach to one pole. However, the monopolar MA seems embarassing for a number of suppositions about the mechanisms of the formation of the MA, those that demand the interplay of two poles. The theory of “orientation by pulling,” requiring two poles for the proper engagement of chromosomes and explaining metaphase as an equilibrium of forces from two poles, would seem to be refuted by the monopolar MA. One could even have predicted that a monopolar metaphase would be impossible

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because the engagement of kinetochores to one pole would immediately pull all the chromosomes to that pole. The facts of monopolar mitosis need not be denying the prevailing and reasonable views about the engagement of chromosomes in normal mitosis. They may be cautioning us that our image of the proceedings has been grossly oversimplified. The evidence for orientation by pulling may represent a second step, after the chromosomes have been positioned by some other means, about which I will speculate below. How simple it has been, from Boveri’s time to ours, to picture the play in a cytoplasmic void! There are centrosomes; chromosomes are lying around; the centrosomes spin out some fibers (or, maybe, the kinetochores spin out some fibers); centrosomes catch kinetochores (or vice versa)! If we advance in history to our times, when centrosomes are MTOCs and kineotchores are captors of MTs (or vice versa) and the background is a buffered pool of tubulin and we can enjoy truly beautiful experiments in which isolated centrosome material can capture isolated chromosomes, we still have to face the problems of the accurate engagement of chromosomes. Perhaps we need to employ more scenery and stagehands in order to explain the puppet play, and we need to examine the roles of the actors more closely. The following speculation is based mainly on our experience with cleavage mitosis in sea urchin eggs-classical and much-studied material but no more nor less typical than any other. The monopolar MA tells us that one pole (which may be embodied in one centrosome or an unseparated pair of centrosomes) can make a complete half spindle that holds the shape of half of a normal spindle. It has the same ground structure as one finds in a normal spindle and that structure is easily distinguishable from the surrounding cytoplasm or even from asters. The half spindle is a body, a structural and molecular domain of the cell. A simple consequence of the observations is: the halfspindle has a boundary at the virtual equator, that is, the boundary facing into the cell. Let us call it the “equatorial boundary”; we are not using the term “equatorial” in a geometric sense but only to compare the monopolar spindle with the normal bipolar.

D. A NEWMODEL Now, the speculation, illustrated diagrammatically in Fig. 3: 1 . Chromosomes are attached to the half spindle at the equatorial boundary. The attachment may precede the formation of MTs connecting kinetochores to centrosomes. One may suppose that only the kinetochore (and not other regions of the chromosome) can enter the margin of the equatorial margin of the half spindle.

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FIG. 3. Speculation: A steric theory of the initial step in the engagement of kinetochores to poles. A kinetochore can be engaged by a centrosome only when it is in contact with the equatorial margin of a half spindle. Because the diameter of the kinetochore site is smaller than the diameter of the whole condensed chromosome, and because sister kinetochores face in opposite directions, it is sterically impossible for both sister kinetochores to enter the same half spindle. The components required for making microtubular connections are confined within the half spindle. Left panel: A monopolar mitotic apparatus. When one kinetochore enters the half spindle, it is engaged and is oriented to the one pole. The sister kinetochore faces away from the margin of the half spindle and cames no microtubules. The image corresponds to all observations of monopolar mitotic apparatus. Center panel: The bipolar spindle consists of two apposed half spindles. The kinetochore that cannot connect to one half spindle connects to the other. When both kinetochores are engaged, the bipolar spindle can be described as in the theory of “orientation by pulling.” Right panel: Meiotic reduction division. In the meiotic division, sister kinetochores lie side by side, as reported in the literature. Engagement of sister kinetochores to the same pole and the engagement of homologous chromosomes to opposite poles is expected.

2. Once a kinetochore is in the half spindle, it can “see” (and be “seen” by) the MT-processing system in the half spindle. Here only the idea of the penetration of the kinetochore into the half spindle is a speculation. The evidence that all the molecules and regulations involved in making MTs are concentrated in the half spindle is very strong. ‘ , 3. This is not the place to discuss a literature that keeps growing: one demonstrating that the structurally coherent half spindle (including the centrosomal region) is the region of the mitotic cell which monopolizes the supplies of MTs,

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tubulin reserves, MAPS, calmodulin, Ca2 -regulating vesicles and pumps, dynein, kinesin, etc. A proper view of the literature would, I believe, sustain the conclusion that every component of the cell that we know to be involved in the assembly of MTs and in MT-based motility is focused in the half spindle. I refer to data on normal MA at anaphase and presume that the same results would be obtained with monopolar spindles. My speculation is that pole-to-kinetochore connections can be made only within a half spindle. 4. Having penetrated the equatorial margin of the half spindle, the kinetochore can connect to the pole. We need not decide at this point whether the kinetochore is an initiator or a terminator of MTs. The difference will matter immensely as we go deeply into the molecular mechanisms of the engagement of chromosomes but, at the level of understanding the puppet show at the microscopic level, what matters is making the correct connections. (In recognizing a connection between Manhattan and Brooklyn, we do not need to decide whether the bridge starts in one city or the other.) 5 . It is not possible for both sister kinetochores of a chromosome to penetrate the equatorial margin of one half spindle. This proposition is the basis of my speculation. It derives the operation of Boveri’s law from the structure of condensed chromosomes and the structure of the half spindle. We are no longer picturing the chromosomes and the centrosomes as free-floating objects, one or the other throwing out ropes that ultimately make the right connection. The proposition deviates considerably from the views that date back to Boveri. It does not say that the initial fibrillar connections of chromosomes to poles somehow determine the exclusive selection of chromosomes by poles. It says that the selection of one pole by one chromosome (and the exclusion of the sister chromosome) is made by the interaction of the one kinetochore with the “equatorial’’ margin of one half spindle. How do we explain the exclusion of the sister kinetochore? Here one appeals to the most fundamental feature of the fully condensed chromosome: the sister kinetochores are on opposite sides; they face in exactly opposite directions. This fact is fundamental for any theory of the engagement of chromosomes and it is a fact, well documented in cytogenetic literature and in recent reviews on the kinetochore (Alov and Lyubskii, 1977; Rieder, 1982; in their Fig. 5, the former authors propose that the kinetochores protrude from the surface of the chromosomes during prometaphase). My basic speculation is that it is sterically impossible for two kinetochores confined on opposite sides of a pair of condensed chromosomes to penetrate into the plane of the equatorial margin of a half spindle. Only one kinetochore can be caught. The phenotypic kinetochores are indeed localized on opposite sides and can project from the opposite surfaces as trilaminar structures, sometimes resolved as hemispheres (Rieder, 1982). With that kind of structure, steric consid+

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erations might forbid the penetration of one half spindle by two sister kinetochores. The argument would also apply to the case where the kinetochore activity is confined to a spot (or line) that is small compared to the width of the chromosome. We may as well note that sister kinetochores would be engaged to the same pole if the kinetochores were side by side rather than on opposite sides of the chromosome pair. That is indeed the case in meiotic reduction divisions (e.g., Wagenaar and Bray, 1977). 6. The penetration of a kinetochore into the half spindle is followed by engagement to the pole, and that is followed by “orientation by pulling.” Evidence is the fact that the engaged sister kinetochore in a monopolar spindle does face its pole and its sister faces in the direction of the missing second pole. The evidence speaks of pull on the one sister, turning her toward the pole, and turning her untethered sister as though the two are quite rigidly attached. 7. The “orientation by pulling” in a monopolar MA draws the chromosomes into a metaphase plane that lies at some distance from the pole and is often seen as curved. The arrangement is easy to understand in terms of the pulling of chromosomes on the surface of the relatively solid half spindle. It is the resistance of the half spindle, not the pull from an opposing pole, that explains why the chromosomes do not move directly to the pole. 8. The normal bipolar spindle is composed of two monopolar spindles. In other words, “. . . the basic autonomous structural unit in mitosis is not a bipolar, but a monopolar spindle” (Bajer et al., 1980). This opinion is not new (cf. Wilson, 1928, p. 143) and is not tautology. If a bipolar spindle is made by uniting two half spindles, we may imagine that the formation of the MA will collect the chromosomes between the equatorial faces of the half spindles. When we appose the equators of two monopolar spindles, the sister kinetochore that does not penetrate one half spindle can penetrate the other, setting up connections to the second pole. The “orientation by pulling,” now working from two poles, will align the chromosome pairs on a flat metaphase plane. When one of the sister kinetochores but not the other makes the correct connection, the subsequent anaphase will result in an aneuploid distribution. It is easy enough to find evidence that bipolar spindles can be made by the joining of monopolar half spindles. That would be inferred from Boveri’s observation (1888) that Ascaris eggs occasionally make two nice monopolar figures instead of the normal bipolar. In echinoderm eggs, one can make cells containing two half spindles, and one can observe the union of the half spindles if there are no impediments. The half spindles go through monopolar mitosis if kept apart by compression of the egg but unite in a bipolar figure if allowed to approach each other (C. Sat0 and D. Mazia, unpublished work), whereas the monopolar MAS are kept apart by the compression of the egg and then allowed to join by relieving the compression.

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To summarize, the speculation I am proposing does not overturn the longprevailing views, but adds some considerations based on more modem views of centrosomes, half spindles and chromosomes, and our growing experience with monopolar mitosis. Fundamentally, it embeds the free-floating puppet play into a more structured intracellular background. a. Each centrosome generates a half spindle, in which are confined all the molecules, molecular systems, and regulating systems for the origination, growth, and anchoring of MTs between centrosomes and kinetochores. b. The kinetochore is the only part of the chromosome that can penetrate into the “equatorial margin” of the half spindle, where it will be engaged to the centrosome in charge of that half spindle. c. For steric reasons, illustrated in Fig. 3, two sister kinetochores cannot enter the same half spindle. d. A bipolar spindle is made of two half spindles that come together at their “equatorial margins” to form a bipolar spindle. e. As one kinetochore can enter one half spindle, its sister can enter the other. The “equatorial margins” of the two half spindles make the equator of the bipolar spindles. f. The subsequent steps are the same as in the most-accepted theory of “orientation by pulling.” The sister kinetochores, each in their half spindle, are connected to the respective centrosomes by microtubules. The behavior is accounted for by the image of “pulling,” whatever that means in terms of mechanism. The ‘‘pull” orients the kinetochores to face the poles. g. In the bipolar situation, the equilibrium of the forces on the sister kinetochores is expressed in a flat metaphase plate, equidistant from the poles. In a monopolar spindle, the metaphase plate is curved, expressing the equilibrium between “pull” by the poles and the resistance of the relatively solid half spindle to the further advance of the chromosomes to the poles. These speculations were motivated by specific questions about centrosomes as organizers of complete half spindles and about the law-abiding behavior of centrosomes even when there is only one pole. The speculation represents an effort to put together one body of experience and especially the problems posed by the monopolar MA. It is not rich enough to deal with objections based on other special cases. My own most serious reservation stems from the failure of efforts to dissociate a bipolar spindle at prometaphase or metaphase into two half spindles or to find, in the many beautiful pictures of bipolar spindles at metaphase, convincing evidence of a boundary between two half spindles. Still, the bipolar MA is seen as two half spindles as soon as anaphase gets under waywould that not indicate that the two half spindles were present and separable before the splitting of the chromosomes allowed them to move apart?

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VII. Conclusion The five chapters of this essay do come together. The chromosome cycle of decondensationtcondensation contains two climaxes of the cell cycle. The ultimate decondensation in interphase is an indispensable condition for the replication of the chromosomes. Now we see that the correct condensation for mitosis is much more than a way of making a compact package for transport in prometaphase and metaphase. It is the indispensable condition for the accurate segregation of the sister chromosomes. What is ultimately important is the “sidedness” that puts the kinetochore on one side of a chromosome so that the sister kinetochores face in opposite directions. The “sidedness” of kinetochores, their engagement to the poles that they face, is demanded by all theories about the establishment of correct chromosome-to-pole connections. The facts about the locations of kinetochores on the opposite sides of condensed sister chromosomes place imperative demands on any model of the condensed chromosome. The very long nucleosome thread must, after replication, start to pack up in such a way (whether as tight coils, solenoids, loops on a scaffold, etc.) so that it forms a pair of tight fat sausages which carry a certain tract of DNA, the kinetochore (centromere) DNA, at matching sites along each sister chromosome and on opposite faces of the chromosome pair. In short, we will understand neither the replication nor the distribution of genetic material without understanding the chromosome decondensation/condensation cycle. At the midpoint of the present century it was understood that we had to know the complete three-dimensional structure of a protein molecule with the accuracy that could then be realized only with simple crystals. That dream having come true, we have to think of understanding the threedimensional structure of an immense mitotic chromosome with comparable accuracy. We are just now overcoming a certain prudishness toward centrosomes and the centrosome cycle. The problem was real enough: how shall we deal with something that declares itself so forcefully in action (after all, one cell always makes two cells) but makes itself visible in such varied forms-or not at all? If there is any merit in the interpretation and model that has been discussed above, the variability of the forms of centrosomes from cell to cell and from stage to stage becomes a necessity. Recognizing their protean character as a necessity, we can look to proteins for more imtimate insight. In my opinion, the hypothesis of the flexible centrosome and the general flow of events by which it divides and produces two poles now has a factual basis. The speculation that the precision of kinetochore-to-pole connection is determined by the sterically limited interactions between kinetochores and the “equatorial margins” of half spindles depends on the evidence that the centrosome is more than an MTOC.

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The evidence that the centrosome is the organizer of a complex half spindle that can be regarded as a distinct structural phase within the cell seems strong enough. (Alternatively, we could stress the “0” in MTOC, recognizing that the centrosome is much more than initiator of the growth of MTs; it also dictates the location of precursors, accessory molecules, and the ion-regulating membranous structures.) It is as though we included the construction of the incubator, the circuitry of the pH meter, the weighing of chemicals, etc. as part of an account of the assembly of MTs in the laboratory; in fact, we might have to describe the architecture and furnishings of the laboratory. In the mitotic cell, the centrosome does all that and more. The centrosome makes the laboratory and the test tube and assembles the reaction system-only then does it initiate MTs. The chromosome cycle and the centrosome cycle interact in the establishment of the MA. We can account for the perfections and the pathologies of the partitioning of chromosomes by seeing the engagement of the chromosomes as an encounter between kinetochores and half spindles. The locations of sister kinetochores have to be determined by the higher-order structure of chromosomes; they are. The structures of half spindles have to be determined by huge and elusive centrosomes; they are. These can seem to be formidable problems, prematurely imposed, better ignored by serious researchers while we await new methods. Allowing ourselves to be less serious, we can take some pleasure in the anticipation of 2 1st-century cell biology.

ACKNOWLEDGMENTS There is no way in which I can thank all of those who have directed my thoughts about the problems I have ventured to discuss in this essay. All too many of them have departed this world and can no longer object to my interpretations and misinterpretations of their teachings. Others, the youngest, are creating the future of a field that is not so tempting to those who need to deal with safe subjects. I do want especially to remember the late James F. Danielli, a founder of the International Review of Cyfology. A friend for over a half a century, he was one of those who believed that fresh ideas should play an important part in the advancement of our science. My current work is supported by NSF Grant PCM840191.

REFERENCES The following brief bibliography does not come close to representing the literature of the subjects discussed in this article, nor does it even cite all the sources used in the preparation of this manuscript. At best, it gives the reader access to the larger literature. The author apologizes to the many authors who have influenced him and who would deserve citation in a proper bibliography of several of the fields discussed in this essay.

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Alov, I. A,, and Lyubskii, S. L. (1977). Ini. Rev. Cytol. Suppl. 6 , 59. Bajer, A. S., and Mole-Bajer, J. (1981). Cold Spring Harbor Symp. Quanr. Biol. 41, 263. Bajer, A. S., De Brabander, M., Mole-Bajer, J., De Mey, J., and Paulaitis, S. (1980). In “Microtubules and Microtubule Inhibitors” (M.De Brabander and 3. De Mey, eds.), pp. 399-425. Elsevier, Amsterdam. Bak, A. L., and Zeuthen, J. (1977). Cold Spring Harbor Symp. Quant. Biol. 42, 361. Boveri, T. (1888). “Zellen-studien 11. Die Befruchtung und Teilung des Eies von Ascaris megalocephala.” Fischer, Jena. Boveri, T. (1901). “Zellen-studien IV. Ueber die Natur der Centrosomen.” Fischer, Jena. Bradbury, E. M., and Matthews, H. R. (1982). In “Cell Growth” (C. Nicolini, ed.). NATO Advanced Study Institute Series, Vol. 38, pp. 41 1-454. Plenum, New York. Brenner, S. L., and Brinkley, B. R. (1982). Cold Spring Harbor Symp. Quant. Biol. 46, 241. Brinkley, B. R., Marcum, J. H., Welsh, M. J., Dedman, J. R., and Means, A. R. (1978). In “Cell Reproduction” (E. R. Dirksen, D. M. Prescott, and C. F. Fox, eds.), pp. 299-314. Academic Press, New York. Broach, J. R. (1986). Cell 44, 3. Church, K., and Lin, H-P. P. (1982). J . Cell Biol. 82, 365. Clarke, L., and Carbon, J. (1985). Ann. Rev. Genet. 19, 29. Clayton, L., Black, C. M., and Lloyd, C. W. (1985). J. Cell Biol. 101, 319. Collarco-Gillam, P. D., Siebert, M.C., Hubble, R., Mitchison, T . , and Kirschner, M. (1983). Cell 35, 621. Cook, P. R., and Laskey, R. A., eds. (1984). J . Cell Sci. Suppl. 1. Endo, S . (1979). Cell Siruct. Funct. 4, 71. Endo, S. (1980). Dev. Growth Difler. 22, 509. Eutenauer, U . , and McIntosh, J. R. (1981). J . Cell Biol. 89, 338. Evans, T., Rosenthal, L. T., Youngblom, J., Distel, D., and Hunt, T. (1983). Cell 38, 389. Flemming, W. (1880). Arch. Microsc. Anat. 18, 151. [Reprint 1965 with English translation, J . Cell B i d . 25,(1), Part 2, 1.1 Fol, H. (1891). Anatomkcher Anzeiger 6, 266. Fuge, H. (1977). Int. Rev. Cyiol. Suppl. 6 , 1. Gerhart, J., Wu, M., and Kirschner, M. (1984). J. Cell Biol. 98, 1247. Gershon, N. D., Porter, K. R., and McNiven, M. A. (1986). Biophys. J. 49, 65. Gilbert, W. (1986). Nature (London) 319, 618. Gollin, S. M., Wray, W., Hanks, S. K., and Rao, P. N. (1984). J. Cell Sci. Suppl. 1, 203. Gurley, L. R., Tobey, R. A., Walters, R. A., Hildebrand, C. E., Hohmann, P. G., D’Anna, J. A,, Barham, S. S., and Deaven, L. L. (1978). I n “Cell Cycle Regulation” (J. R. Jeter, I. L. Cameron, G. M. Padilla, and A. M. Zimmerman, eds.), pp. 37-60. Academic Press, New York. Hadlaczky, G. (1985). Int. Rev. Cytol. 94, 57. Hafner, M., and Petzelt, C. (1986). Proc. Narl. Acad. Sci. U.S.A. 83, 1719. Heath, I. B. (1980). Int. Rev. Cytol. 64, 167. Hepler, P. (1985). J. Cell Eiol. 100, 1363. Hertwig, 0. (1893). “Die Zelle und die Gewebe.” Fischer, Jena. Hildebrand, C. E., Tobey, R. A., Gurley, L. R., and Walters, R. A. (1978). Biochim. Biophys. Acta 517, 486. Hirokawa, N., Takemura, R., and Hisanaga, S . 4 . (1985). J. Cell Eiol. 101, 1858. Karsenti, E., Newport, J., Hubble, R., and Kirschner, M. (1984). J. Cell Biol. 98, 1730. Kirschner, M. (1985). Eur. J. Cell 6iol. 38 (Suppl. 9), 16. Komberg, R. D. (1974). Science 184, 868. Krystal, G. W., and Poccia, D. (1979). Enp. Cell Res. 123, 207.

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Krystal, G. W., and Poccia, D. L. (1981). Exp. Cell Res. 134, 41. Kubai, D. F. (1975). Int. Rev. Cytol. 43, 167. Kuriyama, R., and Borisy, G . (1981). J. Cell B i d . 98, 822. Kuriyama, R., and Borisy, G. G. (1985). J. Cell Biol. 101, 524. Lewis, C. D., Lebkowski, J. S., Daly, A. K., and Laemmli, U. K. (1984). In “Higher Order Structure of the Nucleus” (P. R. Cook and R. A. Laskey, eds.). J. Cell Sci., Suppl. 1. Lohka, M. J., and Maller, J . L. (1985). J. Cell Biol. 101, 518. Lohka, M. J., and Masui, Y. (1983). Science 220, 719. Luykx, P. (1965). Exp. Cell Res. 39, 658. Maro, B., Howlett, S. K., and Webb, M. (1985). J. Cell Biol. 100, 1665. Masui, Y. (1985). Dev. Growth Direr. 27, 295. Masui, Y., and Clarke, H. J. (1979). In?. Rev. Cyaol. 57, 185. Masui, Y., Lohka, M. J., and Shibuya, E. K. (1984). Symp. Soc. Exp. Biol. 38, 38-66. Matsui, S . I., Seon, B. K., and Sandberg, A. A. (1979). Proc. Narl. Acad. Sci. U.S.A. 76, 6386. Matsui, S., Weinfeld, and Sandberg, A. A. (1982). In “Premature Chromosome Condensation” (P. N. Rao, R. T. Johnson, and K. Sperling, eds.) pp. 207-232. Academic Press, New York. Mayr, E. (1982). “The Growth of Biological Thought,” p. 677. Harvard Univ. Press, Cambridge, Massachusetts. Mazia, D. (1961). In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. 3, pp. 77-440. Mazia, D. (1963). J. Cell. Camp. Physiol. 62, (Suppl. I ) , 123. Mazia, D. (1974). Proc. Narl. Acad. Sci. U.S.A. 71, 690. Mazia, D. (1984). Exp. Cell Res. 153, 1. Mazia, D., Harris, P. J . , and Bibring, T. (1960). J. Biophys. Biochem. Cytol. 7, 1. Mazia, D., Paweletz, N., Sluder, G., and Finze, E.-M. (1981). Proc. Nutl. Acad. Sci. U . S . A . 78, 377. Meyerhof, P., and Masui, Y. (1977). Dev. Biol. 61, 214. Mitchison, T. J., and Kirschner, M. (1984). Nature (London) 312, 232. Mitchison, T. J., and Kirschner, M. W. (1985). J. Cell Biol. 101, 766. Mueller, R. D., Yasuda, Hatch, C. L., Bonner, W. M . , and Bradbury, E. M. (1985). J. Biol. Chem. 260, 5147. Mullinger, A. M., and Johnson, R. T. (1983). J. Cell Sci. 64, 179. Nagano, H., Hirai, S., Okano, K., and Ikegami, S . (1981). Dev. Biol. 85, 409. Newport, J., and Kirschner, M. (1984). Cell 37, 731. Nicklas, B. (1985). In “Aneuploidy” (V. L. Dellarco, P. E. Voytek, and A. Hollaender, eds.), pp. 183-195. Plenum, New York. Nicklas, R. B., and Kubai, D. F. (1985). Chromosopa 92, 313. Nicolini, C., and Belmont, A. (1982). In “Cell Growth” (C. Nicolini, ed.). NATO Advanced Study Institutes Series, Vol. 38, pp. 487-520. Plenum, New York. Nishioka, D., and Mazia, D. (1977). Cell Biol. Int. Rep. I, 23. Olins, A. L., and Olins, D. E. (1974). Science 183, 330.. Paweletz, N., and Mazia, D. (1978). In “Cell Reproduction” (E. R. Dirksen, D. M. Prescott, and C. F. Fox, eds.), pp. 495-503. Academic Press, New York. Paweletz, N., and Mazia, D. (1979). Eur. J . Cell Biol. 20, 37. Paweletz, N., Mazia, D., and Finze, E. M. (1984). Exp. Cell Res. 152, 47. Pederson, T. (1972). Proc. Natl. Acad. Sci. U.S.A. 69, 2224. Pederson, T., and Robbins, E. (1972). J. Cell Biol. 55, 322. Peterson, S. P., and Berns, M. (1980). Inr. Rev. Cytol. 64, 81. Peterson, J. B., and Ris, H. (1972). J. Cell Sci. 22, 19. Petzelt, C., and Wiillfroth, P. (1984). CellBiol. Inr. Rep. 8, 823.

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Pickett-Heaps, J . D. (1969). Cytobios 1, 257. Poccia, D., Krystal, G., Nishioka, D., and Salik, J. (1978). In “Cell Reproduction” (E. Dirksen, D. M. Prescott, and C. F. Fox, eds.), pp. 197-206. Academic Press, New York. Poenie, M., Alderton, J., Tsien, R., and Steinhardt, R. (1985). Nature (London) 315, 47. Prentice, D. A,, Robey, R. A , , and Gurley, L. R. (1985). Exp. Cell Res. 157, 242. Rao, P. N.(1982). Rappaport, R. (1971). Int. Rev. Cytol. 31, 169. Rappaport, R., and Rappaport, B. N. (1984). J. Exp. Zool. 231, 81. Rieder, C. L. (1982). l n f . Rev. Cyrol. 79, 1. Sato, C., Kuriyama, R., and Nishizawa, K. (1983). J . Cell Biol. 96, 776. Schatten, G., Simerly, C., and Schatten, H. (1985). Proc. Nut/. Acad. Sci. U.S.A. 82, 4152. Schatten, H., Schatten, G., Mazia, D., Balczon, R., and Simerly, C. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 105. Scholey, J . M., Porter, M. E., Grissom, P. M., and Mclntosh, J . R. (1985). Nature (London) 318, 483. Sedat, J . , and Manuelidis (1977). Cold Spring Harbor Symp. Quant. Biol. 42, 33 1. Setterfield, G., Sheinin, R., Dardick, A , , Kiss, I., and Dubsky, M. (1978). J. Cell Biol. 77, 246. Silver, R. B., Cole, R. D., and Cande. W. Z. (1980). Cell 19, 505. Sluder, G. (1978). In “Cell Reproduction” (E. Dirksen, D. M. Prescott, and C. F. Fox, eds.), p. 563. Academic Press, New York. Sluder, G., and Begg, D. A. (1985). J.Cell Sci. 76, 35. Sluder, G., and Rider, C. H. (1985). J . Cell B i d . 100, 887. Solari, A. (1980). Exp. Cell. Res. 127, 457. Sperling, K. (1982). In “Premature Chromosome Condensation” (P. N. Rao, R. T. Johnson, and K. Sperling, eds.), pp. 43-78. Academic Press, New York. Spiegelman, B. M., Lopata, M. A,, and Kirschner, M. (1979). Cell 16, 253. Suprynowicz, F. A., and Mazia, D. (1985). Proc. Nut/. Acad. Sci. U.S.A. 82, 2389. Thomas, J . 0. (1984). J . Cell Sci. Suppl. 1, I. Wagenaar, E. B., and Bray, D. F. (1977). Can. J . Gener. Cyrol. 15, 801. Wang, R. J., Wissinger, W., King, E. J., and Wang, G. (1983). J . Cell B i d . 96, 301. Wheatley, D. N. (1982). “The Centriole: A Central Enigma of Cell Biology.” Elsevier, Amsterdam. Wick, S. M. (1985). Cell Biol. lnt. Rep. 9, 357. Wilson, E. B. (1928). “The Cell in Development and Heredity,” 3rd ed. Macmillan, New York.

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 100

Cell Reproduction DAVIDM. PRESCOTT Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309

I. Introduction I began the study of cell reproduction as a graduate student with Dan Mazia in 1952. In those days so little was known about the subject that it was difficult to

find a basis from which to start. Many fine observations had been made on mitosis, but biochemical analysis of the process was in its infancy. Mazia and Dan (1952) were pioneering modern biochemical studies of mitosis, isolating the mitotic apparatus in mass from synchronously cleaving sea urchin embryos. In contrast, the general lack of interest in what a cell did between cell divisions is reflected in the terminology of the time: interphase was still called the resting phase. Thought about the control of cell division was dominated by the ideas of Hertwig, who proposed that a cell divided when a critical ratio of cell volume to nuclear volume was reached as a result of cytoplasmic growth (Hertwig, 1903). It is remarkable how those ideas are still applicable to problems of cell reproduction, specifically to the initiations of DNA replication and mitosis (see later). Mazia’s influence led me to examine Hertwig’s hypothesis by determining the pattern of growth of a cell as it proceeded from one division to the next. By modem standards the study of cells in the 1950s was quite unsophisticated because it was so unmolecular. It is easy to forget or ignore that early work led to formulation of the problems we now work with and provided foundation for development of cell research into its current form. A few radioactive tracers were available in the early 1950s, but there were no radioactive nucleosides such as [3H]- or [I4C]thymidine or radioactive amino acids. The technique of radioautography of cells had been developed, particularly in the laboratory of Jean Brachet, but had not yet been applied to problems of the cell cycle. In fact, the term cell cycle had not yet been invented. Swift (1950) had shown by cytospectrophotometry that DNA was synthesized in interphase and not in prophase as some people had earlier supposed. But it was not until 1953 that Howard and Pelc reported their studies using 32P to label DNA in plant root cells and its detection with radioautography. They designed what is now called the labeled mitosis method and showed for the first time that DNA synthesis occupies a fixed interval in interphase. They originated the terms G , , S , G,, and D to describe the 93 Copyright 6 1987 by Academic Press, Inc. All rights of reproduclion in any form reserved.

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temporal relationship of DNA replication to cell division. Their dissection of the cell cycle remains the focus of much research on cell reproduction. However, when a phenomenon is given a label, as in the case of the four sections of the cycle, it often seems to acquire character or definition that it does not merit. Few cell biologists realize that the G in GI and G, originally stood for Gap in time. Thus, the definitions of G, and G, were originally negative; they were only intervals of time between the positively defined events of DNA synthesis and cell division and nothing more. The definitions remain essentially negative because no specific cytological or molecular events have ever been discovered that specifically define their existences or their lengths. In the early 1950s, methods of cell culture and techniques for handling animal cells in culture were still rather primitive, although they were about to advance rapidly at the hands of such people as Harry Eagle and Ted Puck. I chose to use Amoeba proteus to study cell reproduction because it was cultured in Dan Mazia’s lab by Tom James and because it was a large cell and easy to manipulate. I had previously worked in Erik Zeuthen’s laboratory in Copenhagen, and he had taught me to use the Cartesian diver balance. I used this Cartesian diver balance to measure the growth of individual amebae as they grew from one division to the next to see whether the pattern of growth might give some insight about the triggering of cell division (Prescott, 1955). The work showed that the rate of cell growth was not autocatalytic; that is, the normal ameba increases in mass at a constant or even slightly declining absolute rate as it gets bigger. The more rapid absolute growth rate of smaller cells can be seen in exaggerated form in unusually small amebae created by unequal cell division induced by light; small cells grow much faster than large ones, and their growth rates decline as they gain in mass. Growth is also not autocatalytic in 3T3 cells; large cells grow more slowly than small cells relative to their respective sizes (Brooks and Shields, 1985). Amebae stop growing several hours before dividing, and it was clear that attaining a specific size was not an immediate trigger to cell division. Nevertheless, amebae tend strongly to divide at a given size, and size appears somehow connected to cell division. Also, division of an ameba can be prevented for months by keeping size small by cutting away cytoplasm every day from an ameba kept in a continuous state of growth (Hartmann, 1928; Prescott, 1956). However, the connection between size and cell division is not firm. Preventing size increase by starving amebae halfway through the cell cyle results in amebae that eventually divide at a much smaller size-it simply takes the smaller ameba longer to reach cell division (Prescott, 1956). It was more difficult to work on cell reproduction in the early 1950s than today because the black box we were groping around in was a lot darker than it is today. As a measure of how much had been done up until 1954 I can cite the bibliography in my Ph.D. thesis. I wrote the thesis in 1954 and did the expected

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search and reading of the literature that had set the stage for my graduate student research. In the Berkeley library I searched back to 1835 (the year of Schleiden and Schwann and the cell theory) for all published work on what is now called the cell cycle, excluding mitosis, about which much had been written. In the 119-year interval from 1835 to 1954 I found a total of 48 research publications dealing with the cell cycle in bacteria, alga, yeast, and protozoa. There were no papers on plant or animal cells. In 1986 more than 200 papers on the cell cycle and its regulation are published every week. These contain far more information, some of it important, some of it trivial, than one person can assimilate. I still work on cell reproduction, but I spend more time reading about other people’s work than I spend doing my own research. The following pages contain a discussion of other people’s work and ideas with the addition of some ideas of my own about how cells reproduce and how reproduction is regulated. I began with a stack of several hundred reprints that I had selected with care, mostly published in the 1980s, in addition to several symposium volumes and reviews. The more I read, the more difficult it was to cope with and integrate the information. It was necessary to stop and write something down. The rest of this chapter is a progress report in reading and assimilating. I apologize to all those whose work and writing about cell reproduction have not been included, some by necessity and some because I was unaware of it. The older literature is reviewed in books by Mitchison (1971) and by me (1976). The new book by Baserga (1985) and a review by Yanishevsky and Stein (1981) are valuable resources, particularly for more recent work.

11. Cell Growth and Reproduction

The ability to reproduce must have been innate in the origin of the first cell, since only through reproductive replacement can any species of cell escape extinction by thermodynamic forces. Survival depends on numbers and that requires reproduction. The mechanism of cell reproduction, like other cell properties, has been highly polished by four billion years of evolution into an efficient, rapid, and generally mistake-free process. An example immediate to our own interests is the 25 million cell divisions that occur every second in an adult human with only very rare failures. We view reproduction in contemporary cells as a momentous accomplishment. It begins with the coordinated increase in the amounts of the thousands of different kinds of molecules that make up all of the parts and functions of cells and ends with a partitioning of everything into two daughter cells by cell division. We now seek to understand how all of the syntheses, assembly of structures, and partitioning at division are coordinated into a smooth progression to yield two cells from one. Coordination of the many metabolic activities of a cell

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in any moment of time is achieved by a large network of regulatory interactions. The molecular bases of some of these interactions are known; most remain obscure. In any case, these regulatory interactions at the same time are likely to be the ones that maintain balance in the increases in components (growth) over the course of time. But what impels the cell to increase its size is a major question whose answer still remains buried within the network of regulatory interactions. AND DIVISION A. GROWTH,DNA REPLICATION,

The simplest description of cell reproduction is that cells grow and then divide. On the average a cell doubles its size from one division to the next. This implies to those who have thought about it very much that cell growth and cell division are causally connected, i.e., growth to a doubled size in some way triggers cell division. Such a simple interpretation makes sense. However, although growth is ultimately essential for cell reproduction, the connection between growth and cell division is more circuitous and complicated than simple microscopic observation of proliferating cells suggests. For example, closer analysis reveals that, although on average, cells of a species divide at a constant size, the relationship is not precise when it comes to individual cells; dividing cells are not all the same size, but fall into a range of sizes (e.g., Anderson et al., 1969). Growth appears to be linked causally to cell division through an intermediate event, the replication of the genome. There is a lot of evidence that the initiation of DNA replication is related in some unknown way to the size or growth state of a cell. And it is clear that cells can only divide after they have completed DNA replication. Therefore, the problem resolves into two questions: What is the connection between growth and replication of DNA, and what is the connection between replication of DNA and cell division? For a cell unhindered by lack of an essential nutrient or restrained by some other mechanism (as in multicellular organisms), the relationships between growth, DNA replication, and cell division are portrayed by the cell-cycle diagram familiar to all cell biologists. But the diagram displays temporal relationships and nothing more. However, a fixed order of events does imply causal connections, and much effort has been given to defining and elucidating the connections, particularly their molecular bases.

B. A CELLCYCLEWITH THREESECTIONS For some cells the commonly used diagram defines a cell cycle with three time intervals: the period occupied by DNA replication (S period), the interval be-

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tween the end of DNA replication and the visible start of nuclear division (G,), and the interval occupied by cell division (D period). This describes the cycle for some unicellular organisms (see Prescott, 1976), for example, large, free-living amebae, Dictyostelium (Weijer et a l . , 1984), the fission yeast, the micronucleus of Tetruhymenu (McDonald, 1962); for the syncytial organism Physarum (Braun and Wili, 1969); and for cells in at least one simple multicellular organism, Hydra (David and Campbell, 1972). In at least ameba, Physarum, and Tetrahymena, the end of mitosis is tightly coupled both temporally and mechanistically to DNA replication. In ameba and Tetruhymena, arrest of the cell cycle by starvation or experimental treatments that block protein or RNA synthesis cannot separate mitosis from subsequent replication of DNA. Any nucleus that completes mitosis yields two daughter nuclei that immediately and inexorably enter the S period. The observations suggest but do not prove the presence of a tight causal link between completion of mitosis and DNA replication. It appears that chromosome decondensation in telophase is sufficient to initiate the S period, but almost nothing is known about the molecular mechanisms responsible for the tight coupling. In the 30 minutes between the start of chromosome condensation at the end of G , and the end of mitosis and the chromosome decondensation, a crucial change occurs that causes or allows DNA to begin replication. In contrast to the unbreakable temporal coupling of mitosis to DNA replication in ameba and Tetruhymena, mitosis can be temporally uncoupled from the ensuing S period in fission yeast by various experimental procedures that create smaller-than-usual dividing cells (Nurse and Thuriaux, 1977). It appears that smaller daughter cells cannot initiate DNA replication until they attain a critical protein content. The implied hypothesis that cell size is related to the triggering of DNA replication in Escherichia coli is supported by a considerable body of evidence (Donachie e f al., 1976). Similarly, for animal cells in culture cell growth or size appears to be related to initiation of the S period, but the relationship is not obligatory since it can be abolished (see below). In organisms with mitosis immediately coupled to DNA replication the S period is generally short and the G, period is generally long. For example, Amoeba proteus has an S period of 4 or 5 hours and a G, period of 18 or more hours. In Dictyostelium DNA replication takes less than 30 minutes and G, is more than 6 hours. In Physarum DNA replication lasts about 3 hours and is followed by a long G, period. The DNA in the micronucleus of Tetrahymena replicates in the 10 minutes immediately following telophase, and the micronucleus remains in a G , state until the next mitosis several hours later. In ameba, Physarum, and the micronucleus of Tetrahymena, and probably in other organisms with three-part cell cycles, arrest of the cycle by nutrient depnvation occurs in G,. Thus, whatever the train of events that links the end of DNA replication with the induction of cell division, it can be interrupted without jeopardy to cell survival, at least for many days.

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Arrest of the cycle in G, is, therefore, the rule in at least some of the simpler eukaryotes like ameba and Physarum, and presumably Dictyostelium. This location of normal arrest contrasts with the cells of multicellular organisms, in which arrest ordinarily occurs between cell division and the start of DNA replication, i.e. occurs during the G, period (discussed below), although even these cells can safely arrest in G, (Gelfant, 1975). It may be that no specific train of causally related events occupies G , and couples the end of DNA replication with cell division in these simpler eukaryotes. The obvious major event in G, is cell growth. Growth or cell size may provide the impetus for entry into cell division. Some evidence for this comes from studies of the fission yeast (Nurse, 1975; Nurse et al., 1983). A temperature-sensitive mutant of the fission yeast, designated wee-I, divides at nearly the same size as an unmutated yeast at 25°C. When the cells are cultured at 35°C the wee-1 mutant divides at half the size. When mutant cells growing at 25°C were quickly shifted to 35"C, cells in G , were precociously propelled into mitosis. The observations have been explained on the basis of a size requirement for entry into cell division (Nasmyth and Nurse, 1981; Fantes, 1983). The normal product of the wee-1 gene is an inhibitor that prevents entry into division until a cell has reached a critical size. In some way attainment of the critical size results in overriding the wee-I gene product. A ts mutation in the wee-I gene leaves the inhibitor and the control system intact at 25"C, but at 35°C expression of the wee-I gene is impaired or extinguished, the control system is modified, and the cell is allowed to divide at half the size. This is a provocative start on the problem, but as yet nothing is known of how the wee-I gene product works to prevent cell division and how the inhibitory effect is overcome by a critical cell size. A relationship between cell size and division is also apparent in Amoeba proteus. Most of the cell cycle is taken up by G,, about 20 hours in a 24-hour cycle in the fastest reproducing cells. Most of cell growth therefore occurs in G,. If cell size is reduced in mid to late G, by cutting away about half of the cytoplasm with a glass needle, entry into cell division is delayed (Hartmann, 1928; Prescott, 1956). If the size of a growing cell is restricted by repeated amputation of cytoplasm at 12- to 24-hour intervals, it never enters division. An ameba can be kept in this state for months. If the amputations are discontinued, the growing ameba divides within 36 hours. In summary, the integration of growth, DNA replication, and division in some of the simpler eukaryotes may be achieved by mechanisms in which critical cell size triggers cell division and cell division (mitosis) triggers DNA replication. Nutritional shortage blocks cell reproduction by limiting cell size and preventing entry into division. We have only the bare beginnings of an understanding of these connections, and the current hypotheses are very tenuous.

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C. THREE-PART CYCLESIN HIGHEREUKARYOTES It is insufficiently appreciated that some cells of higher eukaryotes have cycles with no G I period. Any explanation of how plant and animal cells reproduce and how reproduction is regulated must take into account the existence of the GI-less cycle. Three animal cell lines have cell cycles that lack G, periods when cultured under optimal conditions. These are the Chinese hamster line V79-8 (Robbins and Scharff, 1967; Liskay and Prescott, 1978), the Chinese hamster line DON, and the mouse teratocarcinoma cell F-9 (cited in Prescott et ul., 1982). The G, periods are short (and absent in V79-8, Liskay, 1977), and for the line V79-8, the labeling index with [3H]thymidine of an asynchronous population proliferating under optimal conditions, the labeling index can exceed 90%. However, by far, most animal cells grown in culture have cycles with GI periods. Beyond the fact that arrest of the cycle by nutrient deprivation or other treatments occurs within the G, period, a spec@ qualitative, molecular basis of G, has not been established. In animal tissues GI-less cycles are rare. One exception is cells in the erythropoietic series. These cells normally proliferate rapidly and apparently at one stage of differentiation without a G I period (Alpen and Johnston, 1967). Of much broader significance is the fact that development of animals in general begins with a series of GI-less cell cycles. This has been described for several invertebrates, for example, for the first five cleavages in sea urchin embryos (Dan et al., 1980), for the initial mitoses in Drosophilu, for the first 12 divisions in Xenopus embryos (Newport and Kirschner, 1982a,b), and for at least the first several cleavages in mouse embryos (Gamow and Prescott, 1970). In some cases G, periods are also absent (Dan et al., 1980). After this early development cell reproduction generally slows, and G , periods appear in the cell cycle at the time of or before the first signs of morphogenesis. Some types of rapidly dividing cells may continue with GI-less cycles; the very first description of a GI-less cycle was for neuroblasts of grasshopper embryos (Gaulden, 1956). The cycle in very early development represents an archetype cell cycle unobstructed by mechanisms that regulate cell reproduction. Whatever the requirements for growth and cell size are, either for the initiation of DNA replication or for triggering mitosis, they do not operate, because the cells of the embryo exceed such requirements by virtue of the prior extensive build-up of the egg cell in the ovary before fertilization. The cells in the early embryo do not double in size between divisions, and they become progressively smaller, until size presumably becomes influential in modulating entry into DNA replication and perhaps entry into cell division. In addition, cell reproduction in tissues is regulated by specific regulatory factors. The influence of these is also obviously absent during early development. Hence, the cell cycles in this stage of animal develop-

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ment are uncluttered by intrinsic or extrinsic restraints. Thus, the cycle can be observed in its elemental form without any nonessential events or periods of time, e.g., a G, period. The dividing cells of the embryo provide the clearest picture of what constitutes the chain of causally connected events that provides an ordered and uninterrupted progression of the cycle. There are only two main events, DNA replication and cell division, one setting the stage for the next. Here it would seem that the molecular connections between cell division and DNA replication could be analyzed without the complicating influence of growth and specific mechanisms that regulate cell reproduction. D. FOUR-PART CELLCYCLES-ADDINGTHE GI PERIOD The first dissection of the cell cycle using radioautography to detect DNA synthesis was done on plant root meristems and identified the periods G I , S, G,, and D (Howard and Pelc, 1953). The experiment has been repeated on hundreds of types of plant and animal cells both in intact organisms and in cultured cells. Almost without exception these analyses have shown the presence of a G, period. So general has been its presence in plant and animal cells that it is usually considered an essential period in the cell cycle. The absence of G, in cells of early embryos, in a few animal cell lines, and in some unicellular eukaryotes seems to have little impact on thinking about the significance of G, . How do the G,-less cell cycles of embryos change into cycles with G, periods during subsequent development? Obviously, there is no other way to achieve this except to introduce a delay between cell division and DNA synthesis. How this is achieved is not known, but the addition of a G, period is a developmental event, and subsequently the property remains a stable part of almost all cells reproducing in tissues or in cultures.

111. Regulation of GI Transit There is one salient feature about G, that draws attention to it: regulation of cell reproduction is achieved by blocking exit of cells from the G, period into the S period. Despite much research, no biochemical event has been identified that uniquely characterizes the G, period, and the molecular nature of the switch or switches that regulate transit from cell division to initiation of DNA replication remains obscure. Most of our understanding about the regulation of initiation of DNA replication comes from genetic studies of the budding yeast, discussed later. The GI is the most variable in length of the four periods in the cycle, and it is variable in two senses. First, in a population of cells proliferating at a constant

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rate the average lengths of G I , S, G,, and D remain the same. The lengths of S, G,, and D remain rather constant from one cycle to the next and from cell to cell. The length of G I varies over a rather wide range from cycle to cycle and cell to cell. Thus, although the average length of G I remains the same, the length of GI in any individual cycle is not predictable except as a wide range of values. This kind of individual G I variation might be due to the variation in size of individual cells at birth. Cytokinesis does not divide cells into two precisely equal daughter cells; one is invariably bigger than the other (e.g., Darzynkiewicz et al., 1982). If the decision to initiate DNA replication is dependent on cell size, then presumably the smaller the cell, the longer the G I period. Measurements on individual cells of the mouse L cell line support this explanation of individual G I variability (Killander and Zetterberg, 1965). Alternatively, each time a cell arrives in G I a putative switch must be turned to the “on” position for the cell to proceed into the next S period. In the transition probability model (Smith and Martin, 1973), which implies such a G I switch, the temporal probability remains constant for the population as a whole (i.e., the average G I remains the same), but turning the switch to “on” is a random event in individual cells. The behavior is like radioactive decay; the half-life is constant, but the timing of the decay of individual atoms is random. Measurements of individual G, lengths in a population do not fit particularly well with the distribution predicted by the transition probability model. In any case, this type of analysis has not provided insight about the nature of GI and transit through it. The second form of variation of G I consists of variation in its average length. For example, the average generation time of animal cells in culture can differ depending on the makeup of nutrient medium (Tobey et al., 1967). In these cases the differences in average generation time are accounted for by differences in the average length of G I . In other words, different rates of cell reproduction can be achieved by a change in the average length of G, . Again, this implies a switch in G I that regulates entry into S, and in this situation operation of the switch is modulated by nutrient conditions. The two kinds of GI variation (variation of individual G I length and variation of average G , length) may be related. Certainly a switch is present in G I by which transit through G I can be regulated. Various conditions may operate the switch. For example, below some critical cell size the switch may remain in the “off” position. Variation in cell size, as suggested above, may account for variation among individual cells in turning the switch on and hence account for individual variation in G, . Nutrient conditions that alter cell growth may change the average time taken for cells to reach the size required to turn the switch to “on” and hence change the average generation time. Nutrient deprivation or absence of specific growth factors may prevent turning the swith on, for example, by limiting cell growth, causing cells to arrest in G I .

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A. THEG I SWITCHIN

THE

BUDDING YEAST

The most complete picture of the cell cycle for any eukaryote is for the budding yeast, Saccharomyces cerevisiae. Only a brief review is presented here (see Singer et al., 1984, for a key to the literature). Unlike the fission yeast Schizosaccharomyces pombe, the cell cycle of the budding yeast does normally have a G , period. Also, instead of dividing by cell fission, reproduction occurs by formation of a bud early in the cell cycle. The bud grows to a full-sized cell (although not to the size of the mother cell), mitosis occurs in the cytoplasmic bridge that connects parent to bud, providing parent and bud with a nucleus, and the two cells then separate. DNA replication begins just about the time of bud emergence, at least in some strains, and is therefore a convenient marker for the initiation of S in living cells. However, yeast lacks the gene for thymidine kinase, and the initiation of S cannot be determined by the very sensitive method of incorporation of [3H]thymidine. Instead, incorporation of [3H]uracil followed by removal of RNA and radioautography can be used to detect DNA synthesis. How precisely this method detects the start of S is not known, but it is unlikely that [3H]uracil incorporation lags more than a few minutes behind the actual initiation. The matter is important in determining the proximity of the G I switch to the initiation of replication (see below). When yeast cells are starved for an essential nutrient, for example a nitrogen source or phosphate, they arrest in G I . Cells in S at the time of nutrient starvation finish the cycle and come to rest in G I . The position of arrest is located no more than a few minutes before bud emergence. In addition, the arrest point, which is called “Start” in yeast, has been defined in two other ways, by mutation and by mating hormone. More than 35 genes have been identified by conditional, temperature-sensitive mutation that are essential for progression of the cell cycle in yeast. These genes code for products that function at a variety of points in the cycle: DNA synthesis, bud emergence, mitosis, and cytokinesis. Several genes code for products that function as part of the “Start” mechanism. Three or more of these genes (Ras-I, Ras-2, cdc-28) are related to protooncogenes that are involved in an as yet undefined way in GI arrest in animal cells. Yeast cells lacking both a Ras-1 and a Ras-2 functional gene cannot initiate DNA replication. Introduction of a normal mammalian Ras gene rescues such yeast cells, demonstrating extraordinary evolutionary conservation of a gene involved in regulating cell reproduction (Kataoka et al., 1985). Mating in yeast takes place between opposite haploid mating types “a” and “a.” Each mating type secretes a different short polypeptide, and these act as mating hormones that arrest cells of the opposite mating type at “Start.” Presumably binding of mating hormone to a specific receptor (Burkholder and

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Hartwell, 1985) creates a signal that blocks the cell at “Start.” Blocked cells of opposite mating type then fuse to form diploid cells that ultimately undergo meiosis to yield haploid vegetative cells. In all three kinds of studies, nutrient starvation, mutation, and mating, the arrest in G , is apparently achieved at a single switch that regulates subsequent entry into DNA replication. Some of the molecular components involved in the operation of the switch have been identified, but how the switch works is not known. Nutrient starvation has been suggested to work by preventing growth to a size that is critical for triggering the “Start” switch to the “on” position. This raises the crucial question of how a cell can sense its own size and use the information to operate a cell-cycle switch. Almost nothing is known about this. Perhaps, in fact, cells do not sense cell size but sense a more specific quantity that might ordinarily be proportional to cell size, for example, the rate of overall protein synthesis or the rate of synthesis of one or more specific proteins, such as ribosomal proteins. What does the “Start” switch control? As long as a cell is blocked at Start, a bud does not emerge and DNA replication does not initiate; one or both of these events might then be immediately controlled by Start. In this kind of thinking it is important to know the temporal relationships. Start is located very close to the G,/S border and no more than a few minutes before the initiation of DNA replication (Rivin and Fangman, 1980). Determination of the tightness of this coupling will require precision in detecting the beginning of DNA replication that is difficult to achieve. Bud emergence is closely linked temporally to the Start switch and cannot occur unless Start is completed, but nevertheless may not be immediately coupled to Start at the molecular level (Singer et al., 1984). Another observation that supports the idea of a critical cell size for opening the Start switch and initiating DNA replication is the effect of the inhibitor of DNA synthesis, hydroxyurea (Singer and Johnston, 198 1). Hydroxyurea blocks DNA synthesis by inhibiting the reduction of pyrimidine ribonucleoside diphosphates to deoxynucleoside diphosphates, which cuts off the supply of deoxycytidine and thymidine triphosphates. The rate of DNA replication is slowed in proportion to the concentration of hydroxyurea to which the cells are exposed. Therefore, the drug can be used to lengthen the S period as desired, without apparent injurious effects. Cells continue to grow during the prolonged S period and presumably produce larger products when cell division (bud separation) is complete. In this experiment with yeast, the more S is prolonged by hydroxyurea treatment, the shorter the G, period in the ensuing cell cycle, until the G, period is completely erased. The authors interpret this result as evidence that the G , period represents “a block at the start event of the DNA-division sequence caused by insufficient growth.” They point out that of the many cell-cycle genes (> 35) identified by mutation in yeast none appears to be necessary for transit through G, except for

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genes involved in Start, which is located at or just before the G,/S border. Cell size thus appears to regulate the cell cycle by regulating operation of Start and entry into DNA replication. In fission yeast cell size apparently regulates the cycle by regulating entry into cell division, as discussed above. The critical size needed to enter mitosis provides for the production of daughter cells whose size is thought to exceed that necessary for entry into DNA replication (Nasmyth, 1979); hence the fission yeast proliferating normally lacks a G I period. Wee mutants have an altered size requirement for cell division and produce smaller daughter cells. These smaller cells do have a GI period, suggesting that a size requirement for entry into S, hidden in wild-type cells, now comes into play (Nurse and Thuriaux, 1977). When the proliferation rate of wild-type cells is slowed by limitation of the nitrogen source, the regulation of cell division appears to be relaxed, and the cells divide at a smaller size, measured as protein content (Nasmyth, 1977). Under this condition the generation time is increased by introduction of a GI period into the normally GI-less cell cycle. Like the events in wee mutants, this finding points to a critical size requirement for initiating DNA replication, a requirement that is unmasked by the condition of slow growth.

B. THEG I SWITCHIN CULTURED ANIMALCELLS Ley and Tobey (1970) were the first to describe the arrest in GI of cultured animal cells deprived of the essential amino acid isoleucine. A trace of isoleucine is probably necessary for cells in S, G,, and D to continue to progress since rigorous elimination of the amino acid results in blockage in these other phases. As long as enough amino acid is present to support a low rate of protein synthesis, cells progress through S (although at a somewhat reduced rate), G,, and D and arrest in the GI period. Clearly transit through GI is far more sensitive to a decreased rate of protein synthesis than transit through any other part of the cycle. Specific arrest in the G I period is also induced in some kinds of normal cells or quasinormal cells (e.g., mouse 3T3 cells) when they proliferate to form a confluent monolayer, a phenomenon called contact inhibition or density-dependent inhibition of cell proliferation. Cells transformed with SV40 or otherwise made turnorigenic do not stop proliferation when they reach confluence, but form multilayered piles. There is a long-standing debate over whether density-dependent inhibition represents a cell-cell interaction, perhaps involving direct cell contact, or is the result of a nutrient deficiency, e.g., lack of serum growth factors in the microenvironment of a cell monolayer. Inhibitors of cell proliferation have been identified in the medium taken from arrested, confluent cultures of 3T3 cells (Hsu et al., 1984; Hare1 et al., 1984) and in the plasma membrane of

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3T3 cells (Wittenberger and Glaser, 1977; Peterson and Lerch, 1983; Vale et al., 1984). Normal or quasinormal or cultured animal cells also arrest in G I when deprived of serum growth factors, and some types of normal cells can be arrested in G , by treatments that raise intracellular cyclic AMP (CAMP) (Pardee, 1974). The molecular mechanisms that underlie induction of G I arrest by amino-acid deprivation in confluent monolayers, by deprivation of serum growth factors, or by CAMPare not known. However, all of these observations clearly demonstrate the presence of a switch in G I that controls entry into DNA replication. The evidence for the existence of a G , switch is made more pointed by the absence of GI arrest in at least some transformed cells (Medrano and Pardee, 1980). Pardee (1974) has designated the switch as the “restriction point.” Others have argued from experiments in which two or more blocking conditions are alternated in succession that more than one arrest point may be present in animal cells, but interpretations of experiments of this design are not straightforward, and the existence of more than one arrest point, or switch, is debatable. A delay in arresting cells in G I by one or another treatment given alone or in succession may simply reflect a distal temporal relationship between the treatment and a single G I switch.

C. LOCATION OF THE G , SWITCHIN ANIMAL CELLS Some insight into the operation of the G I switch in animal cells might well be gained by knowing the proximity of the switch to the initiation of DNA replication. Early approximations obtained by imposing an arresting condition, e.g., serum deprivation, and then releasing cells from the arrest and measuring the time required to reach DNA synthesis were faulty. When cells are arrested in G I , they do not remain suspended in the condition existing at the time of arrest, but undergo poorly understood changes usually described as withdrawal from the cycle and entry into quiescence or a Go state. When cells are released from arrest, at least several hours are required to reverse the quiescent condition and return to the cell cycle. Thus, the length of the time from release of arrest to entry into DNA replication in most cell types in culture exceeds by hours the length of G, in continuously cycling cells. Theoretically a method to define the location of the switch point would be to impose a blocking condition on an asynchronous population of cells and from that moment on measure the percentage increase in cells labeled with [3H]thymidine. The percentage increase can be translated into the proportion of the generation time that makes up the interval between the switch and the start of S. Alternatively, the percentage of cells that enter S after imposition of a block can be measured by double labeling. Both procedures applied to CHO cells show that

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the isoleucine arrest point resides somewhere within the last 40 minutes before the start of DNA replication (Wynford-Thomas et al., 1985a). Forty minutes may be an overestimate of the outside limit because there is likely a time lag between removal of isoleucine from the medium and intracellular depletion, and because cells apparently slip through the restriction switch at a very low rate in spite of isoleucine deprivation. With flow microfluorimetry used to measure exit of cells from G I , the switch appears to be within 15 minutes of the start of S in 3T3 cells (Yen and Pardee, 1978). Inhibition of protein synthesis with cycloheximide instead of isoleucine deprivation gives an arrest point about 12 minutes before entry into the S period (Blair and Roti Roti, 1981). In contrast, inhibition of RNA synthesis with actinomycin D does not block cells in the last 1.7 hours of GI from entry into S. This is not unexpected. The role of RNA synthesis for entry into S is probably mediated through proteins, and the mRNA molecules necessary for translation of proteins essential for initiation of DNA replication have apparently been synthesized by 1.7 hours before S starts. Using serum deprivation to arrest cells suggests a switch located 2-3 hours before S. Either there are two switches, one for serum and one for isoleucine deprivation or, more likely, the switch point for serum has not been measured accurately by the methods used so far. Serum proteins remain bound to cells for a time following removal of serum from the medium, and cells contain growth factor-receptor complexes internalized before serum withdrawal. Not only does inhibition of entry into S show a 2-hour lag on serum withdrawal, but so does hexose transport (Rubin and Steiner, 1974), an activity probably unrelated to the mechanism that initiates DNA replication. This delay in effect on hexose transport suggests a general delay in sensing by the cell of withdrawal of serum proteins bound to the cell surface or already inside the cell. The interpretation of experiments designed to show the relative positions of arrest points for two different blocking conditions in G, , e.g., mutations, nutrient deprivations, high CAMP, is limited by this kind of difficulty. Thus, the actual serum arrest switch is likely located closer to the start of DNA replication than generally supposed. As inaccurate as the measurement of the location of isoleucine and serum arrest points may be, they show that arrest in G, is followed by drift into another state (Go) since cells require four or more hours, depending on the arresting condition and length of arrest, to reach DNA replication after release. The argument made here is that in continuously cycling cells it is difficult to determine precisely the location of the arrest point in G , because of methodological limitations. Therefore, the interpretation that there is more than one arrest point in G, in continuously cycling animal cells is not compelling. The evidence so far points to an arrest point, restriction point, or switch close to the G,/S border, similar to the relationship of the Start switch to initiation of DNA replication in yeast. The term G I switch seems more appropriate than restriction point, arrest point, block point, etc. because it is a point when one of two

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pathways is followed; the state of the switch governs whether a cell proceeds into DNA replication or is shunted into Go. Pardee and his colleagues (Campisi er al., 1982; Pardee et al., 1981) have postulated that the final, proximate event that starts DNA replication is assembly of the necessary enzymes to form a replitase complex. They suggest that the interval between the switch (restriction point in their terminology) and the actual start of DNA replication is the amount of time required to assemble the replitase complex. The conceptual logic that such assembly is initiated at the switch and is the immediate trigger for DNA replication is appealing. Jazwinski and Edelman (1984) have described evidence for such a complex in yeast (a replisome) that includes DNA polymerase, DNA primase, DNA ligase, DNA topoisomerase, and other proteins. But macromolecular complexes usually assemble quite rapidly; ribosomes contain more than 80 macromolecules and assemble in a few minutes. So the idea that it takes an hour to assemble a replitase complex may be incorrect. D. OPERATING THE G, SWITCH

In a general way conditions that impinge on the operation of the G, switch are known. For an animal or yeast cell to be directed into DNA replication it must have adequate nutrients, and its ability to synthesize proteins must be intact. If these conditions are not met, a cell is arrested and switched into Go. In contrast, major inhibition of rRNA synthesis and presumably ribosome production by injection of antibody to RNA polymerase I into the nuclei of quiescent (Go) 3T3 cells does not block entry of such cells into S after stimulation with serum (Mercer et al., 1984). It would be useful to know whether inhibition of RNA polymerase I by antibody injected during G I is without effect in continuously cycling cells. Functioning RNA polymerase I1 is necessary for entry of cells into DNA replication (Rossini et al., 1980), which is consistent with the requirement for protein synthesis. The molecular sensing mechanisms that feed information into operation of the switch are poorly understood. The most specific information concerns growth factors such as epithelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin, etc., but knowledge of the effector chain of events currently stops with the protein-kinase activity of growth-factor receptors. Most of what is known about operation of the G I switch is at the level of phenomena rather than molecular mechanisms. For example, the cytoplasm has some role in initiation of DNA replication. It is an old and often repeated observation that in cells with two or more nuclei both nuclei enter DNA synthesis at precisely the same moment (see for example Ghosh and Paweletz, 1984), although there are occasional exceptions (see Celis and Celis, 1985, for a list). This synchrony is logically presumed to be the result of cytoplasmic influence. Cytoplasmic influence on initiation of DNA replication has been defined most

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closely by the experiments of Rao and his co-workers (Rao and Johnson, 1970). The original observation was that fusion of an S phase mammalian cell with a cell still in G , caused the nucleus of the G , cell to enter DNA replication ahead of the time it would normally do so. Neither fusion of one G , cell with another nor fusion of a G , cell with a G , cell caused precocious induction of DNA replication in G , nuclei, showing that the inducer of DNA replication present in the S phase cell was absent from G , and G , cells. Proof that the factor present in cells in the S period that induces DNA replication by G , nuclei is cytoplasmic was provided by fusing S phase cytoplasts with G , cells and observing stimulation (Myers et al., 1983). The factor is soluble in the cytoplasm, but most of it (70%) is localized in nuclei. Clearly this factor is a crucial element in the operation of the G , switch, and biochemical characterization of it would almost certainly produce new insight about regulation of DNA replication. A step in this direction has been made by Brown et al. (1985). They loaded red cell ghosts with an extract of HeLa cells in the S period and fused the loaded ghosts to G , cells. This brought about precocious initiation of DNA replication in the G , nucleus. This provides a good assay for the S-initiating activity. Microinjection of animal cell nuclei is now a routine technique that could be similarly used to monitor purification of the factor. As mentioned above, many observations have suggested that DNA replication is initiated when cells reach a critical cell size. Now, however, there are many exceptions that invalidate the generalization. Baserga ( 1984) has succinctly reviewed the evidence against the idea that a critical cell size triggers DNA replication. Particularly relevant are ( 1) experiments showing that protein accumulation during G , in continuously cycling cells is not important for initiating S (Rgnning and Lindmo, 1983), and (2) experiments in which 3T3 cells released from quiescence by transient exposure to high pH in the absence of serum progressed to DNA replication and subsequently divided with little or no increase in size (Zetterberg and Engstrom, 1983). Tyson (1985) reviewed several major models for the coordination of cell growth and cell division and concluded that none of them is totally satisfactory. Part of the difficulty in model building is that we do not have the necessary full set of measurements on cell sizes, individual generation times, sister-sister and mother-daughter comparisons of generation time, etc. made on any particular cell type. Although the correlation between cell size and initiation of DNA replication is real, there is probably no cause-effect relationship. Rather, it is likely that some component in the cell responsible for initiating DNA synthesis ordinarily accumulates in parallel with cell size, but its accumulation may be easily dissociated from cell-size increase. Some observations suggest that the component responsible for switching on DNA replication may be a labile protein. For example, partial inhibition of protein synthesis in G , by cycloheximide can block entry into S. When the inhibitor is removed, the cells are delayed in reaching S by a length of time in

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excess of the time cycloheximide was present (Schneiderman et al., 1971). This “setting back” of the cell in G, is consistent with the hypothesis that the block in progress toward S caused by cycloheximide is the result of a block in further synthesis of a labile protein that must accumulate in order to trigger S. During the block the putative trigger protein synthesized prior to cycloheximide treatment decays, accounting for the delay in reaching S after cycloheximide is removed. The idea of an unstable protein that must accumulate to trigger S has been proposed by Pardee and his colleagues (Rossow er al., 1979). They suggest that in transformed cells the putative trigger protein is synthesized at a higher rate and/or is somehow stabilized. They have identified a protein that fits the requirements and suggest that it may be important in the loss of regulation of cell reproduction in tumor cells (Croy and Pardee, 1983). This line of thinking explains how cell size might be correlated with initiation of DNA replication but have no causative role and how cell growth or size can be easily dissociated from initiation of S. It is also consistent with the many observations that protein synthesis must be proceeding normally in order to trigger S. However, if the synthesis and/or accumulation of a particular protein is responsible for triggering S , we are then left with the question of what regulates the synthesis and/or accumulation of the protein. I have one final comment on cell size and initiation of DNA replication to add to Baserga’s list. Tetraploid cells are approximately twice as big as diploid cells, so that immediately after mitosis tetraploid cells are already as big as a diploid cell about to divide. If a critical size triggered DNA replication, then tetraploid cells should always be G,-less, and they are not. To cite a specific example (Graves and McMillan, 1984), instead of a reduced or totally eliminated G , period, polyploid hamster cells actually have a longer G, period than the diploid cells from which they were derived. E. PROPERTIES OF G,-LEss CELLSOF EARLYEMBRYOS The cells in cleavage-stage embryos reproduce rapidly and have cycles that lack a G, period and in some cases a G, period. Moreover, the length of S may be much shortened compared to cells at later embryo stages, in adults, or in cultured cells. For example, the cycle in Drosophila embryos is about 8 minutes. About 4 minutes of this is interphase, which is completely occupied by DNA replication, and mitosis accounts for the remaining 4 minutes (Blumenthal el al., 1974). In cultured cells of Drosophila at the same temperature, the S period is 10 hours, i.e., 150 times longer. (The basis for the length of S is discussed later.) In the first five cleavages in embryos of the sea urchin (Hemicentrotus pulcherrimus) the generation time is 55 minutes; interphase is about 12 minutes and is fully occupied by DNA replication, and the rest is taken up by mitosis (Dan et al., 1980). After the fifth cleavage the cycles become longer-246 minutes by

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the tenth cleavage. G , and G, periods appear and the S period becomes longer, but the time for mitosis remains the same. Therefore, in early embryos the cell cycles are in their most elemental form, consisting of DNA replication alternating with mitosis. Cleavage-stage embryos can be studied without the complications of conditions that delay entry into DNA replication and mitosis. The cell cycles of cleavage stages proceed with little if any transcription (Graham and Morgan, 1966; Forbes et al., 1983; Woodland and Gurdon, 1969). Inhibition of RNA polymerases I1 and 111 with a-amanitin does not change the rate of cleavage (Newport and Kirschner, 1982b). Protein synthesis is required during each cycle (Wagenaar and Mazia, 1978), proceeding with mRNAs produced previously in the developing ovum. Which proteins are important for the cell cycle are not well understood. The cytoplasm of mature egg cells contains a large pool of histones in the cytoplasm (Adamson and Woodland, 1974; Laskey et al., 1977), and therefore histone synthesis is unlikely to be any part of the requirement for protein synthesis. Two proteins, maturation-promoting factor (MPF) and cyclin, that must be synthesized in each cycle are discussed below. These circumstances in cleavage-stage embryos, plus the exceptionally good synchrony of cell cycles, provide a particularly favorable opportunity for analysis of the molecular events that switch the cell alternatively from DNA replication to mitosis and vice versa without G , and G, interpositions. In Xenopus from cleavage 2 to cleavage 11 the cell cycle time remains at 35 minutes, and the blastomeres divide with high synchrony. G I and G, periods are absent (Graham and Morgan, 1966). At cleavage 12 the cycle increases by an average of 25%, and at cleavage 13, by an average of 60% (Newport and Kirschner, 1982b). By cleavage 13 all synchrony of cycles is lost, and individual cell cycles range from 40 to 75 minutes. At cycle 12, when synchrony starts to disappear, RNA transcription begins, and G , and G, periods appear in the cell cycles. The molecular events that underlie the alternation of mitosis and DNA replication are not yet understood, but the experiments of Newport and Kirschner (I982a,b, 1984) provide a substantial start. These experiments revolve around MPF. Maturation-promoting factor was first demonstrated in fusion of mitotic HeLa cells with interphase cells. A factor in the mitotic cell causes the so-called premature chromosome condensation of chromosomes in G I- and S-phase cells. Maturation-promoting factor was subsequently identified in unfertilized Xenopus eggs naturally arrested in metaphase of meiosis and derived its name from those experiments (Masui and Markert, 1971; Wasserman and Smith, 1978; Wu and Gerhart, 1980; Gerhart et al., 1984). Injection of MPF into developing oocytes (arrested in prophase of meiosis) causes release from prophase and continuation of meiosis into metaphase. Maturation-promoting factor is a protein (Wu and Gerhart, 1980) and has been found during mitosis of other eukaryotes including

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yeast and mammalian oocytes and somatic cells (Sunkara et al., 1979; Kishimoto et al., 1982; Nelkin et al., 1980; Sorensen et al., 1985; Weintraub et al., 1982). The level of MPF oscillates over the cycle in the Xenopus embryo, being high during mitosis and absent during the S period (Wasserman and Smith, 1978; Gerhart et al., 1984). When MPF is injected into embryo cells arrested at the end of the S period, it causes breakdown of the nuclear envelope and chromosome condensation. Maturation-promoting factor activity is transiently present during mitosis in mammalian cells (Sunkara et al., 1979; Adlakha et al., 1985). Extracts of mitotic HeLa cells injected into Xenopus oocytes cause breakdown of the nuclear envelope and chromosome condensation. Using this technique as an assay of fractionation procedures, an extract with 200-fold greater MPF activity has been achieved, a major step toward purification. Injection of a low level of MPF into oocytes causes them to enter the first meiotic metaphase. This is accompanied by the appearance of a high level of MPF in the oocyte, even in the presence of cycloheximide (Gerhart et al., 1984). This shows that MPF is present in the immature oocyte in an inactive form that becomes activated by posttranslational modification. This supports the idea that MPF activates itself, i.e., undergoes autoactivation, in this case triggered by a small amount of injected active MPF. What activates MPF normally within the cell, thereby bringing the cell to metaphase, remains a pressing question. Oocytes also contain an MPF-inactivating agent in an inactive form. When the oocyte reaches meiotic metaphase under the influence of MPF, the MPF-inactivating agent becomes active (an activation that does not require protein synthesis) and inactivates (destroys?) MPF, and the cell is released from metaphase and completes mitosis. What controls the timing of activation of the MPFinactivating agent becomes another pressing question. From the fusion experiments of Rao and Johnson (1970) it is clear that progression past metaphase is normally required for MPF-inactivating agent to work since cells held in metaphase with mitotic inhibitors such as colchicine retain high levels of active MPF. The oocyte cannot enter the second meiotic division in the presence of cycloheximide, suggesting that new MPF must be synthesized. Cycles of MPF appearance and disappearance occur during cleavage of the fertilized ovum. In each cell cycle MPF is absent in interphase and appears to be synthesized de n o w (as opposed to posttranscriptional activation) at the end of interphase to induce mitosis. The transition from metaphase to interphase and DNA replication takes place without protein synthesis, indicating that the reciprocal oscillation of MPF-inactivating agent is achieved by activation and inactivation of the agent rather than by synthesis and breakdown (Newport and Kirschner, 1984). The oscillations of the two agents are normally geared to the synthesis of DNA, interposing mitosis between successive rounds of DNA replication to make cell cycles without G, or G , periods. When one round of DNA replication

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is completed, the next round does not normally occur until mitosis has taken place. This is also evident in the fusion experiment with HeLa cells by Rao and Johnson (1970). Unlike the nucleus of a G, cell, the nucleus of a G, cell is not stimulated to replicate DNA when it is fused with an S-period cell. For some reason the chromatin at the end of DNA replication has changed in a qualitative sense that forbids initiation of DNA replication. The transit of chromatin through mitosis, first involving severe condensation and then decondensation, converts the chromatin into a form that now responds to the signal to initiate replication of DNA. Maturation-promoting factor and MPF-inactivating agent regulate condensation and decondensation of chromatin. Another protein whose intracellular level rises and falls in a cell cycle-specific fashion is “cyclin” (Bravo and Celis, 1980; Bravo et al., 1981). This nuclear protein was identified by two-dimensional electrophoretic separation of proteins extracted from mammalian and avian cells. It is present in high amounts in proliferating cells (normal and transformed) but at a low level in quiescent cells (Bravo and Macdonald-Bravo, 1984; Bravo and Graf, 1985). It is identical to the protein known as proliferating cell nuclear antigen (PCNA) (Mathews et al., 1984), originally defined by an antibody present in people with the autoimmune disease systemic lupus erythematosus (SLE) (Miyashi et al., 1978). Nonproliferating cells of normal tissues contain little or no PCNA, but it appears when cells are stimulated to proliferate, e.g., lymphocytes activated by a mitogen. Synthesis of PCNA or cyclin is coordinately induced with DNA replication (Bravo and Macdonald-Bravo, 1984) but is not obligatorily coupled to DNA replication since in quiescent 3T3 cells stimulated by serum cyclin synthesis it is induced even though DNA replication is prevented by aphidicolin (Macdonald-Bravo and Bravo, 1985). In synchronized cells not only is the temporal coordination of cyclin synthesis and DNA replication very tight, but the intranuclear localization of cyclin corresponds to regions in the nucleus where replication is occurring (Celis and Celis, 1985). Nuclei in G, do not stain with anti-cyclin (anti-PCNA antibody), showing that cyclin is probably destroyed sometime between the end of the S period and subsequent entry into G , in the next cycle. A protein that behaves like cyclin of mammalian cells is found in cleavagestage sea urchin embryos, although it has a higher molecular weight (35,000 vs. 55,000) (Evans et al., 1983). It is synthesized continuously throughout S and destroyed at the time of cell division. Its cyclic synthesis and destruction suggest that it might be MPF, although MPF activity only appears at mitosis and not during S. Moreover, vertebrate MPF has a molecular weight of 100,000. Sea urchins contain at least two other proteins that are periodically synthesized and destroyed during cleavage cycles. The significance of mammalian cyclin or sea urchin cyclin in progression of the cell cycle will remain obscure until some kind of functional assay is avail-

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able. Cyclin may be very important for the cycle or it could be trivial. For example, thymidine kinase activity, an activity completely nonessential for cell cycle progression, increases during the S period in mammalian cells and then drops almost to zero in late mitosis (Stubblefield and Murphree, 1967). Blockage of mitosis with colcemid prevents disappearance of thymidine kinase. In short, MPF plays a key role in producing the fixed succession of events in the cell cycle of eukaryotes. In view of its role in meiotic and mitotic cells Gerhard el al. (1984) suggest that a more appropriate name for MPF would be M-phase-promoting factor. Whatever controls the appearance of MPF is controlling the transition from the end of DNA replication to mitosis, i.e., it controls the length of G,. Whatever controls the MPF-inactivating agent controls exit from mitosis. In the absence of other constraints, disappearance of MPF and exit from mitosis may be sufficient to bring about initiation of DNA replication. However, in cells with cycles that contain a G, period, entry into DNA replication must be regulated by an additionally imposed mechanism in the form of a switch regulating transit from the end of mitosis to the start of S. DNA replication and cell division are each accomplished by sequences of molecular events that are tightly scheduled and therefore define fixed time intervals, i.e., S and D periods with little variability in length among individual cells. Hence, the cell cycles in cleavage stages are highly synchronous within embryos and among embryos initiated at the same time. The inclusion of a GI period, with its high variability in length, creates high variability in generation times and prevents achievement of good synchrony for more than a part of one cell cycle. The number of synchronous cycles in embryos is normally constant for a species. In Xenopus embryos there are 11 equal-length, synchronous cycles (Newport and Kirschner, 1982b), in the sea urchin Hemicentrotus pulcherrimus there are 5 (Dan et al., 1980), in the starfish there are 10 (Mita, 1983), and in Drosophila there are 13 synchronous cycles (Edgar et a l . , 1986). Deterioration of synchrony is concomitant with appearance of G, and G, periods and a slowing of cell reproduction. In starfish eggs cut in half before fertilization the number of synchronous cycles was reduced by one. In tetraploid embryos the number of cycles was also cut from 10 to 9 (Mita and Obata, 1984). Similar effects of changing the nuclear-cytoplasmic ratio have been observed in Drosophila, Xenupus, and axolotl (Signoret and Lefresne, 1971). Cycles begin to lose synchrony (gain G, and G, periods) when cell division has increased the nuclearcytoplasmic ratio to some specific values. (At the same time RNA transcription is activated.) The influence of the cytoplasmic-nuclear ratio suggests that throughout the synchronous cycle some factor(s) necessary for GI-less/G,-less cycles is present in excess (Newport and Kirschner, 1982b). The factor is titrated by the increasing amount of DNA per amount of cytoplasm (increased nuclearcytoplasmic ratio), and cycles become longer and asynchronous when the factor becomes limiting and must presumably be synthesized in cycles that follow.

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F. G,-LEss MAMMALIAN CELLSIN CULTURE Three lines of cultured mammalian cells have been found to proliferate continuously and rapidly and without G I periods in their cycles, at least under certain culture conditions. These are the Chinese hamster cell V79-8 line, the Chinese hamster cell DON, and the mouse teratocarcinoma cell F-9. The V79-8 cell has been studied in some detail (Liskay and Prescott, 1978; see Prescott, 1982, for review). V79-8 cells grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 to 15% fetal calf serum and buffered at pH 7.4 with 25 mM Tricine have a generation time of 9.5 hours. In Ham’s F- 12 medium with 10% fetal calf serum the generation time is 8 hours. At both generation times no GI period is detectable by the labeled mitosis method, and the difference in generation time is accounted for entirely by a change in the length of the S period. Careful radioautographic analysis has shown that V79-8 cells also lack a G, period (Liskay, 1977). A 5-minute pulse with [3H]thymidineresults in labeling of 90 to 95% of the cells in an asynchronously dividing population. The cell cycle of V79-8 cells is easily disturbed. With some preparations of serum the generation time may increase to 12-14 hours, and a G, period appears. Sometimes with mitotic shakeoff synchronization the cells enter the next interphase without a G I period. More often the disturbance involved in mitotic shakeoff causes a delay in initiation of DNA replication for the immediately following cycle. Fusion of a V79-8 cell in mitosis with a HeLa cell induces premature condensation of the HeLa chromosomes as expected. Unexpectedly, the same fusion causes induction of DNA synthesis simultaneously with chromosome condensation (Rao et al., 1978). Hence, a cytoplasmic inducer of DNA replication, demonstrable in the cytoplasm of the S period but not in G I , G,, or mitosis, is present in the cytoplasm of V79-8 cells during mitosis. The “constitutive” presence of the inducer presumably accounts for the immediate induction of DNA replication following mitosis. The obvious next question is what is responsible for the constitutive presence of the inducer, but essentially nothing is known about that. The GI-less phenotype of V79-8 cells is dominant in fusions between V79-8 and other hamster cell lines (transformed cells) (Liskay and Prescott, 1978). For example, fusion of V79-8 with CHO cells produces hybrids, all of which have GI-less cell cycles. In some cases the hybrids have generation times and S periods shorter than either parent, suggesting a complementing effect. The dominance of the GI-less phenotype in hybrids suggests that the factor initiating DNA replication constitutively present in the V79-8 cell remains constitutively expressed in hybrids. However, fusion of V79-8 cells with normal Chinese hamster fibroblasts, which have a long GI period (- 15 hours), produced hybrids with G,

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periods, although the G, period was two- to fivefold shorter than in the normal fibroblast parent. The G,-less V79-8 cell is unable to arrest in GI and enter quiescence (Go) in response to isoleucine or serum deprivation (Wynford-Thomas et al., 1985a). Instead, such deprivations quickly result in cell death, presumably because the cells are arrested in S, which is generally lethal for animal cells. V79-8 cells also lack density-dependent inhibition of the cell cycle, as expected for a transformed cell. Fusion of V79-8 with normal fibroblasts produces hybrids that do show density-dependent inhibition, demonstrating that the normal phenotype is dominant to the transformed phenotype (Marin et al., 1984). Although V79-8 cells cannot become quiescent, they do acquire at least a short G , when grown under suboptimal conditions or by addition of a low level of cycloheximide that reduces the rate of protein synthesis by 40% but does not block progress through the cell cycle (Liskay et al., 1980). A G I period can also be introduced into the cycle of V79-8 by mutation (Liskay and Prescott, 1978). Following chemical mutagenesis cells that have acquired a GI period can be selected by adding [3H]thymidine to mutagenized cultures. Cells with G I periods are preferentially protected from the lethal effects of [3H]thymidine compared to cells that spend all of interphase in DNA replication. Cells that remain GI-less and acquire a long G, or a prolonged mitotic period would also show selective survival during [3H]thymidine killing, but no such phenotypes appeared in these experiments. Among the recovered cells the G, periods varied from 2 to 7.5 hours, suggesting cells had undergone different changes in the acquisition of G, periods (Prescott, 1982). This was borne out by complementation testing using cell fusion. Fusion of different GI-plus isolates produced hybrids that were G,-less. By this procedure many different complementation groups were identified. Each of these is interpreted to represent creation of a G I period by mutation of a different gene. Thus, mutation of any one of many genes can convert a G,-less cell into a G I plus cell. Even fusion between G ,-plus cells of long-established lines from the Chinese hamster produced GI-less hybrids (Liskay et al., 1979). For example, hybrids made by fusing a CHO cell with a G , period of 3.0 hours with a CH-I11 cell with a G, period of 2.3 hours only yielded hybrids that were G,-less. This complementation means that different cell lines have G, periods for different reasons, which complicates the task of formulating a general explanation for the existence of the G, period. A hope in the mutation experiments was that creating a G, period by mutation might be a way of identifying genes involved in the switch mechanism controlling entry into DNA replication-in vague analogy to identification by mutation of genes involved in the Start switch in yeast. The finding of so many genes that

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individually cause a transient delay between mitosis and DNA replication was inconsistent with such an expectation. Liskay et al. (1980) clarified the situation by showing that at least some of the GI-creating mutations decrease the rate of protein synthesis and therefore slow down cell growth. The degree by which protein synthesis is inhibited is roughly in proportion to the length of the G , period induced by a particular mutation. This picture fits with the induction of a G, period in GI-less cells by growing them continuously in the presence of a low level of cycloheximide sufficient to slow protein synthesis by 40%. The creation of a G, period by mutagenesis of Gl-less V79-8 cells does not restore to the cells any competence to arrest in G, and enter a state of quiescence in response to serum or isoleucine deprivation (Wynford-Thomas et al., 1985b). The Gl-plus mutant cells, like the G,-less parental cells, arrest randomly in the cell cycle and die. The two ways of inducing a G, period, i.e., by mutation or cycloheximide treatment, focus attention on the rate of protein synthesis as an operator of the switch that regulates entry into DNA replication. The rate of protein synthesis may ordinarily be a function of cell size and may be a link in the chain that relates cell size to initiation of DNA replication. How rate of protein synthesis is sensed and the information used to regulate a G, switch is not known. Observations on diadenosine tetraphosphate (Ap,A) provide a model for thinking about connections between the rate of protein synthesis and control of initiation of the S period. The level of Ap,A is high in proliferating mammalian cells and low in quiescent cells (Rapaport and Zamecnik, 1976). Diadenosine tetraphosphate is synthesized in the back reaction of the amino acid activation required for protein synthesis (Zamecnik et al., 1966) and its rate of protein synthesis. Diadenosine tetraphosphate is unstable, and maintenance of a high concentration requires a continuous high rate of protein synthesis. Addition of Ap,A to permeabilized quiescent cells causes initiation of DNA replication (Grummt, 1978). This action is apparently mediated by binding of Ap4A to one of the subunits of DNA polymerase (Grummt et al., 1979; Rapaport et al., 1981). Diadenosine tetraphosphate acts as a primer for DNA replication in virro by DNA polymerase from HeLa cells with double-stranded synthetic polymer as a template (Zamecnik et al., 1982). Stimulation of quiescent 3T3 cells or baby hamster kidney fibroblasts with serum results in a steady rise in Ap,A concentration, reaching a 1000-fold increase by the start of DNA replication (WeinmannDorsch et al., 1984). Two GI-less cells, V79-8 and Physarum, have constitutively high concentrations of Ap,A throughout the cell cycle, including mitosis. Since the level of Ap,A is highest during S, it could play a role in the precocious initiation of DNA replication observed when a cell in S is fused to a cell in G , (Rao et al., 1978). Finally, treatment with cycloheximide is accompanied by a drastic reduction in the intracellular concentration of Ap,A, which

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could account for the setting back in time of cells in G I exposed to the drug (Highfield and Dewey, 1972; Schneiderman et al., 1971). GI-less mammalian cells in culture are transformed cells and in many respects are not comparable to G I-less blastomeres in cleavage-stage embryos, although the study of GI-less cultured cells is likely to give information that will be useful in learning how the cell cycle is regulated. THE G I PERIODIN MAMMALIAN CELLS G. SHORTENING

If it is correct that DNA replication is triggered by some element whose appearance is normally correlated with cell size, DNA replication should be triggered sooner ( G , should be shorter) in cells that have a larger size at entry into the cycle, i.e., at the end of mitosis. One way to achieve larger size is to slow the rate of progression through the cycle without reducing the growth rate. Cells reaching mitosis will then be larger. This effect has been achieved by culturing CHO cells in the continuous presence of hydroxyurea. At a sufficiently low level of hydroxyurea the cell cycle time is not affected but the rate of DNA replication is reduced (Stance1 et al., 1981). Initially, after the addition of hydroxyurea, cells already in S are slowed in progress through S, presumably allowing for more cell growth and creating larger cells at mitosis. Although cell size has not been measured in these experiments, treatment with hydroxyurea has been reported to create abnormally large cells (Ross, 1976), an effect blocked by inhibitors of protein synthesis. In any case, hydroxyurea has a clear effect on the length of G I . Under culture conditions that give a 3-hour G, period the addition of a low level of hydroxyurea reduces G I to 1 hour. The shorter G I is then maintained in subsequent cycles, presumably because the average cell size is now higher. In HeLa cells the G I period of about 10.5 hours could only be reduced about 3 hours with hydroxyurea (Rao et al., 1984). Unlike the similar experiments with yeast cells (Singer and Johnston, 1981), the G I period in mammalian cells could not be totally erased with hydroxyurea treatment. This implies that a part of the sojourn of a cultured mammalian cell in G I cannot be ascribed to a limitation on cell size. Raising the level of hydroxyurea to lengthen S caused the generation time to be increased without shortening G, below 1 hour. However, cell size is only indirectly related to initiation of DNA replication, and other events unaltered by hydroxyurea treatment may still limit initiation. Alternatively, hydroxyurea may have untoward side effects. In Physarum the drug not only slows DNA replication but also decreases the rate of protein and RNA synthesis (Pierron and Sauer, 1980). Brief rather than continuous treatment with hydroxyurea also produces a shortening of the G I period in the cell cycle following the treatment (Cress and

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Gerner, 1977). Continuous treatment with hydroxyurea also reduces G, (as well as G2) in plant root cells but does not totally eliminate it (Navarette et al., 1983).

IV. The Relationship of Go to the Cell Cycle The arrest of cultured normal cells and of some partially transformed cells (e.g., 3T3 cells) in G, initiates ill-defined changes that bring the cells into a state of quiescence (Go) in which they may remain viable for weeks. Fully transformed cells (loss of density-dependent inhibition, loss of anchorage dependence, and acquisition of immortality) are incapable of arresting in G, and entering Go in response to serum or amino acid deprivation. The molecular basis for the failure of transformed cells to arrest is not known. In part, at least in some cells, it is due to production of serum growth factors, freeing the cells from serum dependence, But this does not explain failure to arrest in response to deprivation of essential amino acids. The induction of quiescence by deprivation of growth factors in serum is assumed to reflect an important element in the regulation of cell proliferation in tissues. The molecular train of events by which growth factors exert their influence inside the cell is not known. Events are understood up to the point of binding of growth factors such as PDGF and EGF to specific receptors in the cell membrane (e.g., Stiles, 1985; Olshaw er a l . , 1983; Heldin and Westermark, 1984). The receptors are transmembrane proteins that transduce a signal generated by binding of growth through the membrane to activate a tyrosine-specific protein-kinase activity in the cytoplasmic portion of the receptor protein. While the protein-kinase activity suggests intriguing possibilities for multiple effects inside the cell, leading to DNA replication and cell proliferation, the specific protein targets of the protein kinase are not known. Thus, the train of events extending from the kinase activity to initiation of DNA replication remains obscure. The study of protooncogene and oncogene functions provides the most promise of deciphering the molecular mechanisms connecting growth factors to the initiation of DNA replication. It seems reasonable to suppose that growth factors and their receptors represent externalization of part of the circuitry that operates in regulation of G, arrest and release. The externalized segment of the circuit is available for modulation, allowing specific regulation of cell proliferation by factors outside the cell. When a cell moves into quiescence following G, arrest, clearly it enters a different metabolic state. This state (Go) has not yet been given a precise molecular, biochemical, or genetic definition. Cytoplasts prepared from quiescent cells inhibit initiation of DNA replication when fused with cycling cells (PereiraSmith er al., 1985). The longer the cells are held in quiescence, the greater the inhibitory activity of cytoplasts derived from them. At least one protein has been

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found in the nuclei of quiescent cells that disappears rapidly when the cells are stimulated to reenter the cell cycle (Wang, 1985a,b). Exit from quiescence following release from a G I block by readdition of serum factors occurs over many hours. The interval from release to DNA replication is typically several times longer than the length of G , in continuously cycling cells. It is currently unknown what proportion of the reentry period is part of the cell cycle and what proportion represents reversal of the quiescent state. While release from quiescence is an important phenomenon, it is not reasonable to assume that events occurring during this time are cell-cycle events.

V. The G,/S Border The G,/S border is a crucial point in the cell cycle. It is defined by the initiation of DNA replication, the first clearly recognizable sign that the cell has become committed to another division. The process of initiation of DNA replication has been studied extensively by molecular and genetic methods, especially in bacteria but also in eukaryotes. An understanding of the full set of molecular events that accomplishes initiation, an understanding that is still incomplete, is important because it could allow identification of the penultimate event before initiation. This type of cause-effect working backward into G I might achieve an unraveling of the essentials of operation of the decision switch that regulates entry into DNA replication. Expression of a number of genes is closely coordinated with the start of DNA replication. These include enzyme activities for synthesis of deoxynucleoside triphosphates, notably thymidine kinase (e.g., Schlosser et al., 1981; Johnson et al., 1982), thymidylate synthase [in yeast (Storms et al., 1984), Physarum (Grobner and Loidl, 1982), and fibroblasts (Jenh et al., 19831, ribonucleotide reductase (e.g., Kucera et al., 1983), and dihydrofolate reductase (e.g., Johnson et al., 1978). In general, the evidence points to regulation of these enzyme activities at the transcriptional level of the corresponding genes. Specific signals must be generated at the G,/S border that activate transcription of these genes. The transcription of histone genes is also turned on at the G,/S border in mammalian cells (e.g., Stein and Stein, 1984), yeast (e.g., Hereford et al., 1982), and other eukaryotes. The close coupling of transcription of histone genes with DNA replication is also shown by inhibition of ongoing DNA replication with drugs. Such inhibition results in rapid turndown of transcription of histone genes (e.g., Sittman et al., 1983) as well as rapid destruction of existing histone mRNA (see Stein and Stein, 1984). Sequences 5' and 3' to histone genes that are important for cell cycle-specific transcription (Artishevsky et al., 1985; Osley and Hereford, 1982) and regulation of histone mRNA stability (Morris et al., 1986) have been mapped. Identification of the signals that interact with these

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regulatory sequences and the origin of such signals would likely give major insight into the mechanisms of cell-cycle progression.

VI. Nuclear Structure and DNA Replication The misconception still lingers in the research literature that initiation and progression of DNA replication are associated with the nuclear envelope. The idea took impetus from the evidence that initiation of replication and replication forks are bound to the plasma membrane in bacteria. Both the initiation of replication and its continuation occur internally within the nucleus, remote from the nuclear envelope (Wise and Prescott, 1973; Huberman et al., 1973; Fakan et al., 1972). It is now clear that replication occurs in association with the nuclear matrix (e.g., Pardoll et al., 1980; Berezney and Buchholtz, 1981; Smith et al., 1984; van der Velden et al., 1984). DNA polymerase is bound to the nuclear matrix during DNA replication (Foster and Collins, 1985). The proteins that make up the replication complex are apparently assembled on the nuclear matrix, where they remain bound during replication. The chromosomes of eukaryotes contain a single, long DNA molecule. The lengths range from less than a million base pairs (bp) (less than 300 pm) to many billions of base pairs (several meters) in some amphibians and plants. The DNA molecules in mammalian chromosomes range from 25 to several hundred million base pairs in length (one to several centimeters). Altogether, a diploid mammalian cell contains about 6.0 pg of DNA (2 m), all of which replicates in an S period of about 8 hours. The radioautographic observations by Huberman and Riggs (1968) and subsequent studies by others (see Hand, 1979) showed that replication begins at many points along each DNA molecule and proceeds bidirectionally at a rate of about 3000 bp per minute at each replication fork. On the average the origins of replication are about 90,000 bp apart, which defines the average length of replicating units or replicons. Therefore, the mammalian diploid nucleus contains about 30,000 replicons, all of which replicate once (and only once) within an 8-hour S period. The replication of each 30-pm replicon is accomplished by two replication forks moving at 1 pm per minute. The average time to replicate a replicon is therefore 15 minutes. Since the S period lasts 480 minutes the firing of repiication of replicons must be staggered. Even assuming that replicons occur in families in which the members initiate synchronously, there must be a minimum of 32 time points at which replicons are initiated to replicate in order to maintain continuous DNA synthesis. It is generally assumed that replication begins at origins that are defined by a specific sequence, as in viruses and in those few bacteria that have been examined. The best-studied case is for the budding yeast, in which autonomously

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replicating sequences (ARS) have been identified by functional assay (e.g., Fangman et al., 1983). ARSs are likely to correspond to origins of replication. Mammalian (Montiel et al., 1984) and other eukaryotic DNAs contain sequences that behave as ARS in yeast, and it is assumed that these represent origins of replication. Evidence for specific origins of replication has been obtained for rDNA genes in the sea urchin (Lytechinus variegatus) (Botchan and Dayton, 1982) and for the replicon containing the gene for dihydrofolate reductase in hamster cells (Heintz et al., 1983). The staggered initiations of the 30,000 replicons in a mammalian cell are highly ordered. Gross reflection of this is seen in the replication of euchromatin in early S and replication of heterochromatin in the last half of S, as in the case of the heterochromatinized X chromosome in a female cell. Highly repetitious, simple-sequence DNA (density satellites) tend strongly to replicate late in S (e.g., Bostock and Prescott, 1971), although there are exceptions (Matsumoto and Gerbi, 1982). Late replication of satellites is expected since such sequences are generally in a heterochromatin state. More refined information about the order of DNA replication has been obtained recently for specific sequences and genes. In yeast ARSl replicates early, followed sometime later by ARS2 and then a sequence known as 1OZ (Fangman et al., 1983). In synchronized hamster cells rDNA and the gene for dihydrofolate reductase (D’Andrea et al., 1983) and genes for a- and P-globins in murine erythroleukemia cells replicate early in S (Epner et al., 1981; Furst et al., 1981). In Physarum the replication of the four actin genes occurs in an invariant temporal order (Pierron et al., 1984). Three of the genes replicate early, during replication of the first 10% of the genome. The fourth gene replicates when 75% of the genome has replicated. In contrast, the DNA sequences coding for rRNA in Physarum, which exists as a family of extrachromosomal molecules, initiate replication randomly and continuously (as a family) through interphase (Vogt and Braun, 1977). The generalization has emerged that “housekeeping” genes, which are transcribed more or less continuously in all cells, replicate in the first half of s; “differentiation” genes replicate early in cells in which they are transcriptionally active, but late in S in cells in which they are not transcribed (Goldman et al., 1984). Thus, the timing of replication of particular replicons is not invariant but depends on the transcriptional status of the gene or genes it contains (Calza et al., 1984). In keeping with this, “3-4 times as much early-replicating DNA is transcribed as late-replicating DNA in exponentially growing KB cells” (Bello, 1983). This is also consistent with earlier observations that DNA in heterochromatin, which is transcriptionally inert, replicates late, while euchromatin, which is transcriptionally active, replicates early. In some manner the transcriptional state of a particular segment of DNA profoundly influences the timing of

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its replication. Transcription and replication are linked-an idea also suggested by the work of Osley and Hereford (1982) showing that the region at the 3’ end of one of the histone genes that regulates the timing of transcription appears to coincide with an ARS. The rapid replication of DNA in early embryos introduces another complexity about timing and ordering of replicons. For example, in Drosophilu cells in culture the S period is about 600 minutes (Blumenthal et ul., 1974), and sites of initiation of replication are 10 to 60 pm apart. In cells of the cleavage-stage embryo the S period is condensed 150-fold to 4 minutes. Condensation is achieved by increasing the number of sites of initiation of replication with consequent shortening of the distance between sites to less than 3 pm. In addition, the initiation events along the DNA molecule are staggered over a period of 10 hours in cultured cells, but in blastomeres all initiations occur within the first minute or less. Differences in DNA replication occur between somatic and embryonic cells of Triturus (Callan, 1972). If replication begins at specific sites (origins), many of them can be inactivated without hindering total replication of the genome, and the timing of firing can be sharply altered. An explanation of how order is imposed on replication of replicons must also explain the rule that every replicon is replicated and that normally no replicon replicates more than once. The latter rule can be abrogated with inhibitors. Transient inhibition of DNA replication already in progress in mammalian cells with drugs that directly affect replication or by blocking protein synthesis results in rereplication of some segments of DNA after removal of the inhibition (Woodcock and Cooper, 1981). A similar effect is achieved in the replication of polytene chromosomes of Drosophilu with an inhibitor of protein synthesis (Mukherjee and Chatterjee, 1984). Inhibition of DNA replication with hydroxyurea in early S of mammalian cells followed by release from the block results in an extra replication of the early-replicating gene encoding dihydrofolate reductase (Mariani and Schimke, 1984). Apparently, after release from the hydroxyurea block, the cells revert to the beginning of S and start over. All of these studies show that the S period is highly ordered and that ordering involves an interplay between the transcription of a particular DNA segment and its replication. The ordered structure of S is therefore flexible at least to the extent that transcription is a flexibly regulated process.

VII. Events of the G , Period The G, period links the end of DNA replication with the initiation of mitosis, but the molecular events that underlie this linkage have not been identified. RNA or protein synthesis is required since inhibition of synthesis in GI in mammalian

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cells blocks entry into mitosis (e.g., Tobey et al., 1966; Westwood and Wagenaar, 1983). Maturation of promoting factor, which induces chromosome condensation, appears transiently between the end of DNA replication and the start of mitosis as discussed earlier. Two other proteins have been described to appear specifically in G, in hamster cells and then to disappear in postmitotic cells (Westwood et al., 1985). These presumably are involved in transit from S to M. Mammalian cells have also been blocked in G, with antitumor agents (Barlogie and Drewinko, 1978), with glucocorticoids (Das et al., 1983), or with deprivation of iron (Reddel et al., 1985). The plant hormone (trigonelline) arrests plant cells in G, (Evans et al., 1979). Gelfant ( 1 963) was the first to describe arrest of animal cells in G , in normal tissues. Such arrest has subsequently been observed in a wide variety of normal, untreated animal and plant cells (see review in Prescott, 1976). These various observations imply ordered events in G,, some of which involve RNA and protein synthesis. The progression can be interrupted by normal physiological mechanisms to produce G, arrest.

VIII. Concluding Remarks The composition of the cell cycle has advanced to the point that the cause-andeffect progression that drives the cycle forward is now understood in general terms. Bits and pieces of the molecular events that underlie the cause-and-effect progression and normal interruption of this progression in the regulation of cell proliferation are now known. Eventually, the picture will be completed and will show how cells arrange their growth, DNA replication, and cell division to achieve the remarkable accomplishment of self-reproduction.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 100

The Early Days of Electron Microscopy of Nerve Tissue and Membranes J. DAVIDROBERTSON Department of Anatomy, Duke University Medical School, Durham, North Carolina 27710

I. Introduction This review is an autobiographical account of the development of ideas about the molecular structure of membranes containing some anecdotes about scientists I have encountered along the way. Within this framework, I shall discuss the origins of some of the major ideas about nerve and membrane structure with which I have been directly concerned.

11. Formative Years

I was born in Tuscaloosa, Alabama and was educated there through the second year of medical school. My father was a city policeman and my mother a county elementary school teacher. There were over a hundred Robertsons in the county descended from a Scottish blacksmith, James (Horseshoe) Robertson, who had entered Washington’s army in North Carolina as a private and, according to family lore, had distinguished himself and retired as a major. He built a house on a horseshoe bend of the Black Warrior River near Tuscaloosa. I can remember it vaguely as an empty rotting two-story French colonial house buried in Spanish moss that I saw once a year on the fourth of July, when the family gathered there for a graveyard cleaning. There were some branches of the family that were fairly well off, but not mine. I always wanted to be a scientist, and when I was quite young I read avidly all the science and science fiction magazines and books I could find and was particularly fascinated by astronomy. My earliest ambition was to go to the moon, an aspiration that my mother’s brothers, good naturedly, but quite firmly, tried to convince me was impossible. One of them lived long enough to tell me a few years ago how wrong he was.

129 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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111. Early Academic Training A. UNDERGRADUATE STUDY

I graduated from Tuscaloosa High School and entered the University of Alabama in 1939 at age 16, having decided that I wanted to be a physician and do medical research. During my first year, to pay my fees, I worked in the federal National Youth Administration program that President Roosevelt had started to help college students during the Great Depression. I spent part of the year with a ditch-digging crew working at 15 cents an hour. As a pre-med student in biology, I attracted the attention of the chairman of the department, Dr. Charles Pomerat, who offered me a job as a teaching assistant in biology for the following year. I accepted with alacrity. Charlie Pomerat was in his first year at Alabama. He had just finished his Ph.D. in biology at Harvard where I learned much later he was a classmate of Keith Porter, who was later to play a part in my scientific life. Pomerat, who had just returned from a postdoctoral training period with Houssay in South America, was a very charismatic and fascinating teacher. He was also a talented ambidextrous artist. His lectures in biology were beautifully organized, and he always filled the blackboard with marvelously detailed colored drawings that he drew with both hands while he talked. He gave me a sense of excitement about biology that I have never lost. He was just starting his research in tissue culture, a field in which he was later to achieve considerable stature. He had a great deal of influence on me and a small group of students who were also teaching assistants. I remember that he took a group of us, including John and Jimmy Gregg, who are now Professors of Zoology, Emeriti, at Duke and Florida, respectively, to a marine biology lab on the Gulf of Mexico one summer. I became fascinated then by invertebrate marine animals and still like marine biology and working with invertebrates. I did not receive a particularly good education at Alabama, through no fault of the school, but because I knew that in order to get through medical school I would have to have an almost perfect academic record. I came close to achieving that, but I never dared take some courses that interested me and would have been very good for me. Instead I took a lot of watered-down science courses designed for premedical students, such as physical chemistry and physics for pre-meds, both taught without calculus. I also dropped a course in music appreciation because I could not recognize symphonies from the playing of a few bars and knew that this would result in a low grade. Toward the end of my college career, the Japanese bombed Pearl Harbor and I joined the Navy. I was given a commission as an Ensign HV(P) early in 1942 and told to continue in school. That fall I entered the University of Alabama Medical School. This was only for the first 2 years of basic science; everybody had to

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transfer to another school for the clinical years. About a year later, the Navy put me on active duty as an Apprentice Seaman. However, they put me in a Midshipman’s uniform, as they did with all medical students, presumably because they thought this would make a better impression on patients. This caused a great deal of confusion when I had to spend 6 weeks at Chelsea Naval Hospital in Boston in 1943, where I was the only person whose uniform and rank did not match.

B. MEDICALSCHOOL

I was admitted to the 2-year Alabama medical school and did well there. By that time I was living away from home and working as an orderly in the nearby Drvid City Hospital for my room and board. My fees were paid by a scholarship. I was tied for second place in my class of 50 and was able, with three of my classmates, to transfer to Harvard for the clinical years. There was a 6-week hiatus between the terms at Alabama and Harvard, and I spent this period at Chelsea Naval Hospital in Boston. I was assigned to radiology at first but spent most of my time on a medical ward where I saw some unique clinical cases, such as a young sailor with lobar pneumonia in whose chest I heard egophony clearly for the first and I believe the only time. I never did any real research in school; all my energies went into my coursework. The only extracurricular activity that I engaged in was acting in Blackfriars, the University of Alabama dramatics society. I had played Sir Toby Belch in “Twelfth Night” in high school and had a few minor roles in Blackfiars plays. In Blackfriars I met George Miller, who was a few years ahead of me and majoring in psychology. He and I crossed paths again many years later at MIT, but in recent years I have seen little of him. He is now a professor of psychology at Princeton and married to Katherine James, who was prominent in my high school dramatics group. I entered Harvard Medical School in 1943 as a U.S. Navy V-12 student and got the M.D. degree in 1945. I must confess that 1 enjoyed living in Vanderbilt Hall and being free of financial problems since the Navy took care of all of my expenses. I enjoyed the clinical coursework, but did not find it particularly challenging, and I am afraid I did some coasting because, for the first time in my life, it was not essential that I always get top grades. In fact I was very happy to finish in the top third of my class because everybody was a good student. I enjoyed for a time living the life of a young gentleman in Vanderbilt Hall, even though most of the amenities there had been wiped out by the war. In my senior year I was offered a job as an extern with room, board, and laundry at Massachusetts Women’s Hospital, where there were a lot of patients from the Leahy Clinic. I decided to take it and moved in there with a classmate, Fred Snell, with whom I later was a Ph.D. candidate at MIT. Fred is now professor of biophysics at SUNY/Buffalo.

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C. INTERNSHIP After graduation, I interned at Boston City Hospital (BCH) in internal medicine on Harvard’s famed IVth Medical Service. The war had resulted in internships being confined to 9 months. The war ended just as I got started as an intern, and the government allowed us to have an additional 3 months as interns. Initially I had wanted to intern in pathology and actually had accepted an internship at Columbia Presbyterian Hospital in pathology while in medical school. However, the Navy had objected and encouraged medical students in the V-12 program to intern in surgery or medicine. So I withdrew from the pathology internship and got one in internal medicine at BCH. When we were given an additional 3 months, I naturally chose to work at the Mallory Institute of Pathology at BCH, where I had already done a fourth-year elective under Fred Parker, the Chief of the laboratory, who was a reclusive but respected and stimulating teacher. One of the sons of F. B. Mallory, Kenneth, was on the staff, and I also was acquainted as a student with his other son, Tracy, who was the Pathologist-in-Chief at Massachusetts General Hospital. I had a very good experience at the Mallory Institute, but I became increasingly frustrated by the limitations of light microscopy.

IV. Military Service and Medical Practice I had 2 years of obligated service in the Navy as a Lieutenant fig) in the Medical Corps after my internship and was assigned to the U.S. Veterans Hospital in Montgomery, Alabama, where I was soon put in charge of the clinical laboratory and eventually made Acting Chief of Pathology. The chairman of the Pathology Department at the University of Alabama Medical School in Birmingham, J. F. A. McManus, came in about once a week to check out my cases with me and this was a good learning experience. McManus was noted for his role in developing the periodic acid Schiff stain for carbohydrates, used widely in histology, so I considered myself lucky to have him as a supervisor. I also set up and ran a bacteriology service for the hospital. Within a short time I married Dody Kohler, a staff nurse on Peabody I, one of the wards of the IVth Medical Service at Boston City Hospital. We had a hard time finding housing in Montgomery near the Veterans Hospital and finally got an apartment in a farmhouse owned by a lady in Lowndes County on Highway 80 halfway to Selma, where Mrs. Liuzzo was later to be murdered and where Martin Luther King conducted his famous march. We soon had our first child, Karen Lee, who is now an assistant professor of English at Vassar College. Nights and weekends I practiced medicine in Hayneville, the nearby county seat, and later also in Lowndesborough. These two towns were about 5 miles apart and each had a population of about 500. Lowndesborough had many fine antebellum plantation houses, most of which were badly in need of paint and repairs.

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My practice was done under rather primitive conditions in wooden-frame offices that had been occupied by two general practitioners who had recently died, which I rented from their widows. In Hayneville, where I began practicing, I built my own examining table from pine boards, had an old-fashioned iron potbellied stove for heat, and my water supply was a bucket hanging from the ceiling that I filled with water from the fountain in the town square in the evenings when I opened the office after supper. I remember making house calls in places where my car could not go and white people normally did not venture. I would be met by some negroes with a mule-drawn wagon and escorted to the patient. I was told that these people were living on parts of farms that their families had lived on as slaves of plantations before the War between the States. Even the sheriff did not venture into these out-of-the-way places. These people had an organization they called “clubs” that functioned as simple health insurance agencies. They each paid a small amount regularly to the club and the club always paid my bill. I tried to practice good medicine, but I do not think it was what the community needed. 1 had a borrowed microscope and did blood counts and urinalyses. I even occasionally did blood transfusions. I always did a physical examination and had a standard office fee of two dollars. I think some of my patients thought I was not a good doctor because I had to examine them to find out what was wrong. I was referred to as “that Yankee doctor who always had to ‘xamin you.” One night while I was Officer of the Day at the Veterans Hospital, a fire started in Haynesville and half the town burned down, including my office with my Latin-inscribed Harvard diploma, which I still have not replaced. My borrowed microscope and the stove were recognizable, but nothing else was. Of course, I had no insurance.

V. Ph.D. Training

In 1948, upon discharge from active naval duty, taking advantage of the GI Bill and using some savings from my practice and what I could earn as a staff physician in the Student Health Department, I enrolled as a graduate student at MIT, where I expected to learn about the electron microscope (EM) and how it could be applied to biological problems. After the first year I was awarded a $3000/year American Cancer Society Fellowship and my wife enrolled at Radcliffe College as a student in English literature. She also had the GI Bill, since she had been a First Lieutenant in the Army Nurse Corps during the war, so we were well supported through the next 3 years of graduate school. The period including the latter half of the 1940s and the first half of the 1950s was historically a very interesting time in cytology, for this was the period when the electron microscope first began to be employed effectively in the field. In the mid-1940s F. 0. Schmitt, Cecil Hall, Marie Jakus, and Richard Bear, among others, had begun to show what could be done with EMS in biology by studying

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structures like skeletal muscle fibers and collagen fibers, in which one could find out things from the native material by X-ray diffraction and then look at the structures with the EM and make correlations. They had obtained some of the first electron micrographs of isolated skeletal muscle fibers and collagen fibers and there was great interest in Schmitt’s department in finding out something about the structure of nerve fibers. Schmitt, Bear, and Clark (187) in the 1930s had obtained the first X-ray diffraction patterns of myelinated nerve fibers and defined thereby the dimensions of the radially repeating lipid and protein units that W. J. Schmidt’s work with polarization microscopy in Germany in 1936 had predicted (185). So the time was ripe to apply the EM to studies of nerve fibers and find out something directly about the structure of myelin. At that time, nobody had yet succeeded in cutting sections of biological material thin enough to be really useful in realizing the resolving power of the EM. To be sure, Fullam and Gessler (73, 82), Claude and Fullam (34), and Pease and Baker (129), using modifications of light microscopic methods, had cut some sections of tissues and taken some electron micrographs, but they had not seen much more than had been seen before with light microscopes. I remember being struck in 1949 by a picture of a Drosophila giant chromosome that Pease and Baker (130) had sectioned and published; in one place they had put an arrow pointing to one of the bands which they called a gene. I thought that was extremely bold; however, they were very nearly right. The difficulty, of course, was that a light microscopist could have done the same and been as nearly right. This was, however, indicative of the times; everyone expected great things of the EM, and so seeing genes seemed quite reasonable. Such a claim in those days could get by a journal referee as easily as Oscar Miller’s beautiful paper with Beatty (1 18) two decades later reporting the first real visualization of a gene by electron microscopy. Everybody expected great discoveries and the journals were eagerly waiting for the papers reporting them. In this atmosphere the group at MIT, particularly those directly under F. 0. Schmitt, became very conservative. As I look back upon it, I now see that we failed to publish many things that we could easily have published that were probably worth recording. The group of postdoctoral fellows and graduate students under Schmitt at that time included Jerry Gross, Bert Valle, Jesse Scott, Betty Geren (later Uzman), David McCullough, Harrison Latta, Frank Hartman, Brian Finean, Alan Hodge, David Spiro, Martin Lubin, Myles Maxfield, Fred Snell, Roland Beers, and several other M.D.s. Martin Lubin, Fred Snell, Myles Maxfield, and I were classmates from Harvard Medical School. Jean Hanson and Hugh Huxley arrived as fellows just after I left, but I made frequent visits to Boston and we became friends. From 1948 to 1950 I spent my time as a graduate student taking courses to help remedy the deficiencies of my science education, and I began to do some electron microscopy and even tried to get some X-ray diffraction patterns from dried

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pieces of the crystalline lens of the eye. I got nothing from the latter, even though, as recent work from my own laboratory has shown (38, 194, 239), I certainly might have. I soon became tired of trying to get somewhere with X-ray diffraction and decided that I wanted to concentrate on electron microscopy. I had just completed Cecil Hall’s course and learned how to take my first electron micrographs with his RCA model EMB electron microscope. This instrument was said to have been the third EM produced by RCA and dated from about 1942. I think Schmitt had bought it for about $3000. The department also had one of the later RCA EMU-2 models dating from about 1944, and there was talk of getting the new Philips EM 100 that the Dutch were beginning to produce and were selling for $20,000. I worked mostly with the EMU and later with the Philips, which the department finally purchased after I got some acceptable pictures with it. I soon chose F. 0. Schmitt to be my thesis advisor and began to work on nerve tissue. At that time we were just beginning to fix tissues in osmium tetroxide and embed them in methacrylate (122, 123) for sectioning, but we had not yet learned how to cut thin-enough sections. We were using steel knives and microtomes designed for light microscopy. The sections were so thick that the electron beam could not penetrate them, so we deposited them on a thin plastic (collodion or formvar) support film on a nickel mesh grid and soaked the grid in toluene to remove the plastic. Then we sprayed the grid lightly at a small angle with chromium to produce contrast. One of the very first experiments I did was to set up a series of dilutions of OsO, in water and use each dilution as a fixative on a frog sciatic nerve fiber. I also tried a time series with a constant concentration of 1% OsO,. In the latter, the myelin sheath was completely tom up after 4 hours of fixation. In that material, nevertheless, I made an observation that should have been reported because I believe it was one of the first observations of intermediate filaments and, most importantly, it showed very clearly that the well-known neurofibrils of light microscopy were comprised of bundles of these filaments, which I measured to be l l .4 nm in diameter. The original micrograph showing this, which was published only in my thesis, is reproduced in Figs. l a and lb. These are direct copies of the original prints with my original notation in ink of the diameter of a filament (in angstrom units) in the enlarged area in Fig. Ib. This dimension is well within the range now accepted for the diameter of an intermediate filament. I should mention that these observations of mine in the laboratory were made about the same time that Betty Geren and F. 0. Schmitt also made similar observations in sections of axons. Schmitt and Geren published a paper in 1950 (191) reporting these filamentous and rather beaded structures in axons in sections, and Schmitt wrote a separate paper the same year on similar observations on squid and myxicola axoplasm (186). These papers were aimed in part at

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FIG. I . Electron micrograph of longitudinal section of myelinated nerve fiber fixed with I % OsO4 in water for 4 hours, embedded in methacrylate, treated with toluene, and lightly shadowed with chromium. Preparation was made in about 1949 and included in Ph.D. thesis of author (143). (a) Note the neurofibrils identifiable from light microscopy produced by clumping of neurofilaments. Note the lack of preservation of the myelin sheath, which appears around the axon as irregular dense clumps of material. X9300. (b) An enlarged area from (a) showing the neurofilaments measuring 11.2 nm in diameter and showing in some places suggestions of an axial periodicity of about 35 nm. x 53,000.

retracting a mistake that had embarrassed Schmitt a few years earlier when he had published some papers with de Robertis reporting the finding of tubular structures in homogenates of nerve fibers that they thought were in the axon and which they called “neurotubules” (45, 46). They then infected some nerve fibers with poliomyelitis virus and found dense granular particles apparently lying within the neurotubules that they identified as virus particles being transported up the nerve fiber along the “neurotubules” (47). This concept had attracted considerable attention, but it had become apparent as soon as we began to look at sections that the “neurotubules” were most likely collagen fibrils. So the 1948 papers were aimed at clearing up this matter.

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FIG. 1. (Continued)

In the same experiment, in myelin fixed for a shorter time (about 30 minutes), I saw a radial periodicity of about 10 nm, but the period was very faint and so I did not publish the picture. In one of the nerves that was fixed for 4 hours in 0.15% OsO,, I saw a very striking phenomenon. It was clear that the myelin had swollen markedly, but one could still recognize the axon and the myelin sheath. However, the individual myelin lamellae had separated and, after the plastic was removed, surface tension forces from the evaporation of toluene caused the lamellae to flatten out on the support film. I was quite sure I was looking at individual myelin lamellae in a surface view, but again I published this only in my thesis. The original figure is shown in Fig. 2. I showed this picture to Marie Jakus at the time and she pointed out some faint suggestions of periodic striations in the lamellar surfaces that I had not seen. Many years later I did some experiments on swelling myelin that made me sure that what I was seeing at that time was pairs of myelin unit membranes adherent along their cytoplasmic surfaces but separated by imbibition of water along the external surfaces (155, 165). Even today I believe nobody has observed the cytoplasmic surfaces of myelin mem-

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FIG. 2. Cross section of myelinated nerve fiber dating from same period as Fig. 1 using same technique except that fixation was in 0.15% Os04 for 4 hours. The axon is recognizable in the right center as dense granular material. The myelin sheath was greatly swollen with the individual lamellae separated. After the methacrylate embedding medium was removed with toluene and the specimen air dried, the individual lamellae, for the most part, were flattened on the support film and their width gave the thickness of the section (about 0.2 km). x 15,500.

branes so extensively and directly, and I have always intended to do this experiment again. It is still worth following up with modern techniques. About this time one of my lab partners, Harrison Latta, a pathologist who was a postdoctoral fellow with F. 0. Schmitt, had a brilliant idea. He decided that we

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were all wasting our time trying to sharpen steel knives to make sections and proposed that instead we use pieces of broken glass. I remember clearly one Monday morning that Harrison came into the lab with a milk bottle from home and stated that he was going to make a glass knife. We all laughed at this strange idea, but I followed him up to the machine shop where he got a hammer and smashed the milk bottle and chose a small piece to use as a knife. He mounted the fragment on a dummy steel knife using a black glue, and he and Frank Hartman proceeded to cut very thin sections that one could use without taking out the plastic. I believe this was the first time anybody had succeeded in getting high-quality sections of biological material routinely thin enough to use for direct study without removing the plastic. This remarkable technical advance probably can be said to have triggered the start of the modem period of EM study of tissues in a histological sense, with realization of a significant gain in resolution over that of the light microscope. Latta and Hartman, in their paper (109) reporting this advance, pointed out that the use of precious stones such as industrial diamonds or artificial sapphires would be a better way to make a knife. The latter, however, did not prove to be easy, but Fernandez-Moran succeeded and developed the technique to make a diamond knife (62). This is, of course, the method of choice today for sectioning, although sapphire knives are also now available and may prove to be useful. Glass knives are still used for much routine work. It should be said that, despite the fact that the glass knife quickly became the universal tool for making sections for electron microscopy, Fritiof Sjoestrand, who had worked at MIT with Schmitt as a research associate, along with Eduardo de Robertis just before I arrived, stubbornly persisted in Stockholm in trying to sharpen steel knives and finally succeeded with Schick razor blades. In 1953 (198), he published the first micrograph of a section of the myelin sheath that revealed the repeating units as we now recognize them. He defined the major dense line as a structure about 2 nm thick repeating radially at a period of about 12 nm with an intraperiod line bisecting the intervening light zone as a row of broken lines and dots. Fernandez-Moran had published the first pictures (60), showing a radially repeating structure in frozen sections of Os0,-fixed material. But he had not seen the details Sjoestrand found and probably was seeing the half-period, failing to differentiate between the major dense and intraperiod lines. Later on Fernandez-Moran and Finean (63), in a combined electron microscopic and X-ray diffraction study, worked out the reason why the 12-nm period seen by Sjoestrand was the real radial repeat. The problem was simply one of shrinkage caused by the preparatory methods, as they were able to assess by following changes in the radial repeat period by X-ray diffraction during the various steps in preparation. Most of us were reasonably sure that Sjoestrand’s picture of the radial repeat was correct at the time, but it was not until much later, after the introduction of epoxy embedding (83, 84), combined with glutaraldehyde fixation (184), that we

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began to see the radial repeat in very nearly its native state. The development of polyglutaraldehyde-embedding methods independently in Barnett’s (91) and Pease’s (13 I , 132) laboratories finally made it possible to obtain thin sections of material that had been treated only with glutaraldehyde. In this case the most faithful reproduction of the repeat period was obtained. We see it now in such material as in Fig. 3. The repeat period is about 18 nm, as it is in the native state as judged by X-ray diffraction. The major dense line is still about as Sjoestrand first saw it, but the intraperiod line is split into two continuous dense strata under 2 nm thick, separated by a space of about equal thickness. I believe this is as close visualizing to the native structure as we have been able to achieve with this kind of technique. In 1949, when I was choosing a Ph.D. thesis topic, I knew of nobody who had managed to get any meaningful pictures of synapses with the EM. So I decided that this would be a good topic. My first inclination was to try to get some sections of the stellate ganglion of the rat or mouse. However, Professor Schmitt discouraged me because he said that I would not be able to identify synapses with certainty. He insisted that I should find a well-defined system in which I would be able to know exactly where I was in sections for the EM. I finally settled on

FIG. 3. Section of myelin in a mouse sciatic nerve, prepared by contemporary techniques, showing radial repeat period of about 18 nm. Note the splitting of the intraperiod line. The section was fixed in glutaraldehyde, embedded in epoxy resin, and sectioned with a diamond knife. Section stained with uranyl and lead salts. X200,OOO.

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the giant nerve fiber median-to-motor synapse in the crayfish as the best thing to work on. The light microscopy on this synapse had been done in 1924 by Johnson (103). It was known to be a one-way conducting synapse and I felt sure I could locate and identify the pre- and postsynaptic fibers in electron micrographs. There are four giant nerve fibers in the crayfish nerve cord, each running the entire length of the animal. There are two median giants, each of which may measure up to 100 k m in diameter, and two lateral giants of about the same size. The lateral giants are interrupted in each segment by a diagonal septum, but the two median giants are free of such interruptions. These giant fibers all carry sensory impulses from the periphery. In each segment of the animal, two giant motor fibers synapse with each of the afferent giant fibers. In each abdominal ganglion, two neurons in the ventral cell mass extend axons dorsally that expand to become giant motor fibers. They decussate by crossing over one another and, as they do so, synapse successively with the two median giant fibers. Each then proceeds to synapse with the contralateral giant fiber and exit through the contralateral third root of the ganglion to innervate the muscles of the tail. Figure 4 is a transection of an abdominal ganglion showing some of these features, taken from my Ph.D. thesis (143). The septae in the lateral giant fibers are really zones of contact between axons of separate neurons in each segmental ganglion, and Johnson recognized that they were synapses. Structurally, they appeared to him to be connective tissue septa just like the zones of contact between the afferent giants and the motor fibers. However, Wiersma (230-232) showed that these zones of contact, although looking the same morphologically, were quite different physiologically. The afferent median-to-motor and lateral-to-motor synapses were polarized, conducting impulses only in the afferent-to-efferent direction. Furthermore, there was an appreciable synaptic delay of 0.3-0.5 ms. The septa1 synapses conducted equally well in either direction and the synaptic delay was very short, about 0.1 ms. Thus, the latter were regarded as electrical synapses rather like the artificial “ephapses” that Arvanitaki (4) had obtained by simply putting carefully dissected giant nerve fibers into apposition. I soon found something by light microscopy that had been missed in previous work. I discovered that the postsynaptic giant fiber extended numerous small fingerlike synaptic processes through the combined sheaths of the two giant fibers that made intimate contact with the median giant axon. Figure 5 is a photomicrograph of a transection through an abdominal ganglion showing these synaptic processes. These minute extensions of the motor fibers were only a few micrometers in diameter. I thought this was an original discovery until I read a paper by J . Z. Young (235), who described essentially the same thing in the squid giant fiber synapse in the stellate ganglion of Loligo in 1939. The nice thing was that these synaptic processes were so much smaller than the presynaptic giant fiber that there was no doubt that I could unambiguously identify the

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FIG. 4. Cross section of abdominal ganglion of crayfish (Cumbarus clarkii) from Robertson (143). The section was fixed by the method of von Rath, embedded in paraffin, and sectioned with a steel knife. Note the four giant nerve fibers cut in cross section. There are rwo median giant fibers and two lateral giants, each measuring 50-80 pm in diameter. Two giant motor fibers are seen just under the median giant fibers. These have much more dense axoplasm and are decussating and synapsing with the two median giants in this section. More caudally they move laterally and synapse with the two lateral giants before entering the third root of the ganglion joining the large nerve fibers seen gathered above the two lateral giants. X 160.

pre- and postsynaptic structures with the EM. It was quite clear in my first electron micrographs that the pre- and postsynaptic membranes were intimately apposed but continuous structures. I think it is fair to say that this was the first confirmation by EM of Cajal’s neuron doctrine. I first presented this work at Cold Spring Harbor in 1952 (142). Alan Hodgkin, Bernhard Katz, Andrew Huxley, K. S. Cole, H. J. Curtis, and many other people whose work I knew about were in the audience. I gave a 10-minute presentation and remember that I got a standing ovation. This was my first presentation at a scientific meeting and it had a profound effect on my subsequent career because it led Bernhard Katz to invite me in 1954 to come to London to set up a laboratory at University College. I published these findings in two papers in 1952 (144) and 1955 (147) as well as some more information in a review article in 1961 (162).

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FIG.5. Preparation similar to Fig. 4. Note the small processes (Proc.) of the motor fiber (M.F.) that are penetrating its sheath (Sh.) and that of the median giant fiber (M.G.) on the right. Only one of the motor fibers is seen in this section. XSOO.

VI. The Kansas Period In 1952 I received the Ph.D. degree in biochemistry from MIT. I then accepted a position as an assistant professor of pathology and oncology at the University of Kansas Medical School, where I was put in charge of an EM lab with an old RCA EMU-2 instrument that was in very poor condition. The objective pole piece had been badly damaged and I did not have grant funds to buy a new one. So I spent a lot of time in the departmental machine shop lapping the pole piece surfaces. I thoroughly enjoyed learning how to be a machinist and I finally got the pole piece to work, but by then I had an NIH grant and could buy a new one. A. THE MOTOR-ENDPLATE At that time I intended to pursue my interest in pathology, and so I worked essentially as a resident in pathology for 3 years. It soon became apparent to me, however, that I could not do an adequate job of routine pathology and do the kind of research I wanted to do. Robert E. Stowell, the Chairman of the department, was quite adamant in insisting that I carry a full load of clinical work as well as keep up my research program. I tried to do the clinical work, but my heart was not in it, and I put most of my time into my research efforts, incurring Stowell’s thorough disapproval. Besides continuing my previous work, I thought it would be a good idea to try to get some electron micrographs of motor-end plates, since nobody had done that. I settled on the lizard, Anolis carolinensis, as a suitable substitute for the European adder that my reading suggested would be the best animal. I soon got

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some sections through lizard motor-end plates and sent off an abstract to the American Association of Anatomists (AAA) for presentation at the annual meeting in Galveston, Texas in April, 1954 (145). I presented essentially the same material later that year at the annual meeting of the Electron Microscopy Society of America (EMSA) (146). When I wrote the abstract for the AAA meeting I had not seen the synaptic vesicles in the endings, but by the time I got to the meeting I had quite clear pictures of them. George Palade (126) and James Reger (136) also reported on EM studies of motor-end plates at that meeting and Palade and I had similar pictures. Reger was still working with thick sections with the plastic removed and so his pictures, though very clearly displaying important new features of motor-end plates, did not show such details as the vesicles. Palade had seen the vesicles before I had, because he had described them in his abstract. However, neither he nor Palay, who assisted him, went on to publish the pictures. Reger published his findings in 1955 (137), but since he still had used the old thick sectioning methods his paper did not reveal the vesicles or other fine features of the endings. When my micrographs were published, I believe they were the first ones recorded showing synaptic vesicles in motor-end plates. To be sure, Palay (127, 128), De Robertis and Bennett (48), Fernandez-Moran (61), and Sjoestrand (197) about that time published pictures of synaptic vesicles in central nervous system synapses and their probable function as carriers of chemical transmitters was immediately recognized. At the meeting in Galveston, I became acquainted with George Palade and Keith Porter. I had met Porter earlier but hardly knew Palade. I first encountered Keith Porter’s work at the meeting of the International Society for Cell Biology at Yale in 1951, where he had a poster exhibit showing the early pictures of the endoplasmic reticulum. I first met him personally one afternoon in the spring of 1952 on Long Island when I was taken to his house by Betty Geren, who knew his wife, Elizabeth. Betty and I were at a meeting in Cold Spring Harbor that day, but I do not remember which meeting. In any case, Keith appeared for tea but said he had to go to the lab that evening and, somehow, I ended up there with him before getting a late train to Boston. Porter showed me some bunk beds provided by the Institute and said he planned to spend the night in the Institute. I watched what he was doing and we had a chance to talk science. I recall that Don Fawcett was working in the lab that evening on another microscope, but we did not talk much. It seemed to me that Keith and I got along very well and he inquired about my plans after I left MIT. I told him that I had accepted a job in Kansas and he said it was too bad they had not found out about me earlier, because he would have liked to have had me at The Rockefeller Institute for a postdoctoral fellowship. I have often wondered what my career would have been like if that had happened. At the meeting in Galveston, Keith Porter and George Palade had a group gathered around them that included Don Fawcett, Sandy Palay, and others whose

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names were later to become very prominent in the application of the EM to studies of cells. I remember that Keith and George had us all gathered at a separate table at the atmual banquet, where we all behaved rather badly by being noisy and disrespectful of the speakers. I got the distinct impression that evening that Porter and Palade did not like being associated with the classical anatomists who dominated the AAA, and I think this was the beginning of the American Society for Cell Biology, which Keith and George in due course to a large extent founded. Some of us have maintained our connection with the parent society, but I must say that it has sometimes been difficult. The AAA simply could not, or would not, move fast enough to keep the cell biologists coming to its annual meetings. I find this unfortunate for the discipline of anatomy. For example, I regard my Department of Anatomy today as one of the better ones, but I have had very little success in getting my younger staff members to become members of the AAA. They find their outlets in cell biology, biophysics, and anthropology. Last year I was nominated for President of the AAA but it was not surprising to me that I was not elected. In the winter of 1953-54 I got a letter from Bernhard Katz asking if I would like to come to University College, London and set up an EM laboratory jointly in his Department of Biophysics and Professor J. 2. Young’s Department of Anatomy. I was invited to come to London to talk about this and in November, 1954 I flew to London with my micrographs. When I showed the micrographs of motor-end plates to Katz, I remember that he exclaimed that I was showing him something, the vesicles, that he would have had to invent to explain the miniature end plate potentials that he and Fatt (58, 59) and he and del Castillo (50) had just described. Katz asked me to let him include one of the micrographs showing the vesicles in a review article he and del Castillo were then preparing (51). I did this and, even though the same micrograph was published in 1956 in my paper on motor-end plates (150), the first published picture showing synaptic vesicles in motor-end plates was my picture published in that article by del Castillo and Katz (51).

B. SPIRALMYELIN Two other discoveries that are worth describing were made during that period in Kansas City while I worked on lizard muscle fibers. Perhaps I should begin with some background. In the late 1940s Herbert Gasser, who was then the Director of The Rockefeller Institute, began working on peripheral nerves by EM. He was specifically interested in the structure of “C” fibers and came to MIT to ask for some technical advice from F. 0. Schmitt, who had been one of his colleagues. Schmitt asked Betty Geren to show him how we did things and Betty worked with him to some extent, although not to the point of being a coauthor. I was not involved in any of this directly. Gasser presented a preliminary

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paper at the Cold Spring Harbor Symposium that I have already mentioned in which he gave a new concept about how “C“ fibers were constructed (76). This was followed by a complete paper in 1955 (77). It had been thought previously that “C” fibers were bundles of axons completely embedded in syncytial Schwann cells. Gasser realized this was not so because he saw membranous structures leading from each axon to the surface of the Schwann cell. He thought the axon was simply embedded in the Schwann cell and was related to it rather like the gut in the body cavity of a vertebrate is related to the mesentery. So, he coined the term “mesaxon” for each of the paired membrane structures connecting each axon to the surface of its Schwann cell. It would be hard to overestimate the importance of this concept because it was the fundamental key to understanding what we now know about the relationships between peripheral and central nerve fibers and their satellite cells. This was a most impressive accomplishment that excited my respect and admiration. Here was a person who had spent his life as a neurophysiologist, earning a Nobel prize decades earlier with Erlanger, and in his later years had the administrative responsibility for a major research institute, who, nevertheless, ventured into a field like EM and succeeded in making one of the most important advances made by anybody at the time. I had the privilege of getting to know this remarkable man during this period and the last time I saw him, not long before he died, I remember spending an afternoon with him at The Rockefeller Institute poring over my micrographs of nerve fibers and being subjected to the most penetrating and insightful questions I had ever encountered. When we parted that afternoon, he invited me to stay and have dinner with him, but I declined because I had to get the train to Boston. That was the last time I saw him. Betty Geren, my former colleague at MIT, began studying developing sciatic nerve fibers in 1953 in her new laboratory in the Pathology Department at the Children’s Hospital in Boston. She observed that before myelin appeared there were nonmyelinated nerve fibers having a one-to-one relationship with Schwann cells that had long mesaxons wrapped in a spiral around the axon. This led her to suggest (80, 81) that the myelin sheath might be simply a spirally wound, tightly compacted mesaxon. She told me about this privately but I was quite skeptical. Nobody had ever seen any other evidence of this and there was nothing else to suggest it. However, while I was looking for motor-end plates in lizard muscle, I naturally saw many transections of small myelinated nerve fibers near the nerve endings. In one of these sections I took the micrograph in Fig. 6. I shall never forget the elation I felt when I examined this micrograph with a hand lens and realized what it meant. It showed exactly what would be predicted by Betty’s theory-an outer and an inner mesaxon running into the outer and inner surfaces of the myelin sheath-just as it should if myelin were simply a spirally wound, tightly compacted mesaxon. I immediately realized that she was right and telephoned her to tell her. I stopped off in Boston on my way to London in Novem-

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FIG. 6 . Myelinated nerve fiber from lizard muscle showing the outer ( 1 ) and inner (2) mesaxons and the axon-Schwann cells paired membranes as described in text. X85,OOO.

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ber 1954 to show the micrograph to her and F. 0. Schmitt and published it in 1955 (148). While it seemed that Geren was right, my micrograph did not prove the main point because, while it seemed clear that myelin must form by some mechanism involving spiral evolution of the mesaxon around the axon, the micrograph was of such low resolution that one could not be sure that there were not some other components added between the spiral loops of the mesaxon to make myelin. The essential details had to await elucidation for some more technical advances as I shall describe below. Before turning to this I will describe another discovery made with the lizard muscle while I was looking for motor-end plates.

C. THETRANSVERSE-TUBULE SYSTEMOF MUSCLE In looking at longitudinal sections of lizard skeletal muscle fibers I observed the relationships shown in Figs. 7-8. These are the original micrographs that I published in 1956 (149) and they show the presence of membranous elements located between the myofibrils regularly at the A-I junctions. I recall vividly that I was working late at night in my laboratory in Kansas City sometime during the winter of 1953-54 when I first saw these structures. Everything suddenly clicked in my mind as I realized what these structures meant, and I got that exhilaration that comes so rarely to a scientist when he makes a truly new and important discovery. I conceived of these tubules as parts of a continuous system of transverse membrane sheets running all the way across the muscle fiber from one surface of the plasma membrane to the other, enveloping the myofibrils. I realized that this explained an anomaly that A. V . Hill had pointed out earlier (94), i.e., that it was quite impossible for any diffusible agent released by the action potential at the surface of a muscle fiber to diffuse fast enough to be responsible for the uniformly synchronous contraction of all the myofibrils that occurred. He suggested that there must be some structure running across the muscle fiber that was responsible for this. As soon as I saw these transverse membranes, I concluded that they constituted the structure Hill had predicted and that they probably were continuous with the surface membrane and conducted an action potential across the fiber. Of course, this was jumping far ahead of the evidence, but the clues were all there and I was right. I was invited to give a lecture in a summer course in June 1954 that F. 0. Schmitt had organized at MIT shortly FIG. 7. Longitudinal section of lizard muscle fiber showing A and I bands with clear H and Z bands. Note the paired membranes (M) seen between the myofibrils. To the upper right they are cut in cross section and appear tubular. They are located regularly at the A-I junctions. To the left center the plane of section passes between myofibrils and the tubules are seen clearly in the thin layer of sarcoplasm between the myofibrils. The section was stained with phosphotungstic acid and the roughly 40-nm axial periodicity is very clearly shown. The inset to the lower left shows a Z band at higher power. X28,500. From Robertson (149).

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FIG. 8. Longitudinal section similar to Fig. 7 showing the paired membrane structures (M) in register at the A-I junctions between the myofibrils. X75,OOO. From Robertson (149).

after this, and I presented these micrographs and my interpretation of them to the group. Nobody claimed to have thought of this or heard of it before. Shortly after this, I was invited to give a paper at the annual meeting of the International Association of Medical Museums (later the International Academy of Pathology) in 1954 in Houston, I believe. 1 wrote an abstract giving the essential findings presented in the material. Again nobody claimed to have had this idea before. Unfortunately, these proceedings were not published and I have not even been able to find a record of the meeting, so my first two presentations of what seemed to me to be a new way of looking at muscle fibers were not recorded and this proved to be important. I did, however, send off a manuscript describing my finding and interpretations that 'was received by the Journal of Biophysical and Biochemical Cytology on May 9, 1955 (149). This paper originally contained the diagram in Fig. 9. FIG. 9. Diagram from the original copy of the manuscript of reference submitted to J . Biophys. Biochem. Cytol. in May, 1955. The diagram was intended to convey an interpretation of the paired membrane structures arranged regularly across the muscle fibers at the location of the junctions of the A and I bands. The drawing has not been altered and reveals the lack of understanding of membrane compartmentalization at the time. In the text it was said that the membranes sometimes appeared tubular but they were not shown this way in the diagram because the unit membrane structure had not yet been seen and the fundamental compartmentalization provided by the internal membranes of cells was not at that time understood by the author.

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VII. The London Period

A. THE TRANSVERSE MEMBRANES OF MUSCLE In 1955 I accepted the job in London, refusing a substantial salary increase at Kansas and a better position as an assistant professor of pathology at Western Reserve in Alan Moritz’s Department of Pathology. My wife and I by then had a second daughter, Elizabeth Ann, now an assistant professor of English at Colorado, and a son, James David, Jr., now a wood-working artisan in Boston. We liquidated our meager assets and accepted a considerable income reduction to go to London to live on the British salary that went with my appointment as an honorary research associate at University College London. I soon had a laboratory operating in the Anatomy Department headed by J. Z. Young. My office was in Katz’s Department of Biophysics. I bought a new Siemens Elmiskop lb, bringing the third one of these new instruments into Great Britain. The lab was in operation by the end of 1955. I had the micrographs of all the things I had seen in Kansas City with me, of course, when I moved to London and, in January or February 1956, I took some of them to Cambridge when I went there to give a lecture at the invitation of Alan Hodgkin. Andrew Huxley was at the lecture and I remember discussing the pictures of the transverse membranes in lizard muscle with him and Hodgkin after the lecture. Huxley had recently done a classical piece of work with Taylor on frog skeletal muscle fibers (97) showing that if a micropipette were placed on the surface of a muscle fiber just over the Z line and a stimulating current were then passed through its tip, a sharply localized contraction would occur involving only a few myofibrils and only the sarcomeres immediately under the microelectrode tip. He was receptive to the idea that transverse membranes might be responsible for this wave of contraction, but he tried to convince me that the Z line structure itself in my micrographs looked membranous and might be the membranous structure responsible. I did not agree because I did not believe the Z line was a membrane. Some months later, he came by my laboratory in London to tell me that he had been working on an invertebrate muscle with a 10-pm sarcomere length and that it showed contraction when the micropipette was placed on the surface opposite the A-I junctions. He suggested, therefore, that my interpretations on the lizard muscle might be right and the differences between my findings and his might simply represent a species difference; later on this was found to be true. By that time I had received the editorial comments on my paper from the journal. Two anonymous editors gave the manuscript very bad reviews. They said my pictures were overinterpreted and suggested that I remove the diagram of the transverse membranes and tone down the discussion. They were particularly concerned with my terminology regarding the sarcolem-

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ma, stemming from my failure to designate the thinnest dense line next to cytoplasm as the cell membrane and call it the sarcolemma. At that time I had not yet seen the unit-membrane structure and was uncertain exactly where the cell membrane was located. The editors clearly did not like my suggestion that the transverse membranes were responsible for electromotor coordination of contraction across the muscle fiber. I was completely intimidated and, despite receiving a telegram from George Palade soon after I got the reviews in the fall of 1955 saying that I should send the paper on for publication, presumably as it stood, I proceeded to remove the diagram and much of the discussion. The original manuscript contained the following sentence in reference to the transverse membranes as the last sentence in the Discussion: “It seems appropriate to indicate nevertheless that such a membrane system could obviously play an electromotor role in the synchronization of muscular contraction (21).” The reference “21” was to A. V. Hill. In the final version this sentence was eliminated and I put in a reference in the Results section to the work of Huxley and Taylor (97) saying, “. . . which have revived interest in the old idea that some kind of physical element probably exists to convey the influence of surface membrane depolarization across the myofibrils (21, 45).” Evidently I put this in at the gallery proof stage because it is not in the copies I have of the original manuscript. I must have done this after my discussion in Cambridge with Andrew Huxley. This, coupled with removal of the sentence about the electromotor role of the transverse membranes, completely distorted the meaning I originally wished to convey. About all that was left when this paper finally appeared in 1956 (149) were the pictures and the reference to A. V. Hill (94), to indicate that I had understood the findings. Partly as a result of this experience I stopped working on muscle and concentrated on nerve structure. In preparing this paper I was stimulated to reread the literature of the period on this topic. I had at the time read carefully a paper by Bennett and Porter (8) on chicken breast muscle published in 1953 and was certain that I had not got the idea about transverse membrane conduction from it. However, on going over the old literature again I reread some papers by Porter (134), Bennett (9), and Edwards et al. (56) that shook my faith in my memory. These were given at a symposium at Arden House in New York held in January 1956 which I did not attend. In each of these papers the idea of the sarcoplasmic reticulum’s being concerned in transmitting a signal across skeletal muscle fibers was mentioned. Bennett even said “Retzius (12) suggested that the sarcoplasmic reticulum might transmit an excitatory impulse from the sarcolemma to myofibrils deep within the muscle fiber” [p. 172 in (9)]. The paper of Retzius in 1881 (139) and of Veratti in 1902 (218) were said to have given excellent descriptions of what we now call the sarcoplasmic reticulum (SR). A translation of the 1902 Veratti paper was published in the J . Biophys. Biochem. Cyrol. in 1961 (219) and it is perhaps worth quoting the last item in Veratti’s own summary of his paper: “7. There are

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no data on which to base any hypothesis on the functional significance of the reticular apparatus of muscle fiber” [p. 54 in (219)l. Both Bennett and Porter referred in their discussions in 1956 to involvement of the SR in transverse nerve impulse conduction and I thought at first that they must have discussed this point in the 1953 paper and I had simply missed it. However, I then reread this paper carefully and found no hint of this idea. They did quote Retzius but not in this context. I still have not been able to locate the Retzius paper, which I have never seen because the journal is not available to me. I believe that my various presentations on this topic around the country had stimulated people to think about the mechanism. It seems quite likely that what I had said simply got around and when people who had not thought of it before heard the concept it seemed so reasonable that they unconsciously forgot where they got the idea. After all, with hindsight it is rather obvious. By that time my work was 2 years old and it still had not appeared in print. In 1957 a paper by Porter and Palade (135) on skeletal muscle appeared in which the probable transverse conduction function of the transverse membrane system, now referred to as the “T” system (68), was discussed in more detail. My paper was referred to among others that had reported morphological features of muscle fibers related to the structures they called “triads,” but not in the context of function. Incidentally, they also had not demonstrated direct connections of the T system membranes to the surface membrane. It remained for Franzini-Armstrong and Porter (69) in 1964 to demonstrate such connections morphologically in a fish muscle and for Hugh Huxley (98) to show that they are present in frog muscle by an indirect morphological method. In the former case the actual connections were demonstrated in thin sections in a fish muscle and in the latter the transverse tubule system was filled with ferritin molecules in the unfixed state and also after a few minutes of fixation in glutaraldehyde, strongly supporting continuity with the outside world. The only person who has ever credited me with having anything to do with this major step in thinking about how muscle fibers work is Andrew Huxley, who mentioned in his Croonian lecture in 1971 (96) my having shown him my micrographs and discussed their significance during the above period. In fact there is no reason why I should have been given any credit for this idea. I was simply naive in having started talking about it in public without having published it. I have told this story for the benefit of the young and naive who have not yet learned how science evolves. Good original ideas may give personal satisfaction but if they are to be credited to an individual they must be recorded in the literature in a timely fashion. Verbal communications are simply too easily forgotten. Another moral of this story is that a young person must have the strength to stand on convictions and not let editors deflect him or her beyond legitimate editorial matters. Certainly one should not be deflected from working on a topic.

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B. THEMYELINSHEATH In the 1950s I observed and finally published [Fig. VI. 1 1 in (165)] a picture of the kind of structure of the myelin sheath now recognized as least derivative as in the micrograph in Fig. 3, prepared recently using current methods. This micrograph, which is reproduced in Fig. 10, was far ahead of its time but did not receive proper attention from me. At the time I set up my EM laboratory at University College, my chief technician was a young lady named Rose Smith (later Wheeler), who had been making various kinds of silver preparations of nerve fibers for Professor Young. Rose rapidly learned the techniques of EM and after some time, thinking about her proficiency with the classical silver techniques, I suggested that she make one of these preparations for me and embed it in epoxy resin for sectioning and examination with the EM. She did and I found that most of the myelin sheath was destroyed except in a few patches in which it appeared as in Fig. 10. Unfortunately, I did not follow this up with further study. In fact, I had seen another kind of radial repeat at that time that I published immediately but also did not understand (153). This was a picture in which the myelin lamellae were very obliquely sectioned, but I did not realize that at the time. Since I did not understand either of these pictures, I did not try to interpret them. This kind of thing often happened in those days when we were being flooded with so much new information that there just was not time to assimilate it.

C. THEUNITMEMBRANE In 1956 John Luft (1 14) rediscovered KMnO, as a fixative for cytology and almost simultaneously Glauert, Rogers, and Glanert (83, 84) introduced the

FIG. 10. Portion of myelin sheath prepared as described in the text. Note the major dense lines (MDL) and the double intraperiod lines (IPL). Compare with Fig. 3. x555,OOO.

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epoxy resin Araldite as an embedding medium. I tried both of these methods together on peripheral nerve fibers and almost immediately began to see membranes in a new way. In the past, cell membranes had always appeared as in Fig. 6, as a single rather hazy and indefinite dense line. Now they appeared in my micrographs as sharp triple-layered structures about 7.5 nm thick made up of pairs of dense strata each about 2.5 nm wide separated by a light zone about 2.5 nm thick, not too different from the way they appear today in material accepted as superior. They gave the triple-layered appearance shown by the human erythrocyte membrane in Fig. 11 after fixation in glutaraldehyde followed by embed-

FIG. 11. Section of human erythrocyte membrane fixed with glutaraldehyde, embedded in polyglutaraldehyde, and sectioned with a diamond knife. The triple-layered pattern is similar to that shown by membranes fixed with KMnO, during the 1950s. X202,OOO.

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ding in polyglutaraldehyde and staining of sections with uranyl and lead salts. The only essential difference is that the thickness in the KMn0,-fixed material was somewhat less, and more uniform. All the plasma membranes in sciatic nerve fibers gave this triple-layered appearance and I also noted that the membranes of the membranous organelles looked the same way. I then applied the technique to sciatic nerves from newborn and somewhat older rats and soon found out what the structure meant. I saw all the stages in myelination that Geren had observed, but now the triple-layered structure of the membranes gave a much more clear picture. There was a gap of 15 nm or so between the two triplelayered membranes of the axon and the Schwann cell, but there was no such gap between the two apposed Schwann cell membranes making the mesaxon. Here the two triple-layered structures in close contact made a pentalaminar structure about 15 nm in overall thickness. The mesaxons were seen making less than one and up to three or more loops around the axon in a simple spiral. In some places one could see the mesaxon loops in contact along their cytoplasmic surfaces to make two lamellae of compact myelin. Finally, there were many fibers showing several layers of compact myelin with the outer and inner mesaxons related to the myelin just as I had observed them earlier in lizard nerve fibers except that now I could resolve the substructure of the membranes. Figure 12 is one of the original micrographs showing the triple-layered structure of the Schwann cell membranes of the mesaxon and how the outer and inner mesaxons are related to compact myelin. The findings are summarized in Fig. 13, which shows diagrammatically how myelin originates. The important point was that I could now say with certainty that, to the resolution of the electron micrographs (about 2.5-3 .O nm), there was nothing added to the membrane components as compact myelin was formed. The repeat period in the compact myelin of permanganate-fixed nerve fibers was about 16 nm. This was closer to the repeat period of 17.1-18.6 nm measured by X-ray diffraction in fresh intact nerve fibers of amphibians and mammals (190) than the period of about 12 nm that Sjoestrand had observed in Os0,-fixed myelin (198). So I believed that the triple-layered structure was a better representation of the real membrane structure than the fuzzy dense line seen in Os0,-fixed material. Having established these morphological relationships I was now in a position to make a deduction about the molecular structure of the triple-layered structure. As mentioned already, W. J. Schmidt had done a study of myelinated nerve fibers by polarization optical methods in the 1930s (185). He had found that fresh fibers showed radially positive intrinsic birefringence in the myelin sheath but that the positivity reversed to negativity after lipid was extracted. This led him to propose that myelin consisted of layers of lipid molecules in a smectic or nematic fluid crystalline state with the long axes of the lipid molecules oriented in the radial direction alternating with layers of elongated protein molecules with their long axes predominantly oriented tangentially as in Fig. 14. Schmitt et al. (187)

FIG. 12. Section of a developing mammalian sciatic nerve fiber showing a Schwann cell nucleus (nucl.) and a myelin sheath around one axon. The inset enlargement shows the relationships of the unit membranes of the outer mesaxon to the compact myelin sheath as described in the text. The outer end of the mesaxon shows a poorly defined occluding junction, which is frequently seen in this location. x32.000. Inset X 178,000.

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n

unii gap membrane

a

membrane

b

. -

UIllT

(lap

membrane C

FIG. 13. Diagrams of the mechanism of formation of the myelin sheath as described in the text. Abbreviations: m, myelin or mesaxon; mx, mesaxon. Myelin is present after the first junction of the cytoplasmic surfaces of the mesaxon to make one major dense line. The first intraperiod line begins in the mesaxon with the apposition of the two external membrane surfaces.

established the dimensions of the radially repeating unit by X-ray diffraction studies of fresh myelin. Since the repeat period of smectic fluid crystals of the kinds of lipids in myelin repeated at a period of approximately 4 nm, it was clear that the repeating unit of myelin, if it also incorporated protein, could have no more than two bimolecular layers of lipid and only a few monolayers of protein. There were several ways the lipid and protein molecules could be arranged, as in Fig. 15 from Schmitt et al. (190). Brian Finean, a British X-ray diffractionist who was a postdoctoral fellow in F. 0. Schmitt’s laboratory at MIT when I was

FIG. 14. Diagram from W. J. Schmidt (185) showing his conception of the radially repeating layers of lipid (L) and protein (Pr) with the long axes of the lipid molecules radially disposed and those of the protein molecules tangentially placed.

0

m

tllflf

FIG. 15. Four possible conceptions of the radially repeating unit in myelin as defined by Schmitt et al. (190). Protein molecules are illustrated as polypeptide chains and lipid molecules as tuning fork symbols with the polar heads as dots and circles.

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there, had made an educated guess about the most likely repeating structure and published the diagram shown in Fig. 16 (65). It was apparent to me that, if Finean’s diagram were correct, I could deduce the molecular structure of the triple-layered structure of the Schwann cell membrane. This was true because I could put the molecular diagram alongside the EM picture and establish identities between the various layers of the two repeating units, neglecting the small differences between the dimensions. This seemed reasonable because it was easy to account for the slightly smaller dimensions in the EM sections by shrinkage. I did this as indicated in Fig. 17 and concluded that the triple-layered structure of the Schwann cell membrane represented the underlying molecular pattern in Fig. 18. This depicted the core of the membrane as a single bilayer of lipid with the polar heads directed outward and covered by monolayers of protein. It was obvious from the EM that there was a chemical difference between the protein in the outside surface and that on the inside because the reactivity to fixatives and stains were clearly different. This was also apparent from the X-ray diffraction work of Finean since the repeat period would have had to be half the value

FIG. 16. Finean’s conception of the radially repeating unit in myelin. From Finean (65)

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FIG. 17. Diagram showing how the X-ray-derived radially repeating unit could be related to the radially repeating unit seen with the electron microscope so as to derive a molecular model for the unit membrane.

FIG. 18. The original unit membrane model derived as in Fig. 17. The main new ideas were the concept of chemical asymmetry and the restriction of the thickness to one bilayer of lipid with the polar heads pointing outward and each covered with one monolayer of nonlipid. The chemical asymmetry was attributed to the presence of carbohydrate in the outer nonlipid monolayer.

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obtained if there were not a significant chemical difference between the two surfaces of the bilayers. Finean referred to this as the “difference factor.” I made a guess that the “difference factor” was the presence of carbohydrate on the outer surface of the membrane. I indicated this chemical asymmetry in the molecular diagram in later papers by filling in part of the protein layer on one side with ink. I showed the protein as a zig-zag line because I was thinking of the dimensions of the repeating unit. A lipid bilayer is about 5 nm thick and a fully spread denatured protein monolayer is about 1.2- 1.5 nm thick. The repeating unit needed to contain two bilayers and four monolayers of protein according to Finean’s analysis. The overall thickness of the lipid would be about 10 nm. This leaves about 7.0-8.5 nm to be occupied by protein. In my initial analysis I supposed that this protein would be fully spread out like protein in a monolayer at an air-water interface and hence about 1.5 nm thick. This much protein would fit comfortably into the repeating unit with a little room left over for some carbohydrate residues which I supposed would be covalently bound to the outer protein monolayer. Of course, one could suppose that the protein molecules were comprised of ahelices, @-sheets,or random coils and then, depending on the exact thickness of the polypeptide chains, more than one chain could possibly be fitted into two or even all four of the monolayers. Of course if the protein were integral each molecule could be thicker. Today we know that the two principal myelin proteins are the Folch-Lees proteolipid protein (66) and the basic protein (108), both of which seem to be integral although most of the mass of the Folch-Lees protein seems to be in the external surface of the glial cell membrane and most of the mass of the basic protein in the cytoplasmic surface. Together those two proteins make up about 85% of the protein of central nervous system myelin. We still do not know exactly how these proteins are arranged in the myelin membrane and how much they increase the thickness of the bilayer, although Blaurock has some recent evidence from X-ray diffraction studies of myelin (13, 14) bearing on the problem. However, at the time the concept of integral proteins had not been introduced and I was thinking only of how protein monolayers could be fitted into the structure without penetrating the lipid; it seemed likely that the molecules were spread out into layers about one polypeptide chain thick. In any case the diagram in Fig. 18 was meant to represent all this rather crudely (156-161, 165). The important point was that it was possible to propose a general molecular architectural pattern for the triple-layered structure. The triple-layered structure had been found in plasma membranes, mitochondria, Golgi membranes, and endoplasmic reticulum membranes, and it now appeared to be the fundamental repeating unit in the myelin sheath. So I first began to refer to it in 1957 (152, 154) as the “unit membrane” and then used this in several papers over the next few years (155, 156, 161, 163, 165). This somewhat ungrammatical but graphic term caught on and came into widespread use. The general conception of membrane structure that it represented was ad-

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vanced as the “Unit Membrane” theory. It constituted an advance over the older (1935) paradigm of Danielli and Davson (43) called the “pauci-molecular” theory because it added two new features. First, it restricted the lipid core to a single bilayer and second, it introduced the completely new idea of chemical asymmetry. This seemed a fairly satisfying argument, but I was well aware that it was a very flimsy one because it all hinged on the correctness of the X-ray.diffraction analysis done by Finean. We all knew very well that Finean’s analysis was only one of several possibilities because the X-ray phase problem had not been solved and his particular choice of phases was therefore arbitrary. This meant that the repeating units could be any of the ones specified earlier by Schmitt et al. (190) (Fig. 15). I thought that we might get some useful information in relation to this problem by looking at lipid model systems. I had met Dr. M. McFarlane in London and knew that she had some very pure preparations of lipids of the type that occurred in membranes. She kindly gave me some highly purified samples of phosphatidyl choline, phosphatidyl ethanolamine, and some others. I also made some crude ethanol-ether (50:50v/v) extracts of rat brains. I dried down samples from these various preparations and treated the residues like pieces of tissue, fixing them in OsO, or KMnO,, embedding them in epoxy resin and cutting thin sections for study. Right away I saw a layered structure, as in Fig. 19, consisting of dense and light strata repeating periodically at about 4 nm. Betty Geren had done a similar thing earlier with a phospholipid (79) and noted that the repeat period checked out very closely with the repeat that was measured in such systems by X-ray diffraction. So it was quite clear that we were looking at lipid bilayers in a smectic fluid crystalline state having the molecular pattern in cross section shown in Fig. 20a. The problem was that we did not know whether the dense strata represented the lipid polar heads as in Fig. 20b or the carbon chains as in Fig. 20c. Clearly, on symmetry grounds it had to be one or the other. The problem was to identify a single bilayer in isolation and see how the densities were distributed. It was clear that if the nonpolar carbon chains were responsible for the dense strata, a single bilayer should appear in cross section as a single dense stratum; if the polar heads were responsible then each bilayer should appear in transection as a pair of dense strata making a triple-layered structure like a unit membrane but thinner. I shall never forget the feeling of exhaltation I had late one night in London when I first saw what I thought was a single bilayer in isolation. It was triple layered like the unit membrane but only about 6 nm thick. I immediately knew that this effectively solved the X-ray phase problem. This first observation was made on the crude-alcohol-ether brain extract I had

FIG. 19. Appearance of egg lecithin fixed with Os04, embedded in epoxy resin, and sectioned with a glass knife. The repeat period is about 4 nm. X 1,250,000.

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FIG.20. Diagram showing smectic fluid crystal of lipid in (a) assumed to underlie Fig. 19 and the two possible interpretations of the dense and light strata in (b) and (c).

made. This preparation turned out to contain scattered micelles which appeared in cross section as stacks of bilayers of unequal width. Each whole micelle appeared like an isolated spicule tapering at both ends, perhaps a micrometer or so in length and 0.1-0.2 micrometers in thickness in the middle. At high power the middle part was made of a lattice of repeating dense and light strata like the ones in Fig. 19. However, at the tapering ends of the micelles the last one turned out to be a single pair of dense strata with a uniformly wide light zone in the middle making a triple-layered structure like a unit membrane but thinner. It was immediately clear to me that this triple-layered structure was a single bilayer since I never saw one of the dense strata continuing on beyond the tip of the tapering micelle without the other. This meant that the polar heads of the lipid molecules were responsible for the dense strata in Fig. 19. Although it was enough for me, by itself this might not have been completely convincing to someone else; I then went on to do the same thing with the very pure preparations I had from Dr. McFarlane. Here I found areas in which the bilayers of the smectic fluid crystals were hydrated and I could clearly see several individual bilayers, all of which appeared triple layered. One of the original micrographs showing this (165) is reproduced in Fig. 21. Although I had not consciously set out to do the experiment, as soon as I saw these triple-layered structures I knew that in effect I had done the experiment reported by Schmitt et al. in 1941 (190) in which they showed by X-ray diffraction methods that a smectic fluid crystalline system of lipids could be hydrated reversibly, splitting off individual bilayers as in Fig. 22, taken from their paper. This gave me great confidence in my analysis. At this point I believed I was no longer dealing with a theory of

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FIG. 21. Part of a preparation of egg lecithin like that in Fig. 19, but partially hydrated. Note that individual triple-layered structures about 6 nm thick are seen. x 265,000.

membrane molecular organization, but with an established fact. So I began to refer to the “Unit membrane concept” instead of “Theory.” In 1958 I went to the European Regional Electron Microscopy Congress in the Dahlem section of Berlin, where I reported these findings. I met Walther Stoeckenius there and found that he had done a similar study of lipid model systems but had come to a totally different conclusion. As I interpreted his pictures he had observed single bilayers in isolation and had even succeeded in coating them with a thin layer of protein. However, he had concluded that each of these triple-layered structures was two bilayers covered with monolayers of protein. He adopted this interpretation because he believed that the dense strata seen in the smectic fluid crystals must represent the lipid-carbon chains, since this was where the double bonds were known to be concentrated and it was known from the work of von Criegee (223, 224) that OsO, reacted with double bonds. Stoeckenius was basing his interpretation mainly on the chemistry of the reaction of OsO, with bilayers and reached the wrong conclusions; my interpretations were based mainly on morphology, not only of bilayers but of nerve

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PPPPPP

FIG. 22. Molecular interpretation of Fig. 21 based on X-ray diffraction experiments done by Schrnitt et al. (190), from whose paper the diagram is taken.

fibers. We still do not know precisely why the density of the polar heads becomes higher than the carbon chain regions of bilayers, but there are some plausible reasons and there is no doubt at all about the observed fact that this is true. We can rationalize the findings by saying that, while some osmium is deposited in the centers of the bilayers as a result of the Criegee reaction, more is deposited in the polar regions for two reasons. First, OsO, reacts directly with some of the polar groups. Perhaps as importantly, OsO, is a product of the Criegee reaction and is relatively hydrophilic. Hence it tends to be driven out of the hydrophobic core of the bilayer and deposited preferentially in the polar regions. These are rationalizations to explain the facts, but even if they are not correct the facts remain. The polar heads are regularly more dense than the carbon chains in preparations of the kind we were using. Stoeckenius and 1 had an extensive public discussion on the floor at one of the

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plenary sessions at that Congress on this fundamental disagreement about the interpretations of our micrographs of models. I presented my finding in a Symposium at the meeting and my views were finally published in the Proceedings volume that came out in 1960 (157). Stoeckenius published his findings and interpretations in 1959 (207). Some time later Stoeckenius followed up Vittorio Luzatti’s work with Husson on controlling lipid phase changes in model systems (115). These workers in France had found that it was possible to get lipid molecules to orient in hexagonal cylindrical columns in which the lipid polar heads were together with little or no water or, alternatively, with the polar heads surrounding columns of water. Stoeckenius fixed such lipid phases with OsO,, sectioned them, and found that the polar heads were stained but not the carbon chains. He published these findings (208) in tandem with a paper by Luzatti and Husson (1 15) in 1962, and this constituted elegant support of my position in our previous debate. One of the problems that I had to solve as soon as I began to see the triplelayered pattern in cell membranes was the question of the generality of the unit membrane structure. During that period I had become acquainted with J. B. S. Haldane and his wife, Helen Spurway, by occasionally having coffee with them after lunch in the Mixed Commons Room at the College and occasionally a beer in the nearby Marlborough Pub. Haldane was retiring and he and Helen were planning to go to India. There is a rather funny story connected with their departure that is amusing. I should point out that my knowledge of this is secondhand, but I believe it is accurate. I learned one day that Helen was in jail and that the Provost was very concerned about getting her released, but Haldane refused to do anything, saying that she would never forgive him if he did. According to the story, Helen walked up to a policeman outside the Marlborough Pub one evening and proceeded to stomp on the tail of a police dog sitting beside him. The policeman is alleged to have said, “Why did you do that, Madam?” and she replied, “Because I do not like British cops.” So she was arrested and

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FIG. 23. Diagrams of three possible interpretations of the triple-layered unit membrane structure as discussed in text.

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jailed to her considerable satisfaction, because what she really wanted to do was register a protest against the British police, presumably because she thought they were oppressing the Indians. This may not be exact, but it was what I understood at the time. I heard that a few days later somebody paid Helen’s fine and got her released. They went off to India and the next and last time I saw them was at a party in Jerry Lettvin’s apartment in Naples several years later, when Haldane had come to Rome to collect a large prize that the Italians award every few years to a biological scientist. He was in fine form, wearing a white Indian robe and surrounded by a retinue of Indians. Helen was also in excellent shape, dressed in a rather svelte bright red cocktail dress-not at all Indian in style. What brought the Haldanes to mind is the fact that when they left London they had six very large orange cats that they could not take to India with them. They asked the Department of Anatomy to dispose of the cats and they were given to me. I proceeded to anesthetize them one by one and perfuse their systemic circulation with KMnO,, fixing the major organs fairly well. I removed samples of all these, embedded them in Araldite, cut sections, and studied them. I was able to find the triple-layered unit membrane structure in all the cells I examined, both in the plasma membranes and the membranous organelles. On this basis I made the generalization that this structure applied to all biological membranes in whatever location. Other people working in my laboratory immediately began to see the unit membrane pattern in their material. Ruth Bellairs saw it in chick embryonic tissue; Mary Whitear saw it in mouse cornea; George Gray found it in locust nerve tissue. Others about the same time reported the same structure in other vertebrates (12, 201, 240) and protozoa ( 1 16, 179, 180), usually making no molecular interpretation, certainly not like the one I made. On the basis of all this evidence coupled with m y observations on the various tissues in the cats I felt justified in saying that the unit membrane pattern was a general one for cell membranes of all kinds in all animals. So, unbeknownst to the Haldanes, they made another contribution to science through their cats. About this time a curious phenomenon occurred. Somehow the word got around that I thought that all biological membranes were the same and this soon got translated into “identical.” To this day there are people who believe this is what I was saying. This, of course, completely distorts my meaning and misses the point. I was saying that all membranes are constructed on the same architectural principle. Perhaps I did not point out explicitly enough that this architectural principle could include very wide variations in chemical species to account for the obvious diversity of different kinds of membranes, but I thought this was obvious to anyone. Perhaps my use of the word “unit” caused this misunderstanding. In fact, in a paper I wrote in 1961 entitled “The Unit Membrane” (161) I said the following, which I quote verbatum from p. 88: “The attachment of substances such as RNA granules to the unit membrane does not alter the

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general conception nor does wide variation in the molecular species composing the lipid core or the non-lipid monolayers. There is ample room for specificity within this structural pattern.” During this period a group in the Microbiology Department across the street in University College Hospital Medical School (a separate institution for reasons too complicated to review here) was working on influenza virus and I was given a sample of the pure virus to examine with the EM. I fixed some of the viruses in KMnO, and examined sections. To my surprise I found the unit membrane structure at their surfaces. I found this disturbing because I did not know that the virus contained lipid. I asked about this and was told that there was lipid present. I was given some figures and just after I received them I happened to be seated with Hugh Davson at lunch in the faculty dining room. I told him about what I had found and my concern that there might not be lipid in the organisms. However, we looked at the chemical data I had just received and together proceeded to calculate whether or not there was enough lipid. We concluded that there was almost exactly enough lipid to make a bilayer at the surface of each particle. This incident may serve to indicate that I was acquainted with Hugh Davson and we got along well. I did not at first know Jim Danielli, but I met him on the occasion of presenting an invited paper at a symposium run by the Biochemical Society in London in 1958 that was published in 1959 (156) as, I believe, my most quoted paper (173). At this meeting there was a rather large audience and I gave about a 40-minute presentation in which I developed the theme of the unit membrane concept for the first time in complete detail. Jim Danielli was in the audience and at the end of my talk rose to make some lengthy comments. The essence of what he said was that he believed that most of what I was looking at and calling cell membranes represented “soap bubbles.” I recall vividly that his opening statement was to the effect that I had just illustrated to the Society that EM was then about where histochemistry had been 10 years earlier. I attempted to answer his criticisms as politely as possible but I did not regard them as very friendly comments. Later on I got to know Danielli and found him a very nice person whom I grew to like, but on that occasion my reaction was very negative. This experience may perhaps explain the irritation I have sometimes manifested when people refer to the unit membrane model as the “Danielli” model. The least they could do is call it the “Danielli-Davson” model. But even that is not accurate. The point is that the pauci-molecular model could not specify with any confidence that the bilayer was the universal structure, not to mention saying anything about chemical asymmetry. To be sure, Danielli (39-42) and Danielli and Davson (43) had mentioned the bilayer earlier as one of the possible structures, but there was no way that the earlier evidence could in any rigorous way justify putting forward the bilayer as a general structure. This is quite well

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illustrated in their 1935 paper (43), in which the core of the membrane is presented in their diagram as a “lipoid” one of indefinite thickness. There was in fact some evidence for restriction of the lipid to one bilayer in the red blood cell (RBC), but the evidence was equivocal and there was no evidence at all for any generalization beyond the red cell. The work of Gorter and Grendel in 1925 (89) had suggested that the RBC membrane contained a single lipid bilayer and the capacitance measurements of Fricke in 1925 (70, 71) had suggested that the thickness of the RBC membrane was less than 10 nm, in fact much less. The measurements of Waugh and Schmitt in 1940 (228) with an instrument they called the analytical leptoscope could be interpreted as supporting the presence of only one bilayer, but this was not the only possible interpretation and, in any case, this was dealing with only one particular membrane. It should be mentioned here that Schmitt et al. (188, 189) found that there was radially positive intrinsic birefringence in the RBC membrane. This is consistent with the presence of a bilayer but says nothing about the number of bilayers. Some of these various studies could have been interpreted as evidence for a single bilayer in the RBC but certainly not as a general structure. Even such a conclusion for the RBC was doubtful. For instance, the techniques used by Gorter and Grendel were suspect because it was obvious by the 1950s that the method they had used to extract lipids was not adequate to get them all, and the method they had used to calculate the area of the cells was flawed. The capacitance measurements, although correct, were not supportive of any generalization to other membranes because Cole and Curtis (35) published measurements of membrane capacitance that varied from the value of somewhat less than 1 microfarad (kf) per cm2 that Fricke had obtained to as much as 6 mf/cm2 for various other membranes. This meant that either any measurements of thickness based on capacitance measurements alone were suspect or membranes varied greatly in thickness. This ambiguity in capacitance measurements remained a problem until Lord Rothschild found out what was wrong. Capacitance values in membranes are always calculated in terms of membrane area, and if the area figure used is wrong the capacitance value will be wrong. Victor Rothschild showed that in marine eggs there were many infoldings of the surfaces of the eggs that made area calculations based on the superficial surface area of the egg wrong (183). My work (149), referred to above, and that of Porter and Palade (135) and Franzini-Armstrong and Porter (69) later on the transverse tubule systems of muscle gave a similar explanation for the erroneous values of muscle capacitance. The point is that in the late 1950s there was no way that the generality I made about the bilayer could be made with any certainty except as I had done it. The unit membrane concept was the first one to establish the lipid bilayer as the general core structure of all biological membranes. This has stood the test of time, for there is now essentially no debate about any other core structure.

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D. THE CRAYFISH SYNAPSE

During the time I was working out the unit membrane concept I continued to work on the crayfish synapses. In the second journal paper I ever published (147) I included a micrograph of a synaptic process in the median-to-motor synapse that showed an important membrane relationship. This was one of the first thin sections I had obtained in Os0,-fixed material embedded in methacrylate. It showed the pre- and postsynaptic membranes in close apposition measuring about 15 nm in overall thickness. However, it did not show the unit membrane structure and, therefore, I could not interpret it beyond saying that the micrograph showed the pre- and postsynaptic membranes were intact as Cajal predicted and as my first paper had established (144). Looking at the picture today, I wish I had said more. The clear space between the presynaptic axon membrane and the Schwann cell membrane and that between the postsynaptic axon membrane and the Schwann cell membrane were at least twice as thick as the clear space between the pre- and postsynaptic membranes. Here the overall combined thickness of the two membranes was approximately 30 nm. The problem was that the membranes appeared only as single dense strata each less than 10 nm thick. In retrospect I can now say that this was so because only the cytoplasmic halves of the membranes were visible in the micrographs. But this is hindsight. At the time I did not understand this. However, as soon as I looked at these synapses in KMn0,-fixed material I knew how to interpret the micrographs. There was simply no space visible between the membranes. Ed Furshpan and David Potter were, at that time, in Bernhard Katz’s laboratory as postdoctoral fellows working on the crayfish synapses. They succeeded in placing microelectrodes in the pre- and postsynaptic fibers and showed that the median-to-motor synapse operated by a purely electrical mechanism and that the synaptic membrane complex acted as a rectifier (74), since current would flow only from the median giant fiber to the motor fiber. We were all familiar then with a paper by Frankenheuser and Hodgkin (67) and another one by Ritchie and Straub (141) in which it was concluded that currents must flow from the surfaces of axons through clefts between the axon’s surface membrane and the membrane of the satellite Schwann cells. Betty Geren and I had independently done some work in the early 1950s on squid giant nerve fibers that had given some clues about how this might occur. She and I with F. 0. Schmitt had tried to write a paper together on this subject in 1953, but Betty and I could not agree on how to interpret our electron micrographs with respect to the definition of the metatropic sheath of Goethlin (85). We were in general agreement on the more important points about the relation of the Schwann cells to the axon membrane and the fact that mesaxons were present delimiting Schwann cell boundaries. We also agreed that there was a hydrated space 10 nm or more thick between the axon and

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Schwann cell membranes and between the two Schwann cell membranes in the mesaxon. Geren and Schmitt published separately (81) because of Betty’s and my disagreement on the nature of the metatropic sheath, and I didn’t publish on this at all. The concepts in this paper by Geren and Schmitt concerning the nature of the gaps between membranes were used by Frankenheuser and Hodgkin (67) to explain how ionic currents between the axon surface and the outside world were sufficiently retarded to give rise to the effects on the negative after potentials that they observed. This was one of the topics of discussion between Hodgkin, Huxley, and myself after the lecture I gave in Cambridge in 1956, mentioned above. So we were all attuned to the idea that the gap between the axon membrane and the Schwann cell membrane in nerve fibers was probably high in water content and provided a low-resistance pathway for ionic currents to flow. As soon as I applied the KMnO, and epoxy-resin techniques to the crayfish synapses I observed that the gap was closed between the pre- and postsynaptic membranes in the synapse, as in Fig. 24. I then understood the earlier pictures I had published showing a narrowing of the intermembrane cleft. In fact, there was no resolved intermembrane cleft; the membranes were in very intimate contact. As soon as Furshpan and Potter showed that this synapse was electrically conducting, it was natural for all of us to conclude that it might be lack of the lowresistance pathway that led to excitation of the postsynaptic fiber. Ed Furshpan, I recall, illustrated this graphically by drawing a diagram in which he showed the current flowing out of the presynaptic fiber into the axon-Schwann membrane cleft everywhere except at the synapse, where it had nowhere else to go but into the postsynaptic fiber. I am not sure how well I understood the electrical arguments, but I certainly understood that the closure of the intermembrane cleft looked like a very good morphological fingerprint for electrical transmission. Again, my failure to pay attention to publishing things when I should led to my not getting out a proper journal article on this matter in timely fashion. Instead I incorporated the material in a review article I wrote in 1961 (162), mentioned above. I think I had developed a certain complacency in those days because new ideas seemed to come so easily that I thought there was no need to rush them into print and often threw away important material in review articles that should have

FIG. 24. Electron micrograph of postsynaptic process in contact with the median giant fiber in the crayfish giant fiber synapse. The Schwann cell is seen to the right and left and the synaptic process axoplasm contains a mitochondrion and simple and complex vesicles. Note that the axon and Schwann cell membranes are separated by a gap of over 10 nm but that no such gap is seen where the pre- and postsynaptic membranes are in contact (arrowheads in inset). Here the unit membranes are in intimate contact. x69,OOO. Inset X 180,000.

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been published in a regular journal article. Young people in the forefront of rapidly moving fields are often pressed to write review articles and tend to regard material included in them as having been published. If they cannot refrain from doing this, as they should, they ought to at least publish the material again in a suitably referred journal. There may be some value in this but I overdid it. It is relevant at this point to mention a controversy that was going on in the literature between me and Fritiof Sjoestrand because it illustrates graphically the importance of paradigms. Sjoestrand, as mentioned above, got the first good pictures of the myelin sheath and correctly interpreted them (198). He regarded the dense strata in the sheath as representative of protein and the light zones between them as lipid. However, he then made a mistake in interpretation that had continuing and enlarging consequences. In his papers with Rhodin (199) on the kidney and Zetterqvist (201) on intestine it was observed that the intercellular boundaries of epithelial cells consisted of a dense stratum next to the cytoplasm on each side and a light space in between, He and his colleagues drew on his interpretations of myelin to make a molecular interpretation of these facts and considered the dense strata to represent protein and the light interzone to represent lipid. The main problem here was that the dimensions were not comparable to those in myelin. The repeat period in myelin was 12 nm, whereas the overall thickness of the intercellular boundaries was 25-30 nm. Furthermore, and most importantly, the thickness of the intercellular boundary was not constant as was the spacing in myelin. Many observers were seeing intercellular boundaries in this way, but some of us had emphasized that the width of the interzone of such “double membranes,” as we called them then (151), was quite variable and, mainly for this reason, had regarded them as extracellular material lying between the cell membranes. This was the basis of the interpretation that Geren, Schmitt, and I had made on the squid giant fibers. Sjoestrand interpreted this space as a lipid layer analogous to the light zones of the myelin lamellae and this really led him astray when he observed the triple-layered unit membrane structure in some of his Os0,-fixed material, bounding the light intercellular material. Instead of accepting my interpretation that this triple-layered structure included the whole membrane with both its lipid and protein components, he interpreted the triplelayered structure as a single protein monolayer with heavy metal stain absorbed on its two surfaces (199). This matter became particularly important in his work on the myocardium. He first published a preliminary paper with Ebba Andersson in 1954 (200), in which they showed the boundary between two cardiac muscle cells as a structure about 12 nm in overall thickness, but they did not venture any molecular interpretation of this at the time. Later on they continued this work with Maynard Dewey and published with him a complete paper on cardiac muscle (202) in which they observed the unit membrane structure and noted that the gap between two such membranes was sometimes variable and sometimes closed. However, while

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mentioning my interpretations of such triple-layered patterns as including the whole membrane, they preferred to interpret the triple-layered pattern as a single monolayer of protein. They referred to the clear space in between when the triple-layered structures were separated as lipid. I tried to convince Sjoestrand that this interpretation was wrong in several review articles (160, 165) during this period, but he would not accept this point of view (204, 205).

VIII. The Harvard Period A. THE SYNAPTIC DISK(GAPJUNCTION) In 1960 I left University College and took a position at Harvard as an assistant professor of neuropathology in the Department of Neurology and Psychiatry in the Research Laboratory at McLean Hospital (the psychiatric division of Massachusetts General Hospital) in Belmont, Massachusetts. Ed Furshpan and David Potter about the same time joined Steve Kuffler’s group in the Pharmacology Department at Harvard Medical School. Ed and I occasionally saw one another despite our being located on opposite sides of the Charles River and the Harvard campus. One day he told me that he and Furakawa, a postdoctoral fellow from Japan working with him, had found evidence in studying goldfish medullae by microelectrode techniques that there was an electrical synapse on the Mauthner cell (75). He did not know where it was exactly, but I believe he suggested it might be on the lateral dendrite. The lateral dendrite has on it numerous endings of eighth nerve fibers that were described in 1934 by Bartelmez (5) as the distinctive “club” endings. These were illustrated very well in a widely used drawing by Bodian (16, 17), reproduced in Fig. 25. At the time I had two Harvard medical students, David Stage and Tom Bodenheimer, working with me in my new EM laboratory at McLean Hospital. I told them about my conversation with Ed and expressed my belief that the club endings on the lateral dendrite might be electrical endings. If so, I said that we should be able to find the preand postsynaptic unit membranes in close contact in these endings in the kind of relationship found in the crayfish giant synapses. So we proceeded to fix some goldfish brains by perfusion with permanganate and in due course found exactly what we were looking for. This did not produce that rare feeling of exhilaration that I had experienced before with completely new discoveries because I was not at all surprised, but we were all very happy. Figure 26 is a light micrograph of a cross section of the lateral dendrite of the Mauthner cell showing several club endings from our original papers reporting our findings (164, 177). Figure 27 is a low-power electron micrograph of one of the endings. We found that the club endings were characterized by disk-shaped regions about 0.3-0.5 km in diameter scattered over the extensive contact regions of the synaptic endings on the

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FIG.25. Diagram of a Mauthner cell showing the lateral dendrite with many club endings, taken from Bodian (17).

lateral dendrite. In cross section these disks were separated by regions about the same size as the disk in which the pre- and postsynaptic membranes were separated by the usual 10- to 15-nm gap seen in other synapses as in Fig. 27. Furthermore, there were accumulations of synaptic vesicles in the terminal axon, and in material fixed in formaldehyde followed by OsO,, there was some increase in density of the pre- and postsynaptic membranes in these intervening regions (Fig. 27). In the disks, which we began calling “synaptic disks,” we saw some very interesting substructure, first in the permanganate-fixed material but later also in formaldehyde-OsO,-fixed material. Figure 28 shows the original high-power electron micrograph of a synaptic disk in cross section taken from my 1963 paper (164). A frontal view of the membranes of the disk showed an almost crystalline pattern of dense bordered facets spaced about 9 nm apart as in Fig. 29 (upper left inset) (164). The facets were closely packed with dense

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FIG. 26. Light micrograph of cross section of lateral dendrite of a Mauthner cell in goldfish medulla as described in text. X900.

straight boundaries measuring about 2 nm in width. Each facet had a central dense spot about 2 nm in diameter. In cross section there were dense spots repeating at a period of about 9 nm between the two membranes, and in some places there were vague densities running across the two membranes roughly to dimples in the cytoplasmic surfaces that seemed to repeat at about 9 nm as in Fig. 28. We wondered whether or not the transverse densities and the scallops in the cytoplasmic surfaces represented transmembrane channels. However, it seemed easy to account for the transverse densities as image overlap artifacts produced by the lattice that we interpreted as being localized mainly in the external surfaces of the membranes. At the time we did this work there was a lot of speculation about the possibility that some kind of phase change might occur locally in the lipid akin to the ones that Luzatti and Husson (1 15) had described. If so, this might convert the lipid in the bilayer into aggregates of spherical micelles with the polar heads pointing outward, creating aqueous pathways across the bilayer. Sjoestrand (203) described a globular appearance of membranes in sections that seemed compatible with this. Lucy (1 1 1, 1 12) and Lucy and Glauert (1 13) explicitly formalized this concept into a theory about membrane structure, and a lot of other people took this sort of thing seriously. I did not believe this was reasonable because if the bilayer were capable of breaking up into spherical lipid micelles in the manner

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FIG. 27. Low-power electron micrograph of a club ending on a Mauthner cell lateral dendrite. Preparation fixed in formaldehyde-0s0, as described in text. X20,OOO. Insets X 133,000.

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FIG. 28. High-power cross section of a synaptic disk in preparation fixed with KMnO, as described in text. X 1,300,000. From Robertson (164).

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FIG. 29. Oblique section of a club ending showing a synaptic disk in frontal view in the center of the field. This is enlarged to the upper left to show the substructure. Some synaptic vesicles are enlarged to the middle left and an obliquely sectioned synaptic disk (2) is enlarged to the lower left. Sections are KMn04 fixed and epoxy embedded. X42,OOO. Inset: top X 120,000; middle X90,OOO; bottom X 140,000. From Robertson (164).

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postulated, I could not see how membrane transport could be controlled since the hydrophobic barrier provided by the bilayer would be destroyed. But most importantly, any such change, if of any generality, would have to show up in X-ray diffraction studies of membranes and no such evidence had ever been found. Nevertheless, I was worried about some of the images that seemed to show regular repetitive transverse densities crossing the bilayer of the kind that one would expect from such a globular transformation of the lipid. However, I thought the transverse densities could be explained by densities in the membrane surfaces such as we saw in the synaptic disk membranes. At this juncture I made a very bad blunder in interpreting some micrographs I had obtained of retinal rod outer segments. I saw what looked like continuities between the membranes of adjacent lamellae of rod outer segments. I believed these images were real, so I published the results (166). Unfortunately, despite editorial review, the paper was published as I submitted it. The results meant that one of my most firmly held concepts about cell structure would have to be abandoned. This was the general concept that unit membranes were always bounded on one side by cytoplasm and on the other by either the outside world or material that at some time was in continuity with the outside world. My pictures of retinal rod outer segments indicated that the rod membranes were rather like a Moebius strip, having no sidedness. I did not like that, but the pictures seemed so convincing that I went ahead and decided I would just have to rethink all my basic concepts. It turned out that this was not necessary. Just after I published the paper, I showed the pictures to a former student of mine in London, Michael Moody, and he immediately saw what was wrong. He had been studying bacteriophage and analyzing image-overlap artifacts. He realized that what I was seeing was an image-overlap artifact produced at the incisures because the membranes in cross section were slightly out of register. At first I could not believe that such clear-cut images could be produced that way. However, I then set up some model experiments that proved that such effects not only could occur but could give precisely the kinds of pictures I had taken (167, 169). This experience reinforced my belief that the so-called “globular substructure” that we were seeing in membranes simply represented image-overlap artifacts. I still believe this is the best interpretation of the images we saw of transverse densities in the synaptic disks. In 1980, two former students of mine, Guido Zampighi and Joe Corless, and I published a paper (238) presenting experimental evidence and a thorough analysis that I believe strongly supports this point of view about such images in the synaptic disk. This has been a very difficult matter, however, because we know that there really are protein-lined channels that traverse these membranes that one might expect to be visible. I still believe that the apparent visualization of these channels in transverse view in electron micrographs of sections or in negatively stained preparations is not valid. I have elaborated on this point several times in various review articles (174-176) and I shall return to

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this further on because I believe the failure to visualize these channels says something very important about their structure. In 1962, just after we had done our work on the synaptic disk, I was invited to give a lecture at the University of Michigan in Ann Arbor. I presented the work on the synaptic disk as well as a general review of membrane structure which was published quite some time later in a review article (168). Maynard Dewey and Lloyd Barr were in the audience and invited me to visit their lab after the lecture. We had a lively discussion about the synaptic disk structure and they showed me some micrographs they had of close contacts between unit membranes of smooth muscle cells. They thought the contacts were mediating electrical coupling between smooth muscle cells that they were measuring with microelectrodes. So I believed that Dewey and Barr were right even though they had not seen the substructure that we had seen in the synaptic disk. They moved very quickly after we talked and published a paper in Science that appeared in 1962 (52) giving the name “nexus” to the kind of close contact that we had all seen. The full papers from both our labs appeared in 1963 (53, 164, 177), but all the work was done completely independently. B. THE LANTHANUM TRACERTECHNIQUE Shortly after we did the synaptic disk work I received a telephone call from Jerry Lettvin at MIT in which he told me about some work he had been doing with lanthanum ions on the electrical properties of nerve membranes. He said that lanthanum seemed to lock up membranes so that no ions could get through them. He suggested that I try fixing some membranes in lanthanum permanganate. He said he had a postdoctoral fellow from Chile in his lab who could make some of the material, which he incidentally pointed out was explosive. He said he also had another Chilean postdoctoral fellow, Samy Frenk, who would like to participate. I too happened to have a Chilean postdoctoral fellow, Carlos Doggenweiler, and said that I would be glad to participate in this if Carlos wanted to work with Samy Frenk. Carlos was agreeable and so Samy Frenk came along with the lanthanum permanganate and we tried it as a fixative on several tissues. It worked and in addition to beautifully clear-cut unit membranes we got a remarkable staining of the intercellular material in the gaps between membranes (54). Jean Paul Revel and Morris Karnofsky, across the river at the Harvard Medical School quadrangle, subsequently devised a way to introduce lanthanum ions into a glutaraldehyde fixative. They applied their method to a lot of tissues and saw structures like the synaptic disks in many locations such as liver, intestinal epithelium, kidney, etc. I recall that they came out to my lab and showed me their material before they published it in 1967 (140). They had taken advantage of the staining of intercellular membrane gap material and had sur-

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veyed a number of tissues. In many places they had seen close contact between membranes in disk-shaped regions which in frontal view showed the same kind of paracrystalline hexagonal arrays of facets as the ones we had seen in the synaptic disk. We agreed that these structures must be the same as the synaptic disk, but it was clear that the term synaptic disk was not general enough and they did not want to use the term “nexus.” They had seen a small gap about 2 nm wide between the membranes, which had not appeared in our material, and had begun to call the structure a “gap junction.” I did not like the term but they used it in their paper and it caught on and is still used. I objected because I thought the term put the emphasis incorrectly.It was not the gap that was important but the places where there was no gap. However, the structure is now known as the “gap” junction despite an abortive attempt by George Palade and his colleagues (193), with my support, to get people to call the structure a communicating junction, “macula communicans.” In 1965 a paper by Benedetti and Emmelot ( 6 ) appeared in which they described a membrane structure they had isolated from liver that looked in negative-stain preparations very much like our pictures of the synaptic disk. The authors, however, while referring to our papers and those of Dewey and Barr, seemed to regard the structure they found as representative of the kind of globular substructure that Lucy and Glauert (113), Lucy (1 11, 112), and Sjoestrand (203) had discussed. In 1968 these authors published another paper (7) relating all these structures, applying the unfortunate term “tight junction” to them, but this time relating them more directly to the synaptic disk (164, 177) and the junctions described by Revel and Karnofsky (140). In retrospect we all now realize that they were looking at essentially the same structure we had all seen, now called the gap junction.

IX. The Duke Period In 1966 I accepted the chairmanship of the Anatomy Department at Duke University. At that time John Heuser, who had worked in my lab periodically from his undergraduate days at Harvard, was working in the lab and moved to Durham with me for a brief period. He had just finished his M.D. degree training at Harvard and I hoped that he would stay with me in Durham. However, he liked big cities and I could not keep him. He is now a professor of biophysics at Washington University in St. Louis. I should mention also that Nicholas Spitzer and Stephen Waxman also worked in the lab with John. Nick is now a professor of biology at the University of California at San Diego and Stephen is chairman of Neurology at Yale. Nick works with his wife Janet Lamborghini, who was my technician at McLean Hospital and now has a Ph.D. degree in biology and a faculty position.

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A. THEUNIT-MEMBRANE CONCEFTUNDER SIEGE During the late 1960s some efforts were made by various people to break down the unit membrane paradigm. Most of these had to do with placing protein molecules within the bilayer in various ways, like the models proposed by Lenard and Singer (1 lo), Wallach and Zahler (226), Chapman and Wallach (31), and Sjoestrand (203). The most extreme model was that of Benson (10, 1l), who conceived of the membrane simply as a monolayer of globular protein molecules with lipid molecules tucked in here and there at random. There was no way that such a model could be reconciled with the body of facts about membrane structure and so I ignored it. Ed Korn (105) attacked the unit membrane model on various grounds in an article in Science, but it seemed to me that he was not convincing. I replied to many of his arguments in a review article some time later (170). All these models it seemed to me could be easily proven or rejected by Xray diffraction or other experimental techniques, but the essential evidence was simply not at hand to support the proposed models. In 1969 a review paper was written by Walther Stoeckenius and Don Engleman (209) going into the various reasons why the bilayer structure was the correct general one for biological membranes. The review was quite comprehensive and I agreed with its conclusions. However, the term “unit membrane” was downplayed to such an extent that it seemed as if the authors considered it hardly relevant. Throughout the paper the authors repeatedly referred to the bilayer model as the “Danielli” model. In the end it seemed to me that the idea was conveyed that most of the work that I had done to establish the bilayer as a general membrane paradigm had already been done by Danielli before I started. Perhaps the reader will understand from what I have said above about my early interaction with Danielli and my understanding of how the field actually developed that I found this review, despite its many excellent qualities, rather curiously distorted.

B. THEFREEZE-FRACTURE ETCHTECHNIQUE In 1963 Moor and Muehlethaler (120) published a paper giving results of the application of a new technique using an instrument that they had developed (1 19) based on a method first introduced by Steere several years earlier in 1957 (206). Steere had found that he could visualize virus particles that had been frozen in water by putting them in a vacuum chamber on a cold stage, etching away some of the ice, and shadow-casting them with metal, using the metallic shadowcasting technique that had been introduced much earlier by Williams and Wyckoff (233). Moor and Muehlethaler developed an elaborate apparatus that essentially was a standard shadow-casting machine with a vacuum chamber containing a microtome and a cold stage. They were able to mount a piece of frozen tissue

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on the cold stage and keep it frozen while evacuating the chamber. The stage was perfused with liquid nitrogen to keep the specimen frozen and it also contained a heater and a feedback mechanism to alternate heating with cooling so the specimen temperature could be kept constant at any chosen value between room temperature and about - 180°C. They cooled the microtome knife with liquid nitrogen and proceeded to cut away some of the surface of the tissue, discarding the sections shaved off. They then deposited metal on the exposed fresh surface either immediately after making a pass with the microtome knife or after a minute or so while holding the specimen temperature at about - 100°C to permit some sublimation of ice to occur, essentially as Steere had done before. It was soon realized that the knife did not cut the tissue but simply fractured it away. They observed membranes in cross fracture and found that they showed the unit membrane pattern. This pleased me and I did not pursue the matter, partly because I did not have the apparatus and partly because nothing new seemed to have been revealed. However, Daniel Branton, after a period working in the laboratory of Moor and Muehlethaler, developed the techniques in his laboratory at Berkeley and soon, alone or with colleagues, published a series of papers (1822) in which the important concept was advanced that membranes could be fractured along the bilayer into two halves which would not etch away as did the surrounding water table. It was concluded that these were the hydrophobic surfaces of the two lipid monolayers making the central bilayer of the membrane. On these etched membrane inner surfaces rather large particles about 10 nm in diameter were seen and interpreted as protein molecules. This, of course, caught my attention and I immediately built an apparatus that would allow me to do some crude freeze-fracturing in my standard Kinney vacuum evaporator. I soon found that I could produce particles like those that Branton and his colleagues were seeing by doing the procedure in a poor vacuum. If I took the trouble to work at higher vacuum many of the particles disappeared. Mistakenly ignoring the fact that all the particles did not disappear I concluded that the particles represented artifacts produced by deposition of material from the vacuum chamber on the cold-fractured membrane surfaces. I did not believe that the interpretation of these particles as protein molecules was correct. After a number of years we now know that, while some such particles are indeed, as I believed, artifacts not only of deposition of gas molecules but also of plastic deformation, some of them do indeed represent protein molecules disposed across the bilayer. I was wrong about this but there were others who were also skeptical, and it took a long time for the techniques to become advanced enough to be sure. In fact, in the late 1970s we succeeded in showing that one such intramembrane particle 12 nm in diameter in mammalian urinary bladder epithelial cell membranes (175, 178) is truly an artifact probably both of plastic deformation and decoration. Ken Taylor and I (210) have done a three-dimensional reconstruction to less than 2 nm resolution using a computer image analy-

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sis method. Wade and Brisson (225), by a similar method on frozen hydrated material, have produced evidence that the particles represent transmembrane proteins. I believe this is correct, but the large globular particle 12 nm in diameter seen by the freeze-fracture etch method is, nevertheless, an artifact. We are still working on this topic and looking forward to a complete analysis. There is no doubt that some intramembrane particles do indeed represent transverse membrane proteins, but the representation is often far from a direct one. Jonathan Singer, not to my surprise, apparently believed that Branton’s original interpretation was literally correct and used this concept to support the advancement of the fluid mosaic model of membrane structure with Nicholson (195) in 1972 [see also (196)l. At first I resisted this conception because I did not believe there was enough hard evidence for it. There was considerable doubt about the nature of intramembrane particles (49, 17 1) and the biochemical evidence involving labeling proteins in membranes did not seem convincing to me. Bretscher reported on labeling experiments (24-26) that at face value seemed in keeping with the fluid mosaic ideas, but I did not find his papers completely convincing even though later on the work was confirmed (21 1). At this point I found myself in the position that Thomas Kuhn has so clearly discussed in his book “The Structures of Scientific Revolutions” (106). I did not realize exactly what was going on at the time, but I was in fact in the impossible position that Thomas Kuhn described when he said something like woe unto him who dares to defend an old paradigm when the field is ready for a new one. I was naive enough to think that the fluid mosaic model simply represented a refinement of the unit membrane model and that after its details had been discussed and evaluated any of the new features that it emphasized would simply be incorporated into the unit membrane concept. After all, there was nothing new about the idea of fluidity, which had been accepted as a feature of membranes since the work of Chambers and others (29, 30) and was discussed in the 1945 edition of “The Physical Chemistry of Cells and Tissues” by Hoeber (95) that I had used as a text as a graduate student. To be sure, the work of Frye and Edidin (72) and of Cone (37) had put a new emphasis on this feature of membranes and added some new quantitative and graphic data. Even though there had never been any doubt in my mind that membranes were fluid, it seemed as though this idea to some was a great new revelation. Also the idea of patches of protein and lipid in a kind of “fluid mosaic” model had been proposed in the 1930s by Collander and Baerlund (36) and was discussed in detail in Hoeber [pp. 234, 275, 277, in (95)]. It was discounted on surface-tension arguments by Davson and Danielli (44) because it was said that such a membrane would self-destruct, to apply modem jargon, because the surface tension of the lipid part would be so different from that of the protein part that the membrane would be unstable and fall apart. Of course, this kind of fluid mosaic model was not exactly what was

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being proposed. The Singer-Nicholson model proposed that single protein molecules were embedded in isolation in a naked lipid bilayer (195). Under these conditions of course the surface-tension argument would not apply and the overall surface tension would be that of a lipid bilayer. In any case, I completely underestimated the degree to which the scientific community was ready for a change of paradigm. So I just continued to examine each piece of evidence critically and discarded it if I found a flaw in the arguments. The early doublelabeling experiments published my Bretscher (24-26), as mentioned, did not convince me because the interpretations seemed to depend on the assumption that no molecular rearrangement occurred when ghost membranes were produced. I regarded this as a false assumption for some of the reasons I will mention further on. I could see nothing about the Singer arguments that I could regard as more than theoretical possibilities. However, in 1974 a double-labeling experiment was published by Whiteley and Berg (229) that I could not break down, and at that point I accepted publicly the transmembrane protein concept. However, it was now too late. Most people in the field had been persuaded by Singer’s arguments and, since I had opposed them, I think most people simply discarded the whole unit membrane concept and put it out of mind. I was trapped in the change of paradigm and thoroughly discredited. I can look at this dispassionately now and realize how wrong I was. The fact that the Singer model did not change the two main features of the unit membrane model, the ubiquity of the bilayer and the idea of chemical asymmetry, and the fact that several of the main features of the Singer model are incorrect will eventually become clear. But at the moment the pendulum is still swinging very much in the same direction that was started by Singer. In fact, Singer deserves a lot of credit for what he did, because the unit membrane model simply was not adequate to deal with many things that membranes do. The concept of transmembrane proteins is extremely powerful and quite rightly pervades every aspect of modern membrane biology. There are, however, some features of the fluid mosaic model that I believe are exaggerated to an extreme degree and some that are quite wrong. First, the model in its original form was proposed for the erythrocyte membrane. It proposed that the bilayer was mostly naked on both sides. This could not possibly be right because if this were so the membrane would have the mechanochemical properties of a lipid bilayer. La Celle (107) and Evans (57) have independently studied erythrocyte membranes, retinal rod outer segment membranes, and pure lipid bilayers with respect to such properties as elasticity, viscosity, surface tension, etc. and found that there are very significant differences between native membranes and lipid bilayers. Singer has realized this and has proposed that the cytoplasmic surface is to a considerable extent covered with spectrin and the proteins associated with it (27, 124, 192). In fact, he and Nicholson knew about the spectrin association (124) before they published their model in 1972. It is clear that some integral proteins move around in the erythrocyte mem-

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brane, but not all move as freely as rhodopsin in the rad outer segment membrane (37) and some receptor proteins in some membranes may under some conditions be quite restricted (92). Furthermore, it is necessary that the outer surface, at least of the erythrocyte membrane, be more or less completely covered with something that limits the access of phospholipases to the outer monolayer of lipid. This is clearly indicated by the fact that Ottolenghi (125) has found that a highly purified phospholipase A does not attack the phospholipids of intact human erythrocytes at all; it does attack the lipids of erythrocyte ghosts, which supports the point I made above about Bretscher’s findings. Furthermore, Zwaal and Roelofson (241) found that some phospholipases are active or intact erythrocyte membranes and some have little effect, but all are active on erythrocyte ghosts. Others (1, 117, 217) [see (172, 21 l)] have found similar differences in the accessibility of the phospholipids of erythrocyte membranes to phospholipases and the difference is always toward an increase in accessibility in ghosts. I interpret these findings to indicate that the external surface of the erythrocyte membranes is not a naked lipid bilayer. Instead it suggests to me that the lipid head groups are covered with something, protein and/or carbohydrate, that limits the access of the phospholipase until this is disrupted when the membrane is tom open to make ghosts. Perhaps there are some membranes to which this concept applies, but I think it is clear that this concept cannot be a general one. Thus, the fluid mosaic model seems to have some very serious flaws. However, I am not likely at the present time to be heard on this point. Perhaps I can, however, stir some interest in this important point. This brings us to the present day, in which I again find myself out of step with much of the scientific community because I do not see evidence that should be in hand for another important concept. Perhaps I am again wrong, but I can only read the evidence as I see it and so am compelled to make interpretations that are not in line with those of many. This problem concerns the fundamentals of how transmembrane channels are constructed molecularly. Many people seem to regard this as a very easy problem. They take the maximum size of molecule or ion that can go through a transmembrane channel and conclude that there is a hydrophilic protein-lined, sewer-pipe-like structure of that diameter filled with water, that exists as a permanent stable structure in the membrane. I take the position that if this were so, somebody would have been able to get a heavy metal stain into one of the channels and visualize it with the EM. I say this not only for theoretical reasons but because of an experiment that I have frequently done and indeed repeated recently. I often use tobacco mosaic viruses (TMVs) as test objects for various reasons and I routinely do negative staining (23, 90) with either uranyl acetate or phosphotungstic acid to assure myself that I have the right concentration of virus. Tobacco mosaic virus is a protein particle 18 nm in diameter that has a sewer-pipe-like hole down its center that is lined with protein in which is embedded a coiled strand of RNA (121). The RNA strand is not in

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direct contact with the lumen of the hole; it is covered by protein. The lumen of this hole is 4 nm in diameter and it is clearly hydrophilic because it invariably becomes filled with the negative stain and is readily seen without any particular care with microscopy. Even though this channel is complicated by containing an RNA strand embedded in the protein and the hole is larger than the usual postulated transmembrane channel, it is protein lined, and I believe it is analogous enough to make the point that an aqueous pore of molecular dimensions traversing a membrane should be demonstrable. To be sure, there have been claims that such transmembrane pores have been demonstrated by filling them with heavy metal stain, and I dealt with one such claim (86, 87) by showing that what was seen was a simple optical artifact (175, 176). I have never seen any such demonstration that I found acceptable and I have tried very hard to produce such a picture myself. So I am driven to take the position that the nature of transmembrane channels must be different from the popular concept of a simple water-filled pore. Guido Zampighi, now a professor at UCLA, as a graduate student of mine chose as his thesis topic the structure of the gap junction. I have already referred to the paper he and I did with Corless (238) in which we analyzed what could be seen in thin sections or negative-stain preparations of gap junctions in relation to the problem of channels. Guido and I wrote an earlier paper concerning negative staining that seemed to me to be convincing (237). This reported that isolated liver gap junctions tend to break up into fragments when dialyzed against EDTA. Some of the fragments contained only a small number of the repeating subunits of the junction and some of these were seen edge-on in negative-stain preparations. The negative stain did not penetrate the channels. It became clear as Guido’s work progressed that he needed to do the kind of analysis on isolated purified gap junctions that Nigel Unwin and Richard Henderson (93,2 15) had done on the purple membrane of Halobacterium halobium. We were not at that time equipped to do this and so I suggested that he go to the MRC Laboratory in Cambridge and do the analysis with Unwin. Unwin was agreeable to this and they proceeded to do a minimal-dose computer analysis of the gap junction structure in negative stain to a resolution of about 1.8 nm (216, 236). They got some interesting evidence about the arrangement of the transverse protein in the bilayer that gave them a very nice idea about how a channel might be opened and closed. They found there were six protein subunits making up each channel unit and that the subunits were slightly tilted. Their evidence suggested that a slight twist, occurring simultaneously in all six subunits, might open or close the channel (216), as indicated by their model that is reproduced in Fig. 30. I think this is a likely mechanism and I agree in general with their conclusions. There is just one point of emphasis that I would put differently. They found no evidence that the negative stain entered the channel. Figure 31, taken from their work (216), is a composite of two forms of one membrane of the

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FIG. 30. Diagram from cover of Nature as described in text.

FIG. 31.

Diagram from Unwin and Zampighi as described in text

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junction showing the electron density distribution across the bilayer (arrowheads) including one subunit. Note that the negative stain (stippling) is concentrated in both forms in pools in indentations in the outer surface of the membrane (E) but it does not extend across the bilayer. In their papers they attribute this fact to their not having high enough resolution to detect the stain if it were there. To me the important point is that stain is not found at a resolution of 1.8 nm. If one bears in mind that, at a resolution of 1.8 nm, structures much smaller than this can be detected, it becomes clear that there may in fact be no stain there to detect. Unwin et a/. have proceeded with a similar analysis of isolated junctions embedded in ice (214). This has already revealed many fascinating facts about the disposition of the connexon subunits across the bilayer in forms that are presumably open and closed (216). However, the exact nature of the channel in the hydrophobic part of the bilayer has not yet been clarified. The channel clearly consists of some kind of protein structure defining a pathway that can, when open, permit free flow of ions and small molecules. In this state it behaves as though it were a cylindrical structure 15 8, or more in diameter lined with hydrophilic amino acids. But if it were like this when prepared for observation by EM methods, it should be easily demonstrable with heavy metals, but this has never been accomplished convincingly. One plausible explanation is that the structure is in a closed state when prepared, behaving as though the channel were only a potential opening lined by hydrophobic amino acids. This would explain the failure of the EM to reveal it. This implies a structure that can change its properties greatly during preparation as the result of subtle molecular alterations. Such a structure is plausible. In Fig. 31 the central pool of negative stain between the external surfaces of the membranes penetrates the bilayer appreciably, depending on its exact definition. Thus, the hydrophobic region is less than the thickness of the bilayer. The water-free region in the middle of bilayers has been estimated from NMR data to be only about 26 8, thick. Thus, the hydrophobic part of the channel might be only -26 8, long. One kind of plausible channel structure is a P-pleated sheet rolled into a cylindrical (3barrel. This could conceivably be made of the six connexon proteins of the channel. The spacing between amino acid residues in a P-pleated sheet is 3.5 A. Thus, a sliding change in the relative positions of <8 amino acid residues in each of the six polypeptide chains of the six connexons could change local net hydrophobicity to hydrophilicity. This mechanism is quite compatible with the one postulated by Unwin and Zampighi. If the closed state is the lowest energy state, the channel would become hydrophobic spontaneously when removed from the living cell. This general kind of structure would be compatible with all the data and it might be called an “amphipathic” channel. Such a channel, delicately balanced between net hydrophilicity and hydrophobicity, would have a definite biological advantage by providing a useful safety factor. It will be some time yet before enough channel structures have been worked out to replace such speculations with facts.

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In the 1950s the unit membrane structure was best seen in material fixed with KMnO,, but today it is easily seen in glutaraldehyde-OsO,-fixed material that is block-stained with uranyl acetate before embedding. A few years ago I used one of the polyglutaraldehyde embedding methods introduced independently by Pease and Peterson (13 1, 132) and by Heckman and Barmett (9 1) to show up the triple-layered structure in erythrocyte membranes as in Fig. 11. This preparation was fixed in glutaraldehyde and embedded in polyglutaraldehyde by the GACH method (91). Sections were cut and stained with uranyl and lead salts. The membrane is somewhat thicker (approximately 10-12 nm) because the lipid is largely retained and there is a tendency for the two halves of the bilayer to separate during sectioning. However, this technique gives a very clear representation of the triple-layered structure. The fact that the same triple-layered structure is demonstrable this way as seen earlier using KMnO, or OsO, makes arguments against the unit membrane concept such as some put forward by Chapman and Wallach (31), seem irrelevant. Recently I have put together all of my thoughts about membrane structure up to the present time in a model that I have diagrammed in Fig. 32. This model attempts to show all the essential points about membrane structure that seem, on current evidence, to have general significance. It shows the bilayer core of the membrane with the asymmetry of the lipids indicated by showing the polar heads of the inner monolayer as filled circles. This is meant to indicate that the amino lipids, phosphatidyl ethanolamine, and phosphatidyl serine are located in the inner monolayer ( 15, 88, 117, 138, 181, 182, 222). The polar heads are left open in the outer monolayer to indicate that the sphingo- and choline-containing lipids, phosphatidyl choline and sphingomyelin, are confined to the outer monolayer. Cholesterol is shown in the diagram confined to the outer monolayer (28). The fact that carbon chains in the outer monolayer tend to be longer and less unsaturated than those in the inner monolayer is not indicated. A few glycolipids are shown in the outer monolayer. Some transmembrane proteins are shown with the polypeptide chains in the core of the bilayer diagrammed as relatively hydrophobic a-helices. Sugar residues are shown on some of the external proteins. The polar lipid surfaces on both sides are shown as completely covered with either protein or sugar residues. I conceive of this model as possessing fluidity of two kinds. I believe many of the lipid molecules are free to rotate and translate although generally not to flip-flop from one side to the other. Also I believe some of them are bound to protein molecules in the surfaces mainly by head-group interaction (175). I see the proteins as having varying degrees of mobility. Some are relatively free to move about, but some are anchored most likely by attachments on the cytoplasmic side to cytoskeletal elements and on the outside to extracellular proteins such as fibronectin (64, 32, 33, 133, 234). The model possesses fluidity but it is a controlled and regulated fluidity. To emphasize the feature of this model that I consider to be its most important one, I call this the

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FIG. 32. Diagram of cross section of present-day unit membrane sometimes called the hydrophobic barrier model. See text for details.

hydrophobic barrier model. I do not expect this to be adopted as a new paradigm. The field is not yet ready for this and, after all, it is not all that different from the old unit membrane model.

X. Conclusion I could go on with descriptions of the work in my laboratory with Jakoi and Zampighi (100- 102) involving discovery of a new intrinsic receptor protein in intestinal epithelial cell membranes of neonatal rat that we call “ligatin” and its occurence in developing chick retinal cells (99) and synaptosome membranes (78), work on the crystalline membranes of urothelial cell membranes with Vergara and others (104, 178, 221), and work on the crystalline membranes of the lens of the eye with Costello, MacIntosh, Zampighi, and Simon referred to above. I could include the recent work with my colleagues Beatrice Anner and Hie Ping Ting-Beall on Na K ATPase (2, 3, 212) and work of Ping Ting-Beall and of Taylor et al. on Ca+ATPase (55, 213), as well as many other exciting things going on in my laboratory, but I think I have covered the most important incidents of my earlier career. Presently, I am starting a new project on the morphological and biochemical basis of learning and memory in octopus jointly with J . Z. Young that represents a return to my early interest in the nervous +

+

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system. I hope I can write another review in the future that will describe some exciting discoveries in this field, but again I expect to be right only part of the time.

ACKNOWLEDGMENT The author wishes to acknowledge support provided by a gift from RJR Nabisco, Inc.

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E. Lux, V. T. Marchei, and C. F. Fox, eds.), pp. 515-521. Liss, New York. 2. Anner, B. M., Robertson, J. D., and Ting-Beall, H. P. (1984). Biochim. Biophys. Acta 773, 253-261. 3. Anner, B. M., Ting-Beall, H. P., and Robertson, J. D. (1984). Biochim. Biophys. Acta 773, 262-270. 4. Arvanitaki, A. (1942). J . Neurophysiol. 5, 89-108. 5. Bartelmez, G. W. (1915). J. Comp. Neurol. 25, 128. 6. Benedetti, E. L., and Emmelot, P. (1965). J. Cell Biol. 26, 299-304. 7. Benedetti, B. L., and Emmelot, P. (1968). J . Cell Biol. 38, 15-24. 8. Bennett, H. S., and Porter, K. R. (1953). Am. J. Anat. 93, 61-105. 9. Bennett, H. S. (1956). J. Biophys. Biochem. Cytol. 2(Suppl.), 171-173. 10. Benson, A. A. (1964). Annu. Rev. Plant Physiol. 15, 1-16. 11. Benson, A. A. (1966). J. Am. Oil Chem. SOC. 43, 265-270. 12. Birbeck, M. S. E., and Mercer, E. H. (1957). J. Biophys. Biochem. Cytol. 3, 223-230. 13. Blaurock, A. E. (1980). Brain Res. 210, 383-387. 14. Blaurock, A. E. (1986). In “Progress in Protein-lipid Interactions 2” (Watts and De pont, eds.), pp. 1-43. 15. Bloj, B., and Zilversmit, D. B. (1976). Biochemistry 15, 1277-1283. 16. Bodian, D. (1937). J . Comp. Neurol. 68, 117-159. 17. Bodian, D. (1952). Cold Spring Harbour Symp. Quant. Biol. 17, 1. 18. Branton, D. (1966). Proc. Narl. Acad. Sci. U.S.A. 55, 1048-1056. 19. Branton, D. (1967). Exp. Cell Res. 45, 703-707. 20. Branton, D., and Park, R. B. (1967). J . Ultrastruct. Res. 19, 288-303. 21. Branton, D. (1971). Philos. Trans. R . SOC. London Ser. B. 261, 133-138. 22. Branton, D., and Deamer, D. W. (1972). In “Protoplasmatologia” (M. Alfert, H. Baner, W. Sandritter et a / . , eds.), pp. 1-70. Springer-Verlag, Berlin and New York. 23. Brenner, S., and Home, R. W. (1959). Biochim. Biophys. Acta 34, 103-110. 24. Bretscher, M. S. (1971). Nature (London) 231, 229-232. 25. Bretscher, M. S. (1972). J. Mol. Biol. 71, 523-528. 26. Bretscher, M. S. (1973). Science 181, 622-629. 27. Byers, T. J., and Branton, D. (1985). Proc. Nail. Acad. Sci. U.S.A. 82, 6153-6157. 28. Caspar, D. L. D., and Kirschner, D. A. (1971). Nature (London) New Biol. 231, 46-52. 29. Chambers, R., and Kopac, M. J. (1937). J. Cell. Comp. Physiol. 9, 331-345. 30. Chambers, R., and Pollack, H. (1927). J . Gen. Physiol. 10, 739-755. 31. Chapman, D., and Wallach, D. F. H. (1968). In “Biological Membranes” (D. Chapman, ed.), pp. 125-202. Academic Press, New York.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 100

Ionized Groups on the Cell Surface: Their Cytochemical Detection and Related Cell Function SATIMARU SENO Division of Pathology, Shigei Medical Research Institute, Research Center for Renal Diseases, 2117 Yamada, Okayama, Japan

I. Introduction The cell membrane with a cell coat is a biologically important barrier separating the matrix of living cytoplasm from the nonliving environment which makes the life of cells possible. All the somatic cells of multicellular organisms live in the tissue fluid or blood plasma, i.e., an aqueous solution of various inorganic and organic molecules, whose pH is kept stable at near 7. The cells maintain their own metabolism which is independent of the extracellular aqueous environment by means of the hydrophobic cell membrane; it has a polysaccharide coat decorated with numerous hydrophilic groups through which the cells take up nutrients from the aqueous environment and excrete metabolites. Many receptors are found on the cell surface taking up specific molecules that cells need. It is well known that polysaccharides of the cell coat are acidic, having numerous anionic groups, mainly glycoproteins (Cook and Stoddart, 1973; Murphy et al., 1983); however, some cells have ionized cationic groups on their surface (Seno et al., 1983a). Therefore, many ionized molecules in the environment should interact nonspecifically with ionized groups on the cell surface by forming ionic bonds, but their physiological functions have only recently attracted the attention of biologists, mainly in relation to the permselectivity of endothelial cells of vessels (Michael et al., 1970; Luft, 1971; Nicolson, 1973; Caulfield and Farquhar, 1976, 1978; Brenner et al., 1977, 1978; Kelley and Cavallo, 1978, 1980; Rennke et al., 1978; Caulfield, 1979; Kreisberg et af., 1979; Kanwar et al., 1980; Reeves et al., 1980; Melnick et al., 1981; Olson et al., 1981; Rosenzweig and Kanwar, 1982; Simionescu et al., 1982; Vogt et al., 1982; Dermietzel et al., 1983; Nagy et al., 1983; Schneeberger et al., 1983; Seno et al., 1983a-c; Sibley et al., 1983; Ukita et al., 1983; Weening and Rennke, 1983; Bulger and Dobyan, 1983; Lawrence and Brewer, 1984; Rollason and Brewer, 1984; Tsujii et al., 1984a,b). The average bond energy of ionic bonds in aqueous solution is about 5 kcal/mol. Thus, their bond energy is so small that the bond will be broken down easily at 37°C (average thermal energy at 25°C is about 0.6 kcallmol), and the 203

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ionic binding seems unimportant in the biological reactions in tissues (Watson, 1977). But a fairly strong binding can be expected between the anionic macromolecules on the cell surface and the cationic macromolecules brought into the environment. For example, if 10 ionic bonds are formed between two molecules, the binding force is calculated to be about 50 kcal/mol, which is comparable to the bond energy of a covalent bond. This will mean irreversible neutralization or inactivation of ionized groups on the cell surface, even partially. Such a loss of electric charge on the surface may seriously affect cell function, e.g., the increased vascular permeability by cationic proteins released from neutrophil lysosomes (Ranadive and Cochrane, 1968, 1970, 1971; Ryan and Majno, 1977). In the following sections I will describe (1) the polyanionic characteristics of cell surface and interstitial tissues, (2) the cytochemical and histochemical detection of ionized groups by cationic and anionic dyes, (3) the reaction between polyanionic and polycationic macromolecules, (4) the ionization of ionic groups on macromolecules estimated by the colloid titration method, (5) cytochemical and histochemical detection of ionized groups on the cell surface by colloidal probes, (6) the effect of aldehyde fixation on ionized groups of cells and tissues, and (7) the reaction of cells to polyions.

11. Ionic Groups on Eukaryotic Cell Surfaces and Interstitial Tissues A, CELLMEMBRANE AND CELLCOAT To better understand the reactions among ionized groups on macromolecules in living tissue, I will give a brief description of the ionized groups on cell surface and interstitial tissues. It has been well established that the essential building blocks of the cell membrane are lipids arranged in bilayers with their polar groups on surface (Robertson, 1965; Danielli, 1975; De Robertis and De Robertis, 1981; Alberts et al., 1983). The Singer-Nicolson fluid mosaic model (Singer and Nicolson, 1972; Singer, 1975), in which hydrophobic protein molecules float in a lipid membrane like icebergs (protein) in the sea (lipid) (Singer, 1975), is well documented and generally accepted (Glick and Flowers, 1978; Edidin and Van Voris, 1983), though the mobility may depend largely on lipid composition of cellular membranes (Yechiel et al., 1985). 1. Ionic Groups of Lipids Most of the lipids composing cytoplasmic membranes are phosphoglycerides (Chapman, 1975; Stryer, 1981). They have two different fatty acids, which vary in carbohydrate chain length and degree of saturation on the first and second glycerol carbons and a phosphate group on the third glycerol carbon. In some

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phosphoglycerides phosphate groups may be free or conjugated with glycerols, glycerolphosphate, or inositol. Accordingly, they have one or two phosphate groups and have net negative charges. But the main components of membrane lipids are phosphatidylcholine (lecithin), phosphatidylethanol amine, phosphatidylmonomethylethanol amine, or phosphatidyldimethylethanol amine, in which the phosphate groups of phosphatidates become esterified to the hydroxyl groups of the alcohols. Therefore, they have cationic groups in the alcohol moieties bound to phosphate groups and are dipolar zwitterions having no electric charges or slightly negative charges at around pH 7. Other minor components are phosphatidylserine and phosphatidylthreonine in which the phosphate groups of phosphatidates are esterified to the hydroxyl groups of amino acids. They have net negative charges as their amino acids are zwitterions. Another important group is sphingomyelin. Sphingomyelins contain phosphorylethanol amine or phosphorylcholine as their polar head groups being esterified to the C- 1 hydroxyl group of ceramide. Sphingomyelins have physical properties very similar to those of phosphatidylethanol amine, and phosphatidylcholine. They are zwitterions at pH 7. They have no electric charges or slightly negative charges as with phosphatidylcholine. Great differences were found among the fatty acids of lipids in different tissues, but the changes found in ionizing groups were not so marked. Therefore, the lipids composing cell membrane do not contribute to the surface charge of the somatic cells. 2. Ionic Groups of Glycolipids Glycolipids constitute about 5% of lipid molecules in the outer monolayer of the cytoplasmic membrane, though they vary markedly among species and also tissues. Among the most widely distributed neutral glycolipids, the plasma membranes have gangliosides (about 6% or less), which are glycosphingolipids having polar oligosaccharide heads and carrying a net negative charge with one or more sialic acid residues (Glick and Flowers, 1978; Hakomori, 1981, 1984; Alberts et al., 1983; Kaufer and Hakomori, 1983). In brain and nerve tissues the sulfate-containing glycosphingolipids carrying a net negative charge, sulfatides, are found (Kaufer and Hakomori, 1983). 3. Ionic Groups of Glycoproteins The most conspicuous carriers of negative charges on the cell surface are the membrane proteins (Cook and Stoddart, 1973; Murphy ef al., 1983). They are glycoproteins having a number of anionic groups at the end of the oligosaccharides attached to the linear polypeptide chains, which elongate from the nonpolar helix floating in the lipid layer (Glick and Flowers, 1978). Many of them contain receptor sites for plant lectins, viruses, and blood groups (NicOlson, 1974).

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The glycophorin, a specific glycoprotein of the erythrocyte membrane, may serve as a model for the characterization of membrane glycoproteins (Nicolson, 1973; Click and Flowers, 1978; Alberts et al., 1983), as the sequence of its 131 amino acids has been completely determined. The polypeptide chain has nine basic groups, including the terminal amino group, and six carboxyl groups. Therefore, the polypeptide core itself is slightly basic but the cationic groups should be uncharged by the neighboring carboxyl groups of the peptide and attached oligosaccharides. The linear hydrophilic polypeptide chain carries about 100 sugar residues on 16 oligosaccharide side chains. The great majority of the total surface carbohydrates are sialic acid and includes more than 90% of all the acid polysaccharides. Therefore, most of the negative charges of the red cell surface are of the carboxyl groups of glycophorin; some are of Band 111 protein and others (Click and Flowers, 1978). As a result the proteoglycans have a net negative charge as a whole by an excess of anionic groups. No sulfate-containing glycolipids have yet been found on the red cell surface (Click and Flowers, 1978). In somatic cells, however, hyaluronic acid, chondroitin 4-sulfate (chondroitin A), and chondroitin 6-sulfate (chondroitin C) are major structural components of cell coats (Lehninger, 1975), and heparan sulfate and other acid mucopolysaccharides have also been found in membrane preparations from normal and malignant cells (Kraemer, 1971; Satoh et al., 1973; Kraemer and Smith, 1974; Roblin et al., 1975; Underhill and Keller, 1975; Click and Flowers, 1978; Oldberg et al., 1979, 1982; Kjellen et al., 1981; Norling et al., 1981; Oohira et aE., 1983; Yanagishita and Hascall, 1983; Rapraeger and Bernfield, 1983, 1985; Iozzo, 1984; Hook, 1984; Jalkanen et al., 1985; Koda et al., 1985; David and Van den Berghe, 1985; Woods et al., 1985; Kim et al., 1985; Rapraeger et al., 1985). Thus, charged groups on the cell surface are carboxyl groups of hyaluronic acid and sialic acid, and sulfate groups of chondroitin sulfate, heparan sulfate, and other sulfated glycosaminoglycans. All of them give net negative charges to the cell surface.

B. IONICCROUPS IN INTERSTITIAL TISSUES The interstitial tissues surrounding cells are also rich in proteoglycans. Almost all of them are synthesized by their parent cells and their polypeptide cores have covalently linked polysaccharides: hyaluronic acid, chondroitin sulfate, keratan sulfate, heparin, dermatan sulfate, and heparan sulfate (RodCn, 1980; Linker et al., 1981; Vasan and Tesoriero, 1985). The collagen networks composing the skeletal structure of interstitial tissues are intermingled with these proteoglycans giving them a net negative charge (Johansson, 1983). As are those found on the cell surface, all the amino groups of the tissue proteoglycans are uncharged by acetylation or sulfated as those of heparin and heparan sulfate. The most conspic-

IONIZED GROUPS ON THE CELL SURFACE

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uous difference between glycoproteins of the cell coat and interstitial tissues is that the latter are attached to the hyaluronic acid core (Hardingham and Muir, 1972, 1974) by the aid of nonpolar linked proteins (Hardingham, 1979). Accordingly, they form large aggregates of polyanions decorated with numerous sulfate groups on their surface. The function of these polyanions in the intercellular environment may be seriously impaired by polycations released by inflammatory cells (Ranadive and Cochrae, 1968, 1971; Ryan and Majno, 1977; Camussi et al., 1982; Cotran and Rennke, 1983) or by foreign basic macromolecules forming polyion complexes.

111. Cytochemical and Histochemical Detection

of Ionized Groups by Ionic Dyes A. INFORMATION OBTAINED BY STAINING WITH IONICDYES FOR LIGHTMICROSCOPY It has long been believed by morphologists that basic and acidic dyes stain the acidic and basic components in tissues, respectively. Some dyes were used for the detection of basic and acidic macromolecules in tissues, for example, Alcian blue for acidic mucopolysaccharides (Mowry, 1963; Btlanger, 1963), methyl green for DNA (Kurnick, 1950), and pyronin for RNA (Kumick, 1955; Sano, 1977). Eosin stains zinc-containing basic protein of eosinophils (Wintrobe, 1974). Therefore, staining of tissues with suitable cationic and anionic dyes at optimal pH will give us some information on the site and quantity of ionized groups in tissues, although some nonspecific staining may occur. Ehrlich was the first to recognize the existence of two different compartments in a cell, one having affinity to basic dyes and the other to acidic dyes. According to von BoroviczCny (1974) young Paul Ehrlich, when he was a student at the Freiburg Medical School, often visited his uncle Karl Weigert, professor of anatomy at the Frankfurt Medical School, and a pioneer in tissue staining with anilin dyes. One day he had to wait a while in Weigert’s laboratory. He happened to look into Weigert’s microscope on which a blue- and red-stained microscope slide had just been set. Although he had no histologic knowledge yet, he was deeply impressed by the fact that some parts of the cell were stained red and others were stained blue. Looking at this, young Ehrlich realized that some parts of cells had an affinity to acidic red dyes and others had an affinity to basic blue dyes. Therefore, each part can be distinguished from the other by the two different kinds of dyes. This was the starting point for the development of Ehrlich’s Affinitatslehre or affinity theory in immunology and our present knowledge on ionic bondings. In 1877, when Ehrlich was 23 years old and a medical student at Freiburg, he

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published his first paper: “Beitrage zur Kenntnis der Anilinfarbungen und ihre Venvendung in der microscopischen Technik” (Contribution to knowledge of anilin staining and its application in the microscopic technique) (Ehrlich, 1877). However, the binding is not stable because the dyes are small molecules having one group or a few ionized groups in a molecule. Therefore, only weak binding can be expected between the dye molecules and target polyionic tissue components of opposite charge. For example, toluidine blue chloride, with a molecular weight of 29 1, has one dimethyl amino group on one benzol nucleus and two amino groups on another one, but its cationic weight is 256, showing the dimethyl amino group is only the ionized one (Gurr, 1971). Accordingly, the binding is very weak and the dye staining tissue sections may be removed easily by washing. As is well known, however, the acid polysaccharides can be stained beautifully by toluidine blue with metachromasia (Pearce, 1968). This means that some other weak bondings such as hydrogen bonds between nitrogen atom of dye and oxygen atom of polysaccharides and other van der Waals bonds join forces with ionic bonds, i.e., the binding is not purely ionic. This should be the principle of routine tissue staining with basic hematoxylin and acidic eosin or blood smear staining with Giemsa, a basic methylene blueAzur I1 acidic eosin mixture. Therefore, these basic and acidic dyes stain some acidic and basic macromolecular components of cells, respectively, but it may be accompanied by nonspecific staining or fading with washing. Cationic Alcian blue 8GS, a copper-phthalocyanine compound, has been widely used for staining acid polysaccharides (Mowry, 1963; Scott and Dorling, 1965; Pearce, 1968). The dye was first introduced in 1950 by Steadman as an empirical stain for mucin (Steadman, 1950). Later the staining method was improved by Mowry (Mowry, 1963) and his method is now used widely. He stressed that Alcian blue stains acid polysaccharides specifically without staining RNA when the cells are stained at pH 2.6 in 30% acetic acid. But the dye can be removed easily by washing with water. DYESFOR ELECTRON MICROSCOPY B. USE OF CATIONIC Alcian blue 8GX has also been used for the detection of anionic sites by electron microscopy (EM) (Behnke and Zelander, 1970; Michael et al., 1970; Schofield et al., 1975; Caulfield, 1979; Reale er al., 1983; Rollason and Brewer, 1984; Van Kuppevelt et al., 1984). The dye was used by mixing with fixatives, e.g., a 0.5% solution of Alcian blue 8GX in Karnovsky’s aldehyde fixative (Caulfield, 1979), and the organs were perfused with the dye-fixative solution. But the dye may stain some’ nonanionic polysaccharides. Our observation showed that the dye-glutaraldehyde (GA) complexes had an affinity for the cationic ion exchange resin, DEAE- and QAE-Sephadex, at pH 7 (Akita and Seno, unpublished data). Therefore, the staining of tissues with this dye at around pH 7 is not recommended.

IONIZED GROUPS ON THE CELL SURFACE

209

Ruthenium red is also used for the staining of anionic sites for EM (Luft, 1971; Latta et al., 1975; Caulfield, 1979; Kanwar and Farquhar, 1979a; Reeves et a!. , 1980; Chien et al., 1982; Reale et al., 1983; Baldwin and Winlove, 1984; Rollason and Brewer, 1984; Van Kuppevelt et al., 1984), generally by perfusing with fixatives as in the case of Alcian blue, e.g., a 0.2% solution of ruthenium red in Karnovsky’s aldehyde fixative (Luft, 1971; Reale et a l . , 1983; Van Kuppevelt et al., 1984) or an osmic, acid-containing ruthenium red solution (Latta et al., 1975; Baldwin and Winlove, 1984). As ruthenium red and its GA complex show distinct affinity for the anionic Sephadex particles but not for cationic ones at pH 7 (Akita and Seno, unpublished data), ruthenium red seems to be a more reliable probe than Alcian blue in staining the ionized groups in tissues by perfusion. Both Alcian blue- and ruthenium red-fixative staining reveals the distinct fibrillar structure of the basement membrane under the EM. It seems to reveal the detailed structure of polyanionic fibrils, but the EM pictures show some rough structures. Recently, Reale et al. (1985) used safranin 0 in place of Alcian blue and ruthenium red and succeeded in revealing fine fibrous structures. This small molecular dye does not seem to induce gross aggregation of the polyanions in tissues. However, when GA is mixed with these cationic dyes, GA will bind to amino groups of the dyes with its aldehyde group or groups forming a GA-dye complex, e.g., Alcian blue forms a gross precipitate by mixing with GA. In the excess GA, which is needed for tissue fixation by perfusion, there may be some free aldehyde groups of GA, whose other aldehyde is bound to an amino group of dye. Thus, when tissues are perfused with a GA-dye mixture there is the possibility that some dyes bind to basic groups of tissues bridged by GA molecules instead of binding to anionic groups.

IV. Reactions between Polyanionic and Polycationic Macromolecules To detect ionized groups on the macromolecular component of cells and tissues, staining with polycationic or polyanionic macromolecular or colloidal probes will be more reliable than staining with small molecular dyes, if the probes can be seen clearly under the light microscope (LM) and the EM. Because the binding between polyions having opposite charges is stable and specific to ionized groups, I will briefly discuss the specificity of the reactions between polyelectrolytes. The reaction of polyanionic macromolecules with polycationic ones produces polyion or polyelectrolyte complexes (Michaels, 1965). They may be highmolecular-weight salts having no electric charge or cationic or anionic highmolecular-weight complexes, depending on the types of polyions to be reacted, the mixing rate of the cationic molecules with the anionic ones, and the pH of the solvents. They may be sol, gel, or some colloidal precipitate. The reaction

210

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between polymers having strong acidic groups and strong basic groups is stoichiometric (Nakashima, 1974). The ionic bonding is very fast and the hydrophobic bondings between two molecules may support the ionic conjugation in some high molecules (Osada et al., 1973a,b; Tsuchida and Osada, 1974). The reaction between cationic and anionic groups on two macromolecules will work cooperatively to form interpolymer complexes (Tsuchida and Osada, 1974). By drying the thin layer of polyionic complexes formed by mixing artificially synthesized polyanionic and polycationic macromolecules, we may get hard papers of the polyion complexes. They become soft by adsorbing water and have the characteristics of an efficient ultrafiltration membrane, comparable to blood capillaries (Michaels and Miekka, 1961; Nakashima, 1974). Among these artificial polyion complexes it has been demonstrated that the anionic complexes have anticoagulant activity, which is the function of the density of the sulfate groups (Nakashima, 1974). Platelets do not adhere to the anionic polyionic complexes (Nakashima, 1974; Kataoka et al., 1978). In contrast, cationic macromolecules promote blood coagulation (Terayama, 1979). On the theoretical basis of the stoichiometric reaction between strong acidic and strong basic polyions, Terayama devised the colloid titration method which is useful in estimating ionized groups on macromolecules and is used widely in chemical engineering (Terayama, 1948, 1949; Mizote et al., 1975). The principle and method of colloid titration, by which we can get reliable information of the ionized anionic and cationic groups in tissues, are important to research on ionized biological polyions in vivo as well as in vitro.

V. Ionized Groups of Acid Polysaccharides and Proteins Estimated by the Colloid Titration Method A. COLLOID TITRATION METHOD Since the colloid titration method may not be very familiar, the principle and the methodology are discussed first, with some historical events. The colloid titration method was devised by Hiroshi Terayama in 1946 (Terayama, 1947a,b, 1948, 1949, 1952). Soon after World War 11, when he was a postgraduate student at Tokyo University and working on the blood coagulation-inhibitory activity of an anionic macromolecule, charonin sulfate, a sulfated polysaccharide from a kind of trumpet shell, the idea came to him that polycationic macromolecules may promote blood coagulation (Terayama, 1979). He wanted to obtain some polycationic macromolecules but found it was impossible, since almost all the Japanese chemical industries had been completely destroyed in the war. Trying to get polycations, he obtained the shells of crabs and lobsters and finally succeeded in getting chitosan, a polycationic polysaccharide, by

IONIZED GROUPS ON THE CELL SURFACE

21 1

treating the shells with concentrated sodium hydroxide. He found that the coagulation of blood, which had been inhibited by charonin, was restored by adding chitosan. Quantitative estimation made on sulfate groups of polyanions and amino groups of chitosan revealed the equivalent of one-to-one binding between sulfate groups and amino groups. This finding led him to a stoichiometric study of the reaction between cationic and anionic macromolecules. Later he used methylated chitosan, his “Macramin,” whose methyl amino groups are dissociated at higher pH, in place of chitosan (Terayama and Terayama, 1948). He also reported that protamine is useful as the polycation for colloid titration (Terayama, 1957). In 1953 Senju introduced the highly polymerized methylglycolchitosan as a useful polycationic probe for colloid titration (Senju, 1953a,b, 1969). Later in 1972 Tbei stated that polydiallyldimethyiammonium chloride (Cat-Floc) is useful as a polycation for colloid titration (Tbei and Kawada, 1972). Terayama used toluidine blue, which shows metachromasia by binding with potassium polyvinyl sulfate, as the indicator for titration (Terayama, 1947a,b), and Tbei used conductometric (Michaels et al., 1965; Tbei and Kohara, 1976) and turbidimetric methods (Tbei and Sawada, 1977; Ono et al., 1979). Thus, at present, Cat-Floc or methylglycolchitosan is generally used as polycations, potassium polyvinyl sulfate as polyanions, and toluidine blue as an indicator. Terayama showed a number of titration curves obtained on horse hemoglobin, horse catalase, diastase, and several acid mucopolysaccharides (Terayama, 1949). Furthermore he observed ionized groups on the bacterial surface by colloid titration, successfully revealing their negative surface charges with marked difference between gram-negative and gram-positive bacteria (Terayama and Arakawa, 1950; Terayama, 1954). Terayama also studied the surface electric charge of ascites tumor cells by colloid titration (Terayama, 1962). Recently, Kanemasa and associates estimated the negative charge of liposomes (Noda et al., 1982) and also several bacteria by colloid titration (Noda and Kanemasa, 1984; Noda et al., 1984). The fundamental knowledge of the reaction mechanism between macromolecules having opposite electric charges increased the understanding of the complex chemical reactions in living tissues and also produced the theoretical groundwork for the cytochemical detection of ionized groups on cell surface and interstitial tissue by using colloidal iron and other macromolecular probes.

B . IONIZATIONOF THE IONIC GROUPSOF ACID POLYSACCHARIDES AND PROTEINS The ionization states of ionic groups of some acid polysaccharides on the cell surface and interstitial tissues and some proteins in relation to the pH of the

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solutions as revealed by the colloid titration method of Terayama as modified by Tdei and Kawada (TGei and Kawada, 1972) are presented here (Seno et al., 1983a). Polydiallyldimethylammonium chloride (Cat-Floc) and potassium polyvinyl sulfate (PVSK) were used for the titration of anionic and cationic groups, respectively, using the metachromatic dye toluidine blue, as the indicator. The carboxyl groups of hyaluronic acid were fully ionized in an alkaline medium at a pH higher than 3.5. At a lower pH the ionization was gradually reduced with the decrease of pH to around 2 and completely suppressed at a pH below 1.8. The carboxyl groups of colomic acid, the polymer of sialic acids, showed the same tendency as hyaluronic acid. They were fully ionized at a pH higher than 5 and the ionization was gradually reduced with the decrease in the pH value to 2.1 and suppressed completely below 2. Carboxyl groups of chondroitin sulfate showed nearly the same tendency as hyaluronic acid, though it had sulfate groups ionized below pH 1.8. Actually, sulfate groups were ionized in the entire tested range of pH, from 1.6 to 9.0, as revealed on sulfate groups of PVSK titrating with Cat-Floc (Fig. la). Anionic groups of casein, carboxyl groups including phosphate groups, and the carboxyl groups of fibrinogen were ionized at a pH higher than their isoelectric points, 4.5 and 5.3, respectively. Their cationic groups, including amino, guanidino, and imidazol bases, were ionized in an acidic medium with a pH lower than their isoelectric points. Amino groups of glycolchitosan, a polycationic polysaccharide, were ionized fully in acidic media, but ionization was gradually reduced at a pH higher than 5.5 and was minimized at pH 7 (Fig. lb). The data indicate that if we could get a suitable cationic macromolecular or colloidal probe, we would be able to detect sulfate groups histochemically by treating tissue sections or fixed cells with the probe at pH 1.8, the carboxyl groups of acid polysaccharides together with sulfate groups at about pH 4,and the carboxyl groups of proteins together with sulfate and carboxyl groups of acid polysaccharides at pH 7. In a similar way we could detect the ionized cationic groups of proteins by treating the tissues with anionic macromolecular probes at pH 2-3. We now have a variety of excellent ferric hydroxide colloidal probes available for histochemical and cytochemical observations of ionized groups by light and electron microscopy.

FIG. 1. Ionization curves of three kinds of acid polysaccharides and polyvinyl sulfate (a), and those of two kinds of acidic proteins and glycolchitosan (b). Note -COO- groups of hyaluronic acid, colonic acid, and chondroitin sulfate are not ionized at a pH lower than 1.8, where - 0 S 0 3 - groups are only ionized, -COO- groups of fibrinogen and casein are ionized at a pH higher than their PI, and their -NH3+ groups at a pH lower than their PI, while -NH3+ groups of glycolchitosan are ionized at acidic media, minimized at pH 7.0 (b). (From Seno et al., 1983a, by permission of The New York Academy of Sciences, New York.)

213

IONIZED GROUPS ON THE CELL SURFACE

Potassium Polyvinylsutfate (-0s0;)

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Chondroitin Sulfate(

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4

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6

7

8

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2 14

SATIMARU S E N 0

VI. Colloidal Probes A. HALE’SFERRICHYDROXIDE COLLOID As understood from the specific reactions between polyanion and polycation (Michaels, 1965; Osada et al., 1973a,b; Nakashima, 1974), the staining of tissues with macromolecular probes or colloidal ones will give reliable information about the localization of ionized anionic groups or anionic sites on macromolecules on the cell surface and interstitial tissues. At present a metal colloid probe, the ferric hydroxide colloid, is widely used for light microscopy (BClanger, 1963; Mowry, 1963; Sano, 1977) as well as for EM (Jones, 1969; Michael et al., 1970; Weiss et al., 1972; Nicolson, 1972, 1973; Blau and Hass, 1973; Dermer and Kern, 1974; Skutelsky and Farquhar, 1976; Seiler et al., 1977; Kreisberg et al., 1979; Reeves et al., 1980; Alcorn and Ryan, 1981; Hiroch et al., 1981; Bakker et al., 1982a,b; Faraggiana et al., 1982; Nagy et al., 1983; Schneeberger et al., 1983; Weening and Rennke, 1983; Rollason and Brewer, 1984). The staining of acid polysaccharides by dialyzed iron (ferric hydroxide colloid) was first introduced by Hale in 1946 (Hale, 1946). In 1951, Rinehart and Abul-Haj obtained better results by using dialyzed ammonium ferric glyceride with distinct, clear, and strong staining of acid polysaccharides (Rinehart and Abul-Haj, 1951). When it later became difficult to purchase iron colloid, the staining procedure became less familiar. Since 1955, however, when Muller reported that ferric hydroxide colloid can be easily obtained by adding a ferric chloride solution to boiling water, the iron colloid staining has again come into general use (Muller, 1955). The nondialyzed ferric hydroxide colloid was decomposed to yield ferric chloride by standing at room temperature for a long time and it had to be dialyzed to become stable. After repeated experiments, Mowry finally recommended staining at pH 1.61.9 in 25-30% acetic acid solution for 1 to 4 hours, 1-2 hours for a 50% solution of original colloid, and 2-4 hours for a 25% solution, and rinsing with 30% acetic acid (Mowry, 1963). He also checked carefully the staining of acid polysaccharides by iron colloid and found that Rinehart-Abul-Haj’s method (195 1) may result in nonspecific staining and take too much time for dialysis (Mowry, 1963). Thus, polysaccharides with carboxyl groups can be stained with iron colloid as revealed by treating the tissues with neuraminidase (Gasic and Gasic, 1962; Defendi and Gasic, 1963; Gasic and Berwick, 1963; Wallach and Esandi, 1964; Benedetti and Emmelot, 1967; Skutelsky and Farquhar, 1976). Benedetti and Emmelot suggested that staining with colloidal iron was the result of the interaction between the positively charged ferric hydroxide complex and the negatively

IONIZED GROUPS ON THE CELL SURFACE

215

charged carboxyl groups of sialic acid, as the incubation of membranes in physiological saline with strongly basic polylysine prevented subsequent staining with iron colloid (Benedetti and Emmelot, 1967). However, carboxyl groups of acid polysaccharides are not ionized at such a low pH (1.6-1.9) as is understood from their ionization curves (Seno et a l . , 1983a). Therefore, ionic binding of iron colloid particles with sialic acids is unlikely. But the absorption test to ion exchange-resin particles revealed that ion colloid particles showed affinity to the resin particles CG-50 having carboxyl groups at pH 1.8 (Seno et al., 1985) where the carboxyl groups are not ionized. So, it is suspected that FeO and Fe3 groups on the surface of the iron colloid particles (Muller, 1964; Geyer and Stibenz, 1974) may bind to oxygen of nonionized carboxyl groups of sialic acid or resin particles by van der Waals forces. Of course, the iron colloid particles will bind with sulfate groups of heparan sulfate, heparin, dermatan sulfate, and chondroitin sulfate by Coulomb’s force, as these groups are ionized at pH 1.6-1.8. Therefore, at that pH iron colloid particles will bind with sulfate groups by ionic bonding and also with carboxyl groups with nonionic bonding. The digestion test with enzymes may serve as a useful method to distinguish the types of ionic groups of polysaccharides, as has been reported by many investigators (Gasic and Gasic, 1962; Defendi and Gasic, 1963; Gasic and Berwick, 1963; Wallach and Esandi, 1964; Benedetti and Emmelot, 1967). Thus, from the standpoint of colloid chemistry, ferric hydroxide colloid is a very useful tool for the detection of anionic groups on polyanions. The particles give a distinct positive Prussian blue reaction for LM and appear opaque under the EM. However, the colloid particles are unstable at a pH higher than 2.0, forming a gross precipitate, and cannot be used for the detection of anionic sites in living tissues. Furthermore, the colloid particles may bind with macromolecules by nonionic bonds at low pH, though they bind with sulfate groups by ionic bondings. These deficiencies in Hale’s iron colloid method have been completely overcome by the appearance of cacodylate iron colloid (Fe-Cac). +

+

B. CATIONIC CACODYLATE FERRICHYDROXIDE COLLOID 1. Stabilization of Hale’s Colloid at pH 1.6-7.6 Hale’s or Muller’s ferric hydroxide colloid was successfully stabilized by adding sodium cacodylate solution (Tanaka, 1974; Sen0 et al., 1975; Seno et al., 1983a-c). The particles kept their original size and positive electric charges even at pH 7.3 (Seno et al., 1983~).The particles were electron opaque and gave a clear Prussian blue reaction and reacted specifically with ionized anionic groups as revealed through the adsorption test to the ion-exchange resin particles at varied pH. The particles were adsorbed to sulfonate groups of IR-I20B, at pH

216

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2.0 and 4.0,and carboxyl groups of CG-50 at pH 4.0but not at 2.0 and were not adsorbed to amino groups of IRA-400 at any pH tested (Fig. 2). The reaction occurred promptly, and the binding to anionic groups reflected their ionization as revealed by colloid titration. Therefore, these colloid particles can be used as ideal cationic probes for the detection of ionized anionic groups in tissues in vivo as well as in v i m by LM and EM. The toxicity of cacodylate is very low with an LD,, of 1.O g/kg in dogs (Merck Index, 1976). Fe-Cac proved to be an excellent cationic probe for the detection of anionic sites as revealed by histochemical observations of various tissues (Seno et a l . , 1975, 1983a-c; Ukita et al., 1983; Tsujii et al., 1984a,b, 1985). The mechanism of stabilization of the ferric colloid by cacodylate is unclear. But it is possible that cacodylate molecules bind with FeO+ or Fe3+ on the

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FIG. 2. Binding test of 59Fe-Cac grains to ion exchange resin having -SO3+ (IRA-400) and -COO+ groups (CG-50). 59Fe-Cac solutions were applied to each resin column at pH 2 , 4 , and 7 and washed three times with cacodylate buffer at pH 2, 4 , and 7, respectively, and the specific activities of resin particles were measured. Note that the binding of 5yFe-Cac to the carboxyl groups of CG-50 is minimized at pH 2.0.

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217

colloid surface (Muller, 1964; Geyer and Stibenz, 1974) with the anionic oxygen directing the methyl groups toward the luminal surface which may inhibit the further binding of OH- ion to the ferric colloid. The discovery of cationic cacodylate ferric hydroxide colloid occurred in 1973, when I was observing EM pictures of a rat lung taken 30 minutes after inhalation of the lead fume (Ogata et al., 1973). I found a few electron-opaque lead particles on the luminal surface of the alveolar epithelium and also endocytic vesicles of epithelial and endothelial cells. The particles were also found in the capillary lumen adhering to the red-cell surface. I thought that such a rapid invasion of lead oxide particles into alveoli and blood vessels might be due to the positive electric charge of the particles. I decided to try to determine whether a similar examination could be made with cationic ferric hydroxide colloid particles; we would then be able to trace the flow of particles in the respiratory tract down to the alveolar lumen by light microscopy on sections treated with potassium ferrocyanide for Prussian blue reaction. This proved impossible because Hale’s colloid was not stable at pH 7 and could not be applied to living animals. I knew that many histochemists worked with Hale’s colloid improving the method, but no one was able to stabilize the positive charge of the ferric hydroxide colloid at a pH higher than 2. At that time one of my co-workers was a young postgraduate student, Akisuke Tanaka. As I wondered how we could keep the positive charge of Hale’s colloid at around pH 7, he suggested that cacodylate buffer might be useful for the purpose. He was then working with cacodylate buffer for tissue fixation with GA. I did not expect his idea to work, but the test proved that cacodylate successfully stabilized Hale’s colloid at pH 7, keeping the positive charge of the particles. Thus, we could use cacodylate ferric hydroxide colloid for observing anionic groups on macromolecules in vivo as well as in vitro. The cacodylate ferric hydroxide particles prepared by Tanaka were rather large, 50 to 200 nm in size (Tanaka, 1974; Sen0 et al., 1975). Later, by improving the method, we obtained colloid of small particles, 3-5 nm in size (CI or Fe-Cac) (Seno et al., 1983a-c).

2 . Cationic Ferric Hydroxide Colloid of Fine Grain (Fe-Cac-f) Since we succeeded in obtaining a ferric hydroxide colloid of small size and stabilizing its positive charge at pH 1.6-7.6 (Fe-Cac), we tried to obtain a cacodylate ferric colloid of fine grain which would be superior to Fe-Cac in diffusibility into the fixed tissues; Fe-Cac was poorly diffusible into GA-fixed tissues for EM. We finally were able to get an ideal one, Fe-Cac-f, by boiling a mixture of ferric chloride, cacodylic acid, and ammonia water at pH 7.3 (Seno et al., 1985). The particles of Fe-Cac-f thus obtained were about 1 nm in size and gave a clear Prussian blue reaction having stable positive charges at pH 1.6-7.6 as in Fe-Cac. The binding test of Fe-Cac-f colloid particles to the ion-exchange

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resin revealed nearly the same chakacteristics as with Fe-Cac, showing the specific affinity to ionized anionic groups. Nonionic binding to resin particles was not found. The staining of fixed tissues with Fe-Cac-f gave a picture of the Prussian blue reaction which was more clearly distinct than with Fe-Cac (Seno et al., 1985). Five to ten minutes were enough to stain acid polysaccharides of the tissue sections by mounting with diluted Fe-Cac-f solution, similar to the case of Giemsa staining of smeared cells, though we stained for 30 minutes in the cases reported (Seno et a l . , 1985). Electron microscopic observation of the fixed tissues stained with Fe-Cac-f revealed the electron-opaque grains of about 1 nm permeated deeply into the tissues. In a perfusion test of living tissues, however, Fe-Cac gave a clearer picture of the vascular endothelium than did Fe-Cac-f. These results seem to indicate that Fe-Cac-f is superior to Fe-Cac in staining of fixed tissues and Fe-Cac is superior to Fe-Cac-f in perfusion tests of living tissues. 3. Cacodylate Ferric Hydroxide Colloid Particles Bind Specifically to Ionized Anionic Groups on the Cell Surface The adsorption test to human red blood cells revealed that Fe-Cac particles were adsorbed well to the red cell surface at pH 4.0-7.0, but the adsorption was minimal at pH 2.0 (Fig. 3). The adsorption test to Ehrlich ascites tumor cells showed a tendency similar to red blood cells, but a distinct adsorption was observed even at pH 2.0 (Fig. 3). The results again indicated that Fe-Cac binds to the ionized anionic groups but not to the nonionized ones, because red blood cells have carboxyl groups on the surface but not sulfonic groups (Glick and Flowers, 1978), while Ehrlich ascites cells have sulfonic groups as well as carboxyl groups as in other malignant cells (Kraemer and Tobey, 1972; Satoh et al., 1973; Yamamoto and Terayama, 1973; Underhill and Keller, 1975).

MOLECULES C. FERRITIN Cationized ferritin molecules are now widely used in viva as well as in vitro for the detection of anionic sites on the cell surface and interstitial tissues (Danon et al., 1972; Rennke et al., 1975; Grinnel et a l . , 1976; Skutelsky and Danon, 1976; Bruyn et a l . , 1978; Venkatachalam and Rennke, 1978; Burry and Wood, 1979; Kanwar and Farquhar, 1979a,b; Kelley and Cavallo, 1980; Reeves et al., 1980; Wright et al., 1980; Raz et a l . , 1980; Christensen e t a l . , 1981; Melnick et a l . , 1981; Simionescu et a l . , 1981 a, b, 1982; Vogt et a l . , 1982; Cavallo et al., 1983; Danon et al., 1983; Dermietzel et a l . , 1983; Johansson, 1983; Pietra et al., 1983; Bliss and Brewer, 1984; Kanwar and Jakubowski, 1984; Marikovsky et a l . , 1978, 1985; Sarphie, 1985). Ferritin molecules show specific figures and give reliable information under the EM, but the molecules cannot be seen by the

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219

1 Ehrlich Ascites Tumor Cells

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200 Human Red

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3

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PH FIG. 3. Binding test of s9Fe-Cac particles to the fixed human red blood cells and mouse Ehrlich ascites tumor cells. Cells fixed in 2% glutaraldehyde for 10 minutes were incubated with 59Fe-Cac solution in cacodylate buffer at different pHs at room temperature for 5 minutes, washed three times with the same buffer solution used for incubation, and the specific activities of the cells were measured. Note the binding of 59Fe-Cac to red cells having only -COO+ groups is minimized at pH 2.0.

LM because of the negative Prussian blue reaction. In addition, the cationized ferritin molecules may also bind to ferritin receptors on some cell surfaces as well as the ionized anionic groups of cell coat and interstitial structures. For these reasons, ferritin may be inferior to cacodylate ferric hydroxide colloid. D. OTHERCATIONIC PROBES Polyethyleneimine (Schurer et al., 1977; Weening and Rennke, 1983; Barnes et af., 1984), lysozyme (Caulfield and Farquhar, 1976, 1978), horseradish peroxidase (HRP) (Rennke et al., 1978; Sibley et al., 1983), catalase (Rennke and Venkatachalam, 1977), and cationized hemeundecapeptide (Ghinea and Simionescu, 1985) are also used as cationic probes.

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Polyethyleneimine (MW 600-60,000) was introduced by Schurer and others as a probe to detect anionic sites in tissues by EM (Schurer et al., 1977) and has been used by several researchers (Vernier et al., 1983; Rollason and Brewer, 1984; Barnes and Venkatachalam, 1985; Koide et al., 1985; Oite and Shimizu, 1985). It is not visible in the EM but can be detected as particles by contrasting it with phosphotangstic acid or OsO, (Schurer et al., 1977). Therefore, polyethyleneimine is a polycation useful for the detection of negatively charged groups on polyanionic polysaccharides. Observations have been made on tissues by intravenous injection or immersion with the probes. However, in intravenous injection there is a possibility that polyethyleneimine may conjugate with anionic serum proteins forming uncharged or anionic polyion complexes and also form fibrin. Lysozyme forms electron-dense deposits through the routine fixation method for EM (Caulfield and Farquhar, 1976). Horseradish peroxidase has peroxidase activity and is visualized by the cytochemical method for peroxidase with diaminobenzidine (Graham and Karnovsky, 1966; Karnovsky, 1967, 1970; Venkatachalam et al., 1969; Gorog and Born, 1983). Hemeundecapeptide is also visualized by its peroxidase activity (Ghinea and Simionescu, 1985). These probes have been used mainly for experiments in vivo, i.e., immersion, perfusion, intravascular injection, etc. These are used in place of cationized ferritin mainly because they are small in molecular weight and well permeable in tissues. Among these cationic probes hemeundecapeptide is the smallest in molecular weight. Simionescu and associates used cationized hemeundecapeptide for the detection of anionic sites (Ghinea and Simionescu, 1985). As the molecules are small they infiltrate the tissues well and can be used to detect anionic groups lying in deeper areas.

E. ANIONICFERRICHYDROXIDE COLLOID;CHONDROITIN SULFATE, CITRATE, AND POLYVINYL SULFATE FERRICHYDROXIDE COLLOIDS In addition to Fe-Cac and Fe-Cac-f, we also have negatively charged ferric hydroxide colloid particles. Chondroitin sulfate ferric hydroxide colloid (FeChS) was devised by Seno and Awai in 1960 (Seno, 1962; Sen0 et al., 1962). The stabilized form, “Blutal, is commercially available from Dainihon Pharmaceutical Co., Osaka, for medical use. These colloid particles bind solely and firmly to the ionized cationic groups on macromolecules or particles as revealed by the adsorption test to ion-exchange resin particles at pH 2.0-7.0. The particles show affinity to IRA-400 (-NH,+) but not to CG-50 (-COO-) and IR-120B (-SO,-) at any pH tested. The size of the colloid particles is nearly the same as those of Fe-Cac and can be seen under the LM by their positive Prussian blue reaction and also under the EM by the electron opacity of the particles. These characteristics of Fe-ChS particles produce an ideal tool for histochemi”

IONIZED GROUPS ON THE CELL SURFACE

22 1

cal and cytochemical detection of cationic sites of the macromolecules on the cell surface and intercellular structures in vivo as well as in vitro. We have recently devised a new anionic ferric hydroxide colloid, citrate ferric hydroxide colloid (Fe-Cit), by mixing one volume of Hale-Miiller’s ferric hydroxide colloid, which was prepared by adding 1 volume of 0.1 M FeCI, solution to 9 volumes of boiling distilled water drop by drop with constant stirring, with 5 volumes of 0.1 M sodium citrate solution. The citrate iron colloid solution thus prepared was nearly isotonic and the pH was around 7.0. The pH of the solution can be adjusted to that desired with NaHCO, or HCI. The particles were 3-5 nm in size, negative in charge, and stable between pH 3.0 and 8.0, and give a strong positive Prussian blue reaction. The test with ion exchange resin having -COO-, -SO,-, and -NH3+ gave results similar to those obtained with FeChS. We also have another anionic ferric hydroxide particle, large in size and suitable for the observation of phagocytosis of macrophages: the anionic polyvinyl sulfate ferric hydroxide colloid (Fe-PVS). This colloid was recently devised by Ono and his colleagues by mixing Hale’s ferric hydroxide colloid with a solution of potassium polyvinyl sulfate (On0 et al., 1983a,b; Ono and Awai, 1984; Ono and Seno, 1986). The particles are large in size, 25-250 nm, but stable at pH 7.0, and give a distinct Prussian blue reaction. They can bind specifically with -NH,+ of IRA-400 but not with -COO- of CG-50 and -SO,- groups of IR-120B and are taken up specifically by macrophages. Native ferritin molecules have a negative charge and can be used for the detection of ionized cationic groups. But again we face the problem of ferritin receptors on the cell surface.

VII. Effect of Aldehyde Fixation on Ionized Groups of Cells and Tissues A. GLUTARALDEHYDE FIXATION Substantial amounts of information on ionized anionic groups have been obtained on fixed cells and tissues by treating them with cationic probes (Hale, 1946; Jones, 1969; Michael et al., 1970; Nicolson, 1972, 1973; De Bruyn et al., 1978; Kreisberg et al., 1979; Reeves et al., 1980; Sen0 et al., 1983a,c; Weening and Rennke, 1983; Tsujii el al., 1984a,b; Sen0 et al., 1985). But, there is evidence indicating that GA fixation results in a marked increase of anionic groups in density (Grinnel et al., 1976; Burry and Wood, 1979) and also a change in distribution of the groups in some membranes. Grinnel et al. (1976) showed that prefixation of plasma membranes of cultured baby hamster kidney cells and human fibroblasts with GA can induce a change in the distribution of

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anionic sites. But they did not see such GA-induced changes in isolated mitochondrial membrane as observed by using cationized ferritin (Grinnel et al., 1976). Burry and Wood (1979) also showed a marked increase in density of anionic groups on the GA-fixed neuronal process of rat cerebellum. Danon and associates (1972) showed, however, no recognizable changes in the number of cationic ferritin molecules adsorbed on the surface of human red blood cells before and after GA fixation. The possible mechanisms of an increase in the density of anionic groups by GA fixation are (1) direct binding of the polycationic ferritin with exposed aldehyde groups of GA bound to the cell surface, (2) GA-induced reorganization of the molecular structure of the membrane resulting in the appearance of new anionic sites (Grinnel et al., 1976), or (3) the action of GA to increase negative charges of neutral proteins by binding to their basic groups (Burry and Wood, 1979). At present, we do not know which of these is true. However, there is much evidence indicating that GA interacts with some basic groups on the protein surface to mask their charges (Hayat, 1981). It is known that GA fixation of tissues is due to reaction with proteins. The fixation reactions of formaldehyde (FA) with proteins is thought to be due to the cross-linking of peptide chains, reacting with their free basic groups, such as amino, amido, guanidino, imidazolyl, and indolyl, or active groups, such as thiol and phenolyl (Hayat, 1981). Similar reactions are expected in GA fixation. However, it was revealed that GA reacts specifically with €-amino groups of lysine (Bowes and Carter, 1966; Habeeb and Hiramoto, 1968; Happich el al., 1970; Korn et al., 1972) but not with hydroxy groups of tyrosine, imidazol groups of histidine (Bowes and Carter, 1966), and SH groups (Hopwood, 1968), though some reactions may occur with tyrosine, histidine, and sulfhydryl residues (Habeeb and Hiramoto, 1968). Thus, in GA fixation of tissues, GA molecules do not react with all the basic groups of proteins, and cells fixed with GA monomers bear positive fixed charges as revealed in GA-fixed sarcoma 180 cells by Skehan (1975). The extent to which cationic groups on proteins are masked by binding with GA can be estimated by the colloid titration method using potassium polyvinyl sulfate. For example, the ionization of the cationic groups of poly-L-lysine was markedly reduced in the presence of GA, especially in the slightly alkaline media (63%) at pH 7.3. In the positive charge of histone 11, which has a quantity of arginine and lysine, the ionized cationic groups were reduced by adding GA, but less than in poly-L-lysine, by about 50% at pH 7.3 (Fig. 4a). In both cases the reduction of positive charge became marked with the elevation of pH but was less conspicuous at low pH. A similar experiment was performed on an acidic protein, fibrinogen, to determine the change in negative charges by using Cat-Floc; there was a large

223

IONIZED GROUPS ON THE CELL SURFACE

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increase (about 200%) in negative charges by fixing with GA at pH 7.3 which was minimized at the low pH of 6 . 2 (Fig. 4b). Therefore, the fixation of tissues with GA in an acidic medium, pH 3-4, results in minimized changes to tissues as far as the effect of GA fixation on the ionization of anionic groups is concerned.

B. FORMALDEHYDE FIXATION As just described, the FA fixation mechanism should be due to the crosslinking of peptide chains reacting with free basic groups. The first step in the reaction of FA with proteins is thought to form methyl amino groups with free amino residues; this is then followed by methylene bridge formation by condensation with other functional groups such as phenol, imidazol, and indol residues (Hayat, 1981). The methylene bridges mean irreversible covalent bonds. However, most of the formol molecules participating in protein fixation can be removed easily by washing with water. This means that FA binds with proteins by weak bonding such as hydrogen bonds and van der Waals bonds. Accordingly, it is thought that in the formol-fixed paraffin sections most of the FA molecules are removed and the cationic groups are unmasked. C. INFORMATION ON IONIZED GROUPSOBTAINED ON FIXED PROBES TISSUESWITH POLYIONIC 1. Information Obtained on FA-Fixed Tissues by Light Microscopy

In FA fixation most of the cationic groups restore their ionized states. Therefore, the staining of the FA-fixed paraffin sections with anionic and cationic iron colloid particles or other macromolecular probes will give reliable information about the distribution and density of ionized groups in tissues. Staining of FA-fixed liver, kidney, and other tissues of rats with the anionic Fe-ChS at pH 2.0 showed an overall blue color by Prussian blue reaction, but the same staining at pH 7.0 showed no blue staining except for some macrophages, which were stained slightly blue owing to their cationic groups on the surface (Seno et a l . , 1983a). Data reflect the results of colloid titration of proteins, in which the amino groups are ionized at pH 2.0 but not at pH 7.0. The findings clearly indicate that in living tissues whose pH is about 7.3, there are no ionized cationic groups except for those on the macrophage surface. The cationic groups of basic proteins like histones, whose basic groups are ionized at about pH 7 as demonstrated by colloid titration, were not stained by anionic ferric hydroxide colloid, showing that their basic groups are masked by the anionic groups of nucleic acids and the associated acidic proteins. In contrast, staining of the FA-fixed tissues with cationic iron colloid, Fe-Cac and Fe-Cac-f, at pH 7.0 gave overall blue staining by the Pmssian blue reaction,

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indicating that in living tissues cell cytoplasm and extracellular structures are filled with ionized anionic groups of proteins and acid polysaccharides. At pH 1.8-2.0 some mucus-producing cells, cartilage, renal glomeruli, axons of nerve fibers, etc. were stained, revealing the sites of sulfate groups of acid polysaccharides. Staining at pH 4.0 gave deeper staining of these tissue components than it did at pH 1.8, indicating the staining of carboxyl groups of acid polysaccharides with their sulfate groups (see Section V).

2. Information from GA-Fixed Sections From the data obtained by colloid titration of histones, polylysine, and fibrinogen with potassium polyvinyl sulfate and Cat-Floc, it might be expected that GA fixation at pH 7.0 will result in a marked increase in intensity of the staining of anionic groups with the masking of cationic groups. The observations on paraffin sections of 2% GA-fixed rat kidney and liver tissues stained with FeCac-f, however, did not reveal much of a difference in the density of ionized anionic groups as compared to those found on the FA-fixed tissues stained with Fe-Cac-f (M. Akita, personal communicaton).

VIII. Reaction of Living Cells to Polyions A. THE REACTION OF CELLSIN SUBCUTANEOUS TISSUEAND THE PERITONEAL CAVITYTO POLYIONIC COLLOID PARTICLES 1. Subcutaneous Injection of Fe-Cac a. Cellular Reaction Induced by Cationic Macromolecules. As suggested by observations on FA-fixed tissues stained with ferric hydroxide colloids at pH 7.0, the environment of living tissues is filled with ionized anionic groups. Therefore, when some cationic macromolecules or colloids are introduced into tissues severe disturbances should occur in the ionic environment. In order to determine possible tissue reactions to cationic macromolecules, 0.1 ml of Fe-Cac (0.008 mg Fe/ml) was injected into the rat subcutaneous tissue. The animals were sacrificed 24 hours after injection and local subcutaneous tissues were observed morphologically, i.e., local cutaneous tissues were cut and small pieces of subcutaneous tissues were pinched off, extended on a glass object by using a needle, formol gas fixed, and observed by LM staining with Prussian blue reaction and poststaining with nuclear fast red. Observations revealed some inflammatory reaction with the infiltration of lymphocytes, monocytes, neutrophils, and a few eosinophils. All the cells including local fibroblasts took up iron colloid (Seno et al., 1983a). The histiocytes also showed a marked Prussian blue reaction but they were not swollen,

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as opposed to those taking up general foreign substances. The local connective tissues also showed a distinct Prussian blue reaction. b. Electron Microscopic Observation of the Reaction of Peritoneal Resident Cells to Fe-Cac in Vitro. We then observed the response of peritoneal cells to Fe-Cac by EM. When rat peritoneal cells were incubated with Fe-Cac solution, 8 pg Fe/ml at 37°C for 1 hour, the colloid particles were found on the surface of all kinds of cells and in their cytoplasmic vesicles, i.e., eosinophils, neutrophils, lymphocytes, macrophages, etc. (Figs. 5a and b). Clusters of colloid particles were found on the cell surface. Some pits with clustered Fe-Cac particles were

FIG. 5 . Electron micrographs of a lymphocyte (a) and an eosinophil (b) incubated with Fe-Cac in RPMI 1640, 8 pg Fe/ml, at 37°C for 1 hour (a) and 2 hours (b). After incubation cells were fixed with 2% glutaraldehyde, postfixed in 2% OsO4 at 0°C for 40 minutes, and show the charge related endocytosis of the colloid particles. N, Nucleus; M, mitochondria; EV, endocytic vesicles; G, eosinophilic granules. (From Ono and Seno, 1986, by permission of the Japan Society of Histochemistry and Cytochemistry.)

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also found. The picture was essentially identical with those of clusters formed by cationized ferritin on blood vessel endothelium of guinea pig (Skutelsky and Danon, 1976), normal and transformed mouse embryo fibroblasts (Borysenko and Woods, 1979), and normal and malignant lymphocytes demonstrated diagrammatically by Marikovsky et al. (1978). Some of them had tubular necks ’connected to the cell surface, but the coated pits were hardly encountered. A number of small endocytic vesicles that did not seem to fuse were also found (Fig. 5b). The findings were almost identical among different kinds of cells. The colloid particles in pinocytic vesicles were found adhering to the luminal surface of vesicles but rarely in the luminal spaces. This is similar to that of the uptake of HRP by Swiss 3T3 cells which have no specific receptors to HRP (Pastan and Willingham, 1985). As there is no specific receptor for Fe-Cac, the adsorption to the cell surface should be due to the ionic bondings. This explains why the cationic particles adhere to all kinds of cells and are endocytosed. This is a kind of adsorptive endocytosis, and we have proposed to call the phenomenon “charge-related endocytosis” (Ono and Seno, 1986). c. Reaction of Peritoneal Macrophages to Fe-Cac, the Charge-Related Endocytosis (CRE). To distinguish CRE from common phagocytosis of macrophages, the early phases of Fe-Cac uptake by rat peritoneal macrophages (Ono et al., 1985) are presented. The resident peritoneal macrophages of adult male Wistar rats suspended in culture medium RPMI 1640 were placed on two series of glass slides, separately by incubating at 37°C for 1 hour. They were then incubated with the Fe-Caccontaining medium (8 pg mg Fe/ml) at 37°C for 5, 10, 30, 60, and 120 minutes and some of them were further incubated in the medium free of Fe-Cac for 2 , 5, 10, and 15 hours. After incubation the cells on one series of slides were methanol fixed, washed, treated with potassium ferrocyanide, poststained with nuclear fast red, and observed by LM. Other slides were fixed with 2% GA for 10 minutes, postfixed with 2% OsO, for 40 minutes, and ultrathin sections were made by the routine method for EM. Light microscopy revealed that all the cells incubated for 5 to 120 minutes were stained blue by the Prussian blue reaction, but they did not show any morphologic changes or distinct swelling even after 2 hours incubation with FeCac . Electron microscopy showed that after 5 to 10 minutes of incubation the colloid particles adhered to the cell surface with a dense distribution at pits on the surface and those in the cytoplasm connected to the cell surface with tubular necks of various length (Fig. 6a). Typical coated pits were rarely encountered. Thirty minutes after incubation the macrophages had Fe-Cac particles in numerous tiny endocytic vesicles. Most of them had tubular profiles (Fig. 6b). One hour later the number of endocytic vesicles with Fe-Cac particles increased.

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FIG.6. Electron micrographs of rat peritoneal macrophages taking up cationic Fe-Cac particles by charge-related endocytosis, 10 minutes (a), 30 minutes (b), and 4 hours (ct.z) after incubation. Endocytic vesicles show canalicular or tubular profiles containing Fe-Cac particles. After 4 hours incubation the endocytic vesicles appear near the Golgi apparatus but they fuse poorly with each other and also with lysosomes. Note that the endocytic process is very similar to that of the lymphocyte and the eosinophil in Fig. 5 , but different from that of phagocytosis of anionic particles by macrophages (Figs. 7 and 8). N, Nucleus; EV, endocytic vesicles. For the method see the legend of Fig. 5 .

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Their sizes were the same as those found at 30 minutes after incubation, showing a poor fusing tendency to one another. After 2 hours of chase in RPMI-1640 medium free of Fe-Cac following exposure to Fe-Cac for 2 hours, many vesicles were found in the area of transreticular (TR) Golgi or Golgi-associated endoplasmic reticulum from which lysosomes arise (GERL) (Novikoff et al., 1980) (Fig. 6c). Large empty vacuoles were encountered occasionally but they were free of Fe-Cac particles. The Fe-Cac particles were always found attached to the luminal surface of vesicles and the central area of vesicles appeared scanty. No vesicles fused with lysosomes as revealed by staining acid phosphatase with modified Gomori’s medium (Barka and Anderson, 1962). After 5 hours of chase in the Fe-Cac-free media some vesicles fused with lysosomes, but acid phosphatase-positive contents were scarcely mixed with Fe-Cac granules. By 10 to 15 hours of chase in the iron-free medium, however, large secondary phagolysosomes appeared, suggesting the transport of ferric hydroxide colloid to lysosomes through TR Golgi or GERL. The movement of endocytosed Fe-Cac particles to lysosomes seemed to be similar to that of the EGF-receptor complex endocytosed by human KB cells as reported by Pastan and Willingham (1985), but the Fe-Cac particles taken up by rat peritoneal macrophages moved slowly compared to the EGF-receptor complex. 2. Reaction to Anionic Colloid Particles a. Reaction of the Cells in Subcutaneous Tissue and the Peritoneal Cavity. As opposed to cationic macromolecules or colloid particles, anionic macromolecules are expected to have no injurious effect, as they do not interact with cells and interstitial tissue components having anionic groups on their surface. Three kinds of anionic ferric hydroxide particles having different coats, i.e., polyvinyl sulfate, chondroitin sulfate, and citrate, were injected safely into the subcutaneous tissues of rats without inducing any inflammatory reaction. The tissues taken 24 hours after the subcutaneous injection, 0.1 ml of 0.1 mg Felml, showed neither edema, cell infiltration, nor cell proliferation, as revealed by LM observation of local tissue pieces. The injected colloid particles were selectively taken up by macrophages. The histocytes gave a deep blue Prussian blue reaction, but other cells and components of interstitial tissues produced a negative reaction (Seno et al., 1983a). Repeated intraperitoneal injections of Fe-PVS or Fe-ChS did not induce any fibrotic changes or adhesion of the organs. The resident peritoneal cells taken 24 hours after a single intraperitoneal injection of Fe-PVS or Fe-ChS had no Prussian blue positive grains except for the macrophages, which were extremely swollen by taking up iron colloid particles (On0 et al., 1983a,b; Ono and Awai, 1984). b. Peritoneal Macrophages. Electron microscopic observations of normal rat peritoneal resident cells incubated with Fe-ChS (2.4 mg Fe/ml) at 4°C for 30

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minutes in vitro revealed the Fe-ChS particles adhering to the surface of macrophages, but not to neighboring lymphocytes, eosinophils, and mast cells. After further incubation of cells in the medium at 37°C for 10 minutes the colloid particles were found in coated pits and coated vesicles (Fig. 7a). This was similar to what was shown by Aggeler and Werb (1982). After 30 minutes of incubation at 37°C the colloid particles were also found in vesicular endosomes attached to the luminal surface of the membrane (Fig. 7b). After 4 hours of incubation large phagosomes filled with Fe-ChS particles appeared, some of them fused with lysosomes (Fig. 7c). Macrophages found 1 hour after intraperitoneal injection of Fe-PVS had numerous large phagosomes filled with Fe-PVS particles and at 5 hours after FePVS injection they had numerous phagolysosomes (Fig. 8). In some phagosomes a direct fusion with the nearby lysosomes was seen (On0 et al., 1983a). The formation of phagolysosomes took place much earlier than the cases of Fe-Cac uptake by macrophages where no morphologic indication of a direct fusion of endocytic vesicles and lysosomes was encountered. Ferritin appeared in phagolysosomes of peritoneal macrophages 24 hours after intraperitoneal injection of Fe-PVS (On0 et al., 1983b). Similar results were obtained on peritoneal macrophages taken 24 hours after intraperitoneal injection of Fe-ChS by Ono and Awai (1984) or Fe-Cit by Sen0 and Akita (unpublished data). Thus, negatively charged particles were taken up selectively by macrophages in a completely different way from their charge-related endocytosis, the uptake of cationic Fe-Cac particles. The fact that they could equally phagocytize FePVS, Fe-ChS, and Fe-Cit with different surface coats indicates that working receptors recognize the surface negative charges of ligands, which are common to these three kinds of particles. The binding is clearly an electrostatic one and the low density of anionic groups on the macrophage surface (Bruyn et al., 1978) may support the binding. The findings are consistent with the view that macrophages recognize various kinds of foreign substances by their negative charges (Seno, 1977). The receptor would be one of the nonspecific receptors of Silverstein et al. (1977) and correspond to the polyanion receptor described by Brown et al. (1980). The characteristics of these receptors are (1) they recognize anionic groups on macromolecules or particles; (2) the particles bound to these receptors are taken into the cytoplasm by endocytosis or phagocytosis (Silverstein et al., 1977); (3) receptor-ligand complexes are easily sorted in phagosomes and ligands accumulate in the phagosome filling the spaces; and (4) the phagosomes show distinct characteristics of tending to fuse with each other and also fuse directly with lysosomes. Thus, phagocytosis by “polyanion receptor” is distinguished from “chargerelated endocytosis” and other receptor-mediated endocytosis (Pastan and Willingham, 1985). The polyanion receptor does not recognize anionic mac-

IONIZED GROUPS ON THE CELL SURFACE

23 1

FIG. 7. Electron micrographs of rat peritoneal macrophages taking up anionic Fe-ChS particles by polyanion receptor-mediated endocytosis. Cells were first incubated with Fe-ChS, 2.4 mg Fe/ml, in RPMI 1640 at 4°C for 10 minutes, rinsed with fresh medium to wash out the unbound Fe-ChS, and then chased at 37°C in iron-free RPMI 1640 medium for 10 minutes (a), 30 minutes (b), and 4 hours (~1.1). CP, Coated pit; CV, coated vesicle; E, endosome; L, lysosome; N, nucleus; M, mitochondria.

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FIG. 8. Electron micrograph of a rat peritoneal rnacrophage taken 5 hours after a single intraperitoneal injection of 3 ml of Fe-PVS, 0.055 mg Feiml showing the formation of large phagosomes and phagolysosomes. P, Phagosome; PL, phagolysosome; N, nucleus. (From Ono et al., 1983, by permission of the Japan Society for Cell Biology.)

romolecules on the living cell surface. The reason is unclear at present and it is a problem to be resolved in the future. B. INTRODUCTIONOF POLYIONS INTO BLOODVESSELS 1. Intravenous Injection or Infusion with Polyions

a. Introduction of Cationic Probes into Vessels. The anionic groups of glycoproteins on the surface of blood cells and the vascular endothelium are all ionized and most plasma proteins are acidic (Blomback and Hanson, 1979), having a net negative charge at pH 7.3. Therefore, some cationic macromolecules introduced into vessels inevitably meet with the excess of anionic serum proteins and combine with them, forming fairly stable uncharged or anionic polyion complexes (Michaels, 1965; Nakashima, 1974). The cationic Fe-Cac-f injected into the vein or peritoneal cavity was taken up by macrophages., e.g., Kupffer cells in liver or peritoneal macrophages, but not adsorbed to the endothelial cells, though some fibrin formation in lung capillaries was found by intravenous injection, as revealed by observing formol-fixed tissue sections

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stained by the immunohistochemical method for fibrin by Sternberger et al. (1970) (Akita, Ono, and Seno, unpublished data). When cationic probes are infused into vessels the excess cationic probes may meet with anionic serum proteins forming cationic polyions (Michaels, 1965), but they may also meet with serum proteins in the capillary wall in situ (Ryan and Karnovsky, 1976; Ryan ef al., 1978; Sonnenberg-Hatzopoulos et al., 1984; Copley, 1985) to form some neutral or anionic polyion complexes. The uncharged poly-L-lysine-serum albumin complexes injected or perfused into vessels of rats were deposited in the subendothelial and subephithelial spaces in the renal glomeruli (Tsujii et al., 1985). It is reasonable to suppose that uncharged complexes receive very few repulsive forces of the anionic barrier of capillaries and leak out into the extravascular spaces and infiltrate the anionic tissue spaces (Weening et al., 1983). According to Bohrer et al. (1978), Rennke et al. (1978), and Kanwar (1984) neutral dextran molecules of about 3 nm in hydrodynamic radius were much more permeable through normal rat glomeruli than anionic ones of the same size, though neutral but globular HRP was much less permeable than neutral dextran of similar size (Skehan, 1975). Thus, there is a possibility that the polycations injected into veins result in the formation of anionic serum protein-polycation complexes or uncharged ones, and anionic complexes will be taken up by macrophages and uncharged ones leak out from vessels. In order to detect ionized anionic sites on the cell surface and interstitial tissues, many investigations were made by injecting cationic probes intravenously, e.g., polyethyleneimine (Batsford et al., 1983; Barnes et al., 1984; Barnes and Venkatachalam, 1985), cationized ferritin (Kanwar and Farquhar, 1979a; Vogt et al., 1982; Tsujii et al., 1984a,b), and others (Vernier et al., 1983; Rollason and Brewer, 1984; Oite and Shimizu, 1985). But it needs to be confirmed in each experiment that the probes keep their positive charges in the blood. b. Intravenous Injection of Anionic Ferric Hydroxide Colloids. Intravenous injection of anionic Fe-ChS, 1 ml of the solution (0.15 mg Fe/100 g body weight), into rats did not induce any serious symptoms, and the liver tissues taken 24 hours after the injection revealed that Kupffer cells gave a distinct Prussian blue reaction but parenchymal cells and capillary endothelial cells did not (Seno et al., 1983a). The intravenous injection of Fe-PVS, 5 ml of the solution (0.1 mg Fe/ 100 g body weight), also induced no recognizable symptoms and the sections taken 24 hours after injection gave nearly the same result as did those from Fe-ChS injection (On0 and Awai, 1984). These facts again show that anionic colloids are taken up by macrophages irrespective of the chemical characteristics of the surface. In the glomeruli of these animals no extravascular deposits or leakage of colloid particles were observed. The results indicate that negatively charged macromolecules or colloids are taken up by macrophages and rarely leak from vessels.

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2. Perjiusion of Tissues with Polyions through Vessels a. Polycations. Perfusion of living tissues with various cationic probes through the aorta or vena cava after washing out the blood and possibly the attached plasma proteins has been done to detect anionic sites on capillary walls (Caulfield and Farquhar, 1976; Kanwar et al., 1980; Kelly and Cavallo, 1980; Kanwar and Rosenzweig, 1982a,b; Rosenzweig and Kanwar, 1982; Reale et al., 1983; Sibley et a l . , 1983; Simionescu, 1985), to follow the pathway of macromolecules through the vascular wall (Karnovsky, 1967, 1968, 1970; Bruns and Palade, 1968a,b; Simionescu et al., 1973, 1975; Sibley et al., 1983), and to analyze the function of the “charge barrier” to protein leakage (Rennke et al., 1978; Kanwar, 1984). Perfusion with cationic probes seems to give the most reliable information on the distribution or the sites of anionic groups on the vascular endothelial cells, since there are no possible artifacts by tissue fixation or by interaction of plasma proteins with the probes by intravenous injection. Reports on LM observation of tissues perfused with cationic probes are few. Our observations on rat tissues perfused with cationic Fe-Cac through the thoracic aorta showed the blue-stained vessel endothelia and capillaries by Prussian blue reaction, but no reaction product was found in extravascular tissues, indicating that the Fe-Cac particles bind firmly to the anionic sites of arterial and venous endothelium and capillary walls but do not infiltrate deeply (Seno et al., 1983a). Electron microscopic observations of the aorta revealed some agglomerated masses of Fe-Cac particles distributed irregularly on the endothelial cell surface (Fig. 9a). This is in contrast to that of the Fe-Cac staining of the endothelial surface of GA-fixed vessels, in which Fe-Cac stained the endothelial surface diffusely without agglomeration (Fig. 9b) (Ukita et al., 1983). The anionic groups in tissues are mainly from proteoglycans and are not fixed on the structure but move laterally in the lipid layer of the cell membrane (Singer and Nicolson, 1972). Therefore, the agglomeration of the proteoglycans seems to occur by binding with cationic particles or macromolecules (Skutelsky and Danon, 1976; Pietra et al., 1983; Marikovsky et al., 1985) by a similar mechanism of agglutination of lectin receptors on the tumor cell by binding the ligands (Nicolson, 1974). In glomeruli perfused with Fe-Cac, the ferric hydroxide particles were found on the luminal and abluminal surface of endothelial cells (Seno et al., 1983a-c). The agglomeration of the particles was not as marked here as in the aortic endothelium, probably being interfered with by fenestrae, but the particles showed some tendency to form small agglomerates. The endothelial cells were irregularly detached from the basement membrane with the deformity and irregularity of the fenestrae, which were seen clearly by scanning EM (Seno et al., 1983a). This resembles what is seen in glomeruli of aminonucleoside-induced nephrosis in rat (Avasthi and Evans, 1979). Such minute detachment of the

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FIG. 9. Electron micrographs of rat arterial endothelium. (a) The endothelium taken after perfusion with 10 ml of Fe-Cac solution, 0.09 mg Felml at 37"C, and fixed with 2% glutaraldehyde. (b) The endothelium fixed with 2% glutaraldehyde and stained with the Fe-Cac solution for 5 hours. Note the agglomeration of Fe-Cac particles on the endothelial cell surface by the perfusion with FeCac before fixation. CL, Capillary lumen.

endothelium from the basal layer is induced by the invasion of cationic probes into abluminal spaces of endothelial cells. Seiler and collaborators (1975, 1977) indicated the deposition of perfused protamine in the glomerular basement membrane with reduced anionic sites and morphologic changes of epithelia. In order to detect the pathway of macromolecules through the capillary endothelium, Karnovsky perfused cardiac and skeletal muscle capillaries of mice with HRP and cytochrome c revealing intercellular spaces of about 4 nm in width, suggesting the pathway for proteins (Graham and Karnovsky, 1966; Karnovsky, 1967, 1968, 1970). On the other hand Palade and co-workers found that the cytoplasmic vesicles or caveolae of endothelial cells worked for transcytosis of macromolecules by perfusion with cationized ferritin, HRP, and cationized hemepeptide (Bruns and Palade, 1968a,b; Simionescu er al., 1973, 1975; Ghinea and Simionescu, 1985). The leakage of anionic serum through capillaries, however, seems to be very small, because the protein contents of the tissue fluid are very low. Even at the excise of the limb, which results in a greatly increased flow of the lymph with the dilatation of arterioles by active hyperemia in the regional capillaries, the total protein contents in lymph did not increase (Anderson, 1980). A massive protein leakage in inflammation does not occur from capillaries but from venules by enlarged intercellular spaces of endothelial cells, as can be seen with histamine injection. Amano and collaborators were the first to show the leakage of carbon particles and trypan blue injected intravenously, which bind with serum proteins (Tasaki et al., 1969), from venules at the site of focal

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inflammation induced by the injection of staphylococci, histamine, sinomenine, terebene oil, and tubercle bacilli extract (Amano et al., 1950; Yasuhira and Yamamoto, 1950; Kawano, 1951; Amano, 1951,1963). Later Palade and Majno showed that the subcutaneous injection of histamine and serotonin induces the leakage of the intravenously injected carbon particles from venules, but not from capillaries (Majno and Palade, 1961; Majno et a l . , 1961). However, it is possible that serum proteins leak from fenestrated capillaries, if their anionic barriers have been inactivated by masking with cationic macromolecules or colloids. Many reports show a decrease in the density of anionic sites of glomeruli in cases of proteinuria (Michael et a l . , 1970; Blau and Haas, 1973; Bennett et a l . , 1976; Kelley and Cavallo, 1978, 1980; Kreisberg et a l . , 1979; Kanwar et al., 1980; Hunsicker et a l . , 1981; Camussi et a l . , 1982; Faraggiana et a l . , 1982; Cavallo et a l . , 1983; Cotran and Rennke, 1983; Mynderse et al., 1983; Vernier et a l . , 1983; Assel et a l . , 1984; Barnes and Venkatachalam, 1985; Koide et al., 1985). A liver perfused with Fe-Cac after washing out blood showed its capillary endothelium lined with Fe-Cac, indicating that anionic groups of the endothelium were masked with cationic ferric hydroxide colloid. Then the liver was perfused with blood from the portal vein to induce the changes comparable to the “serous inflammation” of liver induced by Eppinger in dogs by intravenous injections of a sublethal dose of histamine (Eppinger et a l . , 1935). But unexpectedly, no edematous changes were observed. Histochemical observations revealed the deposition of fibrin at the site of the positive Prussian blue reaction, indicating the formation of a new anionic barrier of the acidic protein, fibrin, at the luminal surface and abluminal spaces of endothelial cells (Seno et a l . , 1983a). The observation is consistent with that of Dvorak and others showing the fibrin formation in increased vascular permeability (Dvorak et a l . , 1985). b. Perfusion with Polyanions. Perfusion of the kidney with anionic macromolecules did not induce any detectable changes in the glomerular capillaries (Seiler et a l . , 1977). The perfusion of rat liver with Fe-ChS from the portal vein had no specific injurious effect to tissues. Particles were not adsorbed to endothelial cells or did not invade Disse’s spaces, but adhered to the surface of macrophages, the Kupffer cells, as revealed by EM of tissue sections fixed with 2% GA (Figs. 10a and b). The particles were in a line at a certain distance from each other and also from the lipid membrane surface, indicating the localization of polyanion receptor near the terminal end of the polypeptide chain of glycoproteins (Fig. lob) (Seno et a l . , 1983a; Ono and Awai, 1984). Recently, the existence of cationic groups on the endothelial cells of liver sinusoids was reported (Ghitescu and Fixman, 1984), but our anionic ferric hydroxide particles did not show the cationic groups. C. Perfusion with Polycation-Polyanion Complexes. The perfusion of rat kidney with a poly-L-lysine-albumin complex through the renal artery showed a

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FIG. 10. Electron micrographs of a Kupffer cell found in rat liver perfused with 5 ml of anionic Fe-ChS (0.2 mg Felml) at low temperature (10°C) through the portal vein. Fe-ChS particles are found on the surface of Kupffer cells at a certain distance showing the sites of the polyanion receptor (a). The enlarged picture (b) shows clearly the particles on the cell coat but not attached directly to the surface of the lipid membrane. K, Kupffer cell; En, endothelial cell; D, dense body; N, nucleus; M, mitochondria; CV, coated vesicles.

distinct subendothelial deposition in glomerular capillaries, indicating that masses of the complexes passed through an anionic barrier of the endothelial layer leaking into the extravascular spaces (Tsujii et al., 1985). It has been reported that as a result of perfusion of a rat kidney with protamin and subsequent perfusion with heparin, heavy subendothelial and subepithelial depositions of polyion complexes were induced in glomerular capillaries (Seiler et al., 1977, 1980; Sharon et al., 1977; Hoyer et al., 1982). The protamin molecules perfused through the artery settled in the abluminal spaces of endothelial cells of glomeruli and also in the subendothelial spaces. Subsequent perfusion with heparin into the vessels resulted in the formation of gross spherical deposits of protamin-heparin complexes in situ. The serum proteins, whose negative charges were neutralized by mixing with a cationic ferric cacodylate complex (a mixture of 10 ml of 0.1 M sodium cacodylate and 0.02 M FeCl, aqueous solution), penetrated through glomerular capillaries to be excreted into urinary spaces (Seno and Akita, unpublished data). All these facts seem to suggest a mechanism of renal deposition and extravascular leakage of the circulating uncharged macromolecules. This may explain the glomerular deposition of immune complexes, which have less ionized groups on the surface and tend to form precipitates (Border et al., 1981). The fact that the intravenous injection of cationic antigens gives rise to the glomerular deposition of immune complexes (Gallo et al., 1981, 1983) supports this view. Thus, results obtained with polyions introduced into vessels may be summarized as follows: polyanions are taken up by macrophages and polycations bind

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with anionic serum proteins, blood cells and endothelial cells with fibrin formation, and the uncharged polyion complexes penetrate into the extravascular spaces, as far as the surface charge of polyionic molecules and their ionic bondings are concerned except for the reactions mediated by specific receptors (Fig. 11). INTO LUNGS PARTICLES INTRODUCED C. IONICCOLLOIDAL

Respiratory tracts and alveoli are also covered with epithelial cells having an anionic coat of proteoglycans and glycolipids. Inhalation of cationic macromolecules or colloid particles may produce injurious effects on the respiratory system. Electron microscopic observations of the lung of a rat which inhaled the lead fume, cationic lead oxide powder (PbO), for I .5 hours, revealed lead oxide particles of 30 to 50 nm in endocytic vesicles of the alveolar epithelium, basement membrane, cytoplasmic vesicles of endothelial cells, capillary lumen, and on the red cell surface (Ogata et al., 1973). The nasal instillation test with cationic cacodylate ferric hydroxide colloid in isotonic glucose solution, pH 6.5 (Tanaka, 1974), gave results similar to the PbO particle inhalation. The cationic ferric hydroxide particles reached the alveolar cavity easily. A paraffin section of tissues taken 30 minutes after the instillation of 0.5 ml of the solution (2 mg Fe/ml) revealed an overall blue-stained alveolar epithelium and capillary walls by Prussian blue reaction. Electron microscopic observations revealed iron colloid particles endocytosed by alveolar epithelial cells and those invaded the basement membrane (Tanaka, 1974). The cacodylate

FIG. 11. Diagrammatic representation of the possible destinations of anionic (-), cationic (+), and uncharged (+ -) polyions introduced into vessels. CL, Capillary lumen; En, endothelial cells; F, fibrin; M, macrophage; PMN, neutrophil; R, red blood cells; BM, basement membrane.

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ferric hydroxide particles of Tanaka were large ones, but some iron colloid particles were found in endocytic vesicles of the alveolar epithelium, basement membrane, and vesicles of the capillary endothelium facing the basement membrane. But no colloidal particles were found in the capillary lumen as opposed to the cases of lead fume inhalation and nasal instillation of HRP (SchneebergerKelley and Karnovsky, 1968). Ferric hydroxide colloid may not enter vessels in a manner different from PbO powder, but heavy iron deposition in the liver and spleen compared to controls suggested the translocation of colloids into blood through a way similar to lead powder (Tanaka, 1974). In contrast, anionic iron colloid particles, Fe-ChS, instillated into the nasal cavity were actively taken up by macrophages but did not adhere to epithelial cells or invade the basement membrane (Tanaka, 1974). Thus, in the lung anionic colloid particles are taken up solely by macrophages, but some cationic ones, such as PbO particles, penetrated into the blood capillaries passing through the anionic barrier of epithelial cells and capillary walls. The PbO and Fe-Cac particles reached the basement membrane probably by vesicular transcytosis of the alveolar epithelial cells and then into the blood capillaries through transcytosis by the caveolae in the capillary endothelium. D. BASK PROTEINSINTRODUCED INTO THE INTESTINE

The intestinal mucosa is covered with thick acidic proteoglycans (Bloom and Fawcett, 1975). Therefore, anionic groups on the proteoglycans should have an affinity for cationic macromolecules. The porcine elastase, a basic protein of pl 9-10, introduced into rat jejunum, was found to be attached to the surface of epithelial cells covering a wide area of the luminal surface of villi (Tsujii et al., 1984). Histochemical observations of the regional intestinal mucosa by LM at 5 to 60 minutes after intrajejunal injection of 0.5% elastase solution in saline revealed that the elastase was on the surface of the apical area of villi 10 to 20 minutes after injection and in capillary lumen 60 minutes after injection. Electron microscopy revealed that the electron-dense elastase accumulated in the crypts at the bottom of microvilli and in the neighboring small vesicles in the cytoplasm 5 minutes after jejunal injection. The particles were also found in the intercellular spaces and basement membrane at this stage. Observations indicated the possibility that some basic proteins can be absorbed through the normal jejunal mucosa by endocytosis without breaking down into amino acids, as in the case of intestinal absorption of lysozyme in adult rats (Yuzuriha et al., 1973, 1975) and protein absorption in the intestine of newborns (Clark, 1959; Cardell et al., 1967; Graney, 1968; Cornell et al., 1971; Hemmings and Williams, 1977; Williams and Hemmings, 1978). This is comparable to that of intestinal lipid absorption by pinocytosis (Palay and Karlin, 1959).

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IX. Conclusion From the point of view that the cellular environment of multicellular organisms is the architecture of anionic polymers, the biological importance of electrostatic bondings between polyanionic and polycationic macromolecules has been stressed. Cells composing our body live in the aqueous environment of interstitial tissues with ionized anionic macromolecules of proteins and polysaccharides. Cell surfaces are also covered with ionized polyanionic acid polysaccharides. Such an anionic environment, which is important for normal life phenomena, can easily be disturbed irreversibly by the introduction of cationic macromolecules. In this article the characteristics of macromolecular anionic groups on the cell surface and the specific chemical reaction between polyanionic and polycationic macromolecules have been described to show how stable electrostatic bondings comparable to covalent bonds develop between these macromolecules. On a colloid chemical basis it is shown that ionized groups on cells and tissue components are detectable in situ by using cationic and anionic ferric hydroxide colloids visible by LM and EM, demonstrating the anionic groups on general somatic cells and the cationic ones on macrophages. The possible artifacts in the processing of cells and tissues for LM and EM have been discussed. Finally, cellular reactions against polyionic macromolecules or colloids introduced into living tissues through various routes are described. That is, subcutaneous injection of cationic macromolecules induces inflammatory changes with the uptake of macromolecules by all kinds of somatic cells by charge-related endocytosis, intravenous injection results in fibrin formation, and those introduced into the respiratory tract and intestinal lumen are easily absorbed through alveolar and intestinal epithelia. Anionic macromolecules injected into subcutaneous tissues are taken up specifically by macrophages without producing any injurious effects on other somatic cells. They are phagocytized by macrophages by polyanion receptor-mediated endocytosis. Macrophages also take up cationic macromolecules by charge-related endocytosis, but the endocytic vesicles fuse poorly with each other and also with lysosomes, unlike phagocytic vesicles. Uncharged macromolecules injected into vessels leak out into extravascular spaces, being deposited there or excreted into urinary spaces. These should be the basic biological reactions to be considered for the analysis of cellular and tissue reactions involved in the pathologic changes induced by the invasion of foreign substances into living tissues. ACKNOWLEDGMENTS

I am grateful to Dr. Hiroshi Shigei, Chairman of the Board of Shigei Medical Foundation, for his support of the research in the author’s laboratory. I also would like to thank Dr. T. Tsujii, Dr. T.

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Okigaki, Dr. K. Watanabe, and Dr. T. Ono for helpful discussions. Thanks are also due to Mr. M. Akita and Mrs. S. Inoue for their excellent technical assistance. In addition, the assistance of Miss N. Nishimoto in the preparation of this manuscript is gratefully acknowledged.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 100

Nucleocytoplasmic Interactions in Morphogenesis J. BRACHET Dkpartement de Biologie Molkculaire, Universitk Libre de Bruxelles, 1640 Rhode-St-GenPse, Belgium

I. Old Reminiscences The role played by the cell nucleus as a whole in morphogenesis is a very old problem; the most direct approach to its solution is merotomy, that is cutting an egg or a unicellular organism into two halves, nucleate and anucleate. Merotomy experiments were done almost a century ago by Verworn (1892), Balbiani (1888), Klebs (1889), Townsend (1897), Yatsu (1905), and others on eggs of various species, protozoa, and plant cells. As pointed out by Mazia (1952), the general conclusion is that “there is not a single case where an activity has not continued in an enucleated cell. However, the life span of anucleate cytoplasm, after merotomy, varies considerably from cell to cell: 1 or 2 days for cytoplasts of mammalian cells, about 10 days for amebae, 3 months or more for the giant unicellular alga Acetabularia. The survival of anucleate fragments of protozoa and eggs for a certain length of time came as a complete surprise to me when I attended, in 1927, the first lecture of a cytology course given by Pol Gerard. Naively, I believed that removing the nucleus should be the same as cutting off the head of a man. I was so fascinated by the problem that I decided to study it in the laboratory headed by my father, Albert Brachet, under the guidance of Albert Dalcq, who was then doing experiments on the effects of X-rays on eggs and sperm of the frog Rana temporaria. He showed, among other things, that injury to the egg or sperm nucleus impeded gastrulation; injury to both the male and female gamete nuclei allowed at best irregular cleavage (Dalcq and Simon, 1932). This suggested an essential role of the nucleus in early morphogenesis. But Albert Brachet was convinced of the major importance of the cytoplasm during these first stages of embryonic development: he was one of the pioneers of experimental embryology, studying at that time the effects of the destruction of a purely cytoplasmic area, the gray crescent (see Section IV) of fertilized frog eggs, on the formation of the nervous system. Earlier experiments on polyspermy in sea urchin eggs had shown him that the nuclei of the supernumerary spermatozoa assumed the same morphology (condensed or swollen) as that of the egg nucleus (see Section IV). He called this phenomenon “la mise 21 l’unisson” ”

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and concluded correctly that the morphology of the nuclei is controlled by cytoplasmic factors (Brachet, 1922). We were therefore convinced of the fundamental importance of the egg cytoplasm heterogeneity (polarity, germinal localizations such as the gray crescent area) for morphogenesis. In 1940, J . Pasteels (another student of Dalcq) showed that centrifugation of a fertilized frog egg disturbs the polarity gradient and leads to the production of microcephalic embryos (Fig. 1). In all these experiments (pricking of the gray crescent, centrifugation, etc.) no harm was done to the egg nucleus; nevertheless, dramatic developmental abnormalities followed. This led us to the belief that genes (if they really existed!) could control only late stages of development. For instance, while the color of Drosophila eyes is obviously under genetic control, the fonnation of the eye itself might not be directed by specific “eye genes.” This idea was made clear by A. Brachet (1930) when he drew a sharp distinction between a “general” heredity controlling early morphogenesis, involving both the nucleus and the cytoplasm, and a “special” heredity of the Mendelian type. It was not realized, at that time, that genes might direct oogenesis and affect the topological organization of the egg cytoplasm: mutations which exert maternal effects are now studied in many laboratories were unknown. I was very excited when I bought T. H. Morgan’s book, entitled “Embryology and Genetics” (1934), because Morgan had been a leading experimental embryologist before he became the father of modern genetics. Disappointment followed excitement until I thought that, after all, I should also read the preface

FIG. 1. Embryos obtained after moderate centrifugation of fertilized frog eggs. Microcephaly in (a) and (b), anencephaly in (c) and (d). The tails are better developed than the heads in embryos (a), (b), and (c) (Pasteels, 1940).

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of the book. There Morgan proposed a very important theory: the nuclei, in the cleaving egg, are genetically identical and have the same morphogenetic potentialities. These equipotential nuclei are distributed, during cleavage, in a heterogeneous cytoplasm. As a result, genetic activity (we would now say gene expression) increases in certain parts of the egg and decreases in others. These changes in genetic activity would modify the chemical composition of the surrounding cytoplasm, with new changes in genetic activity as a consequence. Finally, both the nuclei and the cytoplasm would become more and more differentiated in the various parts of the embryo as a result of continuous nucleocytoplasmic interactions. The final outcome would be organogenesis, followed by cell differentiation. This theory of Morgan impressed me greatly; it is seldom quoted today, although all embryologists now believe that embryonic development results from sequential gene activation. The year 1938 was important for me: while I was dissecting a frog ovary for the isolation of large and small oocytes (I was studying their respiration), I accidentally made a slit in the animal pole of a large oocyte. A translucent spherule popped out of the wound; it was the oocyte nucleus, the germinal vesicle (GV). This opened new possibilities for research and led me to a study (Brachet, 1939) of the distribution of hydrolases in the nucleus and the cytoplasm of frog oocytes and, more importantly, to measurements of the oxygen consumption of isolated GVs (Brachet, 1938). Their respiration was negligible in comparison to that of the enucleated oocytes. This disposed of the old theory of Loeb (1899) who had proposed, on the basis of inadequate cytochemical evidence, that anucleate cytoplasm dies because the cell nucleus is the main center of energy production. In 1938 I also made my first visit to the United States. I met T. H. Morgan in Woods Hole, Massachusetts, E. B. Wilson in New York, and I heard about their ideas on possible interactions between nuclear genes and cytoplasmic factors unevenly distributed in the cytoplasm. E. G. Conklin, in Princeton, New Jersey, told me more about the germinal localizations he had discovered in ascidian eggs. I was then working in E. Newton Harvey’s laboratory in Princeton and I was soon fascinated by the work of his wife, Ethel Brown Harvey, on nucleate and anucleate fragments of sea urchin eggs. Parthenogenetically activated anucleate halves undergo anarchic cleavage (Harvey, 1936) after formation of asters; E. B. Harvey hoped that such activated anucleate fragments could even hatch and form cilia. Although I had good eyesight in those days, I could never see hatching or ciliation, to her great disappointment. However, the possibility of separating on a large scale nucleate and anucleate halves of unfertilized sea urchin eggs for biochemical studies remained at the back of my mind. This possibility had already come to my mind when, at the request of A. Dalcq, I read and summarized for our “Journal Club” the fascinating paper published by J . Hammerling in 1934 on the alga Acetabularia. In this very long

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paper, written in German, he demonstrated that anucleate fragments of this large unicellular alga not only survive for months, but even regenerate species-specific “caps” (umbrellae); this proved that anucleate cytoplasm is capable of typical morphogenesis. During the summers of 1953 and 1954 I collected Acetabularia in the Mediterranean Sea and started cultures of them in Brussels; this is why these lines are written in a house located close to the Mediterranean Sea (where, due to pollution, Acetabularia is now much more difficult to find). In this article, the results obtained by merotomy experiments in Acetabularia and sea urchin eggs will be summarized and compared, and a discussion of the role played by the GV during Xenopus oogenesis and maturation will follow. Finally, the role of cytoplasmic determinants (germinal localizations) and nuclear determinants (genes) in early development will be examined.

11. Nucleocytoplasmic Interactions in Acetabufaria

The early work on Acetabularia of Hammerling and his co-workers was summarized by Hammerling himself in 1953; more recent reviews deal mostly with biochemical aspects (Schweiger, 1977; Schweiger and Berger, 1979; Brachet, 1981) or ultrastructure (Werz, 1974; Spring et al., 1974). Figure 2 summarizes the life cycle of the alga. After fusion of two swimming haploid gametes, the zygote forms a chloroplast-containing stalk that grows in length; it is attached to the substratum by rhizoids which contain the single nucleus. When the stalk reaches its full size, it forms a cap (umbrella) at its apical end; this cap has species-specific characteristics. When the cap has attained its full size, the large vegetative nucleus breaks down in the rhizoids; secondary nuclei divide repeatedly and colonize the cap which subdivides into cysts. When the thick envelope of the cysts breaks down, flagellated gametes are released into the sea. The life cycle takes about 4-6 months under laboratory conditions. The maximal length of the stalk (3 to almost 10 cm in A . mediterranea) depends upon the amount of light received by the algae (Beth, 1953b, 1955). Intense illumination produces algae with a short stalk and a large cap; insufficient light results in the formation of very long algae with small caps, even under natural conditions. The main results of Hammerling’s (1934) experiments are summarized in Fig. 3, which shows that anucleate fragments are capable of regeneration and that morphogenetic substances are distributed along a gradient which decreases from the apical to the basal end of the alga. Interspecific grafts (reviewed by Hammerling, 1953) conclusively demonstrated that the morphogenetic substances display species specificity and that they are produced by the nucleus. They must be “products of gene action, which stand between gene and character.” It is not known for certain how morphogenetic substances produced by the nucleus (in the

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FIG. 2. Life cycle of Acetabularia rnedirerrunea. (1) Zygote: (2-4) growing of the young alga; (4) first verticilla; (5) young cap; (6) adult cap, liberation of cysts; (7) resting cyst with many nuclei (white) and plastids (black); (8) germinating cyst (liberation of gametes); (9) conjugation.

FIG. 3. Diagrammatic gradient distribution of morphogenetic substances in Acetabularia (I. Brachet and A. Lang, Handbuch der Pljlunzenphysiologie 15, 22, 1965).

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rhizoid) accumulate at the apex of the stalk and distribute themselves along an apicobasal gradient. Centrifugation does not modify the polarity of the stalks (Sandakhiev et al., 1972). The migration of the morphogenetic substances toward the apex of the alga results from cytoplasmic streaming which can be inhibited by cytochalasin B. There is a strict correlation between inhibition of morphogenesis and inhibition of streaming (Puiseux-Dao et a l . , 1980). An important finding of Hammerling (1939, 1953) was the demonstration that division of the large vegetative nucleus depends on cytoplasmic factors. If a large cap is removed just prior to division of the vegetative nucleus, nuclear division does not take place until a new large cap is formed; mitosis can be delayed indefinitely if the operation is repeated. Conversely, nuclear division is accelerated if a young rhizoid is grafted on a stalk containing a large cap. Ultrastructural studies by Berger and Schweiger (1975a,b) have shown that an old nucleus is morphologically rejuvenated (there is an increase in the development of the nucleoli) after transplantation into a young cytoplasm; the converse experiment leads to premature aging of the young nucleus. Hammerling (1934, 1953) and Beth (1953a) reported the important fact that anucleate fragments of Acetabularia form caps more rapidly than complete algae: this shows that the nucleus exerts negative as well as positive (production of morphogenetic substances) controls on the cytoplasm. The interactions between nucleus and cytoplasm are thus more subtle than was hitherto believed and they take place in both directions. In fact, the ultrastructure of the perinuclear space is particularly complex (Fig. 4): a complicated ‘‘labyrinthum” surrounds the large vegetative nucleus (Boloukhi?re, 1965; Werz, 1974; Franke et al., 1976). Enucleation has little effect on energy production and photosynthetic activity for at least 4 weeks (Brachet et al., 1955; Craig and Gibor, 1970). Lack of energy production is thus not responsible for the slowing down of regeneration in anucleate halves after 2 to 3 weeks of culture under standard conditions of illumination. Anucleate fragments of Acetabularia retain a photosynthetic circadian rhythm for several weeks. However, it is the nucleus which “sets the clock,” as was shown by experiments in which nucleate and anucleate halves of algae, which were at opposite phases of the cycle, were combined (Schweiger et al., 1964). In other experiments, fragments of algae that had lost their rhythm of photosynthetic capacity were combined with anucleate ones that had retained it, and vice versa (Vanden Driessche, 1967): the presence or absence of the rhythm was nucleus dependent. The rhythm is probably controlled by transcription of nuclear genes, since actinomycin D abolishes it in nucleate but not in anucleate halves (Vanden Driessche, 1966). Inhibitors of chloroplastic and mitochondria1 RNA and protein synthesis (rifampicin, chloramphenicol) have no effect on the circadian rhythm of photosynthesis, but the rhythm is suppressed by inhibitors of cytoplasm protein synthesis (puromycin, cycloheximide) in both nucleate and anucleate fragments. Thus, an apparently typical chloroplastic function (the cir-

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FIG.4. Labyrinthic form of the perinuclear cytoplasm in Acetabularia rnedirerranea. (Electron micrograph courtesy of Dr. M. Boloukhkre.)

cadian rhythm of photosynthesis) is controlled by protein synthesis on 80 S cytoplasmic ribosomes (Mergenhagen and Schweiger, 1975; Karakashian and Schweiger, 1976; Hartwig et al., 1985). The reason I collected Acetabularia in the Mediterranean Sea and grew them in Brussels was that I wished to know the answer to two questions. Are anucleate halves of Acetabularia capable of protein synthesis? If so, can they synthesize specific proteins, presumably encoded by nuclear genes, such as enzymes? As shown in Fig. 5, net protein synthesis takes place in both kinds of fragments; during 2 to 3 weeks, it is even stronger in anucleate than in nucleate halves, confirming the existence of negative controls of the nucleus on the cytoplasm. Afterward, net protein synthesis stops in the anucleate fragments, although incorporation of I4CO, into the proteins persists for at least 7 weeks (Brachet et

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FIG. 5 . Protein synthesis in nucleate (N) and anucleate (A) halves of Acetabularia under optimal conditions. (From Brachet et al., 1955.)

al., 1955). These findings suggest that net protein synthesis is correlated with the initiation and growth of the anucleate caps. When they stop growing after 3 weeks, protein degradation compensates protein synthesis; protein turnover continues for a considerable time in the absence of the nucleus. Among the newly synthesized proteins in anucleate halves are a number of enzymes, as was first shown by Baltus (1959) for aldolase. The early work on enzyme synthesis in Acetabularia has been discussed in a detailed review (Brachet, 1968). The results obtained in different laboratories showed that many enzymes with unrelated functions (phosphatases, UDPG pyrophosphorylase, thymidylate kinase, etc.) increase greatly in activity at the time of cap formation whether or not the nucleus is present. More recent work on the regulation of enzyme synthesis in the absence of the nucleus will be discussed later. The next question was whether or not anucleate fragments of Acetabularia are capable of net nucleic acid synthesis. The question was an important one, because there were great doubts in 1950-1956 about the nuclear origin of cytoplasmic RNA. The problem was discussed at length in my book “Biochemical Cytology” (1957, pp. 349-351). My final conclusion was that “intensive RNA synthesis certainly occurs in the nucleus; part of this RNA probably goes into the cytoplasm. But independent RNA synthesis is also possible.” This last sentence resulted from our own work on Acetabularia (Brachet et al., 1955) showing that there is a net RNA synthesis during the first week after the removal of the nucleus. Our results were not generally accepted before they were confirmed in Hammerling’s laboratory by Schweiger and Bremer (1961) (for further discussion of this early work, see Brachet, 1968). It was a great surprise for me when we found (Brachet et al., 1955) that there is also a net DNA synthesis in anucleate fragments of Acetabularia: both DNA and RNA increase as much as two- to threefold when anucleate fragments form

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large caps. The curves obtained for the two nucleic acids were almost identical to that shown in Fig. 5 for protein synthesis. DNA synthesis in the absence of the nucleus was, around 1960, a baffling mystery. The only explanation I could find was that Acetabularia chloroplasts might contain DNA and that this chloroplastic DNA could replicate in the absence of the nucleus. Our work on chloroplasts isolated from anucleate fragments of Acetabularia unequivocally demonstrated the reality of chloroplastic DNA (Baltus and Brachet, 1962). Soon afterward, Shephard (1965), working then in my laboratory, found that both number and size of the chloroplasts in anucleate fragments of Acetabularia increase; however, this increase is still larger in the nucleate halves of the alga. At that time, we found (Goffeau and Brachet, 1965) that chloroplasts isolated from anucleate halves are capable of protein and even of RNA synthesis. In the 1960s, these findings were both surprising and exciting. But it appeared that chloroplasts in Acetabularia were a serious nuisance for those who tried to identify the mysterious morphogenetic substances. Is the synthesis of chloroplastic macromolecules responsible for cap formation although the biological evidence was that the morphogenetic substances originate from the nucleus? This question was tackled in several laboratories using specific inhibitors (reviewed by Brachet, 1968) and the answer was “No.” Inhibitors of chloroplastic DNA, RNA, and protein synthesis do not affect the initiation of cap formation but slow down the cap growth. Inhibitors of nuclear RNA synthesis (actinomycin D, cordycepin) arrest regeneration in nucleate halves and have little effect on anucleate halves. RNase treatment, which is expected to destroy preformed cytoplasmic RNAs, prevents morphogenesis in both kinds of fragments. The conclusion, as shown diagrammatically in Fig. 6 , was that the integrity of preexisting RNAs is required for morphogenesis in anucleate halves; in their nucleate counterparts, continuous nuclear RNA synthesis is necessary for growth and morphogenesis. Finally, numerous experiments with puromycin and chloramphenicol demonstrated that cytoplasmic, but not chloroplastic, protein synthesis is required for growth and regeneration in both kinds of fragments. These experiments led me to the conclusion (Brachet, 1960) that “specific DNA molecules (or parts of molecules), corresponding to each gene, would act as a template for RNA synthesis; there would be as many specific RNA molecules as there were genes. Finally, each specific RNA molecule would act as a template for a specific protein.” Soon after these sentences were written, the concept of messenger RNAs was developed by Jacob and Monod (1961) working on Escherichia coli. Today it is accepted by all that Hiimmerling’s morphogenetic substances are a family of mRNAs synthesized in the nucleus; they display great stability in the cytoplasm, in contrast to bacterial mRNAs. Are the chloroplasts fully autonomous organelles, uninfluenced by the nuclear genome? Elegant interspecific grafting experiments by H. G . Schweiger and his colleagues have shown that the answer to the question is “No.” The nucleus

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FIG. 6. Diagram of mRNA distribution in Acetabularia (black areas). (a) Normal algae, whole and cut into two halves; (b) algae treated by ribonuclease before cutting; and (c) algae treated by actinomycin.

exerts a control on the chemical composition of the chloroplasts. When a nucleate half of a given species is grafted on an anucleate stalk from another Acetabularia species, the isozyme pattern of lactic and malic dehydrogenases becomes that of the nuclear type after a few days (Schweiger et a l . , 1967). Similar results were obtained for insoluble chloroplast proteins (Apel and Schweiger, 1972, 1973; Kloppstech and Schweiger, 1973). Analysis of protein synthesis in isolated chloroplasts treated with chloramphenicol or cycloheximide further showed that two out of the three major membrane proteins of the chloroplasts are synthesized on both 70 S (chloroplastic) and 80 S (cytoplasmic) ribosomes, while the third is synthesized on the 80 S ribosomes only (Apel and Schweiger, 1973). These experiments demonstrated conclusively that the chloroplastic and nuclear genomes cooperate in order to specify the protein composition of the chloroplasts. We have seen that many enzymes increase in activity when caps are formed, even in anucleate halves where transcriptional control at the level of the nuclear genes is of course impossible. Particularly interesting is the regulation of the enzymes involved in DNA synthesis. Thymidine kinase activity increases considerably at the stage where the large vegetative nucleus breaks down and gives rise to the lo8 daughter nuclei present in the multinucleate cysts of the cap. Surprisingly, the same increase in thymidine phosphorylation occurs when anucleate halves form caps (Bannwarth and Schweiger, 1975), thus under conditions in which multiplication of the secondary nuclei cannot take place. Experiments with inhibitors showed that thymidine kinase is synthesized on 70 S chloroplastic ribosomes (Bannwarth et a l . , 1977). The same situation was found for another enzyme involved in DNA synthesis, deoxycytidine monophosphate

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deaminase (Bannwarth et al., 1982; Bannwarth and Schweiger, 1983). Analysis of the experimental results led Bannwarth and Schweiger (1983) to the conclusion that the enzyme is synthesized in the chloroplasts and that the nucleus suppresses cap-specific information in the cytoplasm; the hypothetical nuclear suppressor is believed to have a short life. However, another enzyme involved in DNA synthesis, thymidylate kinase, is synthesized by the nuclear genome and translated on 80 S cytoplasmic ribosomes. Its activity greatly increases when caps grow, even in anucleate halves where its mRNA is stable for more than 20 days (De Groot and Schweiger, 1983). Finally, De Groot and Schweiger (1985) reported that ribonucleotide reductase activity strongly increases, in both halves, at the time the vegetative nucleus breaks down and gives rise to fast dividing secondary nuclei. The increase in activity is faster in anucleate halves, suggesting again that the nucleus exerts a negative control on enzyme synthesis. Chloroplastic DNA (reviewed by Luttke and Bonotto, 1982) has been well characterized by physicochemical methods and electron microscopy (Green et al., 1967, 1977; Green, 1973; Luttke and Bonotto, 1980; Ebert el al., 1985; Tymms and Schweiger, 1985). It is made up of long (up to 400 pm) fibrous molecules and small circles ( 5 pm) in contrast to the 40 km circles found in the chloroplasts of Euglena and of higher plants. Acetabularia chloroplastic DNA possess about 10 times more DNA than spinach chloroplasts; its kinetic complexity is greater than that of any other chloroplastic DNA so far studied, although it contains tandemly repeated sequences. Thus Acetabularia is not only the largest unicellular organism in the world; it also possesses the largest chloroplastic DNA molecules so far known. Chloroplastic DNA replication is inhibited by hydroxyurea and ethidium bromide; these inhibitors do not inhibit morphogenesis, but slow down the growth of already initiated caps (Heilporn and Limbosch, 1971a,b). Many chloroplasts, especially in the rhizoids, do not contain enough DNA to stain with the highly sensitive DNA fluorescent stain DAPI; the DNAcontaining chloroplasts are distributed along the classic apicobasal gradient (Luttke and Bonotto, 1980; Luttke, 1981). The large (up to 300 pm in diameter) vegetative nucleus of Acetabularia contains much less DNA than the chloroplasts; in contrast, it has very conspicuous, RNA-rich nucleoli (Fig. 7). Spring et al. (1974, 1975, 1976, 1978) discovered that the vegetative nuclei of all Acetabularia species possess Iampbrush chromosomes very similar to the classic lampbrush chromosomes of amphibian oocytes: loops that are 20 pm long extend from condensed chromomeres with a diameter of 1-2 pm (Spring et al., 1975). According to Spring et al. (1978), the gametes and the zygote of Acetabularia contain 0.9 and 1.8 pg of DNA, respectively, whereas the large vegetative nucleus contains 2.6 pg of DNA. Thus, this nucleus, despite its large size, is not polyploid. The moderate increase in DNA content between the zygote and the full-sized vegetative nucleus is due, as we shall see, to amplification of extrachromosomal (nucleolar) genes. The nucleus

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FIG.8. Division of the primary nucleus of Acetabularia in situ (Koop er a / . , 1979). Typical intranuclear spindle in a dividing nucleus of A. clifonii. The inset shows a somewhat different type of spindle (Nomarski interference contrast optics). pg, Polyphosphate granules.

shrinks when the alga forms a cap; this shrinkage is followed by the formation of the giant intranuclear spindle shown in Fig. 8 (Koop et al., 1979). As pointed out by Spring et al. (1975) and Koop et al. (1979), the Acetabularia giant vegetative nucleus, like the germinal vesicle of oocytes, is probably in meiotic prophase and its huge spindle is a meiotic spindle. The haploid nuclei resulting from meiotic division repeatedly divide and ultimately give rise to the gamete nuclei in the cysts. At the time of cyst formation, the overall cell surface increases several times, due to membrane invagination (as in cleaving eggs). At first glance, an Acetabularia and an egg have nothing in common except that they are both living creatures. Yet their initial processes of development are fundamentally the same. Another similarity between the Acetabularia vegetative nucleus and the oocyte’s germinal vesicle is an amplification of the ribosomal genes located in the nucleolar organizers (Spring eta!., 1974; Trendelenburg et al., 1974; Berger and Schweiger, 1975a,b). Electron microscopy of spread nucleoli has shown

FIG.7. Nucleolus of Acetabuluriu mediterraneu. (A) Ribbonlike structure seen with the light microscope after Unna staining (J. Brachet, 1965). (B) Autoradiograph of the rhizoid ([3H]uridine pulse); the nucleolus is strongly labeled. (C) Ultrastructure of the nucleolus. (Panels A and B courtesy of Dr. M. Boloukh2re.)

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that, as in amphibian oocytes, fibril-covered matrix units alternate with untranscribed spacers, giving the classic Christmas tree configuration to the ribosomal cistrons (Fig. 9). According to Spring et al. (1978), an Acetabularia nucleus contains about 4000 copies of the 28 S and 18 S rRNA genes. Amplification thus remains moderate; it probably takes place soon after zygote formation. The spacers are, on the average, much smaller than in Xenopus oocytes: the size of the rRNA precursor is only 2.3 X lo6 daltons, about half of the size of this precursor in mammalian cells. Since the 28 S and 18 S rRNAs have the same size in Acetabularia as in other cells, it follows that processing of the rRNA precursor is much more economical in the alga than in our own cells: only a 300,000-dalton piece of RNA is “wasted” in Acetabufaria as compared to more than 2 million daltons in mammalian cells. The rate of rRNA precursor synthesis is 4 X lo7 nucleotides per second per nucleus in intact algae; after removing part of the stalk, the rate of rRNA synthesis increases 20 times within 3 days and reaches a maximum 5-7 days after section. Ribosomal RNAs are very stable in the alga (half-life of 80 days) and are transferred from the rhizoid to the apex at a rate of 2.4 mm/day (Kloppstech and Schweiger, 1975a; Schweiger, 1977; Schweiger and Berger, 1979). As one might expect, the Acetabularia vegetative nucleus contains the two classic RNA polymerases I and I1 required for the synthesis of the rRNAs and mRNAs, respectively (Brandle and Zetsche, 1977). Kloppstech and Schweiger (1975b) have some insight about the poly(A)+ mRNAs synthesized on the loops of the Acetabularia lampbrush chromosomes. This was possible because chloroplastic mRNAs have no 3’-poIy(A) terminal sequence. They found that, as expected, polyadenylated RNAs are synthesized only in nucleate halves in contrast to total RNA (whidh is mainly chloroplastic and is synthesized in both halves). The poly(A)+ RNAs of Acetabufaria are polydisperse with an M, ranging from 0.5 to 3 X lo6. They migrate from the nucleus to the apex of the stalk at a speed of 5 mm/day, thus somewhat faster than the rRNAs. The rate of poly(A)+ RNA synthesis increases only two- to threefold during regeneration. This led Schweiger (1977) to conclude that transcription of chromosomal DNA plays only a limited role in morphogenesis. Some, if not all, of the poly(A)+ RNAs are authentic mRNAs: Shoeman et al. (1983) obtained their in vitro translation into 77 different proteins. They also found that, at a given stage of development, the translation of some of these proteins is not regulated, while that of others may be turned off or increased. The half-life of the poly(A)+ RNAs that are synthesized in the nucleate halves is FIG. 9. Structure of nucleolar organizer in Acetabularia (Trendelenburg er a / ., 1974). (a) Wellspread nucleolar material showing the regular pattern of matrix units and “spacer” segments. (b) Occasional groups of small fibrils associated with some of the spacer regions (arrowheads). (c) At higher magnification the dense packing of dense fibrils within the matrix is shown. Scales indicate 1 fim .

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about 10 days: they are thus much less stable than the rRNAs. Their stability is greater in anucleate than in nucleate halves, suggesting once more that the nucleus exerts negative effects on mRNA stability (Kloppstech and Schweiger, 1982). It is not known whether poly(A)- mRNAs are produced by the Acetabularia vegetative nucleus; if so, one would expect them to have a still shorter half-life than the poly(A) mRNAs. Neuhaus et al. (1984) isolated the vegetative nucleus and fused it with chloroplast-containing cytoplasts obtained by fragmenting the stalk of the alga. Injection of a DNA-containing virus (SV40) into these reconstituted cells was followed by the appearance of the virus-specific T antigen in the Acetabularia nucleus. If, as concluded by Schweiger (1977), transcription of chromosomal DNA plays only a limited role in morphogenesis, we are left with one hypothesis about the chemical nature of the morphogenetic substances: they would be a store of mRNAs accumulated in a stable form during growth of the alga, along an apicobasal gradient. The unusual stability of the stored mRNAs might be due, as in sea urchin eggs, to binding to proteins that protect RNA against intracellular RNases; mRNA activation could result from protease activity. In agreement with this hypothesis, I found (Brachet, 1977) that protease inhibitors completely prevented cap formation in both kinds of fragments. Experiments where nucleate and anucleate fragments were treated with inhibitors of morphogenesis [continuous darkness (Brachet et al., 1955), inhibitors of polyamine synthesis (Brachet et al., 1978, and unpublished results)] have shown that, after prolonged treatments, inhibition becomes irreversible in anucleate halves. Morphogenesis is resumed in nucleate halves after a lag depending on the length of treatment with the inhibitors. Arrest of development in anucleate halves is probably due to the exhaustion of the stored morphogenetic substances. The experiments showed that arrest of morphogenesis in anucleate halves becomes irreversible if the length of treatment exceeds 2-3 weeks. This suggests that the half-life of the morphogenetic substances is about 10 days, which is identical to the half-life of the poly(A)+ RNAs according to Schweiger (1977). This finding brings circumstantial evidence for the view that Hammerling’s morphogenetic substances are a family of mRNAs and the proteins they express. However, how such a family of mRNAs and their translation products can produce a structure as complex as an Acetabularia species-specific cap remains a mystery. +

111. Nucleate and Anucleate Fragments of Sea Urchin Eggs

Unfertilized sea urchin eggs can be cut into two halves (nucleate and anucleate) by high-speed centrifugation in a density gradient (Harvey, 1933). Both nucleate and anucleate fragments can be fertilized or treated with parthenogenetic agents.

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A description of the morphological (reviewed by Schatten, 1984) and biochemical (reviewed by Epel, 1978; and Vacquier, 1981) changes which take place at sea urchin egg fertilization is outside the scope of this article. The most important ones are the cortical reaction (formation of a fertilization membrane by exocytosis of the cortical granules), fusion of the pronuclei (amphimixy), changes in the membrane potential, increases in free calcium ions, in the intracellular pH as a result of a N a + / H + exchange, and in oxygen consumption, initiation of DNA replication preceding cell division, and strong increase in protein synthesis. Details about these and other events can be found in Brachet (1985). Only few of these events have been studied in nucleate and anucleate fragments of unfertilized sea urchin eggs. Both halves contain, except for the pronucleus and the pigment (Fig. lo), all of the major egg constituents, but their relative proportions are altered by the centrifugation step. The light nucleate halves contain more lipids, ribosomecoated vesicles, and mitochondria than the heavy anucleate halves, which possess most of the yolk. Activation by treatment with hypertonic seawater is followed, in both halves, by a typical cortical reaction. Both can give rise to almost normal larvae (plutei) after fertilization. After parthenogenetic treatment by the method of Loeb (successive treatments with hypertonic seawater and butyric acid), only the nucleate halves can produce larvae. Anucleate halves can only form asters and undergo irregular cleavage (Fig. 11) as the result of anarchic fragmentation (Harvey, 1936); such blastulae do not hatch and never form cilia, suggesting that they are unable to synthesize the hatching enzyme and to assemble correctly the molecular constituents of cilia. Similar results were obtained by Lorch er al. (1953), who removed the nucleus of one of the blastomeres during early cleavage of fertilized sea urchin eggs. As one can see, development of sea urchin eggs deprived of a nucleus is extremely limited as compared to cap formation in anucleate fragments of Acetabularia.

FIG. 10. High-speed centrifugation of unfertilized Arbacia eggs in a density gradient leads to sedimentation of the various organelles in the egg. The centrifuged egg takes a dumbbell shape and finally breaks into two fragments. The light fragment contains the nucleus, lipids, mitochondria, a little yolk, and the major part of the hyaloplasm. The heavy fragment is anucleate; it contains mitochondria, most of the yolk and, at the centrifugal end, red pigment granules. (Drawn by P. Van Gansen, after Harvey, 1936.)

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FIG. 11. Development of parthenogenetically activated anucleate fragments of sea urchin eggs. (a) Anucleate half at the time of activation; (b) anucleate morula after 13 hours; (c) abnormal anucleate blastula after 3 days. (Redrawn, after Harvey, 1936.)

Similar results have been obtained with amphibian eggs, where cleavage in the absence of the nucleus has been reported by Dalcq and Simon (1932), Fankhauser (1934), and Briggs et al. (1951). In general, only partial blastulae, where only the animal pole has cleaved, are obtained when both the egg and sperm nuclei have been destroyed in fertilized eggs. Briggs et al. (195 1) transplanted nonnucleated fragments to inductive sites of normal embryos; the grafts survived up to 4 days but showed no sign of differentiation, and close contact of the anucleate cells with normal nucleate cells did not allow morphogenesis. Both sea urchin and amphibian eggs contain a large store of many kinds of poly(A) RNAs. This large, complex population of mRNAs is theoretically capable of coding for as many as 15,000-30,000 different proteins of average size (reviewed by Hough-Evans and Anderson, 1981; Davidson et al., 1982). These maternal mRNAs differ from classical mRNAs by containing polyadenylated transcripts of repetitive and nonrepetitive DNA sequences: they are more similar to nuclear mRNA precursors than to the mature mRNAs found in the cytoplasm of adult cells. There is little RNA synthesis in freshly fertilized sea urchin eggs; fertilization and early cleavage are not affected by actinomycin D and it is therefore unlikely that newly synthesized mRNAs play an important role in these early embryological events (Gross and Cousineau, 1964). Actinomycin D-treated fertilized sea urchin eggs, like the parthenogenetically activated anucleate halves, form early blastulae that are unable to hatch and form cilia. In view of the great deal of information stored in unfertilized sea urchin and amphibian eggs, it is somewhat surprising that anucleate eggs cannot do better than give rise to partial anarchic morulae or blastulae. Experiments currently being done in our laboratory indicate that the poor development of parthenogenetic anucleate halves is not due to a lack of maternal mRNA sequences (as a result of centrifugation) in the heavy fragments, or to decreased stability of the stored mRNAs in the absence of the female pronucleus. +

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It is clear that, in order for a blastula to hatch and form cilia, fresh information should be added to that which was stored in the unfertilized egg. This information must come from the mRNAs which are synthesized during cleavage and which, as we have seen from the actinomycin D experiments, are not required for cleavage itself. From a philosophical viewpoint, one might say that the maternal mRNAs and their translation products represent, in molecular terms, the “preformation” of the eighteenth century embryologists. It is clear that preformation, which was once believed to allow the full development of the egg into an adult, amounts to very little since it allows, at best, reaching an early blastula stage. Further morphogenetic processes result from “epigenesis,” that is, expression of genes which had not been transcribed during oogenesis. Anarchic cleavage after parthenogenetic stimulation of anucleate halves results from aster formation (Harvey, 1936); anucleate fragments are thus capable of microtubule assembly. Cytasters, centered on a small centriole that has apparently formed de novo,appear in anucleate halves treated with heavy water (D,O) according to Kato and Sugiyama (1971). In intact sea urchin eggs, parthenogenetic agents which act in a single step induce the formation of monasters devoid of centrioles. Two-step methods, for instance that of Loeb, induce cytasters centered on one up to eight centrioles (Moy et al., 1977; Miki-Noumura, 1977; Kuriyama and Borisy, 1983; Kallenbach, 1983). It might be rewarding to extend these studies to anucleate fragments of sea urchin eggs treated with various parthenogenetic agents in order to ascertain that centrioles can form de novo in the absence of the nucleus. Yoneda et al. (1978) have shown by microcinematography that, after activation with butyric acid, anucleate fragments of sea urchin eggs undergo periodic tension and relaxation of their cortex. Although such fragments do not cleave, they display the same changes in contractility of their cortical layer as cleaving fertilized eggs. Similar observations have been made on starfish oocytes and eggs (Yamamoto and Yoneda, 1983) and amphibian eggs (Sawai, 1979; Hara et al., 1980). The same rhythmic contractions were found in anucleate fragments of fertilized Xenopus eggs and in whole eggs, and it was concluded that a cytoplasmic clock controls the cell cycle. However, the rigidity cycle is slower in anucleate fragments of fertilized Xenopus and sea urchin eggs than during the normal cleavage cycle (Sakai and Kubota, 1981; Shinagawa, 1983; Yoneda and Yamamoto, 1985). The factor responsible for the rounding-up-flattening cycle is located in the egg endoplasm (Shinegawa, 1985). Autonomous contractility of the cortical layer for a long time seems to be a general phenomenon; whether it is always due to a cytoplasmic clock that has the same period as the mitotic cycle in fertilized eggs remains to be seen. Shapiro (1935) showed that oxygen consumption of anucleate halves of Arbacia eggs is much higher than that of their nucleate counterparts (Fig. 12). Ballentine (1939) found that anucleate halves possess 70% more dehydrogenases

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10

30

50

70

90

110

Time in minutes

FIG. 12. Oxygen consumption is higher in heavy (anucleate) than in light (nucleate) halves of unfertilized sea urchin eggs. (After Shapiro, 1935a.)

than nucleate fragments of equal volume. This finding provided an explanation for Shapiro’s (1935) results. However, a more likely explanation for the higher respiration of the anucleate halves is the accumulation at the centrifugal pole of echinochrome-containing granules: KCN-insensitive oxidation of echinochrome leads to H,O, production (Perry and Epel, 1981) and it is now well established that the increase in oxygen consumption at fertilization is essentially due to the formation of H,O (Foerder et al., 1978). The work on protein synthesis in anucleate fragments of Arbacia eggs once had its importance and still retains historical interest. As in intact unfertilized eggs, the rate of protein synthesis is very low in both kinds of fragments; if these fragments are activated by treatment with hypertonic seawater, strong protein synthesis is observed in both halves (Brachet et al., 1963; Denny and Tyler, 1964; Baltus et al., 1965). Since this stimulation is even stronger in anucleate than nucleate halves, the results completely ruled out the possibility that the increase in protein synthesis that takes place at fertilization could be due to the de novo synthesis of mRNA molecules by the pronuclei. It was further shown that the number of polyribosomes greatly increases in anucleate fragments after activation with hypertonic seawater (Bumy et al., 1965). This proved that preexisting maternal mRNAs become capable of binding to the egg ribosomes after activation. These experiments were the first to lead to the conclusion that unfer-

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tilized sea urchin eggs contain a store of masked mRNAs that were synthesized during oogenesis. Many proteins are synthesized when anucleate fragments of Arbacia eggs are activated. Our own preliminary results indicate that their number is higher in anucleate than in nucleate halves; the former still synthesize a very large number of proteins many hours after the nucleus has been removed. Two specific mRNAs have been identified among the maternal mRNAs stored in anucleate fragments of Arbacia eggs: they code for tubulin (Raff ef al., 1972) and histones (Gross et al., 1973; Skoultchi and Gross, 1973), respectively. Tubulin mRNA stored in unfertilized eggs is not translated in anucleate halves unless the latter have been activated. It is not known whether translation of tubulin mRNA is required for the formation of asters in activated anucleate halves. Since unfertilized sea urchin eggs contain a large store of unpolymenzed tubulin dimers, it seems likely that aster formation after activation results from the production of microtubule-organizing centers (MTOCs) rather than an increase in tubulin resulting from maternal tubulin mRNA translation. It seems likely that the lack of cilia formation in activated anucleate halves does not result from an insufficient amount of tubulin since, according to Bibring and Baxandall (198 I), only 2-4% of ciliary tubulin is synthesized during cleavage. It is more probable that some minor proteins required for cilia assembly are missing in activated anucleate halves. Arceci and Gross (1977) have demonstrated that histones are synthesized on maternal mRNAs when anucleate fragments of Arbacia eggs are activated. In this case, there is no coordination between histone synthesis and chromosomal DNA replication; the significance of histone synthesis in the absence of the nucleus is an enigma. According to Showman et al. (1982), in contrast to total RNA, total poly(A)+ RNA, total poly(A)- RNA, actin mRNA, and a-tubulin mRNA, histone mRNA is accumulated in the nucleate halves of centrifuged Strongylocentrotus eggs. Accumulation of histone mRNA in nucleate halves was observed even when noncentrifuged, unfertilized eggs had been cut manually into two halves. It is not clear whether histone mRNA is accumulated in the female pronucleus. Showman et al. (1982) found little histone mRNA in isolated pronuclei, but De Leon et al. (1983), using in situ hybridization, observed a strong accumulation of histone mRNAs in the female pronucleus of whole sea urchin eggs; these mRNAs are shed in the cytoplasm at first cleavage. The opposite results of Showman et al. (1982) are probably due to leakage of the histone mRNAs during isolation of the pronuclei. Polyadenylation of the preexisting mRNAs occurs in anucleate halves after activation with hypertonic seawater (Wilt, 1973) or ammonia (Wilt and Mazia, 1974). The enzyme responsible for polyadenylation [poly(A)polymerase] is accumulated in the heavy, anucleate halves after centrifugation (Slater et a l . , 1978); the same maternal mRNAs are polyadenylated in vitro in whole fertilized

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eggs and in anucleate fragments (Slater and Slater, 1979). In the latter, the poly(A) tail cannot serve for the transfer of mRNAs from the nucleus to the cytoplasm. It might, as in Xenopus oocytes (Huez et al., 1974), stabilize the maternal mRNAs and protect them against degradation by cytoplasmic enzymes. Anucleate halves display strong RNA polymerase I1 activity (Morris and Rutter, 1976). The role of this enzymatic activity is unknown. It seems unlikely that it is involved in mitochondrial RNA synthesis since mitochondria possess their own RNA polymerase, which differs from the nuclear enzymes. Anucleate fragments synthesize, after activation, small amounts of mitochondrial RNAs whose sedimentation constants are 11, 13, and 15 S (Chamberlain and Metz, 1972). The 11 S and 13 S species are mitochondrial rRNAs; the mitochondrial 15 S RNA might be either a precursor of mitochondrial rRNA or a mitochondrial mRNA. Selvig et al. (1972) found that, in anucleate fragments of Arbacia, mitochondria synthesize an RNA that can diffuse into the cytoplasm, but it is unable to bind to cytoplasmic ribosomes; therefore it cannot play a role in the increase in protein synthesis which follows activation of anucleate halves. A few observations indicate that the female pronucleus in sea urchin eggs might play a more important role than was previously believed. For instance, Rinaldi et al. (1977) found that mitochondrial DNA synthesis is much more strongly stimulated in activated anucleate fragments than in fertilized or activated whole Paracentrotus eggs. Since fertilization of the anucleate fragments prevents this increase in mitochondrial DNA synthesis, the authors concluded that, in whole eggs, the pronucleus exerts a negative control on the mitochondrial DNA synthesis that occurs in activated anucleate fragments of Paracentrotus eggs. In such fragments, mitochondrial protein synthesis is also increased (Rinaldi et al., 1983). These findings are reminiscent of what was said above about the negative controls exerted by the Acetabularia nucleus on regeneration and macromolecule synthesis. Nothing is known about the molecular mechanisms involved in such negative nuclear controls. Krystal and Poccia ( 1979) have combined polyspermy with ammonia treatment and worked on nucleate and anucleate halves (obtained by either centrifugation or manual dissection) as well as on whole sea urchin eggs. These treatments induce premature chromosome condensation (PCC) in the spermatozoa at the time chromosome condensation takes place in the female pronucleus. Premature chromosome condensation is much less stable in anucleate than nucleate halves, indicating that ‘‘a component of the maternal pronucleus may modulate the stability of the chromosome condensing environment. In more recent experiments, where DNA synthesis was inhibited with aphidicolin, Killian et al. (1985) came to the conclusion that the female pronucleus (which has been long neglected) exerts a negative control on chromosome condensation in the maternal cytoplasm. ”

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IV. Nucleocytoplasmic Interactions in Xenopus Oocytes and Eggs A. GENERAL BACKGROUND Oogenesis Oogenesis is the preparation for embryonic development: there is a progressive accumulation of reserve materials (glycogen, lipids, and proteins), of ribosomes, and of about 20,000 different maternal mRNAs (Davidson and Hough, 1971; Perlman and Rosbach, 1978). Maturation transforms the fully grown oocyte (1.1-1.2 mm in diameter for Xenopus) into an egg that, after fertilization, can give rise to an embryo and finally an adult of the same species. The egg is thus a giant, totipotent cell. Growth of the oocyte is due to both endogenous synthesis of a great variety of molecules and the uptake of the phosphoprotein vitellogenin by endocytosis. Vitellogenin (M, 470,000) is synthesized in the liver under estrogen stimulation, secreted in the bloodstream, bound to oocyte surface receptors, internalized, and finally transformed into phosvitin and lipovitellin, which crystallize in the yolk platelets (Wallace and Jared, 1976). Wallace and Misulovin (1978) cultivated in vifro medium-sized (0.6 mm in diameter) Xenopus oocytes in a medium containing vitellogenin and insulin; such in vitro grown oocytes finally became larger (1.43 mm in diameter) than the largest oocytes present in the ovary. Even “fullgrown” oocytes can take up vitellogenin added to the medium: their volume doubles within 2-3 weeks (Wallace et al., 1981). It is not known whether enucleated oocytes would also increase in size under suitable culture conditions. Small, previtellogenic oocytes synthesize and accumulate the whole mRNA population which is present in unfertilized eggs (Golden et al., 1980). They also synthesize an ovarian type 5 S RNA; it is associated with proteins in the form of 7 S and 42 S ribonucleoprotein particles (Picard and Wegnez, 1979). One of these proteins is the transcription factor TFIIIA, which binds to an internal region of the 5 S gene. This binding results in gene activation (Pelham and Brown, 1980). The biochemical and embryological role of stable transcription complexes (like the TFIIIA-5 S RNA gene), which repress or activate eukaryotic genes, has been discussed by D. Brown (1984). The transcription factor disappears at maturation; this explains why the ovarian 5 S genes become silent and somatic 5 S genes are activated in somatic cells. This factor thus plays a dual role in oocytes: to stabilize 5 S RNA in the cytoplasm and to activate ovarian 5 S genes in the nucleus. Synthesis of the 28 S and 18 S rRNAs does not begin before the onset of vitellogenesis; during vitellogenesis, there is a huge accumulation of ribosomes in the oocyte. This is made possible because the number of the nucleolar organiz-

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ers increases from 30 to 1000 at pachytene. There is a concurrent 1000-fold amplification of the ribosomal genes (rDNA) (Brown and Dawid, 1968). It is remarkable that, despite their amplification, the ribosomal genes are not transcribed in previtellogenic oocytes (Thomas, 1970, 1974). During vitellogenesis, Xenopus oocytes accumulate ribosomes far in excess of their own needs; more than 90% of these ribosomes are free and-only 1-2% of them are in the form of polysomes (Woodland, 1974). The store of ribosomes accumulated during oogenesis is sufficient to ensure organogenesis and even cell differentiation. This was shown by Brown and Gurdon (1964) on the anucleolate mutants ( 0 - n ~of) Xenopus. If both nucleolar organizers of a fertilized Xenopus egg are deleted, the formation of new ribosomes is impossible; nevertheless development takes place and tadpoles with brain, eyes, muscles, etc. can be obtained. The ribosomes are distributed, in full-grown oocytes and eggs, along an animal-vegetal polarity gradient (Fig. 13); the yolk platelets are distributed along an opposite vegetal-animal gradient. One of the factors that establishes this

FIG. 13. Schematic representation of polarity gradients in an amphibian oocyte surrounded by follicle cells. PA, animal pole; PV, vegetal pole. (Left) distribution of the yolk platelets. (Right) distribution of the ribosomes; note their accumulation around the nucleus (germinal vesicle). In the cortex, note gradient distribution of the pigment granules (melanosomes). (Drawn by P. Van Gansen .)

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polarity gradient is, as shown by Robinson (1979), a transcellular electric current due to a movement of ions through the oocyte. The nucleoli are very conspicuous in the large GV, which is limited by a classic nuclear membrane. There are about 1200 very conspicuous nucleoli. The diplotene lampbrush chromosomes are much less conspicuous; about 10,000 loops extend in the nuclear sap from condensed chromomeres (Fig. 14). The genes present in the loops are transcribed during the whole oogenesis; however, according to Sommerville and Malcolm (1976) only 0.1-0.2% of the DNA is expressed in coding sequences. Scheer et al. (1976) found that several transcription units are present on the same loop; highly repetitive DNA sequences are transcribed on many loops of the lampbrush chromosomes (Varley et al., 1980; Scheer, 1981; Jamrich et al., 1983). This disposed of the once-popular “one loop-one gene” theory. The transcription products of the repetitive genes are found in the cytoplasm; the role of these cytoplasmic repetitive RNA sequences (reviewed by Davidson and Posakony , 1982) remains mysterious since these interspersed poly(A)+ RNAs are not translatable (Richter et al., 1984). Although few enucleation experiments have been done on Xenopus oocytes, there is no doubt that oogenesis is the result of a close cooperation between the nucleus and the cytoplasm: the latter is the site of yolk accumulation, while the germinal vesicle supplies the cytoplasm with ribosomes and maternal mRNAs. All kinds of RNAs are synthesized in the germinal vesicle and quickly move out into the cytoplasm through the nuclear pore complexes of the nuclear membrane. This traffic is very intensive, since about 300,000 molecules of rRNA move out of the germinal vesicle every second (Scheer, 1978). But there is also a traffic, in the opposite direction, of proteins which have been synthesized in the cytoplasm (reviewed by De Robertis, 1983; Dingwall, 1985). Experiments where labeled proteins were injected into the cytoplasm of Xenopus eggs and followed by radioautography or chemical analysis (De Robertis et al., 1977) have shown that a distinction should be drawn between karyophilic proteins, which reenter quickly into the nucleus after injection, and karyophobic proteins, which always remain in the cytoplasm. Nuclear membrane permeability is unrelated to size and electrical charge of the cytoplasmic proteins; it depends on the presence, in certain proteins, of karyophilic amino acid sequences which act as signals for entry into the nucleus. The karyophilic proteins penetrate into the nucleus through the nuclear pores (Feldherr et a l . , 1984), but they accumulate in the nucleus even if the nuclear membrane has been punctured with a glass needle (Feldherr et al., 1983). Small nuclear RNAs (snRNAs) injected into the cytoplasm of Xenopus oocytes move into the nucleus, where they become 50 to 100 times more concentrated than in the cytoplasm (De Robertis et al., 1982). When they are injected into enucleated oocytes, they quickly associate with proteins to form snRNP particles. These proteins are translocated into the nucleus when they are associ-

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FIG, 14. (A) Phase contrast photograph of two homologous lampbrush chromosomes in a Triturus oocyte. The chromosomes are held together, at places, by synaptinemal complexes. Many loops extend, in the nuclear sap, from a continuous axis made of chromomeres. (Photograph courtesy of Professor H. G. Callan.) (B) Structure of chromomeres and loops according to J. G. Gall (1956). Loops result from localized uncoiling of a DNA thread in the chromomere; this allows transcription to take place.

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ated to snRNA (Zeller er al., 1983). It is probable that a karyophilic domain of the snRNP proteins becomes exposed when they bind to snRNA (Mattaj and De Robertis, 1985). Thanks to Gurdon and his colleagues, Xenopus oocytes are excellent test tubes for molecular biologists. As found first by Gurdon et al. (1971), injection of rabbit hemoglobin mRNA into their cytoplasm is followed by intensive synthesis of rabbit hemoglobin; each mRNA molecule can give rise to as many as 100,000 hemoglobin molecules (Gurdon et al., 1973). Synthesis continues for weeks even if the oocyte was enucleated before mRNA injection. Since these pioneer experiments, Xenopus oocytes have become classic models for the analysis of all kinds of mRNA preparations. A review where more than 200 references to papers dealing with mRNA translation in Xenopus oocytes can be found has been recently published by Soreq (1985). Injection of globin mRNA into young, vitellogenic oocytes is also followed by globin expression; in such oocytes, in contrast to full-grown oocytes, there is no competition between foreign and endogenous mRNAs for the protein-synthesizing machinery (Taylor er af., 1985). A new development of Gurdon’s initial experiments is the injection of antisense (complementary to the noncoding DNA strand) mRNA into Xenopus oocytes: Melton (1985) showed that coinjection of globin mRNA and antisense globin mRNA suppresses selectively the translation of globin mRNA; this is due to the specific formation of a hybrid between the mRNA and its antisense mRNA. Injection of a pure gene (for instance 5 S DNA) into the nucleus of a Xenopus oocyte is followed by prolonged and faithful transcription of the gene (Metz and Gurdon, 1977; Brown and Gurdon, 1977). Injection of the genes into the cytoplasm is useless. Injection of genes into the GV has brought invaluable information about the regulation of the transcription of many genes [ribosomal (Trendelenburg and Gurdon, 1978), ovalbumin (Wickens et d.,1980), histone (Grosschedl and Birnstiel, 1980) and tRNA (De Robertis et al., 1981) genes]. In vitro induction of site-specific mutations prior to injection of the gene into the nucleus has become a powerful tool for the analysis of the sequences that control gene activity. It has also been possible to study the assembly into nucleosomes of the injected genes: the main factors involved in this process are nucleoplasmin and DNA topoisomerase I, which are both abundant in the GV (Laskey and Earnshaw, 1980). Finally, Gurdon has done experiments where whole nuclei of adult cells were injected into Xenopus oocytes or eggs. His first experiments (Gurdon, 1968) showed that Xenopus adult brain nuclei behave exactly like the oocyte or egg nucleus in both morphology and nucleic acid synthesis after injection. The overall nuclear activities are thus controlled by the state of the cytoplasm during these early stages of development.

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The next step was to inject HeLa cell nuclei into Xenopus oocytes and to follow the mRNA and protein syntheses directed by the injected nuclei (De Robertis et al., 1977). The result was that only three HeLa proteins over 25 were expressed in the injected oocytes whether or not the latter had been previously enucleated. The oocyte cytoplasm thus has the remarkable property of reprogramming the expression of individual HeLa genes. This was still better demonstrated by experiments where De Robertis and Gurdon (1977) injected adult kidney nuclei of Xenopus into oocytes of Pleurodeles. Analysis of the proteins synthesized by injected oocytes demonstrated that Xenopus adult kidney nuclei synthesized some of the Xenopus oocyte’s proteins, which had not been synthesized by the kidney cells for many months. Simultaneously, synthesis of the specific kidney proteins came to a halt. The oocyte thus contains factors that are capable of reactivating certain genes active during oogenesis and inactivating adult genes. Finally, Korn and Gurdon (1981) injected adult Xenopus nuclei into GVs to study the transcription of the ovarian and adult 5 S RNAs (which differ slightly and are coded by different genes). The ovarian 5 S genes were reactivated in certain females but not in others; if the nonhistone proteins were removed from the nuclei by treatment with 0.35 M NaCl before injection, reactivation of the 5 S genes took place in oocytes of all females. Korn et al. (1982) then injected Xenopus erythrocyte nuclei together with extracts from oocytes that reactivate or fail to reactivate the oocyte-type 5 S RNA in adult nuclei. Extracts of reactivating oocytes induced the transcription of the 5 S gene while those of nonreactivating oocytes had no inhibitory effects. Wakefield and Gurdon ( 1983) found that the ovarian 5 S genes are still expressed, but at a low level, in blastulae: there is a 30-fold decrease in transcription during gastrulation. They transplanted neurula nuclei into enucleated unfertilized eggs and observed that the oocyte-type 5 S genes were activated in blastulae and inactivated afterward. Expression of the oocyte-type 5 S genes is thus controlled by cytoplasmic factors (probably proteins) which disappear during development; one of them is the transcription factor TFIIIA (Brown and Schlissel, 1985).

B. MATURATION Full-grown oocytes undergo maturation (reviewed by Masui and Clarke, 1979; Maller, 1985; Masui, 1985) when they are stimulated by progesterone produced by the follicle cells which surround the oocyte; maturation is the progression of meiosis from the oocyte to the fertilizable egg stage. In Xenupus, the egg can be fertilized when the first polar body has been expelled; elimination of the second polar body is induced by fertilization, or parthenogenetic activation. Morphologically, the most spectacular event during maturation is the breakdown of the huge germinal vesicle (GVBD) after a lag of several hours. It starts

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at the basal pole of the GV (Fig. 15) and spreads toward its apical pole. Simultaneously, the lampbrush chromosomes strongly condense; the nucleoli disintegrate except for their nucleolar organizers which fuse together to form large Feulgen-positive bodies (Brachet, 1965). The anastral spindle that carries the condensed meiotic chromosomes moves to the cortex of the animal pole and the first polar body is expelled, reducing by one-half the number of the chromosomes. Only a brief overview of the biochemical changes which take place during maturation in Xenopus can be given here; details are found in Maller’s (1985) recent review. Maturation is easily obtained when full-grown oocytes are cultured in vitro in the presence of progesterone. Under these conditions, maturation takes place even in the presence of actinomycin D, showing that RNA synthesis is not required for this step of development. The hormone must act directly on the cell membrane, where it binds to a receptor (Blondeau and Baulieu, 1984); it is inactive if injected into the oocyte. Maturation can be induced by treatment of the oocytes with substances chemically unrelated to progesterone: organomercurials (Brachet et a l . , 1975). lanthanum chloride, propranolol, insulin, etc. (Schorderet-Slatkine et a l . , 1976, 1977). All of these agents have one thing in common: they set free membrane-bound calcium and this increases the free Ca2+ concentration in the cytoplasm (Baulieu et a l . , 1978). There is indeed a Ca2+ burst at maturation (Wasserman et al., 1980); this Ca2+ release is one of the very early events induced by progesterone addition to the oocytes. Another early biochemical change induced by progesterone is an increase in the internal pH (pHi) of 0.3-0.4 pH units (Cicirelli et a l . , 1983; Wasserman et a l . , 1984; Morrill et a l . , 1984). However, according to a recent report of Stith and Maller (1985), an increase in pHi is not required for GVBD. Another early biochemical change is a 50% decrease in membrane adenylate cyclase activity (Baltus et al., 1981; Sadler and Maller, 1981; Finidori-Lepicard et a l . , 1981). The result is that the cAMP content of the oocytes drops quickly but transiently after addition of progesterone (Maller et a l . , 1979). This drop is probably important for successful maturation, since agents that increase the cAMP content prevent the induction of maturation by progesterone (O’Connor and Smith, 1976; Bravo et a l . , 1978; Schorderet-Slatkine et al., 1978; Mulner et al., 1979; Maller et a l . , 1979). According to Blondeau and Baulieu (1985), progesterone inhibits the phosphorylation of a single 48-kd membrane protein. However, it is not certain that a decrease in membrane adenylate cyclase activity is sufficient to induce maturation. Jordana et al. (1982) have reported that progesterone inhibits the enzyme in small vitellogenic oocytes that do not respond to the hormone by GVBD. These early changes are followed by “late” changes which precede GVBD.

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There is an increase in oxygen consumption (Brachet et al., and an arrest of 5 S rRNA synthesis (Wormington and Brown, 1983). More important is the strong increase in protein synthesis discovered by Smith and Ecker (1970). The rate of protein synthesis steadily increases after progesterone addition and reaches a peak after 6 to 8 hours (thus around the time of GVBD). The synthesis of all the oocyte proteins is not stimulated to the same extent; the synthesis of 5 polypeptides out of 600 selectively increases at the time of GVBD (Younglai et al., 1982). There is a 20- to 50-fold increase in the synthesis of the core histones H,A, H,B, H3, and H4 without a concomitant increase in histone HI synthesis (Adamson and Woodland, 1977). It is likely that the histones synthesized during oogenesis and maturation are used for nucleosome formation when fertilized, and cleaving eggs undergo intensive DNA replication. Although there is no chromosomal DNA synthesis during maturation, progesterone-treated oocytes synthesize the machinery required for DNA replication during cleavage. New forms of DNA polymerase (Grippo and Lo Scavo, 1972), ribonucleotide reductase (Tondeur-Six et al., 1975), and thymidine kinase (Woodland and Pestell, 1972) become detectable; a protein factor that stimulates the initiation of DNA synthesis also appears during maturation (Benbow et al., 1975). A strong wave of protein phosphorylation is observed about 20 minutes before GVBD (Morrill and Murphy, 1972; Maller et al., 1977; Bell6 et al., 1978). Many proteins, both soluble and membrane bound, are simultaneously phosphorylated. There is probably a correlation between this burst in protein phosphorylation and the breakdown of the nuclear membrane: phosphorylation of the pore-lamina proteins (the lamins) would lead to their solubilization and to the dissolution of the nuclear envelope. Progesterone-treated oocytes synthesize two biologically important ‘‘factors” of still unknown chemical nature: the maturation-promoting factor (MPF) (Smith and Ecker, 1970; Masui and Markert, 1971) and the cytostatic factor (CSF) (Masui and Markert, 1971). Injection of cytoplasm taken from oocytes treated during a few hours with progesterone into normal recipient oocytes induces GVBD within 1-2 hours (instead of 6-8 hours) (Fig. 16); this is due to the presence of MPF in the injected cytoplasm. Injection of MPF-containing cytoplasm induces an almost immediate increase in protein phosphorylation and synthesis in the recipient oocyte. Partially purified MPF (Wu and Gerhart, 1980) is a 100-kd Ca2+ -sensitive protein capable of phosphorylating four endogenous FIG. 15. Morphological changes during Xenopus oocyte maturation. (A) Full-grown oocyte with intact germinal vesicle; (B) breakdown of the nuclear membrane at its basal pole; (C) first maturation spindle; (D) second maturation spindle under the first polar body (arrowhead); (E) at much higher magnification, Feulgen-stained coalescent nucleolar organizers. The Feulgen-positive bodies are made of ribosomal DNA as shown by in siru hybridization at the ultrastructural level. (Photographs courtesy of P. Van Gansen, F. Hanocq, and J. Brachet.)

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proteins in the presence of ATP. The same or similar proteins are found in eggs of many species and even in HeLa cells (Sunkara et al., 1979). Their role is to induce nuclear membrane breakdown and chromosome condensation during meiotic or mitotic prophase. The cytoplasm of MPF-injected oocytes synthesizes more MPF, which can be transferred from one oocyte to another by serial transfers of cytoplasm; it undergoes a kind of autocatalytic amplification (Masui and Markert, 1971) (Fig. 16). CSF appears much later than MPF, after the expulsion of the first polar body. If the cytoplasm from a fertilizable egg is injected into a blastomere of a cleaving egg, mitotic activity is arrested at metaphase in the injected blastomere (Fig. 17). The cytostitic factor is no longer present in fertilized eggs; this is due to the facts that CSF (like MPF) is inactivated by Ca2+ and that there is a free Ca2+ surge at fertilization (Masui, 1974; Meyerhof and Masui 1977, 1979). Cytostatic factor is not responsible for chromosome condensation; it rather plays a role in stabilizing the spindle microtubules and preventing their depolymerization at anaphase

I -

-

1-1

FIG. 16. Schematic representation of MPF formation and amplification. A full-grown Xenopus oocyte undergoes maturation after treatment with progesterone (P). Injection of cytoplasm taken from the maturing oocyte induces maturation in a recipient full-grown oocyte; this demonstrates the production of MPF in progesterone-treated oocytes. The cytoplasm of the injected oocyte induces a maturation in a recipient oocyte in the absence of progesterone. Repeated transfers of cytoplasm demonstrate that MPF-injected oocytes synthesize more MPF (amplification).

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FIG. 17. Injection of the cytostatic factor (CSF) present in unfertilized eggs into one of the two blastomeres of a cleaving frog egg arrests its cleavage. (A) Arrested injected blastomere on top; normal cleavage of the uninjected blastomere below. (B) Arrested mitosis (in metaphase) in the CSFinjected blastomere (Masui, 1974).

(Meyerhof and Masui, 1979). Its function in unfertilized eggs is probably to arrest meiosis in metaphase 11. Gerhart et al. (1984) have shown that MPF appears in the oocyte before GVBD, disappears at the end of the first mitotic cycle, and reappears before metaphase 11. These oscillations during the meiotic cycle do not require protein synthesis; MPF cycling is accelerated by colchicine and injection of CSF. Maturation-promoting factor disappears 8 minutes after fertilization or activation. Fertilized and activated eggs contain an inactivating agent that destroys injected MPF; CSF injection blocks the disappearance of MPF at fertilization. Gerhart et al. (1984) think that MPF activity is linked to a “cell-cycle oscillator” believed to be responsible for the rhythmic contraction waves that take place in activated and fertilized eggs. This cytoplasmic oscillator would be blocked in nonmaturing oocytes by an arrest system that is eliminated by progesterone treatment. Maturation is characterized by GVBD; this fascinating phenomenon is the reason I had already become interested in maturation around 1940. Several experiments suggest that the GV plays only a passive role as soon as the oocyte has been challenged with progesterone: both the overall increase in protein synthesis (Smith and Ecker, 1970) and the selective synthesis of the nucleosome core histones (Adamson and Woodland, 1977) still take place when Xenopus oocytes are enucleated prior to progesterone treatment. Protein synthesis during maturation is thus controlled by selective utilization of stored maternal mRNAs. This conclusion is confirmed by the experiments of Bienz and Gurdon (1982) on the production of heat shock proteins (hsp) after heating Xenopus oocytes, in

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which synthesis of the same hsp 70 protein is induced by the heat treatment in nucleate and anucleate oocytes. The oocytes thus contain preformed hsp 70 mRNA molecules and heating leads to their selective translation. This is an exceptional situation since, in all the systems studied so far, hsp synthesis is controlled at the level of transcription. Synthesis of hsp 70, after a heat shock, is not affected by progesterone-induced maturation or activation of unfertilized eggs by the Ca2 ionophore A23 187 (Baltus and Hanocq-Quertier, 1985). This response to a heat shock disappears during cleavage and reappears at the late blastula stage, where accumulation of hsp mRNA after heating now results from the classic transcriptional activation of the hsp genes (Bienz, 1984; Heikkila et al., 1985). The strong burst in protein phosphorylation which occurs shortly before GVBD takes place in previously enucleated oocytes. Still more important is the fact that enucleated oocytes still synthesize MPF (Masui and Markert, 1971). Enucleated oocytes also display the same changes in ion permeability and the same cortical reactions after parthenogenetic activation as normal progesteronetreated oocytes (exocytosis of the cortical granules, uplifting of a fertilization membrane). This does not mean that the mixing of the nuclear sap and the cytoplasm at maturation is without importance. Proteins which were accumulated in the GV are not degraded when they mix with the surrounding cytoplasm but are taken up during development and are still detectable in the nuclei of tadpole cells (Dreyer and Hansen, 1983). The nuclear sap of the GV is absolutely required for the swelling of injected spermatozoa (Katagiri and Moriya, 1976; Moriya and Katagiri, 1976; Skoblina, 1976) and for the condensation of chromatin into chromosomes in injected brain nuclei (Ziegler and Masui, 1973; reviewed by Masui, 1985). None of these reactions of nuclei injected into the cytoplasm of matured oocytes occurs if the oocytes have been enucleated before progesterone addition. The mixing of nuclear sap with cytoplasm is also important for microtubule (MT) assembly, which can be easily induced in Xenopus unfertilized eggs by treatment with D20 (Van Assel and Brachet, 1968) or injection of basal bodies (Heidemann and Kirschner, 1975, 1978; Heidemann et al., 1977). Figure 18 shows the formation of numerous cytasters (which appear simultaneously and never divide) in a D20-treated Xenopus unfertilized egg. Cytasters never appear when oocytes with an intact GV are treated with D,O (Heidemann and Kirschner, 1975). Basal bodies do not induce asters in enucleated, progesterone-treated Xenopus oocytes (Heidemann and Kirschner, 1978). Injection of nuclear sap from a full-grown oocyte into such enucleate oocytes fails to restore the capacity of the injected basal bodies to induce aster formation. It is clear that the nuclear sap undergoes changes when it mixes with the cytoplasm of progesterone-treated oocytes and that there is a cytoplasmic maturation in addition to the morphologically spectacular nuclear maturation. +

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FIG. 18. Cytasters form when unfertilized amphibian (or sea urchin) eggs are treated with heavy water (D20).The condensed material which can be seen in their center probably corresponds to a “pro-centriole’’ (Van Assel and Brachet, 1966).

C. UNFERTILIZED AND ACTIVATED Xenopus EGGS Xenopus eggs are fertilizable when the first polar body has been expelled; they can also be activated by pricking, electrical shock, or treatment with the divalent ions ionophore A23187. As in sea urchin eggs, the main early biochemical changes in activated or fertilized Xenopus eggs are increases in free Ca2+ and pHi. The most important experiments done on unfertilized amphibian eggs were the nuclear transplantations performed, for the first time, by Briggs and King (1952, 1953) in order to answer the question raised by Morgan in 1934: when does differentiation take place in the nuclei? Their experiment consisted in the transplantation of a cell nucleus removed from a blastula or a later embryonic stage into an enucleated, unfertilized frog egg (Fig. 19). Since the subject has been frequently reviewed (Gurdon and Woodland, 1970; Di Berardino, 1979, 1980; Briggs, 1979; Di Berardino et al., 1984), it will not be dealt with in detail. The experiments showed that blastula nuclei are still totipotent: after injection of such nuclei into enucleated Rana pipiens or Xenopus unfertilized eggs, adults may be obtained. King and Briggs (1954) found a restriction of the potentialities of nuclei removed from advanced gastrulae in Rana. But, in Xenopus, development

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FIG. 19. .Method of Briggs and King for transplanting blastula cell nuclei into enucleated frog eggs. (1) Removal of a blastula cell; (2) the isolated cell breaks in the micropipette; (3) injection of the nucleus into a previously activated and enucleated unfertilized egg; (4,5) removal of the exovate with glass needles (Briggs and King, 1953).

up to the tadpole (but not the adult) stage has been obtained after injection of nuclei isolated from fully differentiated larval or adult cells (Gurdon and Laskey, 1970; Laskey and Gurdon, 1970; Gurdon etal., 1975). The discrepancy between the results obtained with different species is due in part to the fact that chromosomal abnormalities are frequent when “old” nuclei replicate quickly in the “young” cytoplasm of the enucleated unfertilized eggs (Di Berardino and Hoffner, 1970). Gurdon’s experiments strongly suggest that adult nuclei still possess all the genetic information required for organogenesis and differentiation of larval tissues. However, cleavage nuclei have already lost their totipotency at the four-cell stage in mouse eggs (McGrath and Solter, 1984). Other experiments with uncleaved Xenopus eggs are important for cell biologists. For instance, Forbes et al. (1984) injected phage DNA into fertilized eggs and observed the appearance of structures very similar to eukaryotic nuclei in the vicinity of the injected material. These structures were surrounded by a typical nuclear envelope, which broke down when mitosis was induced. The assembly and breakdown of a nuclear membrane are thus independent of specific DNA information. Formation of the nucleuslike structures could even be obtained in vitro with extracts of Xenopus eggs.

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Lohka and Masui ( 1983a,b) treated demembranated Xenopus spermatozoa with extracts of activated R . pipiens eggs in virro. They observed the formation of a nuclear membrane and the decondensation of chromatin in the sperm nuclei. This was followed by DNA synthesis and condensation of chromosomes. To obtain the complete series of events both soluble and particular (membranes) fractions of the cytoplasmic extract are needed. The 150,000 g supernatant produces only the dispersion of chromatin; the cytoplasmic vesicles, which originate probably from the endoplasmic reticulum, are needed to form a nuclear membrane. In later experiments, Lohka and Masui (1984a) found that cytoplasmic extracts from activated frog eggs transform demembranated Xenopus spermatozoa into pronuclei similar to those of fertilized eggs. Similar results have been obtained by Iwao and Katagiri (1984), who worked on toad eggs. They found that the cytosol of unfertilized eggs is inactive unless the eggs have been activated; addition of Ca2 to the extracts of nonactivated eggs activated them. There was no chromatin-decondensing activity in the cytosol of full-grown oocytes and in that of enucleated progesterone-treated oocytes. The ability of the cytosol to decondense the sperm nuclei was suppressed by Ca2+ chelators and by serine protease inhibitors, suggesting that a serine protease is involved in chromatin decondensation. In intact eggs, one of the controlling factors is the increase in free Ca2 concentration which immediately follows egg activation or fertilization (Lohka and Masui, 1984b). The biochemical mechanisms responsible for this Ca2 burst (which causes the exocytosis of the cortical granules) are better known since the recent work of Whitaker and Irvine (1984) on sea urchin eggs and that of Picard et al. (1985) on Xenopus oocytes. They found that injection of inositol 1,4,5-trisphosphate (InsP,) into unfertilized eggs triggers the cortical reaction. InsP, is probably produced from plasma membrane phosphatidylinositol4,5-bisphosphatevia hydrolysis by membrane-bound phospholipase C. Its function is to release calcium from intracellular stores located in endoplasmic reticulum vesicles (Berridge, 1983). Injection of InsP, in unfertilized Xenopus eggs rapidly increases free calcium in the cytoplasm and induces activation; however, injection into Xenopus oocytes fails to release them from the prophase block (Picard et al., 1985). Finally, Karsenti et al. (1984) have studied the role of the centrosomes and of the nucleus in aster formation in Xenopus oocytes and eggs. Purified centrosomes were injected into oocytes or into eggs in metaphase or interphase. No asters formed around the centrosomes in unfertilized eggs arrested in metaphase; activation of these eggs was followed by the formation of asters around the centrosomes. Karsenti et al. (1984) also injected karyoplasts containing a nucleus and a centrosome in oocytes or eggs in metaphase and obtained mitotic spindles with centrosomes. If karyoplasts devoid of a centrosome were injected, only anastral microtubules assembled around the condensing chromatin. Coinjection of a nucleus and a centrosome led to the appearance of spindlelike +

+

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structures with well-defined poles. Injected centrosomes did not induce asters in oocytes, nonactivated unfertilized eggs, or fertilized eggs arrested by CSF injection. The main conclusion to be drawn from all these experiments is that association with a nucleus activates the centrosomes.

V. Cleavage of Fertilized Eggs Various modes of egg cleavages are shown schematically in Fig. 20. They are largely conditioned by the amount and location of the yolk. In amphibian eggs, cleavage is equal and complete during the first two divisions, leading to four blastomeres of equal size, but the third cleavage is not equatorial, dividing the egg into micromeres at the animal pole and macromeres at the vegetal pole. This is due to the polarity gradient already present in iarge oocytes. The yolk, which has accumulated in the vegetal hemisphere is an obstacle to furrow progression. Successive synchronous cleavages lead to the formation of a morula in which a layer of cells (blastomeres) surrounds the blastocoele cavity. At each division the volume of the blastomeres decreases. As a result, the ratio between the volumes of the nucleus and the cytoplasm (nucleoplasmic ratio) steadily increases during cleavage; it reaches the value characteristic of all cells of a given species at the blastula stage. During cleavage, cytoplasmic regions which are important for further development (germinal localizations) may be segregated in one or a few blastomeres.

0

E

E‘

FIG. 20. Schematic representation of the main cleavage types. (A) Radial, equal and total cleavage (Amphioxus; Prochordates); (B) radial, unequal and total cleavage (frog eggs); (C) spiral, unequal and total cleavage (worms, mollusks); (D) partial cleavage (fish, reptiles, birds); (E, E’) superficial cleavage of insect eggs; ventral side (E) and longitudinal section (E’). (Drawn by P. Van Gansen.)

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The cytoplasmic determinants they contain will be discussed in Section VII. Destruction or transplantation of a blastomere containing cytoplasmic determinants leads to developmental abnormalities. Cleavage is a period of intensive DNA synthesis, as I first showed in 1933 (Brachet, 1933). As shown in Fig. 21, there is a correlation between the rate of DNA synthesis and the mitotic index which drops continuously (Parisi et al., 1978). These conclusions, which were derived from experiments on sea urchin eggs, remain valid for all animal species. The cell cycle in cleaving eggs, except in those of the mammals, is very different from that of cultured cells: there are no G , and G , phases, and the S phase of DNA replication is very rapid. It lasts 8- 10 minutes in the large axolotl eggs and only 3-4 minutes in Dramphila. The longest phase in the cell cycle is mitosis, which takes 80 minutes in the axolotl because the progression of the furrow is slow in its large eggs. The speed of the first cleavage in amphibian eggs is controlled by cytoplasmic factors. This was shown by experiments in which Aimar et al. (1981) made reciprocal injections of cytoplasm between rapidly and slowly dividing eggs. The cleavage cytoplasmic clock is controlled by at least two different proteins according to Aimar et al. (1983). The absence of a G , phase and the exceptional speed of DNA replication during cleavage can be explained by the presence, in fertilized eggs, of the machinery required for DNA synthesis. However, there seems to be a need for increased deoxyribonucleotide precursors since there is a synthesis during early cleavage of both sea urchin (Noronha et al., 1972) and amphibian eggs (Tondeur-Six et al., 1975) of deoxyribonucleotide reductase. Inhibitors of this enzyme arrest cleavage at the 8- to 16-cell stage in sea urchins and at the midblastula stage in amphibians (Brachet, 1967). The key enzyme for DNA replication is DNA polymerase a;the amount of the enzyme present in unfertilized sea urchin eggs is sufficient for the production of 1000 nuclei. During cleavage, it moves from the cytoplasm into the nuclei when the chromosomes swell at anaphase (Fansler and Loeb, 1969). The absolute requirement of DNA polymerase (Y for DNA synthesis during sea urchin egg cleavage has been demonstrated by Ikegami et al. (1978): a specific inhibitor of the enzyme, aphidicolin, arrests development soon after amphimixy. Although unfertilized eggs possess a large store of the core histones needed for nucleosome assembly, fertilization of sea urchin eggs results in a strong synthesis of all types of histones, due to intensive transcription of the histone genes. During sea urchin cleavage, there is a switch in histone synthesis: after maternal histones and their mRNAs have been used, “early” or “cleavage” histones are synthesized. In the blastula, “late” or “embryonic” histones that are more closely related to those found in the adult begin to accumulate. According to Maxson and Wilt (1981), the rate of early histone mRNA synthesis sharply

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a

t

Hours of

development

b

HOURS AFTER FERTILIZATION

FIG. 21. (a) DNA synthesis in developing sea urchin eggs (Brachet, 1933). (b) Increase in DNA content (solid line) and drop in mitotic index between morula and mesenchyme blastula (broken line) during sea urchin egg development (Parisi er al., 1978).

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increases at the 16-cell stage, reaches a peak at the 128-cell stage, and decreases until the 300-cell stage; at that time, synthesis of late histones becomes more and more important. When Xenopus eggs reach the blastula stage (1000-2000 cells), a new set of histone genes also begins to operate (Woodland et al., 1979). It is likely that the shift from cleavage to embryonic histones is responsible for chromatin condensation at the blastula stage. The unusual speed of DNA replication during cleavage is due to the close proximity of the replicons in chromatin. This means that a large proportion of the DNA is in single-stranded form during cleavage. Nevertheless, nucleosomes, with a core of cleavage histones, already exist at the two-cell stage in sea urchins (Shaw et al., 1981). The building up of the mitotic apparatus and furrowing have been extensively studied in cleaving sea urchin eggs; since mitosis is the subject of an article by D. Mazia in this volume, the reader is referred to his article for a discussion of these topics. The control of the cell cycle during cleavage of Xenopus has been the subject of important papers by Newport and Kirschner (1982a,b, 1984; reviewed by Kirschner and Newport, 1985). They found that, after 12 rapid (30-minute) synchronous cleavages, the embryo undergoes the “midblastula transition. ” At that time, the G , and G, phases of the cell cycle appear. If the cells are dissociated, they display motility in contrast to the inertness of isolated large blastomeres. Transcription of tRNAs, 5 S RNAs, snRNAs, and 7 S RNA becomes detectable, while that of the 28 S and 18 S rRNAs is still repressed until the late blastula stage. Experiments in which fertilized eggs were ligated with a hair, centrifuged, or treated with cytochalasin have shown that the timing of the midblastula transition depends on a critical ratio between the volumes of the nucleus and the cytoplasm, and not on the number of cleavages, DNA replication cycles, or the time elapsed since fertilization. The midblastula transition is speeded up in polyspermic eggs. This led Newport and Kirschner (1982a) to suggest that the nuclei titrate a cytoplasmic component. To test this hypothesis, Newport and Kirschner (1982b) injected into fertilized Xenopus eggs a plasmid containing a yeast tRNA gene. They found that transcription of the injected DNA quickly stops during cleavage and is resumed at the midblastula transition. But there is no transient suppression of transcription if the amount of DNA injected is equal to the cellular DNA content after 12 cleavages. This suggests that DNA reacts with a cytoplasmic repressor, which is progressively exhausted during the repeated cleavages. Transcription starts when nuclear DNA is no longer saturated with the repressor. More recently, Newport and Kirschner (1984) reported that injection of cytoplasm from unfertilized eggs (which contains CSF) arrests mitosis by blocking an endogenous cell-cycle oscillator. This causes the stabilization of MPF,

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which normally undergoes cyclic activity in cleaving eggs; its activity in the cytoplasm is strong during mitosis and weak during the S phase. Cytostatic factor maintains MPF at a high level and this induces the arrest in metaphase. Ca2+ inactivates CSF, allowing the cell to progress in mitosis. Maturation-promoting factor is probably synthesized periodically; addition and loss of MPF, which acts on the DNA template, drive DNA replication during the cycle. The initial targets of MPF are the nuclear membrane proteins (the lamins). Maturation-promoting factor seems to be a lamin kinase (Miyake-Lie and Kirschner, 1985) capable of solubilizing the lamins and the nuclear envelope itself. A detailed model of the interactions between the two cytoplasmic factors, MPF and CSF, in the control of the cell cycle during cleavage in Xenopus eggs has been proposed by Kirschner and Newport (1985). The main conclusion is that the cell cycle is controlled by cyclic addition and removal of MPF. If one recalls that anucleate eggs of Xenopus and Arbacia undergo repeated cleavages (implying cyclic formation of asters and furrows) after activation, the conclusion that the cytoplasm plays the leading role until the midblastula transition is inescapable. The situation changes completely after this stage: gene transcription begins and the nucleus keeps the upper hand over the cytoplasm until the end of the organism’s life.

VI. A Very Brief Overview of Later Stages of Development The stages which follow cleavage (gastrulation, organogenesis, embryonic differentiation) are characterized by the time- and site-specific sequential activation of sets of genes, For instance, all cells of the embryo contain globin genes, but these genes are expressed only in a localized region of the embryo (the blood islands) at a precise stage of its development. It should be pointed out first that development cannot take place unless the fertilized egg has cleaved repeatedly. Cleavage is necessary to give the cells the plasticity required for the morphogenetic movements characteristic of gastrulation. Differentiation without cleavage in Chaeroprerus eggs (Lillie, 1902) is only an apparent exception. In this species, unfertilized eggs treated with excess KCl undergo first pseudocleavage (abortive attempts to cleave), then pseudogastrulation (the clear cytoplasm surrounds the yolk mass), and finally hatching and ciliation (Fig. 22). However, the ciliated larvae lack the apical tuft of long cilia and the enteric cavity characteristic of multicellular trochophore larvae. This and a number of other observations led me to conclude that differentiation without cleavage only mimics the normal development of fertilized Chaetopterus eggs, since there is no real gastrulation; however, differentiation without cleavage remains an excellent model for the study of cell differentiation, particularly for the analysis of cortical contractile activity and ciliogenesis (Brachet and DoniniDenis, 1978).

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FIG. 22. Differentiation without cleavage in KCI-activated Chaetopterus eggs. (A) Living eggs 12 hours after activation. Note the strong amiboid activity (right) and the segregation between yolk and clear cytoplasm (left). The embryo at the top is cytolyzing (Brachet and Donini-Denis, 1978). (B)Differentiation without cleavage at its final stage, and (C) normal larvae from fertilized Chuetopterus eggs. Note the absence of an apical tuft and intestine in (A). (B) and ( C ) redrawn from Lillie, 1902. (D) Section through a Chaetopterus-activated egg 4 hours after KCI treatment; the nucleus has enlarged and there is no segregation between yolk and hyaloplasm. (E) Incomplete and anarchic segregation between yolk (lightly stained) and basophilic cytoplasm (16 hours after activation). (F) Same as (E) but complete segregation; the basophilic cytoplasm completely surrounds the yolk mass (pseudogastrulation). In both (E) and (F), the large nucleus lies between the basophilic cytoplasm and the yolk mass. (D-F) from Alexandre e t a / . (1982).

Gastrulation is a period of mass cell movements that depend on cell migration and contractility. Cell migration is guided by extracellular fibrils; production of this extracellular fibrillar matrix requires protein glycosylation, production of sulfated glycoproteins in sea urchins (Carson and Lennarz, 1981) and of fibronectin in amphibians (Boucaut et al., 1984a,b). During gastrulation, genes encoding a whole array of proteins are expressed. As shown by McCiay and Wessel (1985) and by Angerer and Davidson (1984), the surface of a sea urchin gastrula is a mosaic of specific molecules; lineage genes are expressed before morphological differentiation. In the amphibians, certain proteins are selectively synthesized in the dorsal halves and in the various regions of gastrulae and neurulae (Smith and Knowland, 1984; Slack 1984a,b). This agrees with a crude model I proposed many years ago (Fig. 23) which assumed that genes are expressed in the dorsal half before the ventral half in the amphibian gastrula.

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FIG. 23. Schematic representation of protein synthesis and RNA distribution during amphibian development. (a) Distribution of the ribosomes (small dots) along an animal-vegetal gradient in a fertilized egg; (b) this gradient remains unchanged during cleavage; (c) during gastrulation, the nuclei become more active on the dorsal side; polysomes (dotted lines) are abundant in this side; (d) at the late neurula stage, the polysomes are distributed along dorsoventral and cephalocaudal gradient. (Redrawn from Brachet, 1967.)

Organogenesis results from the morphogenetic induction of “competent” cells by neighboring inducing cells. The best-studied case remains neural induction by Spemann’s organizer (the chordomesoblast of the early amphibian gastrula). There is little doubt that inductions result from the production of specific inducing substances which bind to the cell surface of the reacting cells. Since no inducing substance has so far been isolated and characterized, it is impossible to say how they act on the reacting cells at the molecular level. The cells respond to the inducing stimulus by activation and expression of sets of genes and frequently by an increase in mitotic activity. The role of the cytoplasm during morphogenetic inductions is probably to establish intercellular communications, to internalize the inducing agents, and to modify the cytoskeleton in order to allow locomotion and changes in cell shape. Embryonic differentiation is a problem of the greatest importance and complexity. It is generally studied on cultures of embryonic cells in vitro, seldom on intact embryos. I do not think that the two systems are identical: in cultures, the cells first have to be dissociated, reaggregate in a haphazard manner, and grow in an abnormal environment. They escape from polarity gradients, morphogenetic fields, and positional information, which are of primary importance in whole embryos. The subject of cell differentiation is so vast that it cannot be handled adequately here. It suffices to say that in all cases studied so far, terminal differentiation is controlled at the level of transcription. Only a few genes are

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activated (for instance, globin and carbonic anhydrase genes during erythrocyte differentiation) in a selective way soon after the cells are committed to undergo a given type of differentiation. How genes are selectively activated still remains a major enigma for molecular biologists. As pointed out by D. Brown (1981), embryonic development is too complicated a process to be explained by a single control mechanism. Many people believe, as was proposed by Scarano (1969), that development is controlled by changes in the DNA molecules themselves. Among the possible changes, it seems unlikely that gene amplification and gene rearrangements are major factors in morphogenesis. Amplification of the ribosomal genes in oocytes and of the genes coding the chorion proteins during oogenesis in Drosophila are exceptions; in fact, Levine et al. (1981) failed to detect selective gene amplification during the development of Drosophila. Comparison of germ line (sperm) and differentiated cell DNA has failed to disclose gross structural rearrangements during development (Haigh et al., 1982). A better case could be made for the idea, first proposed by Scarano (1969), that DNA methylation and demethylation play an important role in embryonic development (reviewed by Razin and Briggs, 1980; Razin et al., 1984; Jaenisch and Jahner, 1984). There is some evidence for the view that DNA demethylation is connected to gene expression and, as a consequence, to cell differentiation, but the presumed correlation between undermethylation and gene expression suffers from many exceptions. The only conclusion one can draw presently from a large amount of work is that local undermethylation of DNA gene sequences is probably one of the factors, but certainly not the only one, required for chromatin to assume an active configuration. The best test we have to date for transcriptionally active chromatin is hypersensitivity to DNase digestion. How nucleasehypersensitive regions form in chromatin is not known. Weintraub, who reviewed the subject in 1983 and 1985, concluded that the major factor in the formation of DNase-hypersensitive structures in chromatin is the secondary structure of DNA, which would be affected by cis-acting factors such as cytosine methylation and B- to Z-DNA transitions. Another possibility is that DNase sensitivity or resistance results from the binding of specific protein factors to certain DNA sequences (trans-regulation of transcription); recent experiments by Kaye et al. (1984) and Wu (1984) bring some support to this belief. But gene activation is such a complex, highly regulated process that both cis-acting and trans-acting regulations probably cooperate in gene expression.

VII. Cytoplasmic Determinants (Germinal Localizations) Cytoplasmic heterogeneity has long been recognized as a major factor in the embryonic development of “mosaic” eggs. Cleavage may lead to the segrega-

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tion, in given blastomeres, of the cytoplasmic heterogeneous regions called “germinal localizations” by the experimental embryologists who discovered them and which are believed to contain “cytoplasmic determinants. Moderate centrifugation of fertilized frog eggs leads to microcephaly (Fig. 1); turning the frog egg upside down leads to twinning, and pricking the dorsal region indicated by a gray crescent (Fig. 24) suppresses the formation of axial organs (nervous system, chorda) completely or partially. This kind of experiment was at the root of T. H. Morgan’s (1934) theory, where equipotential nuclei would be distributed in a mosaic of chemically different territories; specific genes would be activated in a given germinal localization while others would remain silent. Theoretically, two different mechanisms might explain how a germinal localization could give rise to the corresponding organ, a muscle for instance. Either maternal mRNAs, coding for muscle-specific proteins, are accumulated in the region which will later differentiate into muscles or this region contains factors that selectively activate genes coding for specific muscle proteins. We would be dealing with preformation in the first hypothesis and with epigenesis in the second. ”

A . THE GRAYCRESCENT OF AMPHIBIAN EGGS‘ As shown by Roux (1903), the gray crescent (which marks the dorsal side of frog eggs) appears on the site opposite the entrance point of the spermatozoon. Growth of the spermaster induces rearrangements of the animal pole materials; they react with the egg cortex and the yolk, which accumulates in a thick mass (the “vitelline wall” of Pasteels, 1964; Ubbels et al., 1983). However, experiments by Ancel and Vintemberger (1948; Fig. 24c) demonstrated that the dorsal side and, as a consequence, the plane of bilateral symmetry can be determined at will by modifying the orientation of freshly fertilized eggs. With proper design of the egg rotation conditions, the gray crescent can form on the side where the sperm entered the egg. Reinvestigation of the problem by Gerhart et al. (1981) has led to the conclusion that displacement of the egg contents by gravity can determine the orientation of the subsequent dorsalventral axis of the embryo, and induce twinning under certain conditions. If the spermaster microtubules are destroyed by vinblastine, a gray crescent forms under the influence of gravity (Ubbels et al., 1983). The conclusion drawn by Kirschner et af. (1980) and Gerhart et al. (1981) is that the gray crescent reflects a contraction of the animal hemisphere which achieves an asymmetric arrangement of vegetal materials. It would reflect a transient “dorsalizing” effect but would not itself be a dorsal determinant.

‘Reviewed by Brachet (1977), Kirschner et al. (1980), and Gerhart cr a/. (1981)

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FIG. 24. The gray crescent of amphibian eggs. (A) Photograph of a gray crescent (star) induced precociously by placing an axolotl egg at 3 5 . X for 10 minutes. (B) Schematic representation of a frog egg about 2 hours after fertilization. PA, animal pole; PV, vegetal pole; star, gray crescent. (C) A classic experiment by Ancel and Vintemberger (1948). ( I ) An unfertilized egg is placed in an oblique position and then fertilized (A, animal pole). (2) The egg rotates under the influence of gravity, after uplifting of the fertilization membrane, as indicated by the arrow. (3) The gray crescent always appears on the side facing the lamp. [Photograph in (A) courtesy of Dr. M. Namenwirth; (B,C) Brachet, 1977.1

The unknown cytoplasmic determinants present in the gray crescent region are UV sensitive (Grant and Youngdahl, 1974; Malacinski et a!., 1975). If the vegetal pole or the gray crescent region is UV-irradiated, microcephalic embryos (similar to those in Fig. 1) are obtained. This suggests, but does not prove, that ribonucleoprotein particles might be involved. Recent work by Gurdon (Mohun et al., 1984; Gurdon et al., 1984, 1985a,b) on the activation of cardiac- and muscle-actin genes during Xenopus development opens new perspectives. Using specific radioactive probes, they discovered that these genes become active, during gastrulation, in the very limited region of the embryo which will later differentiate into somites and muscles. Transcripts of muscle-actin genes form and accumulate in this mesodermal region several hours before somite differentiation; they are thus markers of muscle determination. The muscle-actin genes are activated as the result of an induction when animal and vegetal cells are fused together. Transplantation of a nucleus from a tadpole muscle cell into enucleate eggs leads to the following events. During cleavage, the synthesis of muscle-actin transcripts by the injected nucleus is suppressed; at gastrulation, the muscle-actin genes are selectively reactivated in the pre-

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sumptive somite region. Selective activation of these genes is thus controlled by the surrounding cytoplasm. Gurdon et al. (1985a) found that all the components required for the activation of the muscle-specific genes are already localized in the subequatorial region of fertilized but uncleaved eggs; these determinants are accumulated on the dorsal (gray crescent) side. These experiments give a concrete basis to Morgan’s theory of development and may lead to the identification of cytoplasmic determinants that control activation of the muscle-actin genes.

B . INSECT CYTOPLASMIC DETERMINANTS Of particular interest among the cytoplasmic determinants is the germ plasm of insects and amphibians, since it is absolutely necessary for the formation of gonads and thus for reproduction of the species. The germ plasm is located at the posterior end of insect eggs (where it is called pole plasm) and at the vegetal pole of amphibian eggs. The germ plasm in both insects and amphibians is characterized by the presence of large electron-dense ribonucleoprotein granules (111mensee and Mahowald, 1974; Williams and Smith, 1971). Destruction of the germ plasm, in both frog and Drosophilu eggs, by localized UV-irradiation leads to the production of sterile adults. If, as was done by Okada et al. (1974), UVirradiated eggs are injected with pole plasm material removed from a normal egg, fertile adults are obtained. Illmensee and Mahowald (1974) injected pole plasm material into the anterior part of a Drosophila egg and obtained germ cells in this area. Cytoplasmic determinants are also important in the development of the midge Smittiu (Kalthoff et al., 1982). Experiments have shown that the development of the head depends on cytoplasmic determinants located in the posterior half of the egg. If the posterior part of the egg is UV irradiated or injected with RNase, “double-abdomen’’ larvae that possess abdominal structures instead of a head are obtained. Pole cell formation is inhibited by UV-radiation and the action spectrum shows a maximum at 260 nm, suggesting that a nucleic acid or a nucleoprotein is involved (Brown and Kalthoff, 1983). It has also been reported by Jackle and Kalthoff (1980) that Smittia eggs synthesize a “posterior indicating” protein. These experiments support the hypothesis that cytoplasmic determinants are specific maternal mRNAs in insects.

C. Zlyanassa EGGS As shown in Fig. 25, cleavage in this mollusk is characterized by the formation of a purely cytoplasmic polar lobe at the so-called trefoil stage; removal of the polar lobe at this stage results in developmental abnormalities (Fig. 25). Isolated polar lobes synthesize proteins and thus contain maternal mRNA’s (Clement and Tyler, 1967). Removal of the polar lobe affects the pattern of

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FIG. 25. (A) Cleavage stages of llyanassa (mollusk eggs). (a) Fertilized e g g ; (b) formation of the first polar lobe; (c) trefoil stage: the material of the polar lobe is incorporated into one of the two blastomeres (called CD); (d) formation of the second polar lobe at the four-cell stage. (B) Left, a normal veliger larva after 9 days of development. Right, abnormal “lobeless” embryo. The first polar lobe had been removed and the egg had then been cultured during 9 days (Clement, 1952).

protein synthesis in the lobeless embryo (Newrock and Raff, 1975) as well as the rate of RNA and DNA synthesis (Collier, 1975, 1977). However, all these changes appear relatively late in development and coincide with the first developmental abnormalities. More recent studies (Collier and McCarthy, 1981; Brandhorst and Newrock, 1981) confirmed that synthesis of polar lobe-specific proteins cannot be detected and that more than 98% of the proteins synthesized during early embryogenesis are translated from maternal mRNAs.

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Some proteins are no longer synthesized, and new ones are synthesized between early cleavage and 24-hour embryos. Surprisingly, the same course of events occurs in nonnucleate isolated polar lobes (Brandhorst and Newrock, 1981). Among the proteins synthesized at the same rate by intact eggs, lobeless eggs, and isolated polar lobes are the histones (Collier and McCarthy, 1981). The conclusion drawn by Collier and McCarthy (198 I ) is that development up to the mesentoblast stage is not the result of differential gene transcription, but of the translational control of precociously transcribed genes.

D. ASCIDIANEGGS^ Ascidian eggs are the prototypes of mosaic development. Shortly after fertilization, segregation of cytoplasmic constituents leads to the appearance of five distinct ‘‘plasms’’ that differ in their fates (ectoplasm, myoplasm, endoplasm, chordoplasm, and neuroplasm). Of particular interest is the myoplasm (Fig. 26) that can be recognized as a yellow crescent. It gives rise to the muscle cells of the tadpole larva. Myoplasm-containing cells isolated at the 4- or 8-cell stage develop typical myofilaments (Crowther and Whittaker, 1983); myofilaments may even differentiate in this region in cleavage-arrested eggs (Crowther and Whittaker, 1984). A biochemical marker for muscle differentiation in ascidians is acetylcholinesterase; alkaline phosphatase and tyrosinase are marker enzymes for endoderm and brain melanocytes, respectively. Whittaker (1973, 1979) has followed, with cytochemical methods, the appearance of these three marker enzymes in eggs where cytokinesis had been suppressed by cytochalasin B treatment and in blastomeres isolated at the 8-cell stage. He used actinomycin D to establish whether the enzymes were synthesized on preformed or newly synthesized mRNAs, and puromycin to ascertain that they were synthesized de novo. Suppression of cytokinesis did not modify the general pattern of enzyme localization and appearance; the three enzymes are newly synthesized. Actinomycin D experiments demonstrated that alkaline phosphatase is synthesized in the endoderm on a maternal mRNA. In contrast, cholinesterase, tyrosinase, and ectoderm alkaline phosphatase are synthesized on newly synthesized mRNAs. Acetylcholinesterase mRNA is not synthesized before gastrulation, although cytoplasmic determinants needed for its transcription are already segregated in the myoplasm of fertilized, still uncleaved eggs. Meedel and Whittaker (1984) have separated the blastomeres of ascidian eggs, extracted total RNA, and injected it into Xenupus oocytes in order to detect acetylcholinesterase mRNA translation. The gene is active only in muscle and mesenchyme tissues, and its transcription does not begin before gastrulation. No cytoplasmic acetylcholin*Reviewed by Whittaker (1979) and Jeffery (1985).

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esterase mRNA is segregated in the cleaving egg. There is a striking parallelism between these results and those of Gurdon et al. (1984, 1985a) on the appearance of muscle-actin mRNA in Xenopus embryos. The conclusion of all these experiments (Whittaker, 1979) is that synthesis and localization of the enzymes depends either on a local accumulation of preformed maternal mRNA (endoderm alkaline phosphatase) or on the activation of specific nuclear genes by cytoplasmic determinants as had been postulated by Morgan in 1934. Thus, preformation and epigenesis coexist in the same egg during early development. Jeffery et al. (1983), who used in situ hybridization techniques, found that 45% of the poly(A)+ RNAs are localized in the ectoplasm and 50% in the endoplasm of fertilized egg; only 5% of the total poly(A) RNAs are located in the myoplasm. In contrast, 45% of the actin mRNA sequences are accumulated in the myoplasm, 40% in the ectoplasm, and only 15% in the endoplasm, while the histone mRNAs are ubiquitous. These findings demonstrate the heterogeneity at the molecular level of ascidian eggs. Centrifugation experiments suggest that the muscle cell determinants are anchored to the actin cytoskeleton of the myoplasm (Jeffery and Meier, 1984). This agrees with the fact that segregation of the myoplasm, soon after fertilization, does not take place in ascidian eggs treated with cytochalasin B, which disrupts the actin cytoskeleton (Zalokar, 1974). The analysis has gone one step further with the work of Satoh and Ikegami (1981a,b). Satoh (1982a) and Mita-Miyazawa et al. (1985) used aphidicolin to arrest DNA replication in normal and cytochalasin B-treated ascidian eggs and in isolated blastomeres. Embryos that had been permanently arrested by treatment with aphidicolin up to the 64-cell stage were capable of acetylcholinesterase synthesis. It was concluded that the eighth DNA replication cycle is of crucial importance for synthesis of this enzyme. Similar experiments (Satoh, 1982a) have shown that tyrosinase is not synthesized if DNA replication is halted before the gastrula stage. Endoderm alkaline phosphatase is not synthesized if aphidicolin is added at the 16-cell stage; it is synthesized if the inhibitor of DNA replication is added at the 32-cell stage. Thus, several DNA replication cycles are required for histospecific enzyme synthesis and the number of these cycles varies for each enzyme. The existence of a crucial mitotic cycle during cleavage has also been found in Chaetopterus (Alexandre et al., 1982) and in the mouse (Alexandre, 1982). Such crucial mitotic events have been called “quantal mitosis” by Holtzer et al. (1972). In a quantal mitosis, the two daughter cells are not identical and the difference between the two is hereditary. Unfortunately, we do not know the molecular mechanisms of these crucial mitotic events. In one review Satoh (1982b) concludes that the early morphogenetic events are controlled by a cytoplasmic clock, while the later events are associated with the DNA replicating cycles. +

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VIII. Nuclear Determinants (Genes) of Early Embryonic Development If the cytoplasm plays a leading role during cleavage, the importance of the nucleus, i.e., of gene expression, becomes primordial after the blastula stage. This is shown by the fact that haploidy, aneuploidy, in some cases hybridization, and many mutations sooner or later prove lethal for the embryo. The cases where lethality stops development at gastrulation or neurulation demonstrate that even primary morphogenesis is under nuclear control. These cases will be briefly discussed as follows. A. HAPLOIDY,POLYSPERMY, A N D ANHJPLOIDY IN AMPHIBIANS Haploidy can be obtained by a variety of means in amphibian eggs, such as pricking the unfertilized egg, fertilization with UV-irradiated sperm, removal of the maturation spindle from a freshly fertilized egg, etc. The result is always the same: cleavage is normal but gastrulation and further development are slowed down. Finally the embryos die at an early larval stage from the so-called “haploid syndrome,” including microcephaly , reduction of the gills, edema, and ascites. The reasons for haploid lethality remain obscure, but it is probably not due to the presence of lethal genes that are not balanced by their normal alleles since parthenogenetic homozygous diploids are viable in amphibians [but not in the mouse (reviewed by McLaren, 1984)l. In polyspermic eggs, only one of the nuclei of the supernumerary spermatozoa fuses with the female pronucleus; the other nuclei remain haploid. If polyspermy is not too heavy, cleavage follows, giving rise to a mosaic of haploid and diploid cells. Although part of the embryo is diploid, polyspermy is usually more lethal than haploidy. Aneuploidy (unbalanced chromosome complements) reduces viability, and morphogenesis is abnormal if only one or two chromosomes are added to the normal complement. If chromosome imbalance is marked, development stops at the blastula stage as in enucleate embryos,

B. LETHALHYBRIDS Many hybrids between sea urchins and amphibians stop development and die at the onset of gastrulation. The interpretation of the experiments is difficult due to the frequent loss of paternal chromosomes during the repeated cleavages. FIG. 26. Development of an ascidian Cynrhiu egg. (A) Side view of a fertilized egg showing the formation of the yellow crescent (cr, myoplasm) from the yellow hemisphere (yh); cp, clear cytoplasm above the yellow crescent. (B) Eight-cell stage. A, anterior, and P, posterior side of the embryo. pb, polar body marking the animal pole; cr. yellow crescent (myoplasm). (C) Young tadpole showing neural grove (np), mesenchyme (mch) and three rows of muscle cells (ms) deriving from the yellow crescent (Conklin, 1905).

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Lethal hybrids between sand dollar (Dendraster) eggs and sea urchin (Strongylocentrotus) sperm are the best-studied combination from the biochemical viewpoint. Both the paternal and maternal genomes are transcribed (Lee and Whiteley, 1982); surprisingly the proteins (esterases, malate dehydrogenase, hatching enzyme) are mostly maternal. Most of the antigens are also maternal in the hybrids (Badman and Brookbank, 1970), but there is a substantial synthesis of paternal histone H, during their early development (Easton and Whiteley, 1979; Maxson and Egrie, 1980). A gross underrepresentation of paternal proteins is also found in sea urchin hybrids that can reach the pluteus stage (Tufaro and Brandhorst, 1982). Clearly, sea urchin hybrids raise interesting questions about the control of transcription and translation. A tentative explanation for the variety of results on paternal genome expression in hybrids has been proposed by Crain and Bushman (1983), who found that transcripts of both paternal and maternal actin genes are present in hybrid blastulae. They suggested that the paternal genes for universal, highly conserved proteins (histones, actin) would always be expressed. On the other hand. specialized paternal genes whose expression is regulated by interaction with specific cytoplasmic factors would be poorly (or not at all) expressed in the hybrids. The development of many lethal hybrids between anurans is only slightly better than that of completely anucleate eggs (arrest at the blastula or early gastrula stage). However, if the dorsal lip of the blastoporus (organizer) of an arrested lethal frog hybrid is grafted into a normal gastrula (even from a newt), it is “revitalized”; it differentiates into chorda and somites, and it induces a secondary nervous system in the host (Brachet, 1944; J. A. Moore, 1947, 1948). It seems that lethal hybrids are incapable of synthesizing diffusible substances (which have no species specificity) that are required for further morphogenesis. Nuclear transplantation experiments by King and Briggs (1953) and Hennen (1974) have shown that if nuclei from a lethal hybrid arrested at the early gastrula stage are transplanted into recipient enucleate unfertilized eggs, development is normal until the onset of gastrulation and then stops abruptly. This shows that the nuclei of the hybrids are still unchanged at the time development stops; repeated replication in the egg cytoplasm does not revitalize them. In experiments where a diploid nucleus of the toad Bufo bufo was transplanted into a Bufo calamita egg, development stopped at gastrulation (Delarue and Aimar, 1984). Injection of hyaloplasm taken out of centrifuged B . calamitu eggs into fertilized B . bufo eggs also arrested development during gastrulation: the hyaloplasm contains at least two inhibitory proteins, which have not yet been characterized. Only crude biochemical parameters (respiration, overall nucleic acid and protein syntheses) have been studied in amphibian lethal hybrids. Only two facts deserve mention here. One is that arrest of DNA synthesis is not the cause of developmental arrest since DNA replication remains identical to that of the controls for many hours after gastrulation has stopped in the hybrids (Gregg and Lcivtrup, 1955). The other finding is that the surface glycoproteins of hybrid and

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normal gastrulae are not identical (Johnson, 1981). It is probable that changes in surface glycoproteins and cell adherence play an important role in the arrest of several hybrid combinations at the early gastrula stage. Treatment of cleaving Xenopus eggs with lectins (which bind to glycoproteins) prevents gastrulation (Moran, 1974). Normal frog gastrulae possess extracellular fibrils which promote cell migration, and these fibrils are reduced in number or even absent in lethal hybrids (Nakatsuji and Johnson, 1984). One of the major components of the extracellular fibrils is fibronectin. Antibodies against fibronectin arrest gastrulation (Boucaut et al., 1984a,b) and it has been shown by Delarue et al. (1985) that toad lethal hybrids lack a fibronectin-rich extracellular matrix.

C . A FEWEARLYLETHALMUTATIONS The field of developmental genetics is so broad that we must limit ourselves to the presentation of the mutations which arrest development at an early stage, or strongly modify morphogenesis. The ideal material for these studies is of course Drosophilu, although several mouse and amphibian developmental mutants have also been studied. The first evidence for a role of genes in early morphogenesis came from the work of Poulson (1940, 1945) on Drosophilu. He showed that complete loss of X chromosomes (nullo X condition) results in early arrest of development. The migration of the nuclei toward the periphery in order to form the blastoderm (Fig. 20) becomes abnormal, and development stops because the nuclei accumulate at the two poles of the egg. Ede (1956) and Counce (1956) later found that small deletions or even point mutations have the same effect on the migration of the nuclei as the absence of whole X chromosomes. I was greatly impressed when I became aware of Poulson’s observations because they led to the conclusion (in opposition to the distinction between general and special heredities proposed by my father) that individual genes control development at a very early stage. However, the contradiction was only apparent since it is now clear that “maternal effect” genes control the organization and chemical composition of the egg cytoplasm. In nullo-X unfertilized eggs, the cytoplasm is accumulated at the two poles instead of being evenly distributed around the inner yolk mass. Thus, it is not surprising that migration of the nuclei during cleavage is abnormal in nullo-X eggs and that this leads to early arrest of development. Another interesting early lethal mutation in Drosophila is deep orange (dor) (Garen and Gehring, 1972). In the homozygous condition (dorldor), development stops at gastrulation. Interestingly. injection of cytoplasm from a normal wild-type egg into dorldor eggs suppresses the lethal effects of the mutation and development may proceed until an advanced stage of embryogenesis. The lethality is due to the absence, in the dorldor egg cytoplasm, of a substance (or substances) required for development beyond the blastoderm stage. Comparable results have been obtained by Santamaria and Nusslein-Volhard

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(1983), who studied the dorsal locus in Drosophila. By a maternal effect, this locus induces dorsalization of the embryos, The abnormal dorsoventral pattern can be partially corrected by injecting cytoplasm from wild-type donor embryos. Further studies disclosed that mutations with maternal effects at 10 different loci give completely dorsalized embryos (Anderson and Niisslein-Volhard, 1984). Injection of poly(A) RNA from wild-type embryos into the eggs partially reversed dorsoventral polarity at 6 loci. It completely restored the normal dorsoventral pattern in one of the mutants (shake).These experiments prove that the information for this pattern is stored in maternal mRNAs synthesized and distributed during oogenesis under the control of the dorsal locus. If the maternal-effect Toll gene is inactive, the embryo is dorsalized; injection of cytoplasm from wild-type embryos corrects the abnormality (Anderson et al., 1985). Drosophila has long been an important material for developmental geneticists because mutations in the so-called homeotic genes affect late morphogenesis. For instance, Bender et al. (1983) have studied homeotic mutations in the bithorax (bx) complex; these mutations transform segments of the embryo into other segments. An extreme case is the bx3 mutation, which transforms halters in wings. The result is production of adult flies with four wings instead of two. Bender et al. (1983) found that mutations in the bithorax complex are due to DNA rearrangements. Most of the mutations have insertions of a particular genetic mobile element called gypsy which affects DNA sequences distant from the insertion site. Another homeotic mutation of interest is Kruppel; it leads to the deletion of the thoracic and anterior abdominal segments. It has recently been shown by Rosenberg et al. (1985) and Preiss et al. (1985) that injection of antisense Kruppel mRNA produces phenocopies of the Kruppel phenotype. A repetitive DNA sequence, which has been called homeo box by W. Gehring, is raising considerable interest today (reviewed by Gehring, 1985). This sequence was first found in the 3’ portion of several Drosophila homeotic gene complexes that are required for correct segmental development (bithorax, antennapedia) by McGinnis et al. (1984a). A homeo box is also present in the fushi tarazu (ftz) locus and mutations at this locus reduce the number of larval segments. Still more important is the fact that homeo box sequences are present in many invertebrates and vertebrates including man (McGinnis et al., 1984b; reviewed by Ruddle et al., 1985). A homeo box is also present in embryocarcinoma stem cells (Colberg-Poley et al., 1985). The homeo box DNA sequence codes for a highly conserved, very basic sequence of 60 amino acids which probably binds to DNA. Xenopus possesses a gene homologous to the Drosophila homeo box, which is expressed during gastrulation and gives three different transcripts under strict temporal control. This gene also codes for a basic, chromatin-binding polypeptide of 60 amino acids, which presumably controls the activity of a battery of genes (Carasco et al., 1984). Another Xenopus gene also containing a homeo box domain is abundantly transcribed in full-grown +

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oocytes (Miiller et ul., 1984). It might play a role in the early determination of embryonic cell types. In mice, a homeo box is located on chromosome 6 , which contains several genes which, if mutated, cause developmental abnormalities (McGinnis et al., 1984~).It is generally supposed that homeo boxes play an essential role in all the metameric organisms (in the separation of the somites in vertebrates, for instance). This seems likely in Drosophilu, but it is still too early to form a hypothesis about the role (presumably regulatory) played by homeo boxes in vertebrate embryology. Fushi turazu homozygotes are lethal before hatching. Theftz gene codes for a 1.9-kbRNA, which is expressed during early cleavage; it is no longer expressed during gastrulation (Kuroiwa et al., 1984). Elegant hybridization experiments by Hafen et al. (1 984) have demonstrated that theftz transcripts are already detectable during cleavage. At the blastoderm stage, they are restricted to seven evenly spaced bands of cells, corresponding to the future segments of the larva; they disappear at later stages. All this speaks for a key role offtz in the segmentation pattern of the Drosophilu embryo. Recently, Mlodzik et al. (1985) discovered another homeo box gene (dorsal) of still unknown function. its transcripts accumulate in the oocytes and generate an anteroposterior gradient at the syncytial blastoderm stage; they accumulate later in the posterior region of the blastoderm. The expression of this gene is maternal. Several mutations in amphibians deserve mention here. We have already mentioned the famous anucleolute (nu-o) mutation of Xenopus. The deletion of the nuclear organizers in homozygous nu-olnu-o eggs and embryos results in an inability to synthesize 28 and 18 S rRNAs and to accumulate new ribosomes after fertilization (Brown and Gurdon, 1964).Such Momozygous mutants hatch but die at the time control animals begin to feed. Heterozygotes have a single nucleolus instead of two. They are viable and have a normal rRNA content and a normal load of ribosomes because regulatory mechanisms allow them to synthesize as much ribosomal RNA as normal controls. Pierandrei-Amaldi et al. (1985) recently studied the synthesis of ribosomal proteins in nu-olnu-o embryos. They are normal until hatching but ribosomal proteins synthesized in the absence of rRNA production are unstable. Thus, their synthesis is regulated at the level of mRNA stability. Another interesting mutant is the nc (no cleavage) mutant of the axolotl (Raff et a f . , 1971).Fertilization is normal but eggs are unable to cleave because they cannot assemble their large tubulin store into spindle and aster microtubules. Repeated cleavages occur after injection of basal bodies into the cytoplasm of nc fertilized eggs. Another maternal effect mutation is the o mutation, which affects the cytoplasm of axolotl eggs. Homozygotes of olo develop normally until the blastula stage but are unable to undergo gastrulation (as in many interspecies hybrids). If

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one injects nuclear sap removed from the GV of an o / o or o f lo oocyte into one of the blastomeres at the two-cell stage, the injected blastomere develops much further than the uninjected one (Briggs and Cassens, 1966; Briggs, 1972). Injection of the same material into a fertilized uncleaved mutant egg allows normal development (Fig. 27). The GV thus contains a gene product (presumably a karyophilic protein since the oocyte cytoplasm is inactive) necessary for development beyond gastrulation. This product has no species specificity and is already present in the GV of young oocytes. It may play a role in RNA synthesis, which is very low in o / o mutants. Brothers (1976) transplanted nuclei from o/o morulae into anucleate unfertilized eggs and found that they can support full development. In contrast, nuclei from arrested o / o blastulae had lost this capacity. Thus, irreversible alterations of the nuclei occur between the morula and blastula stages in the mutants. Finally, in mouse eggs, interest has been mainly focused on the complex Tlt locus, where many different alleles are known (reviewed by Frischauf, 1985). This complex is localized on chromosome 17 and is the equivalent, for mouse embryos, of the H-2 complex which, in the adult mouse, controls the synthesis of +

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FIG.27. A maternal effect mutation (0, for ova deficient) in the axolotl. Females homozygous for o produce eggs which always arrest at gastrulation. This gastrular arrest can be corrected by the injection of o+ substance. This o+ substance is produced during oogenesis under the direction of the normal allele of the o gene. The substance is found in the germinal vesicle and in the cytoplasm of normal full-grown oocytes (Brothers, 1976).

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the histocompatibility antigens. Many t mutants are known and have been studied from both genetic and embryologic viewpoints. TIT is lethal on the tenth day of development and Tlt mice have no tails. The tI2/tJ2embryos stop developing already at the morula stage (Smith, 1956). The tJ2mutation is probably a classic one affecting a gene required for early development, already active during cleavage, rather than a maternal-effect gene affecting the composition of the egg cytoplasm during oogenesis . The intensive work done on transgenic mice today, obtained by injecting pure genes in one of the pronuclei of a fertilized mouse egg, deserves the greatest admiration because it has fulfilled an old dream for all biologists to create new strains of animals with predetermined genetic changes. The subject, which has been recently reviewed by Palmiter and Brinster (1985) and by Gordon and Ruddle (1985), is still in full expansion. It lies outside the scope of this article since it deals with adult mice and their offspring. All I can do is express admiration for those who are working in this exciting field.

IX. The Past and Future of Molecular Embryology This article is a brief and incomplete account of our present knowledge of nucleocytoplasmic interactions in morphogenesis . Recent progress in this field is closely linked to the discoveries made by molecular biologists. This was not always the case and one may argue that work done on eggs and embryos has played an important role in the beginnings of molecular biology. When I started working in A. Dalcq’s laboratory almost 60 years ago, it was believed that animal cells contained thymonucleic acid (our DNA) in their nuclei, while plant cell nuclei had a pentose nucleic acid. My early work with cytochemical and biochemical methods clearly showed that sea urchin eggs possess a store of “plant” nucleic acids (RNAs) and that RNA is mainly localized in the cytoplasm (Brachet, 1933). My later work (1942) as well as that of Caspersson (1941) strongly suggested that RNAs are involved in protein synthesis in all cells. These ideas were iconoclastic proposals at the time. Before proposing that a plant nucleic acid is present in sea urchin eggs, I sought the advice of Joseph Needham. He was so surprised that he consulted his Professor, Sir F. G . Hopkins, Nobel Prize winner. “Hoppy’s” verdict was that I should do experiments to prove my contention. This led me to measure the pentose content of sea urchin eggs with a method devised for the measurement of pentosanes in . . .straw. This was done despite the ironic comments of my French friends, B. Ephrussi, A. Lwoff, and J. Monod. When Caspersson and I proposed that RNAs are required for protein synthesis, we received irate letters from leading protein chemists who refused to believe in all this nonsense. Yet we were right and we already had at the back of

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our minds all the elements of Crick’s fundamental dogma of molecular biology; i.e., DNA makes RNA, and RNA directs protein synthesis. However, I fully realized that what seemed to be sound hypotheses should be proven and this was done by molecular biologists working on bacteria and phages. Once J. Monod told me: “You will never prove that RNA plays a role in protein synthesis if you stick to your eggs. You should work on E . coli like we do.” He was right and I watched with growing enthusiasm the progress made in molecular biology. I immediately believed that Avery and his colleagues were right when they showed that genes are made of DNA. Skepticism was widespread at the time because it was generally believed that DNA is a small molecule with an M, of about 1200. I also immediately believed in the one gene-one enzyme theory of Beadle and Tatum, partly because previous experiments by my friend Ephrussi on Drosophila had already convinced me that genes direct the synthesis of specific proteins. When I read for the first time the short note where Watson and Crick proposed a double-helix structure for DNA and its implications for the mechanisms of replication and mutation, I thought “I hope they are right!” The doublehelix structure was not accepted immediately by all crystallographers and I remember vividly a terrible duel in Brussels between two famous Nobel Prize winners, Bragg and Pauling. Bragg, who headed the Laboratory where Crick and Watson were working, won, in the presence of a nervous and anxious Jim Watson. I rated-and 1 still rate-the complete deciphering of the genetic code as one of the major successes for human intelligence. I followed with interest the development of the Jacob-Monod theory of gene regulation in bacteria. However, this interest was mild because I never believed (despite Monod’s dictum: “what is true for a bacterium must be true for an elephant”) that elephants or men are bacteria. We have seen that the multiple and complex mechanisms of gene regulation in eukaryotes are not yet fully understood and that they certainly differ from those discovered in bacteria. Many embryologists tried to explain development by the Jacob-Monod model. They failed but it should be admitted that we have no model yet to work with. The messenger RNA story was of course very close to my heart in view of my work on Acetabularia. Like Jacob and Monod, I hoped that mRNAs would be found in all living organisms. This hope has been fulfilled. I should like to mention two anecdotes about mRNAs. Once I visited Jacques Monod at the Pasteur Institute. This was before the mRNA hypothesis at a time when it had been found by the Pasteur group that introduction of the P-galactosidose gene in a recipient E. coli is almost immediately followed by synthesis of the enzyme. Monod said to me, “My poor friend, I am sorry for you. You believe that RNA is required for protein synthesis and we have evidence that DNA makes proteins directly.” I answered that if proteins are made so fast, an RNA copy of the gene could also be made very fast. This led later to the proposal by Jacob and Monod that mRNAs have a very short half-life, and to the second anecdote. At a

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symposium, Franqois Jacob asked me, “Whenever I speak of mRNAs, somebody objects. And what about Acetabularia? What should I answer?” The answer is simple, I said, “Just drop the short life of the mRNAs in your messenger story and everything will be all right.” Today everything is all right, I even have an answer to the question that aroused my scientific curiosity almost 60 years ago: variations in the life span of anucleate cells result primarily from differences in the stability of their mRNAs. During the period which saw the amazing growth of molecular biology, embryology has been lagging behind. Today more and more molecular biologists are interested in problems of development and embryologists are aware of the importance of the molecular events which underlie development. Despite the marked progresses described in this article, many basic problems remain unsolved. What are the molecular mechanisms of embryonic regulation and determination? What are the molecular bases of the germinal localizations, and what is the chemical identity of the cytoplasmic determinants? How does the nucleus, in certain biological systems, exert a negative effect on cytoplasmic activities (its positive effects are of course largely understood)? What are the molecular mechanisms of cytoplasmic and nuclear clocks, of quanta1 mitoses? What is the chemical nature of the inducing agents and, still more important, how do they act on the target cells? What are the differences between competent and noncompetent cells? As one can see, many basic embryological problems are still unsolved. Their solution will require close cooperation between embryologists and molecular biologists, but the latter are seldom aware of the existence of these problems. Several young and bright molecular biologists have asked me the same question, “What shall we do when all the genes have been cloned and sequenced?” I do not think that a complete knowledge of the mouse genome will be enough to understand fully how a mouse egg gives rise to a mouse. Franqois Jacob was once asking me the question, “How long will it take before we know how a mouse egg gives rise to a mouse? Thirty or 300 years?” I am sure that I do not know, but I am convinced, from everything I have seen happening during half a century of research, that we are on the right track and that the day will come when we shall have the answer to that question.

REFERENCES Adamson, E. D., and Woodland, H . R . (1977). Dev. Biol. 57, 136-149. Aimar, C., Delarue, M . , and Vilain, C. (1981). J . Embryol. Exp. Morphol. 64, 259-274. Aimar, C., Vilain, C., and Delarue, M. (1983). Cell Difer. 13, 293-300. Alexandre, H. (1982). C.R. Acad. Sci. Paris 294, 1001-1006. Alexandre, H . , De Petrocellis, B . , and Brachet, J. (1982). DiSferenriarion 22, 132-135. Ancel, P . , and Vintemberger, P. (1948). Bull. B i d . 31 (suppl.), 1.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 100

Protistan Phylogeny and Eukaryogenesis JOHN 0 . CORLISS Department of Zoology, University of Maryland, College Park, Maryland 20742

I. Introduction and Overview Well before the modern reemergence of protistology as a bona fide, if interdisciplinary, field in its own right (Corliss, 1986c), biologists had begun to realize that the eukaryotic condition could hardly have appeared full blown in the cells of metazoan and metaphytan tissues directly from a prokaryotic predecessor. Some intermediate stage or state in such a momentous evolutionary step must have existed, and it was reasonable to postulate that it involved-as the best link containing extant representatives-the unicellular eukaryotic microorganisms. Numerous candidates were available for study, but many of the implicated groups of protozoa and algae were either confusingly assumed to be interrelated or, worse, were artificially separated taxonomically on superficial criteria (see below). Furthermore, the bacteria, and sometimes viruses, were often considered “protists” along with the truly eukaryotic assemblages of microscopic forms, even as recently as the 1970s (e.g., see Jennings and Acker, 1970; Poindexter, 1971; Ragan and Chapman, 1978). Lumping minute forms of life together on the basis of size or common habitat, along with lacking appropriate techniques for in-depth study of cells, prevented much success in earlier investigations into eukaryogenesis. Widespread recognition-backed by hard data-of the evolutionary gap between prokaryotes and eukaryotes, discussed below, was a necessary prelude to genuine progress in the area. The evolution of plants and animals themselves from “lower forms” had long been thought about, with generation of various hypotheses based principally on theoretical considerations. Discussions of such topics and additional references to them may be found in various textbooks and specialized treatises (e.g., see Cronquist, 1968; Dougherty et al., 1963; Hanson, 1977, 1981; House, 1979; Stewart, 1983; and such recent papers as those by Bremer, 1985; Dahlgren, 1983; Taylor, 1982), but they are beyond the scope of this article. With the advent of the cell theory a century and a half ago unicellular organisms came to warrant some special attention. This was perhaps climaxed in the stimulating, if (by hindsight) somewhat misleading, essay by the angry young Englishman Dobell (1911) who, in his vitriolic attack on that revered theory, proposed 3 19 Copyright 0 1987 by Academic Press, Inc. All rightr of reproduction in any form reserved.

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complete separation of (unicellular) protists from (other) eukaryotes with his insistence on the non- or acellular nature of the former. The relationship between advances in cell biology and improvements in microscopy-not always occurring in synchrony, incidentally, as pointed out by Hughes (1952) in the first paper published in the first volume of this series-has been treated in a brief contribution by the author to the history of protozoology (Corliss, 1978- 1979). The principal aim of this article is to focus attention on (the nature of) the protists themselves, a group deserving such attention today if for no other reason than their suitability as “model” cells in experimental researches of diverse kinds, and to discuss approaches to a better understanding of their phylogenetic interrelationships. Treating topics of their own origins and evolutionary lines will inevitably impinge on the vast area of eukaryogenesis. But the latter subject per se will not specifically be reviewed in detail, mainly because of the existence of the recent very thorough coverage found in the combination of such works as Cavalier-Smith (1981a), Dodson (1979), Frederick (198 I), Gray and Doolittle (1982), Li (1979), Margulis (1981), and Taylor (1979, 1980a, 1983). Indeed, because of restriction on space, some details and historical aspects of the generally neglected “protist story” itself will also have to be omitted here. References to much of such information, however, may be found in two earlier papers by the author (Corliss, 1984, 1986~). It hardly needs be said that this article, at best, can serve only as a progress report, since it is concerned with a fast-moving area of biological inquiry in which much of our knowledge is currently in a state of flux, with a far greater number of questions than answers available.

11. Reflections on the “Protist” Concept

The words and concepts of “protists” (or “protoctists”) and “protistology” are not new to biology. They have existed, with one meaning or another, for scores of years, essentially since the times of the seminal works by Hogg (1 860) and Haeckel(l866, 1878). Until recently their popularity has waned more than it has waxed, however, and their exact meanings are still hotly debated today (e.g., see Corliss, 1984, 1986~;Margulis and Sagan, 1985; Margulis and Schwartz, 1982; and references within those works). In my opinion it is neither a difficult nor unreasonable decision to employ the more euphonious and perfectly clear words “protist” and “protistology” as the terms of preference (over “protoctist” and “protoctistology”), especially since our modern concepts conform exactly to neither Hogg’s nor Haeckel’s original boundaries of groups involved. (Hogg’s “Protoctista” was first, but priority is not binding at this level.) In any case, there is little disagreement over the proposition that the protists

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sensu luto, at least as a vernacular term or with reference to a grade of organization, embrace groups of both unicellular and multicellular algae and protozoa plus at least the so-called “lower” fungi (zoosporic forms, slime molds and relatives), perhaps other fungi, and maybe such anomalous taxa as placozoa and sponges. Rather, the major problem not yet resolved to universal satisfaction is whether or not these “lower” eukaryotes truly represent a single integrated assemblage of forms, a unique taxon (see Corliss, 1986c, and references therein), or whether they are better dispersed among a number of separate eukaryotic kingdoms, some of which may also contain nonprotist groups (again, see references in Corliss, 1986c; but particularly see papers by Cavalier-Smith, 1978, 1981b, 1983; Leedale, 1974; Starobogatov, 1984; and the compendium by Mohn, 1984). Then there is still another school of thought, following a (second) proposal by Leedale (1974), in which the protists are best considered as simply a grade or level of organization, an evolutionary stage generally intermediate between prokaryotes and the “higher” eukaryotes, with no independent taxonomic status of their own. Figures 2-4 illustrate diagrammatically (and simplistically) the three principal ways of treating the protists systematically/evolutionarily today. Figure 1 is included to contrast the original Haeckelian approach, bold advance though it was over the entrenched two-kingdom system, with the phylogenetic concepts of the present time. Resolution of this controversy, fortunately, is not a prerequisite to discussion of the other topics included in this article. The modern or neo-Haeckelian concept that the protists (however defined: see below) may possibly comprise a (separate) kingdom of eukaryotes serves a

FIG. 1. The 120-year-old Haeckelian concept of a “third kingdom,” Protista, at the base of the evolutionary “tree” of the entire biotic world. An, animal kingdom; PI, plant kingdom; Pr, protist kingdom.

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FIG.2. A modem “five-kingdom’’ concept of the biotic world, essentially as proposed by Whittaker (1969). An, kingdom Animalia; Fu, kingdom Fungi; PI, kingdom Plantae; Pr, kingdom Protista; PROK, the prokaryotes. The last is probably best considered a superkingdom, the PROKARYOTA, with several kingdoms of its own. Then the other four kingdoms would comprise the superkingdom EUKARYOTA (see further discussion in the text).

heuristic role in contemplating differences of evolutionary significance between them and the prokaryotes on the one hand, and in considering both similarities and differences between them and the other eukaryotes-the widely endorsed kingdoms of the (“higher”) fungi and the multicellular/multitissued/vascularized plants and animals-on the other hand. The fact that the whole field of

FIG. 3. A “multikingdom” concept of the biotic world, inspired by one of Leedale’s (1974) suggestions. The protists, as a single, discrete taxonomic “unit,” disappear, replaced by numerous separate independent kingdoms of ‘‘lower’’ eukaryotes. The representation here is highly diagrammatic, and there is no significance to be attached to the sizes, numbers, or positions of the ‘‘clubs.’’ In fact, some such multiple kingdoms involving protists would probably also embrace all or parts of the other three eukaryotic assemblages (too difficult to depict here in a simple way; see text). Abbreviations as in Fig. 2.

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FIG.4. A “four-kingdom” concept also based on (another) one of Leedale’s (1974) stimulating suggestions. Here the protist groups have no taxonomic identity of their own. They have “disappeared” except as a grade or “level of organization” (delimited by the dotted line) in the evolution of the three eukaryotic kingdoms shown from a prokaryotic ancestry (see text for further discussion). Abbreviations as in Fig. 2.

systematic and evolutionary protistology is in a state of ferment and foment these days, I insist, may be considered great news, a sign of progress!

111. Bases for Renewed Interest in “Lower” Eukaryotes As suggested above, reflection on the groups of protists-especially on their evolutionary history-may well throw light on aspects of the fascinating phenomenon of eukaryogenesis sensu lato. Furthermore, the value of these mainly microscopic eukaryotes as ‘‘model” cells in experimental work-as already mentioned-is beginning to gain wide appreciation. But such usages depend on recognition of protists, first of all, and on a general acceptance of the protist concept as well as on development of properly sophisticated methods for their thorough study as cells. As I view the situation, four basic events or developments, “happenings” in the growth of the biological sciences overall, had to occur before there could be a substantial revival of interest in the ‘‘lower’’ eukaryotes, viz., the protists. Briefly mentioned elsewhere by the author (Corliss, 1986c), these factors may be considered here in a little more detail.

DISTINCTION A. THEPROKARYOTE-EUKARYOTE Although the proposal that the biotic world can be divided into two major categories, the prokaryotes and the eukaryotes, is hardly new (or “news”) any

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longer, this revolutionary concept has had a most profound and salutary effect on evolutionary ideas for all living things, and it has served as an important prelude to the “new protistology.” In a repeatedly overlooked work published by Edouard Chatton more than 60 years ago (Chatton, 1925) one will find the first use of the terms “procaryote” and “eucaryote” (though only in tables appended to a long paper devoted to the biology of an amazing parasitic amoeba, Punsporellu perplexu, thus explaining, if not forgiving, people’s failure to make the discovery earlier). Credit for formal recognition of this split among living organisms is generally bestowed on Roger Stanier, not without a degree of justification, for he was an indefatigable disciple and promoter of the concept from the 1960s until his own quite recent passing (e.g., see Stanier, 1970; Stanier and van Neil, 1962; Stanier et ul., 1976, 1979). While recognition of the prokaryote-eukaryote division made possible the whole idea of (and even the word, of course) “eukaryogenesis,” it did not directly help focus taxonomic attention on the protists. In fact, many biologists until very recent times (see citations in Section 11) followed the lead of Chatton and others in employing the vernacular term “protist” to include both “lower” (prokaryotic) and “higher” (microscopic eukaryotic) microorganisms, despite the acknowledged (from 1962 on) great evolutionary gap between the two. On the other hand, the growing appreciation of microorganisms as opposed to macroorganisms generally did represent a significant step forward with respect to the groups under consideration in this article. €3. REVIVALOF A KINGDOMFOR PROTISTS

Revival of the seemingly ill-fated Haeckelian (or Hoggian) view(s) of a third kingdom to embrace the numerous microscopic eukaryotic organisms represents the next important development leading to the present high interest in the biology of protists. At last, new champions were coming forth and rechallenging that terribly entrenched and persistent notion that algae are, in effect, merely “miniplants” and protozoa “mini-animals” (Corliss, 1983). New discoveries as well as fresh thinking began to make clear that imposition downward of plant and animal characteristics fails miserably to characterize properly the vast assemblages of diverse microorganisms that share the cellular and nuclear state (while showing other differences of significance) with fellow (macroscopic) eukaryotic groups. Chatton (e.g., see 1925, 1938) and perhaps more so Copeland (e.g., see 1938, 1956), and maybe-to a degree-even Dobell (191 1) still earlier, might justifiably be hailed for resurrecting the “protist concept” after its too-early general demise following the efforts of pioneers Haeckel and Hogg in the middle of the preceding century. Indeed, Soyer-Gobillard (1985) equates the life of Chatton directly with “l’essor de la protistologie moderne. ”

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But what I have considered to be a bona fide fully refined and defined neoHaeckelian approach to protistology did not occur until the 1970s, in my opinion (Corliss, 1986~).Of course, by that time-50 years since some of Chatton’s major monographs-more data of relevance and investigative methods of greater sophistication (see below) had become available. Taking advantage of the situation and often giving practically undivided attention to the protists-their systematic standing, their phylogenetic interrelationships, their overall biology, even their conceptual definition or characterization-were such workers and leaders as the late R. H. Whittaker, Lynn Margulis, G . F. Leedale, L. S . Olive, and F. J. R. Taylor, followed shortly by T. Cavalier-Smith, K . D. Stewart, K. R. Mattox, A. R. Loeblich, Jr., HelenTappan, M. A. R’agan, D. J. Chapman, I. B. Heath, M. A. Sleigh, J. D. Dodge, K. Hausmann, G . Brugerolle, J.-P. Mignot, M. Melkonian, 0. Moestrup, D. J. S . Barr, D. J. Patterson, J. 0. Corliss, and still others. See the pioneering publications by Leedale (1974), Margulis (1970, 1974a,b), Olive (1979, Taylor (1974, 1976a, 1978), Whittaker (1969, 1977), and Whittaker and Margulis (1978). Such early modern comprehensive accounts stimulated further production of an unprecedented number of papers in what I have heralded as “the new protistology,” considering it a unique interdisciplinary field coming into its own only some 10-12 years ago (Corliss, 1986~). C. THE SERIALENDOSYMBIOSIS THEORY

Hand in hand with the growing general interest in the protists (see above) has come resuscitation of hypotheses of eukaryogenesis that have specifically implicated the phenomenon of endosymbiosis as responsible for the origin of some of the major cytoplasmic constituents recognized in cells today. Like the acknowledgment of protists and the separation of prokaryotes and eukaryotes, the idea of an exogenous origin for certain cell organelles, notabIy mitochondria and chloroplasts, is hardly a new one (e.g., see Mereschkowsky, 1905; Wallin, 1927; and other references available in Margulis, 1970). Yet the celebrated serial endosymbiosis theory (SET) may be said to have been clearly enunciated, and in a modem cell-biological context, only recently, in the highly stimulating if somewhat controversial books by Margulis (1970, 1981). While contemporary species of protists are frequently involved in symbiotic associations as either host or symbiont, much of the emphasis of the SET theme is on the early origin of eukaryotic cells (or organisms) via the engulfing by or the invasion of some hypothetical prokaryote, or “primitive” eukaryote, by another similar such organism. The endosymbiotic partner is conceived of as having obligingly evolved into (the progenitor of) today’s mitochondrion or plastid, these organelles retaining (some of) their original DNA while also becoming fully integrated into functional systems of their host cell. lt is worth

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noting that, from the point of view of unraveling evolutionary/phylogenetic relationships, the occurrence of endosymbioses can be a very complicating factor, as most recently stressed and documented by Rothschild and Heywood (1986). As mentioned earlier, it is not a purpose of this article to consider in detail the pros and cons of theories of exogenous (or xenogenous) as opposed to endogenous (or autogenous) means of eukaryogenesis, nor to treat the many fascinating facets of yet another new field of biological research labeled ‘‘endocytobiology” by its founders (see Schwemmler and Schenk, 1980; Schenk and Schwemmler, 1983). The latter closely related area of study could, in its broadest sense, embrace all work related to the SET idea as well as covering other lines of cell research.Taylor (1979) has identified this new field as “cytobiosis,” and Corliss (1985a), emphasizing the presumed original foreign nature of the invasive organism/organelle (hence called a “xenosome”), has suggested “xenosomology” as a possible synonym. Taylor (1974, 1976b, 1979, 1980a, 1983, and surely more in the offing) has probably been the most faithful and most incisive reviewer of the overall topic of endocytobiology and the SET proposition, while contributing new data and ideas to aspects of the field as well. Also, pertinent multiauthored compendia have appeared (e.g., Frederick, 1981) in addition to still other critical reviews concerning eukaryogenesis by such workers, to cite a few, as Cavalier-Smith (1981a), Dodson (1979), Gray and Doolittle (1982), Margulis (1981), Ragan and Chapman (1978), Schwartz and Dayhoff (1978), Uzzell and Spolsky (1981), Van Valen and Maiorana (1980), and Whatley et al. (1979). Books edited by Anderson et af. (1984), Goff (1983), Jeon (1983), and Wiessner et af. (1984) are relevant to endocytobiology sensu lato, too, as are the works of Bannister (1979) and Smith (1979). A short but significant and very recent publication is that by Cavalier-Smith and Lee (1985). Many additional papers, often treating plastids alone or mitochondria alone or restricted to even more specialized topics, are important but beyond direct citation here (for the bulk of them, see the bibliographies of many of the references given directly above, plus Corliss, 1986~).

D. DEVELOPMENT OF NEWTECHNIQUES Finally, in this short list of developments or “happenings” essential (in my opinion) to the rise of the “new protistology,” the advent of modern cytological and biochemical techniques has clearly proven to be an indispensable boon. Without the approaches to cell study possible today at molecular, macromolecular, and genetic levels, little real progress-beyond speculation-could have been made in gaining comprehension, for example, of underlying mechanisms in establishment, maintenance, and integration of endosymbionts (past or present) in their host cells. Such xenosome-cytocosm relationships may often be as

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useful in solving phylogenetic and systematic problems as they are in understanding physiological, behavioral, and genetic/evolutionary parameters of cellto-cell interactions. Even to recognize and describe with accuracy the very existence of xenosomes and organelles-and to be able to identify their internal substructure, their bounding membrane(s), etc. -has required resolution beyond that of the conventional light microscope. The electron microscope is a “must,” as Smith and Patterson (1986) have most recently stressed and elegantly demonstrated. New (i.e., within recent decades) ways of studying cells that yield data of high information content at the descriptive (and sometimes chemical) level include refinements in electron microscopy (ultra-high voltage, freeze-fracture, tiltable stages, back-scatter electron detectors, EM radioautography, X-ray detectors, etc.) as well as improvements in light microscopy (differential interference, epifluorescence, etc.) and development of new staining methods, etc. Comparative approaches combining light and ultrastructural (TEM and/or SEM) techniques yield especially useful data. At the molecular level, biochemical, immunological, genetic, and other kinds of experimental investigations of whole cells-and of subcellular organelles, membranes, etc. -have been aided and abetted by new ways in general for studying enzymes (in this case electrophoresis serves as an example), pigments, chromosomes, metabolites of diverse sorts, etc. Most recently, amino acid, nucleotide, and tRNA and rRNA sequencing techniques, restriction enzyme mapping, hybridization of cloned probes, immunoblotting, and the like are proving to be invaluable (Jeon, 1986). But sequencing work on protists is still in its infancy and has involved very few species, thus curtailing its usefulness until some future date. Unfortunately, there is no single source of information on the kinds of celland molecular-biological methodologies referred to in the brief listing given above. A good start can be found in the pioneering, but now aging, book by Ragan and Chapman (1978). It was extended somewhat in a paper by Chapman and Ragan (1980), but a second full edition is urgently needed! Since the protists represent such a diversity of systematic/evolutionary groups, technical papers are scattered throughout scores of different journals, not all published in the English language. These include systematically oriented subject matter (e.g., protozoological, phycological, mycological, parasitological) outlets as well as specialist journals in fields (biochemistry, physiology, ultrastructure, genetics, ecology, behavior, molecular biology, etc.) that cut across classical taxonomic boundaries. A few references to helpful recent books or review-type papers, themselves rich in citations to the original literature, are offered here, but since most of them are concerned only with the broader cytological methods the interested reader still must often depend on the appropriate specialized journals in hidher library for up-to-date information on particular biochemical or more restricted molecular

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techniques. Nevertheless, it may be useful to consult the following: Bardele (1983), Bold and Wynne (1985), Cole (1979), Corliss (1979a,b, 1986c), Demoulin (1979), Dillion (1981), Dodge (1979), Duckett and Peel (1978), Gantt (1980), Grain (1986), Gray et al. (1984), Hausmann (1978), Hibberd and Norris (1984), Honigberg et al. (1982), Lee et al. (1983, Lynn (1981), Mattox and Stewart (1984), McCutchan et al. (1984), Melkonian (1980, 1984), Moestrup (1982), Patterson and Fenchel (1989, Pickett-Heaps (1979), Ragan and Chapman (1978), Rosowski and Parker (1982), Rothschild and Heywood (1986), Scholtyseck (1979), Smith and Patterson (1986), Sogin et al. (1986), Vickerman and Preston (1976), Yao et al. (1985). As I have pointed out elsewhere (Corliss, 1986c), computerized treatment of data represents yet another technological advance with a positive impact on progress in protistology.

IV. Diversity within the Protist Conglomerate One of the most vexatious problems plaguing protistologists is-and always has been-defining or characterizing their organisms in an all-inclusive taxonomic manner. Because so much is unknown about the vast majority of the described species (possibly numbering nearly 200,000; Corliss, 1984) and because so many of the groups are strikingly different from one another, it is no wonder that the total assemblage has been considered ‘a motley array of poorly understood microorganisms,’ an ‘unnatural rag-bag of organisms,’ a ‘taxonomic wastebasket of little-known microscopic forms,’ ” as lamented in Corliss (1981). In short, perhaps the greatest virtue of the protists-their diversity-is at the same time their greatest weakness or handicap to being better understood. One solution to the systematics of these ‘‘lower’’ eukaryotes, first most clearly expressed (as mentioned in a preceding section of this paper) by Leedale (1974) in one of his two views on the subject, is to consider them simply as a structural grade of organization, an evolutionary “gap bridger” between prokaryotes and the multicellular and multitissued plant, animal, and fungal eukaryotes. The kingdoms containing the latter would have had protistan ancestries, but such involved groups of protists would be suspected to have had (or would have left descendants which have) little or even no phylogenetic homogeneity or cohesiveness among themselves. Taken in its extreme, this notion that protists principally serve only as temporary stages in the evolution of “higher” forms is unacceptable to me, since it seems to fail to recognize the uniqueness of many contemporary protist taxa (i.e., their species certainly are not all en route to evolving into macroorganisms) and to appreciate the fact that there are many shared derived characteristics “

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among protist groups. Also, although the spotty fossil record (despite its help in certain cases; see Loeblich, 1974; Tappan, 1980; and references therein) is of little aid, it is conceivable that a number of major protist taxa did not arise until well after an evolutionary transition to some of the “higher” eukaryotes had already occurred (Corliss, 1981). The matter of kinds and numbers of phyla and supraphyletic assemblages possibly involving/containing lower-level taxa of protists is treated in subsequent sections of this article. But we may consider here that the principal reason for the great diversity shown by the entire protistan conglomeration stems from the broadness of the coverage conceived for it. That is, by virtue of embracing all of the (eukaryotic) algae, the various protozoa, and select taxa originally attached to the fungi proper, a tremendous range of organisms is brought together under one heading, “the protists.” This is true whether or not they, are recognized as comprising a single or several kingdom(s) from a formal taxonomic point of view, or whether they are thought to represent a (mere) structural level of organization in the biotic world. Cytoarchitecturally, the gamut runs from the so-called ‘‘simple” amoebae to the most complex of the ciliates, from the minute unicellular flagellates (pigmented or otherwise) or tiny sporozoan species to the multicellular or coenocytic filaments of certain green or golden-brown algae, and from the motile zoosporic fungal protists to the gigantic thalloidal-branched brown algae. Cell walls of differing compositions may be present or absent, and body shapes as well as sizes are diverse. Physiologically, protists range nutritionally from free-living autotrophic (with a variety of photosynthetic pigments possible) or heterotrophic forms to saprotrophic and truly parasitic forms. Motility, by various modes, may be present temporarily or permanently or absent from the life history. In reproduction, ‘‘simple’’ asexual binary fission or complicated polymorphic life cycles with multiple budding or spore formation may be involved. Meiosis, if/when occurring, may be gametic, zygotic, or sporic. Habitat preferences range from a great number of basically aquatic types to edaphic, terrestrial, and-for symbiotic forms-various sites on or within the integument, body cavities, tissues, or cells of other (eukaryotic) organisms serving as hosts. Of course, this very flexibility or plasticity in characteristics among members or groups within the entire assemblage, perhaps evidence of early evolutionary experiments, has made some of the protists ideal progenitors of the divergent lines now recognized among the several groups of “higher” eukaryotes. On the tentative assumption that the protists may phylogenetically be interlinked into a single, if widespread, conglomerate of organisms with its own taxonomic integrity or reasonable amount of phylogenetic cohesiveness, the following diagnosis, general characterization, or description, based on consid-

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eration of a constellation of characters (and condensed from that appearing in Corliss, 1984, 1986a), is offered here without further comment, sensitive though I am to its limitations. The Protistu are eukaryotic organisms with no more than one “true” tissue at most. The majority are unicellular in organization and microscopic in size. Even in macroscopic multicellular species (e.g., brown algae), clearly differentiated tissue stages may be considered absent, and no vascular forms exist. Motile species (via flagella, cilia, or pseudopodia) are common and widespread among most groups. All modes of nutrition are exhibited. Both pigmented and nonpigmented forms abound, and often are not separable into distinct taxonomic groups. Intra- and extracellular elaborations may show great complexity, and life cycles are sometimes polymorphic and complicated. Cysts or spores are often common. Mitochondria1 cristae are tubular, lamellar, discoidal, or vesicular. When lysine is synthesized, either aminoadipic or diamino-pimelic acid pathway is used. Nuclei are haploid, diploid, or polyploid; one major taxon (ciliates) is totally heterokaryotic. Mitotic mechanisms are diverse, and meiosis may be gametic, zygotic, or sporic.

V. Data of Supposed Phylogenetic Significance To set the stage for the following two sections, we need to review briefly the kinds or sources of information available that will enable biologists to better understand the nature of the protistan cell. From such knowledge, it may be possible to envision evolutionary relationships with allegedly kindred groups on the one hand, and with likely progenitors (among the prokaryotes) and probable descendants (leading to the “higher” eukaryotes) on the other hand. That it is advisable to have a multiplicity of characters and to treat such data comparatively in order to ascertain the most likely phylogenetic lines or associations of known protist taxa is widely agreed. We are naturally hampered by the generally microscopic size of our organisms, the paucity of fossil material, and the frequent absence of sexuality in their life cycles. Modem approaches in cell and molecular biology, however, are overcoming-or have the potential for solving-many of these problems (see treatment of techniques in Section 111,D). Then we also have the handicaps facing the researcher interested in the phylogeny of any large and diverse group of organisms: for example, the absence of intermediate forms among extant taxa and the difficulties in recognizing the occurrence of convergent evolution. However, for protistologists the greatest frustration, to date, remains simply our lack of sufficient information of a truly useful and unambiguous nature on perhaps >99% of the nearly 200,000 protist species named in the literature. In the past (and still today for most workers interested in protistan phy-

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logenetics), the classical approach has largely been employed to determine *‘relatedness” of major protist taxa. That is, a few characters, subjectively selected as being “of greatest significance,” are considered adequate as a basis for drawing inferences concerning probable evolutionary and systematic interrelationships of the groups under study. The many problems associated with recognition of homology, monophyly, convergent evolution, and the like are essentially, although perhaps unwillingly, ignored. The “constellation of characters” approach, originally suggested for use in ciliate systematics (Corliss, 1976), is easier to talk about than to carry out. But, obviously, the first step in comparative phylogenetic studies of high-level protist taxa is to gather data on a large number of the same characters or characteristics occurring in many species well distributed throughout those taxa. This has not yet been accomplished on a large scale, for the equally obvious reason of the inherent difficulties, mentioned above and elsewhere in this article, in such an enormous undertaking. The closest approach to success to date is perhaps represented by the cladistic analysis recently reported by Lipscomb (1983, who used 77 characters in a study of some 35 eukaryotic taxa, primarily protist groups. The favored characters so often used to date-sometimes practically (each one) alone or perhaps in combination with a few of the others in the following list-are these: configuration of the inner membrane of the mitochondrion (a very popular one!), condition of the flagellar hairs associated with flagella (also widely chosen), kind and organization of pseudopodia, choice of lysine biosynthetic pathway, type of nucleus and of mitosis (the latter extremely popular because of its nearly universal availability), kind of photosynthetic pigment(s) and storage products, modes of nutrition and of locomotion (often used together), nature of the subpellicular kinetal system, cell walllmembrane characteristics, and type of lorica (or shell), spore, cyst, or endoskeletal system. A few examples may be offered of papers that have shown an apparent preoccupation of the authors for a limited number of subjectively highly weighted sets of character data in their consideration of the interrelationships of major protist groups. Cavalier-Smith (1978, 198lb) has stressed microtubular organelles and mitochondria1 cristae, while Dodge (1979) favors flagella, plastids, and nuclei. Heath (1980, 1981) emphasizes mitotic figures, and Moestrup (1982) prefers ultrastructural features of the flagellar apparatus. Taylor (1976a, 1978) combines data on flagella, nuclei, chloroplasts, and mitochondria, and, in his second paper, he does utilize a total of some 25 different characters. In treating subgroups within a major taxon, numerous workers have shown a predilection for favoring small numbers of traits in establishing macrosystems for the group. For example, Bovee and Jahn (1973) and Jahn et al. (1974) extol functional morphology (i.e., pseudopodial types and mechanisms of movement) as the best single source of taxonomic and phylogenetic indicators in treating groups of sarcodinid protists; Barr (198 1) and Spiegel (198 I ) stress the rootlet

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system in work on the zoosporic “fungi” and the prostelid eumycetozoa; and Small and Lynn (1981, 1985) employ principally features of only the somatic ciliate kinetid system as reliable in considerations of the phylogeny, evolution, and systematics of the ciliated protozoa. Additional examples could be cited. The importance of attributes related to the (mainly) structural characteristics listed above is by no means denied! Calling attention to properties of the complex microtubular and microfibrillar systems associated with the basal bodies or kinetosomes of many different species, for example, represents a great service to comparative protistology. But three points need to be made: bias toward preselecting and employing only one or two such characteristics, in relative isolation, should be avoided; most of them represent categories that can be broken down into many unique (sub)characters (as some authors have realized) that could be used to advantage; and, finally, several additional major sources of data should be included, a matter considered further immediately below. Recall that the great majority of protist species are unicellular forms. Neglected areas or sources of characters useful in phylogenetic studies may be divided into two principal categories directly related to the “double” nature of these microorganisms: that is, to the fact that they are, at one and the same time, single cells and complete organisms. [The latter condition was first clearly enunciated by Ehrenberg (1838) nearly 150 years ago and emphatically reemphasized by Dobell (191 l), interestingly enough, exactly 75 years ago.] As whole organisms, the protists exhibit broad ecological, functional, and behavioral characteristics frequently neglected in comparative studies. A great many subcategories exist here, ranging from microhabitat preference and adaptive “life style” features, including the complexities of symbiotic relationships, to sexual attractants (e.g., gamones) in reproductive strategies. Numerous others could be mentioned. The major pitfall here is the difficulty in recognizing whether or not convergent evolution has occurred. As cells, many molecular-biological characteristics of protists remain to be investigated. These run the gamut from properties of photosynthetic pigments and of cell walls to specific metabolic pathways and to still other biochemical, immunological, and genetic traits (including DNA base composition, DNA, RNA, or protein sequence data, etc.) that can now be investigated with precision and relative ease. The techniques used by cell biologists with nonprotists are generally adaptable to microorganismic material without difficulty. A great deal is being learned today, in research on plant and animal cells and tissues, about substances ranging from nucleic acids to microtubules, microsomes, and membranes. Such approaches could more often be incorporated into studies on protistan cells, with “spin-off‘ ’ value for phylogenetic and systematic considerations of these “lower” eukaryotic forms. A preliminary list of potentially usable/useful characteristics, including ones

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from the areas mentioned directly above, is being compiled (unpublished data of the author), indicating that more than 350 separate items are available. The next step will be to determine/verify their status in members of as many high-level taxa of protists as possible. The final step would be a computer analysis yielding phenograms, cladograms, and phylogenetic “trees” appropriate for drawing testable conclusions of probable value in both evolutionary and systematic considerations of the entire protist assemblage. To date, with the notable exception of Copeland (1956), there have appeared no books devoted to the overall biology of only (but all) the protists. In recent years, Ragan and Chapman (1978) have perhaps come the closest to such an achievement; a work in press (Margulis et al., 1987) should do even better. Textbooks, understandably, are limited to the (still large) fields of algology, protozoology, or microbiology. Many of these are cited in appropriate places in subsequent sections of this article. Protists in their taxonomic entirety are covered-although often rather scantily-in a number of treatises today that cover the whole biotic world (e.g., see Barnes, 1984; Margulis and Schwartz, 1982; Mohn, 1984; Parker, 1982). A relatively small number of papers published within the past 10-12 years has treated the protists overall in a comprehensive manner, including attention to their comparative cytology, systematics, and possible phylogenetic interrelationships: see Bardele ( 198I , 1983), Cavalier-Smith (1978, 1981b, 1983), Corliss (1981, 1984, 1986b), Heath (1980, 1981, 1986), Honigberg (1984), Jeffrey (1982), Leedale (1974), Loeblich (1974), Margulis (1974a,b), Sleigh (1979), Taylor (1978), Whittaker (1977), Whittaker and Margulis (1978). To this latter list might well be added the unusual microbiology book (limited to marine forms, however) by Sieburth (1979).

VI. Major “Evolutionary Lines” of Protists Using the tentatively proposed 18- 19 “supraphyletic assemblages” of Corliss (1984, 1986c) as a basis for evolutionary groupings within the entire conglomerate of protistan forms, I suggest that it is possible to segregate a collection of more or less established and accepted multiple-group lines from one of seemingly unrelated or much less clearly related-thus “isolated”-groups. Admittedly, there is a degree of arbitrariness in this decision. The second category of assemblages is reserved for treatment in the following section. Both collections of phyla contain pigmented as well as nonpigmented forms. [This may suggest that phycologists, by and large, have done no better (or worse!) than protozoologists in solving problems of phylogeny for their “favorite” groups. In the guise of protistologists, can we perform more successfully in the future?] Here I want to focus our attention on my first grouping, comprising the eight

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major lineages on which we generaily have the most information and thus with which, at this stage of our studies, we feel the most comfortable. My own ideas, along with those of other workers, are naturally still evolving; therefore, a healthy degree of uncertainty accompanies the speculative part of any “conclusions” presented below. A goal of all phylogeneticists is to be able to establish the condition of monophyly for any high-level taxon proposed. At the present state of our knowledge, there is growing evidence that most if not all of the eight major “evolutionary lines” considered in this section seem to meet that important criterion. (I shall beg the question here of whether or not the whole “kingdom Protista” is monophyletic, considering that topic beyond the scope of this article.) Many problems remain, obviously, and these include some that are not specifically treated in this article: for example, the taxonomic nomenclatural matter of proper names for the numerous suprafamilial taxa proposed by workers during the past few years. This not insignificant question-implicating, among other things, the very credibility of protistological systematists-has already been treated in some detail in Corliss (1984, 1986b,c); see Rider (1982), Silva (1984), and Smith and Patterson ( 1986). The arrangement-that is, the ranking, A + H-of my eight groupings here is to be given no special significance. It may be noted, however, that the first two represent (with modifications the two great lines of primarily and/or primitively pigmented forms long studied particularly by botanists/phycologists; the third and fourth, basically “colorless” groups treated by protozoologists and/or mycologists; and the last four, largely aplastidic assemblages investigated mostly by zoologists/protozoologists (including parasitologists for F and G, especially). References, limited principally to recent works, are generally placed at the end of each of the subsections covered below. Despite the remarks made above-that my assemblages are segregated in a way that may appear to be based largely on presence or absence of pigment (therefore, “algal” versus “protozoan”)-the fact is that I have erected no high-level (or, often, even low-level) taxonomic walls between photosynthetic and nonphotosynthetic protists on that basis. Similarly, my classification scheme does not deliberately separate unicellular from multicellular forms, nor microscopic from macroscopic, nor motile from nonmotile. Such characters or character states, by and large, are only of superficial or “identification” value. Yet, in many macrosystems involving protists (even such recent treatments as are to be found in Farmer, 1980; Hausmann et al., 1985; Lee et al., 1985; Parker, 1982; and Levine et al., 1980), presence or absence of color, of locomotory organelles, etc. is still the cause of artificiality in the resulting taxonomic arrangements. The “protozoalgal” interface (Corliss, 1981) has either been ignored or (as by Round, 1980) denied.

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A. THE CHLOROBIONTS, CHLOROPHYTE SERIES,CHLOROPROTISTS This large assemblage essentially represents the “green algae” sensu strict0 (i.e., the euglenophytes are excluded) and includes both (bi- or multi-) flagellated and nonmotile groups, unicells, coenobia, coenocytic filaments, and multicellular forms. This is one of the two principal branches of the photosynthetic protists (see Christensen, 1966; and especially Taylor, 1978). Because of its “popularity” (reviews, etc.), only a brief account is needed here. The contained groups-considered by me to be the phyla Chlorophyta, Prasinophyta, Conjugatophyta, and Charophyta (but to which perhaps a phylum Ulvophyta should be added?)-possess in common at least the following characteristics: chlorophylls a + b, flattened (a primitive condition?) mitochondria1 cristae, cell walls with cellulose, motile cells (primitively thecate?), true starch in their plastids, thylakoids arranged into many-layered grana, chloroplasts bounded by a double membrane, motile cells showing “stellate” structure in flagellar transition region, and cruciate flagellar root system. They are predominantly freshwater forms; sexuality is commonly known for many species; and a phycoplast is involved in cytokinesis (except when replaced by a phragmoplast in charophytes). Ultrastructural studies are providing the bulk of the information for a “new look” at this long-known group. Representing an assemblage most likely serving an ancestral role for the kingdom Plantae, the chlorobionts (neatly termed the “chloroprotists” by Rothschild and Heywood, 1986) have been placed by many systematists in a single kingdom that also includes the “higher” plant phyla. This is a most important topic, but one considered beyond the scope of this article as already indicated elsewhere. The enigmatic glaucophytes, unique cyanelle-bearing forms sometimes appended to the chlorobionts, are excluded from any group in this article. Key publications of recent vintage, particularly ones with valuable overviews of the phylogenetics and systematics of (essentially) the whole group as defined above, are presented in two blocks below. The first contains comprehensive accounts treating assemblages beyond solely the chlorobionts: cross-reference will be made to these on subsequent pages of this article. The second is limited to works that, themselves, are generally restricted to one (or more) of the phyla under consideration in immediately preceding paragraphs. Naturally, some overlapping is inevitable, and the lists are by no means to be considered exhaustive in coverage. Works (some with multiple contributors) rich in treatment of and/or reference to matters of the comparative cytology, systematics, phylogeny, and evolution of all or many of the “algal” protist groups are Bold and Wynne (1985), Bourrelly (1966-1981), Cavalier-Smith (1981a, 1983), Christensen (1980), Cox (1980),

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Dodge (1979), Ettl (1980), Hanel (1979), Jeffrey (1982), Lee (1980), Pritchard and Bradt (1984), Rosowski and Parker (1982), Rothschild and Heywood (1986), Scagel et al. (1982), Silva (1982), Stewart and Mattox (1980), Stewart (1974), Tappan (1980), Taylor (1976a, 1978), van den Hoek and Jahns (1984), and Whittaker and Margulis (1978). For taxonomic nomenclatural reviews, see Silva (1980a,b). Not included are textbooks of botany, although a number of these include comprehensive sections on algae. Some additional recent papers or books with main emphasis on chloroprotist groups are Bourrelly (1972), Bremer (1983, Ettl (1981), Floyd and O’Kelly (1984), Irvine and John (1984), Mattox and Stewart (1977, 1984), Melkonian (1980, 1982, 1984), Mishler and Churchill (1989, Moestrup (1978), O’Kelly and Floyd (1983, 1984a,c), Pickett-Heaps (1975), Round (1984), Sluiman (1983), Stewart and Mattox (1975, 1978), and Taylor et al. (1985). (Hundreds more are available in combined bibliographies of works cited in this and the preceding paragraph.)

B . THE CHROMOBIONTS, CHROMOPHYTE SERIES,“HETEROKONTS” This is an even larger assemblage (than the group in Section VII,A, above) of predominantly “algal” protists that, in turn, are mostly the “golden-brown’’ (plus, for me, also the “yellow-green’’ and the “brown”) forms of the older literature. The included flagellated (typically biflagellated) forms characteristically exhibit the so-called ‘‘heterokont” condition (redefined by Moestrup, 1982, to mean the presence of one “hairy” and one smooth flagellum). Some of the groups that I recognize as chromobionts, however, do not exhibit this trait (e.g., bacillariophytes and haptophytes); yet, it is shown by the oomycetes, members of my group in Section VI,D, below. Furthermore, many (diatom) species have no flagellated stage at all in their life cycle. I include a number of phyla (or divisions) within this single large evolutionary line, with the assumption that it still remains a monophyletic assemblage (Corliss, 1984): the Chrysophyta, Haptophyta, Bacillariophyta (diatoms), Xanthophyta, Eustigmatophyta, Phaeophyta (brown algae, often macroscopic forms), and probably the unique Raphidophyta. Other possible (sub)groups remain uncertain: the bicosoecideans, the heterochlorideans (belong in the xanthophyte phylum?), and, above all, the frustratingly engimatic phylum(?) Proteromonadea (see discussion of these last forms in Section VI,F, below). Characteristics common to most if not all of the major included groups listed above are these: chlorophylls a + c (only a in the eustigmatophytes), tubular mitochondria1 cristae, typically (except for phaeophytes) no cell walls (but often scales), often silicified cysts, pair of heterokont flagella common among flagellated groups, various storage products (e.g., chrysolaminarin), thylakoids stacked in threes, plastid bounded by three membranes (although four in hap-

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tophytes and eustigmatophytes), basal body transitional region showing helical structure. Sex is uncommon in many groups. The majority of species is marine. They include diverse morphological types, ranging from unicells to filaments and multicellular thalli of relatively great size (brown algae). Here, as in the preceding assemblage in Section VI,A, motile and nonmotile, pigmented and colorless, unicellular and multicellular forms are not separated from one another taxonomically on the basis of such characters. Phycologists have long recognized the “naturalness” of this; protozoologists have not. For key publications see the first group of works cited at the end of subsection A, above. A few additional recent references to papers (occasionally books) on specific groups of chromobionts are Andersen (1986a,b), Bourrelly (1981), Clayton (1984), Estep et al. (1985), Green (1980), Heywood (1980, 1983), Hibberd (1976, 1979, 1981), Hibberd and Leedale (1970, 1972), Irvine and Price (1978), Kristiansen and Andersen (1986), Larsen (1985), Lobban and Wynne (1981), Manton (1978), Moestrup (1982), Norris (1985), O’Kelly and Floyd (1984b, 1985), Preisig and Hibberd (1983), Round and Crawford (1981), Silva (1979), Warner (1977), and Wynne (1981). (As in subsection A, hundreds of additional works could be cited on the diverse included phyla.) C. THE RHIZOPODS, ESSENTIALLY THE “RHIZOPOD SARCODINIDS” auctt. A large assemblage, the rhizopods overall are perhaps as “amorphous” as some of their typically included species. Unless one is dealing with unsuspected convergences, however, there do seem to be some significant features possessed in common by the great majority of the groups placed here by Corliss (1984). The principal one of these is the exhibition of “cytoplasmic flow,” often manifested in change of body form and widely used in locomotion and/or food capture. Clearly present in at least one stage of the life cycle are pseudopodia (lobose, filose, or reticulose) or a shuttle-type flow of cytoplasm. A biflagellated stage occurs in many species, alternating, as it were, with an amoeboid stage. Mitochondria, if present, typically show tubular cristae, although there are important (and baffling!) exceptions. Tests or shells are common in some subgroups, and fruiting bodies are typical of most of the forms claimed as “fungi” by many mycologists (but as “protozoa” by zoologists). Sexuality has been described for some species, but is absentiunknown in many others. Included phyla, with often admittedly rather unclear relationships among them, are the Karyoblastea, Amoebozoa, Acrasia, Eumycetozoa, Plasmodiophorea, and Granuloreticulosa (largely the shelled Foraminifera). The enigmatic Xenophyophora-large marine benthic branched-tube organisms-are excluded for want of data. Amoeboflagellated forms are no longer considered to bind this assemblage closely to (other) “flagellated” protists, an arrangement that Levine et al. (1980)

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and others have persisted in maintaining. Yet the fact that some rhizopods have photosynthetic protist endosymbionts and that some have flagellated as well as amoeboid stages in their life cycles reopens the problem of separating them taxonomically from certain chrysomonads (of the assemblage in subsection B, above), on the one hand, and certain heliozoa (subsection E, below), on the other. The solution to this ever-vexatious problem perhaps best lies in systematically shifting about the troublesome subgroups rather than forcing an otherwise artificial union of the high-level (phyletic/supraphyletic) groups themselves (cf. Smith and Patterson, 1986). Much of the polyphyly marking the “sarcodinids” of the older literature has been removed by separating out the actinopods (E, below) and the enigmatic labyrinthulids (Section VI1,H). Some workers (e.g., Margulis and Schwartz, 1982) would isolate the Karyoblastea (essentially represented by a single species, Pelomyxa palustris) from all other protists, holding it to be an extremely primitive form. Another problem, that of the presence of both intra- and extranuclear spindle apparatuses in the same assemblage, may serve more to cast doubt on the high-level evolutionary significance of such “closed” and *‘open” systems than to indicate a definite polyphyletic condition (see Heath, 1986; Oakley, 1978; Roos, 1984). Paralleling the approach used at the end of subsection A, above, where major literature references to the more or less “algal-type’’ protists were given in one place, the following block of citations covers recent works devoted primarily to the largely “protozoan type” (in the conventional but not necessarily correct sense!) of protist assemblages in combination. They are thus useful for C and subsequent subsections E through H (and also for certain parts of Section VII). The second paragraph of references is mostly limited to some recent specific books or papers concerned only or primarily with the present assemblage, the rhizopods. Broader-coverage works include Corliss (1982a), Farmer (1980), Grell(l973, 1980), Hanson (1976, 1977), Hausmann et al. (1985), Krylov (1981), Krylov and Starobogatov (1980), Kudo (1966), Lee et al. (1985), Levandowsky and Hutner (1979-1981), Levine (1978a), Levine et al. (1980), Sleigh (1973), and Westphal and Muhlpfordt (1976). Not included, although often a source of good information or review, are introductory biologytzoology textbooks and especially invertebrate zoology books, which often contain a sizeable section on the protozoa. Key rhizopod sensu lutu references, generally of recent date (let me first cite one classic here: Schaeffer, 1926), which are concerned with the phylogeny and/or the systematics of the whole assemblage or with significant parts (still high-level taxa) of it are Andresen et al. (1968), Balamuth et al. (1983), Bovee and Jahn (1973), Bovee and Sawyer (1979), Carey and Page (1985), Daniels

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(1973), Griffin (1978), Hedley and Adams (1974-1978), Jahn et af. (1974), Jeon (1973), Karling (1968), Loeblich and Tappan (1984), Ogden and Hedley (1980), Olive (1975), Page (1976a,b, 1983), Page and Blanton (1985), Patterson (1983, 1984, 1985), Raper (1984), Sawyer (1973, Sawyer and Griffin (1975), Schuster (1979), and Spiegel (1981). OR D. THEMASTICOMYCETES, PHYCOMYCETES, “WATERMOLDS” sensu lato

Three groups of (formerly) “lower fungi” may be said to comprise this assemblage (Corliss, 1984), but not without misgivings of at least two sorts. The so-called “chytrids” do not seem to be phylogenetically very close to the other two included phyla, and the oomycetes show some striking resemblances (e.g., heterokont flagella) to members of my assemblage in subsection B, above. Nevertheless, the phyla brought together here (the Hyphochytridiomycota, Oomycota, Chytridiomycota) seem to be closer to each other, at a very high level, than any of them are to phyla assigned to other supraphyletic groups. Certainly they do not belong in the kingdom Fungi, where, however, they have long resided. Their evolutionary origin may have been in the chromobionts (B, above) or from some ancient lineage, now extinct, that may have served as progenitors for several (now) diverse protist and fungal assemblages. All species have in common the possession of one or two flagella (there are none in the fungi), with the attendant basal-body complex composed of two kinetosomes (one barren in two of the phyla), the absence of a mechanism for photosynthesis, a preference for fresh water or soil (plus often living as saprobes or parasites on/in selected plants or animals) as habitat. Cell walls contain chitin or cellulose; mitochondria1 cristae are tubular in two phyla, lamellar in the other (Chytridiomycota); lysine biosynthesis is by the DAP pathway in the first two, AAA in the other. Only the Oomycota have a pair of emerging flagella, which also exhibit the typical heterokont condition: the forward one with (tubular) hairs, the trailing one smooth. Zoospores of the Hyphochytridiomycota have a single anteriorly directed flagellum with tubular hairs. In the Chytridiomycota, gametes and (some) zoospores have a single posteriorly directed flagellum with no hairs of any kind. The thraustochytriaceans, typically included here, are treated in Section VII along with the labyrinthuleans as isolated, enigmatic groups seemingly related, at best, only to each other. The literature on this assemblage is to be found primarily in mycological books and journals. This is not surprising, for these protists (designated the “fungal group” in Corliss, 1981) have long been considered “zoosporic” or “lower” fungi, the phycomycetes of Sparrow (1960) and of most mycologists

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since that time. In recent years, they have begun to be treated as “protists”; for such reference, see (certain of) the citations at the end of Section V, above, works in which all protist groups are treated together. Some further sources of important information on the mastigomycetes, mainly of a “mycological” nature, include the following: Ainsworth et al. (1973), Alexopoulos and Mims (1979), Barr (1980, 1981), Beckett et al. (1974), Buczacki (1983), Cole (1979), Fuller (1976), Hawksworth et al. (1983), Heath (1976), Kendrick (1985), Olive (1979, Walker and Doolittle (1982).

E. THE ACTINOPODS, ESSENTIALLY THE “ACTINOPOD SARCODINIDS” auctt. This is a large assemblage of rather unique protists, although whether or not it is, as a whole, monophyletic is open to question. Some of the members of the included heliozoan phylum appear to share characters of importance with species of both the chrysophytes (in my assemblage in subsection B, above) and the amoebozoa (of C, above). As pointed out in subsection C, however, such affinities need not destroy the unity of the overall assemblage: cannot the offending taxa be removed, leaving the rest as (closer to being) a monophyletic grouping? Its ancestry, admittedly, remains unknown. The actinopods are (should be) no longer considered close to or sharing a single taxon with the rhizopods (subsection C). Such a relationship was based on such superficial characteristics as common possession of pseudopodia and of skeletons or tests, but these structures are quite different (nonhomologous) in the two groups. Presence of flagella in some stages in the life cycles of some subgroups in each assemblage is also not a compelling factor for joining the two: these locomotory organelles are common to scores of phyla throughout the biotic world (except for the kingdom Fungi). Five phyla may be included here: the Heliozoa (the one containing the phylogenetic “troublemakers”!), Taxopoda, Acantharia, Polycystina, and Phaeodaria. Bona fide members of these phyla, predominantly marine forms, generally possess in common radiating axopodia with cores of crosslinked microtubles, tubular mitochondria1 cristae, a spherical body plan correlated with a floating predaceous life style, zoospores (often biflagellated) common, and an elaborate endoskeletal system-of varying composition and structure-widely produced. Many fossil species have been described, particularly of the Potycystina, a group having a siliceous endoskeleton. The literature on actinopods is nearly as widely dispersed as their species: sources include paleontological, ecological/planktonic, and biological/oceanographic outlets as well as the zoologicaUprotozoologica1and cell/general biological journals. A few key references of the past 10-12 years include Anderson (1983), Bardele (1972, 1975, 1977), Brugerolle and Mignot (1983, 1984a,b),

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Cachon and Cachon (1974, 1978), Davidson (1982), Febvre-Chevalier (1980, 1982), Febvre-Chevalier and Febvre (1984), Go11 and Merinfeld (1979), Merinfeld (1981), Mikhalevich (1980), Patterson (1979, 1985), Patterson and Fenchel (1985), Rieder (1979), Schulman and Reschetnyak (1980), Smith and Patterson (1986), and Zimmerman et al. (1986). But I must also call attention to the classic by Schewiakoff (1926). F. THEPOLYMASTIGOTES, COLORLESS POLYMONADS, “HIGHERZOOFLAGELLATES” Largely endosymbionts today (with insects and vertebrates commonly serving as hosts), this assemblage of nonpigmented often multiflagellated forms is difficult to relate phylogenetically with other protist groups, perhaps mainly because of its nearly universal adaptation to the parasitic mode of life. Some features seem primitive: for example, the extranuclear spindle in some subgroups, the total absence of mitochondria (or is this a secondary loss?), possession of smooth flagella only, and presence of hydrogenosomes. Others might be advanced (e.g., multiple flagella, tremendous development of endoskeletal organelles, types of meiosis and modes of sexuality, and the elaborate Goigi apparatus in one subgroup). I consider that two phyla, despite their differences, make up the assemblage: the Metamonadea and the Parabasalia. In protozoology textbooks, these “higher zooflagellates” are/would be preceded by a phylum of “lower zooflagellates. But most protistologists today agree that all that the trypanosomes (for example) have in common with the trichomonads (for example) is the possession of a flagellum and existence as obligate parasites. Yet, in recent years, some workers have recognized a different composition for the “lower zooflagellate” moiety. They (see discussion and references in Brugerolle and’Joyon, 1976; Corliss, 1986~;Patterson, 1986; Vickerman, 1976) call it the Proteromonadea (or some variant spelling) and consider it to embrace at least the bi- and quadriflagellated parasitic genera Proteromonas and Karotomorpha, to which some would add another dozen or so difficult-to-classify nonpigmented flagellate genera of various kinds. Perhaps such a group-although very likely polyphyletic in composition-is better associated with the chromobionts (B, above)? Patterson (1986) inadvertently further exacerbates the situation by suggesting that the enigmatic opalinids are quite closely related to (some of) the proteromonadeans. The last-mentioned point above allows the possibility of enlarging the assemblage in subsection F to include the opalinids or “paraflagellates,” a group that I consider phylogenetically “isolated” (see Section VI1,G). But I believe that the known differences involved are far too great to permit such an association-at least not until we have more comparative data on the implicated groups. On the other hand, features particularly of the mastigont systems of the meta”

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monadeans and the parabasalians justify retention of these two phyla in a common assemblage. Partly because of the parasitic nature of these long-recognized organismswith even humans as hosts for a number of them-the literature on them is vast. However, relatively few such papers are directly concerned with the phylogeny or evolution of their included subgroups at suprafamilial levels. Beyond the “zoological/protozoological” block of citations given at the end of C, above, the reader is referred to the following selected pertinent works of significance on members of the two phyla assigned to the polymastigotes in this paper: Brugerolle (1975, 1977), Camp et al. (1974), Hollande and Carruette-Valentin (1971), Honigberg (1963, 1978a,b), Honigberg et al. (1982). G. THE SPOROZOA, “APICOMPLEXANS”

Although I follow my earlier proposal (Corliss, 1984) and include but a single phylum here, the Sporozoa, I consider it to be a monophyletic “assemblage” containing substantial interrelated subgroups, viz., the gregarines sensu lato, the coccidians sensu strict0 (including the adeleans, eimerians, Toxoplasma, etc.), and the ‘‘hematozoa” (including Plasmodium and relatives and the piroplasmids). Thus I have, if arbitrarily, left/placed it here rather than among the “isolated groups” included in Section VII. Special, if brief, mention needs to be made concerning the taxonomically highly controversial protist Perkinsus marinus (syns. Dermocystidium marinum, Labyrinthomyxa marina), an oyster pathogen. Most protozoologists/parasitologists have followed N. D. Levine’s proposal (see Levine, 1978b; and Levine et al., 1980) that the organism be transferred to the genus he created in honor of F. 0. Perkins, a leading student of its ultrastructure and life cycle (e.g., see Perkins, 1969, 1976), and placed in a separate class, Perkinsea, alongside the Sporozoea in the phylum Apicomplexa. Some Russian workers (e.g., Krylov and Kostenko, 198l), while preferring Sporozoa over Apicomplexa, do, however, accept “Perkinsemorpha” as the first of three classes in that phylum. But other workers have questioned Levine’s systematic decisions, especially at the high level, claiming either that too little is known to make such a judgment or that the data available can be interpreted in ways leading to a different conclusion (e.g., see Corliss, 1984, 1986b; Vivier, 1983). Ultrastructural studies by Brugerolle and Mignot (1979) and by Foissner and Foissner ( 1985), on an equally curious flagellate parasitizing other protists, may be cited as having possibly found some “Perkinsus-like” features in forms definitely not “sporozoan-like” in their other characteristics. And the flagella of the zoosporic stage of Perkinsus seem to me to be reminiscent of those of the oomycetes (D, above) or of certain “heterokontic algae” (B, above). Therefore, I am leaving the organism out of the present assemblage at this time but assigning it nowhere else until more information is available.

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Thanks to data accruable through use of transmission electron microscopy, N. D. Levine and others (see references below) have been able to provide an incontrovertible set of characters held in common by practically all species of the assemblage/phylum, at least at one stage in the universally parasitic life cycle. These shared features include tubular mitochondria1 cristae, zygotic meiosis, basal bodies of flagella (found on some microgametes) with nine singlet rather than triplet microtubules, and presence of an “apical complex” composed of these ultrastructures: polar ring, conoid, rhoptries, micronemes, subpellicular microtubules, and often one or more micropore(s). Life cycles are often complex, sometimes involving polymorphism, multiple hosts, sexual and asexual generations, etc. While the ancestors/predecessors of the fossilless sporozoa remain unknown (the fungi are a possibility not to be ruled out), evolutionary lines within the phylum have been proposed, and the group overall is quite well known from the points of view of comparative cytology and supposed taxonomic interrelationships. Many “sponsored” studies have been carried out on members of the assemblage because of the medical and economic importance of some of them (Plasmodium, malaria, is still the “number one” killer of humans in the world today; Toxoplasma also causes a serious disease; Eimeria, and other coccidians, and Babesia cost agricultural industries millions of dollars a year; etc .). As was true for the polymastigotes (F, above), the great bulk of the publications on the sporozoa is to be found in the general parasitological literature and in biomedical and tropical medical outlets. A few selected works, often of a broad nature and with phylogenetic implications but purposely limited (with rare exception) to consideration of the Sporozoa sensu strict0 or to major groups within the phylum, are these: Chobotar and Scholtyseck (1982), Garnham (1966), Hammond and Long ( 1973), Kreier ( 1977a,b) , Krylov and Dobrovolsky ( 1980), Krylov and Kostenko (1981), Levine (1973, 1985a,b), Long (1982), Scholtyseck (1979), Theodoridbs (1984), Vivier (1979, 1983). , HETEROKARYOTES H . THE CILIATES,CILIOPHORANS

Both boundaries and the internal cohesiveness of this assemblage are probably the most widely acknowledged and best understood/accepted among all protist groups (Corliss, 1979a, 1984). That it contains but a single phylum does not (in my estimation) preclude its inclusion in the set of multigroup evolutionary lines considered in this section. Like the Sporozoa (G, above), or even more so, the phylum Ciliophora contains a number of quite clearly defined subgroups, the phylogenetic interrelationships of which are possible to envision (although there is not unanimity among ciliate specialists on the subject). The combination of characteristics that separate ciliates from all other protists are the following: exhibition of nuclear dualism or the heterokaryotic condition (a diploid micronucleus and, commonly, a polyploid macronucleus: one or more

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of each); possession of simple cilia or compound ciliary organelles, often in abundance on the body; the presence of a cortical infraciliature, oral and somatic, of which the kinetid is the main feature, and pellicular alveoli; demonstration of homothetogenic fission and of sexuality by conjugation or autogamy (not syngamy); and the presence, frequently, of a cytostome and cytopharynx, structures used in their heterotrophic mode of nutrition. Ciliates are fundamentally nonpigmented protists, though some species contain “zoochlorellae” that are capable of photosynthesis. Mitochondria1cristae are universally tubular. Habitats and life styles are diverse. Consideration of subphyletic classification with the ciliophoran assemblage is beyond the scope of this article. But phylogenetic lineages with implications for the systematics of the included high-level taxa have been proposed by several workers in recent years. Most of the literature selected for citation below is concerned with the major books or papers produced by researchers interested in these two aspects of ciliatology: phylogeny and/or macrosystems of classification. Since most of the pre-1983 references given below, including those of the present writer, contain misinterpretation of the evolutionary significance of the small marine homokaryotic protist Stephanopogon, a word should be said about it here (see also Section VI1,A). Long classified as a “primitive ciliate” and neatly serving as a mononucleate ancestor of contemporary (heterokaryotic) forms, its ultrastructurai features (first revealed by Lipscomb and Corliss, 1982) make clear that it is not a member of the Ciliophora. Nor is it likely to have been on the direct line of protists giving rise to ciliates, although we do not know what group did play that role in the evolution of unicellular eukaryotes. There is a vast literature on this assemblage, starting with the published observations of A. van Leeuwenhoek nearly 325 years ago. Leaving aside the hundreds of invaluable monographs and treatises of the more distant past and omitting even recent volumes such as those on particular genera (e.g., on Blepharisma, Ophryoglena, Paramecium, Stentor, and Tetrahymena), I am restricting inclusion here to comprehensive works that are primarily concerned with overall systematic or phylogenetic aspects of ciliatology (major exceptions: GrassC, 1984; Nanney, 1980) and that were published within the past 12-15 years (only exception: the classic if modest paper by FaurC-Fremiet, 1950, which stimulated production of nearly all the other works in the list!): Bick (1972), Borror (19731, Corliss (1972, 1974, 1975, 1976, 1979a, 1982b), Curds (1982), Curds et al. (1983), Dragesco and Dragesco-Kerneis (1983, FaurC-Fremiet (1950), Gates (1978), Gerassimova and Seravin (1976), Grain (1984), Grain et al. (19731, Grass&(1984), Jankowski (1972, 1973, 1980), Jones (1974), Lom and Didier (1979), Lynn (1976, 1981), Lynn and Small (1981), MadrazoGaribay and Lopez-Ochoterena (1985), Matthes (1982), Nanney (1980), Orias (19761, Poljansky and Raikov (1976), de Puytorac and Grain (1976), de

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Puytorac etal. (1974, 1976, 1984), Raabe (1971), Raikov (1976, 1982), Seravin and Gerassimova (1978), Small (1976, 1984), Small and Lynn (1981, 1985).

VII. Seemingly Isolated Groups As confessed in the introduction to Section VI, above, 1 have somewhat arbitrarily relegated to this section those assemblages included in Corliss ( 1984, 1986c) concerning which our phylogenetic knowledge seems both less and less certain than it is for the groups already treated. That is, we are not only often unsure as to whether or not the assemblages mentioned below represent monophyletic groups within themselves but are also baffled, at this time, with respect to their interrelationships among themselves and with the eight (A-H) of the preceding pages. (This is not, however, to presume extensive knowledge concerning those “evolutionary lines” of Section VI, either!) In any case, I 1 of the 19 supraphyletic assemblages under discussion in this article appear to be relatively isolated evolutionarily within the entire kingdom Protista conglomeration. Five of these groups contain pigmented (thus truly “algal,” in the old conventional sense) species in abundance; their evolutionary/phylogenetic histories are made complicated by past occurrences of endosymbioses, prokaryotic and/or eukaryotic. Further, some of their own members have themselves become parasites, often in the bodies (cells) of other protists! Three other groups are entirely nonpigmented; two of them bear flagella; and/but there is no hint of relationship among these three. The three remaining assemblages are nonpigmented, nonflagellated, totally parasitic, and unique. The last of these (the “Myxozoa” of the recent literature) is even regarded as being of a completely nonprotistan nature by some workers (see K , below). Before considering these 1 1 cases (A-K, with no special significance to the ordering), the reader should perhaps be reminded that several groups or subgroups of protists are considered by me to be even more vexatious, indeed, to represent collections of forms that are relatively the most poorly knowdunderstood of all members of the kingdom. Commonly, they have been appended by numerous workers including the writer to various of the Corlissian assemblages. Although they have been loosely associated, questioningly, with groups mentioned in parts of this or more often of the preceding section, their ultimate “taxonomic homes” must await considerable further study. Most of these small groups have indeterminate or debatable taxonomic boundaries of their own, adding to the confusion they generate in general. The implicated forms include the glaucophytes, bicosoecideans, proteromonadeans, xenophyophorans, and the single species Perkinsus marinus (see Sections VI,A-C,G). Again, selected references on a given phylum or division are supplied at the end of each lettered section, A-K. Less space is generally given to the groups

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treated below, in comparison with those assemblages of Section VI, because the very uncertainty of their phylogenetic interrelationships makes them of reduced interest to the main topic of this article. On the other hand, our wonderment about such mysteries heightens their attractiveness for future investigation. A. THEEUGLENOZOA, EUGLENOPHYTES PLUS KINETOPLASTIDEANS This assemblage is one of the most iconoclastic within the Protista from all conventional points of view (Corliss, 1984). It has not only been completely separated from (other) green “algal” groups of the phycologist, but has also been removed from the ‘‘phytomastigophoreans” of the protozoologist. Furthermore, it combines the “algallike” (whether autotrophic or heterotrophic) euglenophyte subgroups with a group of totally aplastidic ‘‘lower zooflagellates,” many subgroups of which are vertebrate blood parasites. Finally, there is the possibility that the enigmatic “ciliate-turned-flagellate’ ’ genus, Stephunopogon, belongs here as well. In all, it is a most bizarre combination of forms, unacceptable if we relied on the “phylogenetic” characters of the past, features now viewed as being either superficial or too broad to be of real value in evolutionary considerations. One of the major complicating factors appreciated-but not totally resolvedin modern treatment of the phylogenetics and systematics of protist groups in which some, most, or all of the included species are chloroplast-containing forms is the likely endosymbiotic origin of the plastid (see Section II1,C and the especially illuminating account in Rothschild and Heywood, 1986). The source or original nature of such a xenosome, the recency of its invasion or engulfment, the degree of its genetic and physiological incorporation into systems of the host cell, and the amount of retention of its original morphological/structural attributes are all problems that must be taken into consideration in attempts to trace the phylogeny of the host cell/organism. In the case of the assemblage here under consideration, the presence of three membranes, complete with other properties of the chloroplast, has led Gibbs (1978 et seq.) and others to conclude that the euglenoid plastid has been secondarily derived from the acquisition and degeneration of a eukuryotic, rather than prokuryotic, symbiont. In conclusion on this fascinating subject, there is growing evidence, today, that contemporary plastids have evolved several times independently via symbiosis (see recent reviews by Reisser, 1984; Taylor, 1983; and references therein). At least seven diverse assemblages (or some phyla within them) of this article could be implicated: the chloroprotists, chromobionts, euglenozoa, dinoflagellates (some species), rhodophytes, cryptomonads, and chlorarachniophytes. Characteristics of the euglenozoa distinctive in combination include chlorophylls u + b (in pigmented forms: but recall that even euglenophyte species are nearly two-thirds colorless); unique pellicle underlaid by interlinked micro-

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tubules; single large mitochondrion with discoidal cristae; paraxial rod in flagella; specific, particular properties in cytochrome c and 5 S rRNA; nucleus with conspicuous endosome, permanently condensed chromosomes, and intranuclear spindle; a cytostome present and a contractile vacuolar system; no cellulosic cell wall nor starch as storage product; no sexuality reported. There is variation in numbers of flagella, in pathway of lysine synthesis, and, of course, in habitats and life styles. A prominent kinetoplast (huge concentration of fibrillar DNA in the mitochondrion) is present only in the kinetoplastideans. Phyla included are the Euglenophyta and the Kinetoplastidea. Appended with uncertainty is the Pseudociliata, a taxon erected by Corliss and Lipscomb (1982) for the enigmatic Stephanopogon, as discussed briefly under the ciliates (cf. Section VI,H). It seems to share a number of the characters listed above (see Lipscomb and Corliss, 1982), but more data on these nonpigmented multiflagellated, phagotrophic organisms are needed. Isonema and certain other small heterotrophic flagellates may also be assignable here (Kivic and Walne, 1984). The literature on the groups involved is extensive. Avoiding repetition of references from blocks of the (partially) pertinent citations given at the ends of Sections V and VI,A,C, the following additional papers of recent vintage have been selected for mention here: Cavalier-Smith (1982), Davis and Sieburth (1984), Gallo and Schrkvel (1985), Gibbs (1981a,b) Kivic and Walne (1984), Leedale (1982), Lumsden and Evans (1976, 1979), Patterson (1980), Vickerman (1976), Vickerman and Preston (1976), Walne (1980), Whatley (1983), and Willey and Wibel (1985).

B. THE DINOFLAGELLATES, DINOZOA, PYRRHOPHYTES sensu lato, PERIDINEANS PLUS SYNDINEANS This is another assemblage evolutionarily baffling but extensively studied and subjected to many conjectural phylogenetic hypotheses, including the suggestion (latest paper: Herzog et al., 1984) that it represents a totally separate kingdom (the Mesokaryota) on the basis of the seemingly “intermediate” nature of its nuclear apparatus. It may be related in some way to the ciliates (cf. Section V1,H) because of apparently sharing several significant features (such as pellicular alveoli, tubular mitochondria1 cristae, trichocysts, flagellar/ciliary apparatus similarities, etc.: see review in Taylor, 1986), although there are also vast differences between these two great assemblages. The dinoflagellates, briefly, are set apart from other protists principally by their possession (in pigmented forms) of chlorophylls a and c, with plastids generally bounded by three membranes; two uniquely positioned heterodynamic flagella; “primitive” nucleus; tubular mitochondrial cristae; pusules; and cortical alveoli. Great diversity is shown in various additional characteristics among lineages within the major included phylum (see especially Taylor, 1980b, 1986):

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motility, nutritional mode, storage metabolites, extrusomes, bioluminescence, body covering, aggregation (solitary versus colony), habitat, life style, cyst formation, toxin production, fossil availability (50% of all described species), etc. The major included phylum-the only one, in the opinion of more conservative workers-is the Peridinea (with several popular synonyms equally “legal” to use). I separate the “syndiniophycean” forms off into a distinct phylum, the Syndinea, although with some hesitation (see Corliss, 1984). The latter group, clearly related to the former (as best seen in the motile zoospore, “dinospore,” stage of the life cycle), is totally made up of nonpigmented, endosymbiotic species with many specialized characters of their own, including very low chromosome numbers (only 4-10). There are parasitic peridineans as well, but-in at least some stage in their life history-these dinoflagellates retain characters shared with the more commonly known “typical” maritime or freshwater planktonic forms and do not exhibit any of the unique syndinean features. At least three additional groups of protists are generally-and, I believe, correctly-associated with the dinoflagellates sensu lato, viz., the ebriideans, the ellobiophyceans, and the mysterious, very likely polyphyletic, fossil “groups” of acritarchs. Once again, a vast literature has accumulated on the peridineans and their alleged relatives. Generally not cited elsewhere in this review are the following mostly recent works, the bibliographies of which should be consulted for many additional references: Dale (1978), Dodge (1983, 1985), Evitt (1963), Herzog er al. (1984), Kubai (1975), Li (1984), Loeblich (1970, 1976), Loeblich and Loeblich (1983), Oakley and Dodge (1979), Rizzo (1981, 1985), Roberts (19851, Sarjeant (1974), Sigee (1984), Soyer (1981), Spector (1984), Spector and Triemer (l981), Taylor (1980b, 1986), Williams (1978). C. THE RHODOPHYTES We have here a third prominent assemblage of unique protists, with scant knowledge regarding its probable evolutionary relationships with other groups within the kingdom. Indeed, a number of ‘‘rhodoprotistologists” (personal communications) feel that the red alga may truly be deserving of its own kingdom. In these times of rampant inflationary systematics (see Corliss, 1986b,c), perhaps such a proposal is not out of order (Cavalier-Smith, 1981a, at one time essentially carried this out in his creation of a “kingdom Biliphyta,” although he placed the enigmatic glaucophytes there, too). Primarily because of their body size (they may reach a meter or more in length) and, in some species, a multicellular parenchymatous organization, these mainly marine “macroalgae” are persistently considered to be plants by many biologists. And they, along with the “browns,” are the principal reason for

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Lynn Margulis’ (e.g., see Margulis and Schwartz, 1982) preference of “protoctist” over “protist,” the latter (in her opinion) restricted to small and unicellular members of the kingdom (a view unnecessary to maintain). For me, there is only one phylum in this assemblage, the Rhodophyta. It is characterized principally by the presence in its plastids of chlorophyll a plus biliproteins (phycoerythrin, phycocyanin), with “floridean starch” as the storage product in the cytoplasm, plastids bounded by two membranes and containing single thylakoids, total absence of centrioles and basal bodies (and thus of flagellated stages in the life cycle, a character shared with all “higher” fungi), mitochondria1 cristae lamellar, zygotic meiosis in species exhibiting sexuality, spores in some life cycles, cell walls microfibrillar and containing gelatinous material (e.g., agar). Some fossil species are known. The plastids of red algae are presumably derived from an ultimate free-living cyanobacterial ancestry. Many papers, of an experimental as well as ultrastructural/taxonomic/ecological/evolutionary nature, have appeared that are concerned with rhodophytes. A few examples of significant works not cited in preceding sections are the following: Boney (1978), Cole and Sheath (1980), Demoulin (1974, 1985), Dixon (1973), Entwisle and Kraft (1984), Gabrielson et al. (1985), Gantt and Lipschultz (1977), Garbary (1978), Garbary et al. (1980), Guiry (1982), Irvine and Price (1978), Kraft (1981), Lim et al. (1983), Lobban and Wynne (1981), Percival (1979), Pueschel(1977), Pueschel and Cole (1982), Scott et al. (1980), Sheath (1984), Sheath and Hymes (1980), Takaiwa et al. (1982), Wetherbee and Quirk (1982a,b). (Many more could be added.) D. THE CRYPTOMONADS, CRYPTOPHYTES The protists placed in the single phylum, Cryptophyta, of this assemblage have defied a classification based on degrees of interrelationship with ‘‘neighboring” groups because of our ignorance of such affinities (if they exist!). Thus, the cryptomonads are another phylogenetically isolated group. Most exciting in recent years has been promulgation of a concept which holds that these protists have long contained a (former) eukaryotic endosymbiont, remnants of which are seen in the presence of the single plastid bounded by four membranes and of a double-membraned body, the so-called nucleomorph, occurring in the space between the chloroplast envelope and the chloroplast endoplasmic reticulum. Because of the nature of the accessory photosynthetic pigments, cryptomonads are thought either to have had their own “blue-green’’ xenosomic prokaryote (different from that of the rhodophytes) or to have become associated with a primitive red algal endosymbiont or some kind of dinoflagellate invader, with subsequent degeneration of most of (the constituent parts of) the original xenosome (Cavalier-Smith, 1982; Gibbs, 1978, 198 la,b, 1983; Gillott and Gibbs, 1980; Whatley and Whatley, 1981). The distinguishing characters of the cryptomonads, organisms sometimes clas-

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sified in the past with other protist assemblages such as the dinoflagellates, are today considered in toto to justify complete isolation of the group. As mentioned earlier, the presence of an “organelle” that may have had a long evolutionary history of its own (both before and after gaining its intimate association with the “host” cell) only confounds efforts to trace the host’s lineage. The main features of systematic as well as phylogenetic usefulness are these: chlorophylls a + c and phycobilins (in pigmented species); typically biflagellate, with both flagella bearing (tubular) hairs; starch-producing centers, along with nucleomorphs and plastids, confined within some membrane system; lamellar mitochondria1cristae; distinct gullet and unique extrusomes (ejectosomes); internal cortical plates known in some species; and open mitosis. The literature on these “protozoalgal” forms is dispersed throughout phycological and zoological journals; a few selected recent references on their comparative cytology, biochemistry, systematics, and phylogeny are Gantt (1979), Gibbs (1981a,b, 1983), Gillott and Gibbs (1980), Hibberd (1977), Mignot et al. (1968), Morrall and Greenwood (1980, 1982), Roberts et al. (1981), Santore (1977, 1982a,b), and Sepsenwol (1973). As has been true for all assemblages treated in this article, a valuable section on the cryptomonad/cryptophyte group appears in many of the broader references listed at the end of Sections V and VI,A,C.

E. THECHLORARACHNIOPHYTES This is the smallest assemblage of all, containing but a single species at this time: Chlorarachnion reptans. A new division (here equals phylum) was created for it just 2 years ago (Hibberd and Norris, 1984). A thorough study by both light and electron microscopy revealed characteristics so novel and unique in combination that the authors had no alternative but to acknowledge the justified need for erection of a separate highest-level taxon for this green amoeboid protist with a uniflagellated zoosporic stage. First seen over 50 years ago, it has routinely been assigned to the xanthophytes (of the chromobionts: cf. Section VI,B), although with uncertainty. Chlorarachnion forms large plasmodia, with green-colored individual cells linked by a network of reticulopodia. In this stage it is actively phagotrophic. The motile zoospore, with a single flagellum coiled helically around the cell during swimming, bears unusual hairs, and there is no second vestigial flagellum or even a second basal body. The combination of biochemical (mostly related to the plastid) and ultrastructural features that render the organism unique in the kingdom Protista is best seen in the diagnosis supplied in the paper by Hibberd and Norris (1984), which I am giving below in only slightly altered form. Cells are naked, amoeboid, and interconnected by filose pseudopodia; each cell has a single nucleus and several peripheral chloroplasts, the latter containing chlorophylls a and b; chloroplasts have a wide periplastidal compartment con-

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taining ribosomelike particles and dense spherules and are bounded by four membranes; carbohydrate storage metabolite is not starch; mitochondrial cristae are tubular. The zoospore has a single laterally inserted flagellum coiled around the body in swimming and no eyespot. As the authors point out, such a group of characters, cutting as they do across the boundaries of several major “algal” protist assemblages (e.g., note major features of the chloroprotists and the chromobionts, Sections VI,A,B), with some novel ones as well, which at best are reminiscent of still other phyla (including the enigmatic cryptomonads, considered above), substantiate the high-level uniqueness of the species (and thus justify the new phylum). Hibberd and Norris (1984) suggest that the protist may have arisen “from an original symbiosis between a colorless eukaryote and a chlorophyll b containing eukaryotic alga. But, until more studies, including modem biochemical/molecular approaches, can be carried out, they caution that its “evolutionary origin . . . remains enigmatic. . . .” The best single source of literature on this isolated assemblage is, of course, the comprehensive paper by Hibberd and Norris (1 984). ”

F. THE CHOANOFLAGELLATES , COLLAR FLAGELLATES, CRASPEDOMONADS Another isolated group of unknown protist affinities, this assemblage contains a single phylum, the Choanoflagellata, which may have closest evolutionary ties with the parazoa or sponges, a group conventionally placed in the kingdom Animalia. This important phylogenetic question still awaits a definitive answer. The group’s major features may be considered to be the following: a unique funnel-shaped collar surrounding a single anteriorly projecting flagellum, composed of a ring or sheet of filopodia (also termed “tentacular microvilli” by some workers) without any supporting microtubules; collar functions in filter feeding; flagellum has no hairs (mastigonemes) and is tapered and retractable; unicellular species often have microtubular endoskeleton plus, sometimes, extracellular lorica with siliceous ribs; mitochondria1 cristae are flat. A few species exhibit colonial organization; some are stalked (others are free swimming), with or without a (visible) theca or lorica; most species are marine. The literature on the choanoflagellates is relatively light and/or pre-1970. The following five papers will lead the reader to earlier works, but in themselves present excellent data and represent good up-to-date reviews: Buck (1981), Hibberd (1975), Lava1 (1971), Leadbeater (1983, 1985).

G. THE PARAFLAGELLATES, OPALINIDS auctr. Long considered primitive (“proto-”) ciliates (cf. Section VI,H) on the basis of superficial characters, members of this assemblage have no clear close phylogenetic relationships with other protists. In a general way, they do exhibit basic

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“flagellate” (as opposed to “ciliate”) features, but so do half of all the (other) assemblages that make up the kingdom Protista. Patterson (1986) has very recently suggested that opalinids share important characteristics with the “proteromonads.” Unfortunately, that group itself is currently sub judice taxonomically (see discussion in Section VI,F), and it might also be argued that the characters not shared are of equal if not greater phylogenetic significance. The principal characteristics of the paraflagellates (with the Opalinata as the single included phylum), unique in combination and some no doubt having evolved in response to their symbiotic life style, may be listed as follows: body typically covered with short flagella arranged in diagonal rows; two to many homokaryotic nuclei; nonpigmented, mouthless forms, feeding saprozoically in (amphibian) host’s hindgut; sexual reproduction by fusion of anisogametes; mitochondrial cristae tubular; nuclear divisions acentric; cell division basically symmetrogenic and interkinetal, with bisection of unique falx; pellicle in folds supported by microtubules. The opalinids, as popularly known, are treated in many of the general works (textbooks and the like) in protozoology and parasitology; they are also given (abbreviated) coverage in such recent compendia as Barnes (1984), Lee et al. (1985), and Parker (1982). They are popular experimental organisms in certain lines of cell physiological research. A few papers appearing within the past few years and concerned primarily with ultrastructural cytology, systematics, and evolution of these protists are the following: Chen (1972), Corliss (1979c), Earl (1976), Foissner et al. (1979), Mignot and Brugerolle (1974), Patterson (1986), Wessenberg (1978), and Wilbert and Schmeier (1982).

H. THE LABYRINTHOMORPHS, LABYRINTHULEANS PLUS THRAUSTOCHYTRIACEANS, “NET SLIMEMOLDS” As is true for a number of the “isolated groups” considered in this section, the protists assigned to the labyrinthomorphs possess a number of unique characteristics that are of little help in postulating their evolutionary relationship with other assemblages. And the closeness of the group assigned to a second phylum, below, to the first is also unclear. Protozoologists, mycologists, and cell biologists have long been puzzled over the most appropriate taxonomic spot for these organisms. I believe that the decision made by Levine et al. (1980) represented a major step in the right direction, viz., totally separating them from other protozoan and fungal groups at the phyletic level. I have proposed going a bit further, eliminating their “protozoan” association and treating them simply as an isolated phylum in the kingdom Protista (Corliss, 1984). Contained in the assemblage are two groups, tentatively assigned to separate phyla: the Labyrinthulea and the Thraustochytriacea. Their distinguishing characteristics, in combination, include nonpigmented organisms exhibiting a unique membrane-bounded extracellular network byion

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which movement of spindle-shaped nonamoeboid cells occurs; thin flat scales in cell walls; zoospores have pair of heterokont flagella; unique organelles, sagenogens (bothrosomes, sagenogenetosomes), in surface pits, allegedly responsible for slime secretion; tubular mitochondria1 cristae. Organisms occur primarily as parasites on algae and eel-grass in marine and estuarine habitats. Although often seen-and easily culturable in the laboratory-the labyrinthomorphs have been studied critically rather rarely. Important papers selected from the literature since the time of Pokorny’s (1967) helpful review of 20 years ago are the following: Amon (1978a,b), Gaertner (1972), Perkins (1973a,b, 1974), Porter (1974), Schwab-Stey and Schwab (1973), Schmoller (1971), and Valkanov (1972).

I. THE MICROSPORIDIA, MICROSPORANS This assemblage contains organisms formerly/conventionally lumped with members of the following two groups (Sections VII,J,K) into a single relatively low-level (subclass or class) taxon called “Cnidosporidia” (variously spelled). In turn, the latter category was placed with the “Telosporidia” in the single class or subphylum Sporozoa of the phylum Protozoa. By some authors (e.g., Sleigh, 1973), the two groups (Sporozoa and Cnidospora) were raised to separate subphyletic rank. Six years ago, Levine et al. (1980), in their extensive overhauling of the Protozoa, clearly separated the three groups here under considerationboth from the Sporozoa (called Apicomplexa: see Section V1,G) and from each other, recognizing them as making up the individual phyla also endorsed (with slight name-changing) by the present author (Corliss, 1984). The microsporidia (often written ‘‘microsporidians”) are nonpigmented, minute, intracellular parasites (hosts are principally insects and fishes, but several crustaceans, some other vertebrates and invertebrates, and even a few protists are also infected) having unicellular spores characterized by containing a single large polar filament or tube of some complexity. There are no mitochondria, flagella, or basal bodies in any stage of the life cycle. The polar tube, which is apparently produced by the Golgi apparatus, is reversible, and through it the infective sporoplasm is extruded and inoculated into fresh host material. There is chitin in one layer of the resistant spore wall; the outermost (proteinaceous exospore wall) often shows a sculpturing useful in lower-level taxonomy. The sporoplasm entering a host cell typically develops into a multinucleate plasmodia1 stage, subsequently yielding a new generation of spores. The classic on the systematics of the Microsporidia is Kudo’s (1924) monograph. A brief list of (other) important works mostly appearing within the past 10 years, aside from sections of books or treatises cited earlier in this article, follows: Bulla and Cheng (1976), Canning (1977), Canning and Nicholas (1980), Desportes (1976), Issi (1978, 1980, 1981), Lom (1972), Loubks (1979), Sprague (1 977), VBvra (1976a,b), Weiser (1977), Weissenberg (1976).

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J. THEHAPLOSPORIDIA, ASCETOSPORANS There is a small group of nonpigmented protists endoparasitic in certain invertebrates and “lower” vertebrates that was long classified with the microsporidia (or, at least, the “cnidosporidia”) on a rather superficial basis (e.g., spores with amoeboid sporoplasms developing into a plasmodia1 stage that eventually produces a fresh generation of spores). But there are now known such unique characteristics as the total lack of a polar filament (or of polar capsules: see K, below) and the presence of haplosporosomes, membrane-bounded osmiophilic bodies, in the cytoplasm of the sporoplasm. The spore walls also differ in structure and composition from those of species in the preceding and following assemblages. Some included forms presumably have mitochondria (with flat cristae). All species are parasitic in cells or tissues of mainly marine mollusks or annelids (or of trematode worms themselves in certain marine invertebrates). I follow Levine et al. (1980) in recognizing their high-level separateness (Corliss, 1984). A few enigmatic species included among the haplosporidia by some workers are assigned to the following assemblage by others; further ultrastructural and biochemical observations are sorely needed to resolve such conflicts. Incidentally, the possibility of a (true) fungal evolutionary relationship for (various) members of these last three groups cannot be ruled out. One intriguing common feature that may be of considerable phylogenetic significance is the occurrence of a diplokaryon stage in the life cycle. The literature on solely this group is not abundant. Several recent works are Azevedo (1984), Desportes and Nashed (1983), La Haye et al. (1984), Perkins (1979), Schulman (1966), and Sprague (1979).

K. THEMYXOSPORIDIA, MYXOSPORANS, MYXOZOA The final assemblage to be considered among my group of phylogenetically isolated protist taxa is represented by a large number of species having in common, most strikingly, valved spores containing one or more so-called polar capsules each of which, in turn, contains a coiled, inverted polar filament. Polar capsules are reminiscent of cnidarian cnidocysts. Mitochondria1 cristae are flat, and there are no flagellated stages in the life cycle. A multinucleate plasmodium is part of every life cycle, and there are multicellular developmental stages, including one in the differentiation of a new mature spore. The shell valves often show considerable sculpturing, typically more so than noted on the outside of microsporidian spores (subsection I, above). Species are histozoic or coelozoic endosymbionts of some invertebrate animals (e.g., annelids and oysters) and especially lower vertebrates (notably fishes, in which they are often highly pathogenic to their host).

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Their multicellularity at times, their polar capsules plus filaments, and their parasitic existence could be said to support the suggestion that the myxosporidia are (degenerate?) metazoa of some sort. A clear example of a multicellular stage is found in the development of a polar capsule/filament in sporogenesis. At one time, there may be nucleated cells in close apposition (but a true tissue?) as follows: one or two sporoplasms, up to six capsulogenous cells (destined to become the polar capsules with their internally coiled filaments), and half a dozen to a dozen valve cells (developing into the shell valves, number varying in different species). But protozoologists should have learned from phycological colleagues (and we have seen examples in many of the “algal” groups treated in this article) that neither multicellularity nor even a tissue doth a metazoon/metaphytan make. Today’s protistologists do not restrict their organisms to unicellular (or even microscopic) forms; furthermore, some of the most unique characteristics of the myxosporidia are no more amazing (nor frustrating, from an evolutionary/phylogenetic point of view) than features abounding in other assemblages in both Sections VI and Vll. This group contains one principal (or only one?) phylum, the Myxosporidia. Note discussion of possible appended taxa in Corliss (1984). The recent report that-contrary to all previous findings-some myxosporidia may have a twohost life cycle (Wolf and Markiw, 1984) complicates their systematics/phylogenetics at the highest level. That is, the myxosporidian fish parasite studied was said to have a form in the second host, an oligochaete annelid, very similar to annelid parasites always assigned to the Actinomyxidea, a taxon considered by some workers to represent a separate phylum( !) from the Myxosporidia (see full nomenclatural discussion in Corliss, 1985b). Except for mention, first, of the taxonomic classic on myxosporidia by Kudo (1920) and the relatively recent monograph by Schulman (1966), the following selected references are limited to a few papersheviews that have appeared within the past 10 years: Corliss (1985b), Desportes (1984), Grasse and Lavette (1978), Lom and Noble (1984), Mitchell (1977), Schulman and Podlipaev (1980), Sprague (1982), Uspenskaja (1976), Weiser (1985), and Wolf and Markiw (1984).

VIII. Progress and Prognoses, Problems and Frustrations It can be said without fear of contradiction that most biologists have a much greater appreciation of protists and the “protist concept” today than they had even as short a time ago as 1975, a date that (arbitrarily) may be associated with a full-scale beginning of the “protist revolution” (Corliss, 1986~).This is progress! Even the controversies going on within the protist community need not be

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considered counterproductive. Indeed, they have been having quite the opposite effect, being one of the main reasons for the recent “explosion” in the protistological literature. With the realization that there are “lower eukaryotes,” biologists are now readily admitting that division of the biotic world into simply prokaryotes and eukaryotes is not enough. Furthermore, the latter consist of more than solely plants and animals. Imposition of characters of these two groups downward onto unicellular, microscopic forms (and their close relatives) is not adequate for proper identification of the vast assemblages of such forms. In short, the tremendous diversity of the protists literally demands reflection in their systematics overall and in the schemes of classification proposed to contain them. Euglena, Chlamydomonas, and Chlorarachnion, for example, can no longer be dismissed (taxonomically) as being, in effect, “mere green mini-plants’’ of close affinity. If you’ve seen one green alga, you’ve not seen ’em all! While there is evolutionary unity exhibited at various high levels among the protists, their longstanding distinctiveness, one from the other-now that we have uncovered many of these differences (see below)-obliges us to consider them in the same way biologists have treated diverse assemblages of the multicellular/multitissued organisms in the past (viz., separate them at high taxonomic levels). Because of the antiquity of at least some of the protist lineages, it is likely that our taxonomic fragmentation is fully justified, not a runaway inflation without a sound phylogenetic rationale. Formal recognition of the diversity manifest in the protistan conglomeration represents progress. Finally, the amazing advances made in recent years in development of new technical/technological approaches for (improved) study of protists-both as cells and as whole organisms-have been a boon to protistology that cannot be overlooked. In fact, applications of new techniques, running all the way from improvements in culture methods and electron microscopy to molecular biology, have been indispensable in revolutionizing the whole interdisciplinary field of protistology. In many ways, select protists have become ideal models for numerous kinds of investigations in cell biology today. General prognoses or predictions are easy to make. It is quite clear that a cadre of workers “out there” are putting protists on the map through the results of their continuing research efforts. Further, old “divisions of labor” are falling by the wayside (e.g., “algal” protists are now being studied by all sorts of “nonphycological” folk). I predict that communication and cooperation will both continue to improve. The growing “protistologist image” requires not only an interdisciplinary approach but also a conscientious attempt to learn something about the organisms formerly “divvied up”-and jealously guarded-by authoritarian traditionalists in both camps (botany and zoology). (Speaking as a former protozoologist, it is not easy to absorb all that knowledge accumulated over the centuries by those frightfully industrious phycologists and mycologists.)

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Ingenuity, industriousness, and promulgation and application of new, and sometimes bold and imaginative, ideas will be resulting in new data and new concepts of inestimable value to our understanding of the phylogenetic interrelationships of the major protistan assemblages. I predict that such new knowledge will contain some unimaginable surprises, too. While it is encouraging to envision all the advances one may expect protistologists to be making, it is equally discouraging to survey the roadblocks to progress that still confront us today. We have been reminded time and time again of such problems throughout preceding pages of this article. Opinions may differ on what is the greatest frustration of all. Very likely, it is included in the following list of 10 “shortcomings” of the protists themselves and/or in the knowledge gathered on them to date (there is inevitable overlapping among some of these). 1. Microscopic size of most protists 2. Common absence of fossil material 3. Impossibility of knowing whether “intermediate” forms/lines have become extinct 4. Incompleteness of knowledge (of full life cycles, sexuality, ecology, etc.) of practically all groups 5. Fewness of species overall yet subjected to study, with almost total neglect of some (key?) groups 6 . Shortage of ultrastructural data of comparative value 7. Lack of molecular “dating” information on great majority of forms 8. Ignorance concerning possible occurrences of convergent evolution 9. Complicating factors of parasitism, both of “host” protists themselves and of (some of) their cytoplasmic/nuclear inclusions/endosymbionts 10. Dearth of genetic information in general

These 10 “failings” might be summarized into one brief statement: there is great scarcity of unambiguous data in general, information essential for proper study of the phylogenetics of the protists overall and of major assemblages within the conglomerate as well.

IX. Hopes and Conclusions Naturally, one hopes that the ‘‘problems and frustrations” enumerated above can in due time be overcome, or at least partially resolved, for a gradually increasing number of protist taxa. There is reason to be optimistic, based on progress made within recent years. Several conclusions may be drawn:

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Protistan diversity is a fact, and many biologists are becoming aware of it. As more and more species of “lower eukaryotes” are studied, more investigators from many biological fields are beginning to recognize that some protists represent the “cell” material of choice in experimental research of a variety of kinds. As interdisciplinary approaches are made and sophisticated techniques are applied, our knowledge of the “protistan conglomeration” is growing rapidly, even if we still have far to go. Heuristic concepts are helpful in phylogenetic considerations, including studies on eukaryogenesis. Ultrastructural information is indispensable, and molecular biological dataifiwhen collected in sufficient quantity-should aid in solution of numerous problems now irresolvable by any other approach. Recognition of a kingdom Protista may be of value, at least for a while, untilhnless such a proposition becomes clearly untenable with accumulation and proper analysis of relevant data. A single “protistological perspective” is still eventually desirable. While a good deal of “taxonomic inflation” may be inevitable, systematically inclined protistologists should avoid losing credibility by wholesale production of differing ranks and names with little regard for either historical “priority” or future stability. “Protist awareness” is exemplified by the widespread adoption, already, of such terminology as “protist,” “protistology,” and “protistologist. Formal words like Protozoa, Zoomastigophora, Protophyta, Phytomastigophora, Thallophyta, Algae, and Lower Fungi, used with capital initial letters and as names of specific taxa, are falling into disuse with the growing acceptance of the suggestion that they are misleading in their connotation. ”

ACKNOWLEDGMENTS Support of National Science Foundation grant BSR 83-071 13 is gratefully acknowledged.

REFERENCES Ainsworth, G. C., Sparrow, F. K . , and Sussman, A. S . , eds. (1973). “The Fungi: An Advanced Treatise.” Academic Press, New York. Alexopoulos, C. J., and Mirns, C. W. (1979). “Introductory Mycology,” 3rd Ed. Wiley, New York. Amon, J . P. (1978a). Mycobgiu 70, 1297-1299, Amon, J. P. (1978b). Mycologia 70, 1299-1301, Andersen, R. A. (1986a). Am. J . Bor., in press. Andersen, R . A. (1986b). Am. J . Bor., in press.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. IW

Protozoological Approaches to the Cellular Basis of Mammalian Stress Repair S . H. HUTNER*AND S . L. MARCUS? *Haskins Laboratories of Pace UniversiQ, New York, New York 10038, and ?Department of Oncology, Monte$ore Hospital, Albert Einstein College of Medicine, New York, New York 10467

I. Introduction The 35 years of the International Review of Cytology recounts discoveries of cellular structures and regulatory systems and their linkages, justifying speaking of the “metabolic web.” “Web” implies a comprehensive reductionist understanding of how mammalian cells are integrated into body. Taylor’s (1983) neologism for the eukaryotic cell-cytocosrn-implies, as we extend it, that there remains a hardly yet conceivable plexus of cellular mechanisms still defying detection. Our extrapolation stems from the recent bleak consensus that little is understood about how the body marshals its resources to repair serious injury after the initial 24- to 48-hour shock phase-a problem defined half a century ago. This article was partly prompted by revelations that basic mechanisms of cell repair are part of life itself. Moreover, on evolutionary grounds, some protozoa may prove advantageous probes for dissecting out these deep-seated homeostatic mechanisms and for facilitating translation of such laboratory results to the bedside. Three developments set this problem in new perspective, justifying a cautious long-range optimism: 1. Discovery of the stress proteins signals a startling capacity of the stressed cell to reorganize itself: its life-as-usual economy switches to an emergency protein-synthesis economy. If the stress is invasion by a parasite-sepsis if infected by a prokaryote-an all-out wartime economy soon prevails. Some genes and proteins so expressed are traceable from bacteria to protozoa to Drosophila, and even, on a vast outdoor scale, to crop plants and to mammals. Hence what one discovers in this respect from any one organism might eventually aid patients. 2. Notwithstanding conservatism of the stress proteins, the gap between prokaryotic and eukaryotic organizations looms ever wider. The eukaryotes emerge as a closely knit group despite their stupendous phenotypic differences, i.e., 37 1 Copyright 0 1987 by Academic Press, Inc. A11 rights of reproduction in any form reserved.

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genotypically they are very much nearer to each other than the diverse prokaryote groups are to each other. 3. Much knowledge has accumulated on inborn errors of amino acid metabolism (Nyhan, 1984), thanks to elaboration of powerful analytical methodologies to diagnose these disorders (chapters in Nyhan by M. Bordon on screening for metabolic disease and L. Sweetman on organic acidurias). These analyses increasingly guide successful therapeutic interventions. In some diseases intervention is by vitamin supplementation in the so-called pharmacological range, not the conventional vitamin dosage level. The knowledge thereby acquired amounts to a significant though indirect beginning toward the goal set by Moore (1985)to inventory bodily resources that can be drawn upon for repair of serious injury. Inventorying has not yet been applied systematically to detect stress-prone breakdowns as though they were inborn metabolic diseases; abnormal amino acid fluxes and muscle protein breakdown are conspicuous in stress. Another consideration in preparing this inventory is the feasibility of assembling a battery or panel of protozoa to serve as analytical reagents; their sensitivity and specificity help fill the gap for estimating vitamins which are ubiquitous enzyme cofactors and for which chemical or physical methods are neither sensitive nor specific enough. Radioimmunoassays are filling in the gap for hormones; some are being developed to meet the same needs as the microbiological assays. But many decision points in metabolic pathways are unknown, e.g., the polyamine and biopterin pathways are still being charted. Live reagents, especially the protozoans, have special value: in serving as analytical reagents their own regulatory mechanisms come to light, especially from exploring the specificity of their responses. They often closely imitate the mammal. To discern how the different cellular pathways are linked in the metabolic network is a daunting mega-variables task. Practicality dictates that, with the high costs (Section 11) of resources and time for research with laboratory animals, let alone patients, those protozoa which are the most nearly higher animallike and also easy to cultivate on a large scale should be called upon to prize therapeutically useful secrets from that enigma-packed gray (if not wholly black) box which is the archetypical mammalian cell. Effective therapeutic interventions are likely to depend on low-molecularweight substances for readier permeation of damaged cells and tissues. Where synthetics are used, they too will be preferably of low molecular weight and fairly lipid soluble for ease of synthesis and to favor permeation. Protozoa are known which require lipid factors, but given the primitive state of emulsification and other molecular dispersion methods, such protozoa have not served as reagents for those factors. This article therefore mainly discusses water-soluble low-molecular-weight metabolites; enzymes and informational macromolecules only figure as they govern functions of related low-molecular-weight metabolites. Because of the therapeutic near-impasse in injury repair to be described

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here, we sought to cull from the literature suggestions for lines of inquiry extending even faint hopes of therapeutic gains. Such results, from the point of view of the cytologist, would denote that at least one activity of the cytocosm-repairwas being tackled operationally, however grudging the advances. Shock is a state of impaired circulatory cell and tissue perfusion, commonly induced by extensive hemorrhage superimposed on severe injury. Vascular damage in sepsis, especially by the endotoxins of gram-negative bacteria, is a pressing problem. Massive tissue injury may lead to hypoxia and loss of ATP because of interruption to cells of 0, and nutrients and impaired disposal of wastes. Negative nitrogen balance, outpouring to plasma of catecholamines and glucocorticoids, and hyperglycemia generally characterize the shock phase and are elicited mainly from pituitary-adrenal activity in response to conjoined physical and psychic trauma. Dunn and Kramarcy (1984) hypothesize a third response: activation by the central nervous system (CNS) of catecholaminergic systems. The shock phase, especially its uniformity, whatever the kind of injury provided injury is extensive, is discussed in a symposium (Lefer and Schumer, 1983). Protozoology has a part to play here. For instance, tetrahydrobiopterin, a cofactor for syntheses of catecholamines and indoleamines, is a growth factor for trypanosomatid flagellates. A nonpathogenic trypanosomatid, Crithidia fusciculata, is a widely used analytical reagent for the biopterin series (Section IV). Other protozoan tools will be discussed later. Therapeutic intervention for shock aims at restoring blood circulation, thereby alleviating cellular hypoxia and its sequels of deficiencies in energy generation and, presumably, in synthesis of spare parts for injury repair. Much experience has shaped present treatment of the shock phase supervening after severe injury; it is expounded trenchantly by Abboud (1985). Initial neurohumoral responses to injury and its concomitant psychic trauma are detailed in a symposium (Dunn and Kramarcy, 1984) and a review (Buckingham, 1985). Trauma is partly a synonym for shock, sometimes defined explicitly as traumatic shock, with its connotation of pain and CNS discharges, thereby focusing on the neurohumoral role in immediate response to injury. Trauma seems increasingly to encompass both primary stress and the postshock reparative phase as well. Stress in its widest sense-as in this article-comprises perturbation and restoration (or not) of homeostasis, i.e., stress repair. The postinjury stage presumably carries over some features of shock and generally presents a catabolisminduced negative nitrogen balance, with loss of body cell mass (the energyproducing working cells of the body) (MacLean, 1985) and accumulation of Ca2+ in the cytosol associated with membrane damage, including that of mitochondria, thereby impairing ATP supply. Such Iarge-scale cellular malfunction uncorrected is fatal (Heath, 1985a). Stressor is the agent producing stress. Reversal of the negative nitrogen balance, restoration of normal blood circulation,

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and lowering of fever (fever is conspicuous in the hypermetabolism seen in patients with extensive burns or sepsis) are traditional indices of repair and survival. Mechanisms of repair are “mysterious” (Smith, 1985). This article therefore, as intimated, assembles hints from cytometabolism and cytonutrition, especially of protozoa, on ways to assist repair while fending off exhaustion of the body’s homeostatic resources. It is ‘‘unreasonably optimistic” to suppose that repair can be much speeded by providing nutrient-rich parenteral feeding (Moore, 1985). Repair, Moore argues, requires about the same period as that for synthesis of the most complicated of the body’s molecules. If so, and evidence reviewed below supports it, one may ultimately have to provide the patient with every possible assistance for expeditious removal by macrophages of useless and dangerous cell debris while assisting salvage and recycling of the many kinds of potential rebuilding blocks released from breakdown of cells and tissues. Recommendations of protozoa as stress-repair investigative tools spring, as intimated, from several sources: 1 . Discovery of the inducible stress proteins, notably those conferring transient thermotolerance. 2. As timed by such evolutionary chronometers as their 5 S or 16 S rRNAs, protozoa are biochemically far closer to man than to any prokaryote except perhaps in certain genetic respects to archaebacteria (Woese, 1983, 1985; Woese and Wolfe, 1985). Protozoa have had the same geological time as metazoa in which to radiate and colonize exceedingly diverse ecological niches, hence their extraordinary phenotypic diversity (Lee et al., 1985). No protozoan group can lay exclusive claim to being outstandingly humanoid. Reliance should reside on the broadest practicable battery of protozoa. 3. Animals-the virtuosos of phagotrophy-trap nutrients at myriad receptors. Animal-cell devices for storing and recycling food-vacuole membranes and some receptors are especially elaborate, with heavy traffic among organelles and between organelles and environment. Professional phagotrophs have numerous mechanisms for transport and for interiorizing nutrients by pinocytosis (solubles) or phagotrophy (particles). Protozoa share these hallmarks of animality with mammalian cells, conspicuously so with the leukocytes of the monocyte-macrophage series. Hence our theme: how protozoa repair themselves after injury does not differ fundamentally from how a mammalian cell repairs itself or, for that matter, the whole mammal. The practical problem is how to ferret out those fundamentals. Which protozoa-and why? That is the burden of this article. There has been scant progress before and since Kinney (1981) underscored the

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necessity of distinguishing between nutritional management of the ‘‘acutely ill and injured patient from the chronic, depleted patient whose primary problem is that of an abnormal and depleted body composition. . . . Nutrition in the acute catabolic phase should be supportive but cannot be expected to be curative.” Some samplings of opinion: 1. Elwyn (1985): “The traumatized patient is glucose- and insulin-resistant and is therefore resistant to the administration of exogenous nutrients, particularly glucose; thus the ileus [intesinal obstruction] and anorexia which usually accompany injury may at least in part be adaptive, protecting the patients against an influx of nutrients.” And, as for the efficacy of ‘‘parenteral or enteral feeding to minimize or eliminate the catabolic phase itself, in many patients . . . we do not yet know whether this increases, decreases or has no effect on morbidity.” 2. Stoner (1985): “At best we seem to be holding the position [therapeutic initiatives aimed at improving the metabolic state of the patient with injury and sepsis], but we can still do little to aid recovery.” 3. Buckingham (1983, challenging the view that corticosteroids suppress the normal defense mechanisms, cites the suggestion (Munck et al., 1984) that corticosteroids function not to protect from the trauma, but rather to prevent the response to trauma, producing damage itself and threatening homeostasis. 4. Rennie (1985): “Studies of trauma and disease have so far shown a great variety of protein changes in lean body mass and in the components of muscle turnover. It is vital to try to integrate the results . . . to form a scientific basis for therapy.” 5. Fellows and Woolfson (1985): “There is no convincing controlled evidence that the routine administration of nutrients has any advantage in promoting recovery after trauma.” Aside from when “the catabolic phase of the injury is likely to be protracted, it would generally be considered wise to intervene with nutritional support (which can doubtless help to maintain body mass) rather than wait until the patient has lost a great deal of weight and nitrogen.” 6. Muller et al. (1985): After caring for 500 patients over a 10-year period, they see little progress except that in their intensive care unit in Cologne the median time to death increased from 8 days to 13 days and, where patients survived, duration of hospital stay decreased. Present intractability of the problem as attacked along present lines is, then, clear. The next section documents that the therapeutic impasse is exacerbated by soaring direct and administrative costs of conventional research, including that with laboratory rodents and that in some respects recourse to tissue-cell models is attended with difficulties and, likewise, proportionately, research with large mammals. Research with patients poses difficulties of another order of magnitude.

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Many inquiries into the postinjury phase strove to determine whether fuels and spare parts might be beneficial. There has also been agreement that after injury the main cause of lethality may reside in a breakdown of the defenses against 0, on the one hand (conspicuous as reperfusion damage) and, on the other hand, having to restore that minimal supply of ATP inadequately provided by anaerobic glycolysis while trying to repair the efficient 0,-utilizing mitochondria1 function-a tricky balancing act. In providing emergency fuel and repair parts the body cannibalizes itself, drawing conspicuously on lean body mass-a life-ordeath gamble. This article tries to coordinate clues about spare parts, regulatory metabolities, enzyme cofactors, “cellular mediators,” growth factors, etc. as may derive from studies of our unicellular fellow animals: protozoa. Might remedial metabolities for them against life-threatening stressors, e.g., supraoptimal temperatures, starvation, cytotoxic drugs such as those used in cancer chemotherapy, apply to humans? Might some regulators of intracellular, i.e., organellar, traffic contain rudiments-anfagen-of intercellular regulators, e.g., metazoan hormones, and be more discernible in protozoa than by mammalian endocrinology? Such regulators might be restorative metabolities. Some listings of hormones-or amino acid sequences suggestive of hormones-are therefore listed in Section V on chrysomonads and in Section VI on Tetrahymena. The devices supporting the animal way of life have only lately received the respectful attention merited by its uniqueness in the biological kingdoms. Those fantastically elaborate specializations, above all phagotrophy , are recounted in a symposium volume (Pastan and Willingham, 1984) and a review (Wileman et a f . , 1985). After comparing the body plan of the photosynthetic Euglena with that of the clearly though distantly related trypanosomatid line, which includes the literally bloodthirsty Leishmania and Trypanosoma, and with the very different chrysomonads, Kivic and Walne (1984), joining the consensus that the ureukaryote acquired mitochondria and chloroplasts from phagocytosed prokaryotes, conclude that the first eukaryotes were phagotrophs, i.e., particleingesters and digesters; nonphagotrophic photosynthetic eukaryotes came later, their phagotrophy no longer needed. Extant protozoa, phagotrophic by definition (Lee et a f . , 1985) however innovative their radiative dispersal in geological time, retain a common phagotrophy. But, given the infinite diversity of their prey organisms-bacteria, algae, microfungi, p-flagellates, other protozoa, viruses-no protozoan can choose to have more than a limited assortment of receptors and digestive enzymes. Presumably all are diversely specialized to exploit particular kinds of prey or detritus or to withstand different adversities, including the antibiotics of the microbial jungles of soils, muds, and other surface-rich environments; other protozoa are planktonic. Unfortunately the precise origin(s) of metazoa is a mystery (Sleigh, 1979). In any case, as mentioned, morphological evidence is lacking for any group of protozoa being especially

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akin to whatever metazoan line led to the chordates. If one goes by pattern of its vitamin B 1 2 (cobalamin) requirement, then the chrysomonad Poterioochromonas (Ochromonas)rnalhamensis (Section V) would appear to be uniquely like bird or mammal, i.e., it provides a clinically and industrially accurate assay for B,, (Baker et al., 1986a). This is only one of many chrysomonad attributes. The key cells for repair of injury in the mammal are the professional phagotrophs of the monocyte-macrophage line. To what degree they imitiate protozoa-or vice versa-is another angle from which to address the problem. Interestingly, impaired phagotrophy may partly account for the morbidity and mortality in extensive thermal injury (Duque et uf., 1985). Now we wish to explain how our seemingly disparate predilections evolved from a clutch of varied researches. In 1935, one of us, the senior author, a graduate student in microbiology and biochemistry at Cornell (Ithaca), was parachuted onto the Physics Department at MIT, then sponsoring a Haskins Laboratories team assembled to assess the desirability of million-volt X-rays for deep tumors: the higher the voltage, the smaller the proportion of radiation delivered uselessly to the skin. That X-ray burns were slow to heal was known. Would extrapolation from lower voltages hold? Rather than constructing the apparatus itself-expensive!-the strategy was to design an electron source whose thermal electrons, accelerated in a magnetic field, corresponded to energies of the X-rayinduced photoelectrons. Mold spores were the target. Spore killing followed the expected extrapolation from lower voltages. On return to Cornell I was asked by James B. Sumner (later a Nobel laureate: he demonstrated that enzymes were proteins) to determine whether the porphyrin in his newly crystallized catalase was truly heme-a ticklish problem because catalase was assayed by its rate of destruction of H,02, but H,O, decomposed some of catalase’s porphyrin, confounding the analytical figures for heme. Heme it was indeed, as shown by applying Crithidia’s specific response to heme as demonstrated by Andr6 Lwoff (Hutner, 1971). It was an introduction to microbiological assays and aroused curiosity as to why blood was so rich in catalase. Was H,02 the real substrate for the enzyme? If so, why? Sepsis as a scientific problem influenced my doctoral thesis, inspired partly by a hemolytic streptococcus which had engaged the then Department of Dairy Industry and Bacteriology: an epidemic of grievous septic sore throats had been spread by a milker having a sore finger; the milk had not been pasteurized to satisfy the credo of a “health-food”-demanding clientele. On rejoining Haskins Laboratories, colleagues and I explored use of streptococcus and gram-negative toxin, i.e., endotoxin, to treat solid tumors. The lethality of even small amounts of crude preparations was striking. Sarcoma 180, a transplantable rodent tumor, was cured by endotoxin, apparently by hemorrhage blocking its capillary blood supply, as detailed in a series of papers, one joint with Sloan Kettering people (Zahl et al., 1942). Endotoxin shock must have early beset metazoa because

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photosynthetic gram-negative bacteria were rich in endotoxin. With war looming, and aware that both shock and injury repair had been recognized in World War I as unsolved problems, we extended the endotoxin-shock studies to NobleCollip drum shock in rats. We stumbled upon the efficacy of atropine in alleviating some of the symptoms, of which a conspicuous component proved to be motion sickness. We thereby had inklings (Zahl et al., 1943) of what Hans Selye called the general adaptation syndrome as a stereotypical pituitary-adrenal response to prolonged stress. Cross-resistance between animals recovering of either endotoxin shock or moccasin snake venom had indicated to us that we were dealing with some sort of generalized adaptation to injury. Drum shock, still used today, is thought to be epinephrine shock (DuBose et al., 1985). War over, pursuit resumed of a potent growth factor for the green pond flagellate Euglena. Alerted by a pathology course in Cornell’s Veterinary College to the Euglena factor, we realized that it shared some properties with the then still-crude pernicious anemia factor. The Euglena B,, assay thence served our collaborators at Lederle Laboratories to monitor production of B,, and it was later applied to clinical diagnosis. It remains used in many countries. Euglena and Lactobacillus assays for B I represented a dramatic analytical miniaturization: the original analytical protocol centered on a ward full of pernicious-anemia patients titrated against graded liver concentrates. The miniaturization represented by introduction or refinement by Herman Baker and his colleagues of microbiological, mainly protozoological, assays to assess clinical vitamin status (chemical methods, as mentioned, were too insensitive) (Baker and Frank, 1968) met opposition from one of the nutritional old guard, who asserted that only the rat and chick were legitimate assay organisms. Transition-metal buffering with EDTA partly resulted from the aforementioned MIT research; it had helped monitor removal of radium from a Princeton physicist whose apparatus had blown up in his face. Removal consisted of infusing huge doses of citrate. The bone-seeking radium was excreted as citrate-Ca complex. The patient then had his calcium replenished. After several such wracking cycles his radium was lowered to a tolerable level. Citrate was known to be metabolized about as fast as glucose. After EDTA was synthesized but used mainly as a reagent for inorganic analysis, its potential as a nontoxic, nonmetabolizable chelator for use in biology and medicine was apparent. We thereafter used EDTA to buffer such chelatable essential but easily poisonous elements as copper for the medium for the Euglena B assay, practically eliminating dependence on contaminations of reagent chemicals to supply the essential trace elements adequately but at nontoxic levels. A paper describing theory and reduction to practice of such metal buffering (Hutner et al., 1950) speeded introduction of EDTA to medicine and biochemistry; its introduction to enzymology was effected by a former worker at Haskins, the late Dr. Wolf Vishniac, who demonstrated in Dr. Severo Ochoa’s laboratory its use as probe for metal cofactors in enzymes.

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BIOPTERIN The via-protozoa discovery of bioptin arose from the collaboration with the Lederle group. After my group had devised a defined medium for Crithidia, a puzzle, as noted, was its inordinate folate requirement. Something in a folatefree liver concentrate extremely actively spared folate. Availability of the Crithidia assay spurred a search for a more accessible large-scale source of the folate-sparing factor. Poterioochromonas malhamensis was a good source which then was grown on a pilot-plant scale on a cheap skim milk powder-cane sugar medium. Fractionation of the chrysomonad cells revealed the unfolatelike stability of the factor. A search began for a simpler starting material. Urine did nicely: several thousand liters yielded some milligrams of pure biopterin. It brought home the desirability of concentrating one's efforts, where possible, on organisms cheap and easy to grow in bulk if trace cell constituents might have to be isolated. Enzymologists later identified tetrahydrobiopterin as cofactor in syntheses of catecholamines and indoleamines; biopterin had been simultaneously isolated from eyes of a Drosophilu mutant. The abyss separating prokaryotes and eukaryotes was newly recognized. It explained why it was so easy to find antibiotics and synthetics against subacterial pathogens and hard to find them against eukaryotic parasites, especially against Leishmania and Trypanosoma (and fungi). Realizing that henceforth there would be a steady flow of biochemical information about them, my colleagues, especially Dr. Cyrus J. Bacchi, entered the search for the desperately needed new drugs while hoping for enlightenment on biochemical mechanisms accessible also in the easy-to-handle related nonpathogens. This thrust has, as noted, been successful (Section IV). That tissue repair, reflecting cell repair, was rooted in life itself could hardly have been guessed half a century ago.

11. Economics and Practicalities in Stress-Repair Research: The Protozoological Gambit Therapy in stress repair may involve synthetics to help repair regulatory mechanisms. As with cancer and, increasingly, infectious diseases, multidrug regimens have evolved. Standard protocol for Hodgkin's lymphoma is eight drugs applied first as group A (four drugs), then group B [four other drugs, none with the same modes of action as in group A (Wagener et al., 1983)l-a regimen evolved from knowledge gained from bacterial assays and a rich bacterial folic acid source for initial isolation of folic acid. Later the synthesis of methotrexate came, around which the regimen was developed. Information on lactic bacteria used for making cheese was drawn on for bioassays at all steps. Evaluation of methotrexate was based on work with mouse leukemias and transplantable tu-

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mors and then close monitoring of many patients-in all, many microbiological maneuvers, not a frontal clinical attack. We wish to know how to supply the precursors and coenzymes for making restoring enzymes. What limits the body’s abilities to obtain them exogenously under stress by the so-called salvage pathways? Which preliminary microbiological maneuvers for stress repair? The resources needed, if repair research follows conventional lines-laboratory rodents, dogs, monkeys, then patients-may be grasped from costs of developing a novel drug from idea to market-counter and bedside-in 1976, about $54 million (Hansen, 1979). Goodwin (1978) estimated it at $10 million and requiring 5-7 years for tests of toxicity, teratogenicity, and genotoxicity. Pocock ( 1983) endorsed Hansen’s estimate after detailing the tortuosities of committees and multitudinous reports to government agencies. Trains (1983) ventured no figures but did estimate that ca. 0.9 kg of drug was needed for preclinical safety tests carried out over a year’s span, including a carcinogenicity study of 500 rodents: the pathologist must examine over 18,000 tissue sections and tissue sites. Arbuthnot (1984) estimated the development cost of a new antibiotic at $70 million. A major hazard in extensive burns is sepsis; here, fast-penetrating, nontoxic antibiotics are badly needed probably indefinitely, since resistant strains will almost inevitably emerge. We endorse the position of Desjardins (1984) in emphasizing the need for new antimalarials in the face of spreading drug resistance: that application of in vitro techniques could greatly improve the efficiency of a chemotherapeutic research program, with its conservative requirements for space, equipment, and trained people-our position with stress-repair research. This injunction is borne out by the rapidly increasing reliance in many other fields, e.g., toxicology and cancer chemotherapy, on cell culture, with all its difficulties, for preliminaries. Rational intervention in serious injury may require, as intimated by Moore (1985), inventory of (1) the body’s (and cell’s) stocks of spare parts, ( 2 ) knowing the means of their transport to the injury, (3) knowing where some parts are stockpiled only to a limited extent and where precursors to make the needed parts are themselves stockpiled, (4)failing the above, knowledge of the body’s ability to obtain them exogenously-a cytological problem of almost unimaginable logistic intricacy. The costliness of preliminary research with laboratory animals and then superimposition of administrative complexities and ethical quandaries attending human trials argue for more attention to in vitro preliminary explorations. Trial of a new drug for U.S. human use requires an Investigative New Drug (IND) form to be filled, often at length. Each new drug combination, even though individual drugs had been approved, requires a new IND. Suppose, further, that the final proposed intervention will be, as with the cited instance of Hodgkin’s disease, eight agents. In our research on chemotherapy for African trypanosomiasis, ca. 500 inbred mice, at ca. $4.00 each, are required to plot adequately a two-drug interaction, e.g., for the difluoromethylornithine-suramin combination (Clarkson et al., 1984); such a test must precede human trial. Eight agents . . . ?

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Elucidation of metabolic strategems by which the highly diverse protozoa presented here live as independent organisms, yet unmistakably much like a mammalian cell except for relative indifference to external hormonal controls, is accordingly more desirable than ever. Successful use of Tetrahymena to estimate the biological value of proteins (Baker et al., 1978) indicates that minute size does not rule out important correlations with the intact mammalian body. The efficiency and versatility of food utilization of a Tetrahymena are hardly inferior to, say, the mammalian gut and the enzyme secretions of liver and pancreas. As an expanding literature on the sensing equipment of protozoa, especially ciliates, indicates, they are well equipped to detect food and much else in the environment affecting survival. SOMEDIFFICULTIES OF TISSUE-CELL CULTURE Tissue-cell culture as tool for elucidating regulatory mechanisms suffers from constraints of limited or expensive availability of many growth factors. For example, the hormonal form of vitamin D,-l,25-dihydroxy D, (Koefflier et al., 1985)-is not yet in catalogs of standard suppliers of biochemicals; a special request has to be made. Most peptide growth factors are extremely expensive; many are not generally available (Evered et al., 1985). Since the hydroxylations that lead to hormonal vitamin D from the calciferols are carried out by kidney or liver cells, therein may reside some explanation of the paucity of cells establishable from tissue outgrowths. A drug not known at first to do so may act primarily as an antimetabolite. The classical example is sulfanilamide. Its antibacterial activity in vitro was manifest only in a lean medium; yeast extract had the agent nullifying bacterial activity in high concentrations. On fractionating yeast the factor was identified as p-aminobenzoic acid. Many drugs, old and new, are potential probes for new cellular mechanisms, even though the primary object might be only that of detecting socalled side actions of unexpected either nullifying or synergistic activities. Opportunities for discerning metabolic relationships thus open-tracing the metabolic targets for drug-altered responses of organisms being grown in necessarily defined media under stress conditions. Media for most tissue cells are not yet at the physiologically minimal stage (McKeehan et al., 1977; Ham, 1984) that would avoid most maskings of antimetabolite activity by presence at luxury level of the target metabolities. A resource drain in tissue-cell culture work is the need for vigilance in detecting Mycoplasma and viral contaminations. Sterility testing is simpler with the protozoal reagents. It consists of checks on their quantitative responses to some of their absolute, sensitive vitamin requirements: for trypanosomatids, biotin and fo1ate:biopterin ratio; for Tetruhymena, biotin, lipoate, (and under tria1:folate); for P. malhamensis, B,, and biotin. The basal assay media, except for a few supplements added separately, (e.g., hemin for hemoflagellates), are formulated

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as powders, with a long shelf-life when stored frozen, much as powdered media have displaced liquid media for tissue-cell culture. These procedures stemmed from mapping specificities of the vitamin requirements. The expectation is that a contaminant will excrete one or more of the growth factors needed by the protozoan; contamination is revealed by the vitamin-response curve having a high blank. That all is not well with tissue-cell culture is evidenced by the persistence with which workers use media containing antibiotics, commonly gentamicin + penicillin. A similar test of growth requirements served to detect endosymbionts in trypanosomatids; parallels are the lessened growth requirements which identify cells transformed by oncogenic viruses.

111. Stress Proteins A. GENERAL Preoccupations in research on the postinjury phase mostly dealt with fuels and amino acids. There is now much discussion on whether the crucial metabolic insult after the primary injury centers on breaches in the defense against oxygen, conspicuously evident in reperfusion damage. The body must maintain a vital supply of ATP by glycolysis while trying to reassemble the efficient 0,-utilizing mitochondria1 cytochrome system. In so doing, especially to provide fuel and repair parts, the body cannibalizes itself, especially its lean body mass-a process obviously having limits. Some assumptions: 1. Most metabolites protecting protozoa from life-threatening stressors, from supraoptimal environmental temperatures and starvation to cytotoxic agents such as those in cancer chemotherapy, apply to humans. 2. Regulators of intercellular traffic, i.e., hormones, may have originated as intracellular regulators. Some regulators may be more discernible in protozoa than by the methods of mammalian endocrinology. If certain transport mechanisms for interiorizing metabolites apply to diverse protozoa and, moreover, if they are prone to inactivation under stress, putative spare parts for these mechanisms would have a high priority in experiments.

B. INDUCERSOF STRESSPROTEINS Every cell examined developed at least transient resistance after brief exposure to supraoptimal temperature and, to a somewhat lesser extent, to other insults not lethally prolonged, meanwhile switching off everyday protein synthesis and

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synthesizing a subset of proteins not or hardly detectable before. Long amino acid sequences in these stress proteins are conserved from prokaryotes to metaphyta and metazoa. Adaption to many stressors entails drastic reorganization of cytoarchitecture, especially the cytoskeleton, concomitant with switching on stress proteins. Because of excellent recent monographs and symposia on stress proteins (several noted below) that background is not detailed here. Some genes for stress proteins have been purified but correlations with specific metabolic mechanisms are still few. The protozoan systems selected here are those where some clues to function of stress genes might be discerned by seeing how shifts in nutritional requirements under stress coincide with appearance of particular stress proteins. Sixty-eight heat-shock proteins have been isolated from the stressed rat thymocyte alone (Maytin et al., 1985). As electrophoretic separations improve, the number of detected stress proteins should increase. Heat stress probably evokes the most stress proteins. If one includes other stressor-induced proteins, e.g., arsenite-induced proteins, 100 is about the present total for mammalian cells. Therapeutic dilemmas may arise through the profusion of leads thus uncovered. Suppose that experiments with protozoa point to 10 nutrients, some novel biochemically, which might evoke a favorable response in the stressed rat. Suppose, further that, tried singly, the response to most of the 10 might be barely beyond experimental error. For microbiological assays, error runs between 5 and 15%;rigorous standardization of procedure may decrease it appreciably. A bottleneck, much bewailed in antiprotozoal chemotherapy, is the scarcity of lead compounds, i.e., prototypes of novel therapeutic entities. The investigations outlined here aim to uncover novel or unexpected therapeutic metabolites in some instances, as sketched, by using available antimetabolites as biochemical probes to uncover lead compounds for repair besides those biochemically known. The comparative biochemical approach implies that all relevant information should be drawn on, irrespective of organism-a counsel of perfection. Because of lack of protozoological omniscience, we make only passing mention of protozoa which, for technical reasons, we judge less suitable for stress research than the protozoa dealt with here at some length. An excellent survey (223 references, over two-thirds from 1980 or later) (Craig, 1985), centers on the heat-shock response. The extensive literature on prokaryotes, especially molecular biology (mainly on Escherichiu coli), is covered by Neidhardt et al. (1984); Drosophilu is covered by Southgate et al. (1985), and land plants, by Key and Kosuge, 1985. Cytopathology has been reviewed, with emphasis on thermosensitivity of the plasma membrane, nuclear structures, and cytoskeleton (Nover and Neumann, 1984). The accumulated data on stress-protein inducers calls for a taxonomy-a task fulfilled for the time being by Whelan and Hightower (1985). They group eukaryotic stress proteins into two classes:

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1. Heat-shock inducers. Included here are heat, certain metals (Cu, Zn, Co); amino acid analogs, e.g., canavanine; and sulfhydryl-reactive proteins, e.g., diamidine. 2. Glucose-regulated proteins. Included here are glucose starvation, glucosamines, 2-deoxyglucose, tunicamyin, Ca ionophores, and prolonged anaerobiosis. They surmised that an intracellular oxidizing environment might elicit heatshock proteins; a reducing environment, glucose-regulated proteins. In their work glucose-regulated proteins were induced by 2-mercaptoethanol; heat-shock proteins, by cells exposed to high external pH and to t-butyl hydroperoxide. The heat-stress proteins (hsp’s) include (in kilodaltons) 88, 71, 22; glucoseregulated proteins, 58, 78, 99. The 72-kDa protein is the major hsp, closely related to 71. Glucose-regulated proteins require 12 hours or more for induction; hsp proteins, including reducing agents, 3-4 hours. Can et al. (1985) think that the common denominator of stress proteins in E . coli is response to undue synthesis of abberant polypeptides-a conclusion resting partly on observation of a mutant having diminished ability both to degrade aberrant proteins and mount a heat-shock response. 1. Ethanol

Ethanol induces a hypermetabolic state (Kaplowitz, 1985) with diminished ATP chronic excess consumption-induced adrenocortical overactivity (Fink, 1984). The cytoplasmic inclusions (Mallory bodies), consisting of masses of tubular 10- to 20-nm filaments characteristic of severe alcoholic injury and other derangements, resemble several heat-shock proteins (Denk et al., 1984). Pelham (1985) reported that heat and ethanol stresses were cross protective. Recognition that ethanol induces stress proteins adds interest to the description (Frank et a l . , 1976) of vitamin status in a stressed animal. Rats were given a single oral intoxicating dose (9 g/kg body weight) and examined 24 hours later for content of water and fat-soluble vitamins in liver and brain (Frank et al., 1976). The predominant storage form of folate, 5-CH3-H, folk acid glutamates, showed a net decrease in the liver; B,, increased in the nucleus of brain and liver, leading to the supposition that the nucleus may stockpile B,, and perhaps folates (which showed a complex redistribution) for repair purposes. Pantothenic acid activity markedly increased in the mitochondria and microsomes of the liver, perhaps explicable as increase in CoA-metabolizing acetate from ethanol oxidation. A role for folate in stress is perhaps more clearly indicated in 44 febrile children all with a lowered red cell folate as body temperature rose above 37°C (Osifo et a l . , 1981). It was interpreted as release of red cell folate stores as folate was mobilized. Such release may be a small-scale enactment of the widespread protein breakdown in serious injury.

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2. Arsenite Arsenite induces heat tolerance and most of the major hsp’s, especially in plants (Key et a l . , 1985a,b). 3 . Anaerobiosis in Plants Land plants, including crop plants such as maize and soybeans, produce heatshock proteins during hot afternoons. Large-scale crop adaptation extends to roots subjected to anaerobiosis during temporary flooding with formation of stress proteins (rice: Pradet et a l . , 1985; maize: Sachs e t a l . , 1985). Work on this unexpected economic aspect of stress proteins is likely to expand and provide insights into stress-protein function. Interestingly, the radiation-resistant bacterium Deinococcus is placed as the first offshoot from the branch giving rise to the eubacteria and eukaryotes and nearest to its branching point from the archaebacteria (Stackebrandt, 1985), making it a uniquely interesting prokaryote for work on stress proteins. The importance of stress proteins is manifested by their interactions with other regulatory systems. The classical inhibitor of oxidative phosphorylation-2,4dinitrophenol-applied above 1 mM, induces stress proteins in Saccharomyces cerevisiae, as does raising the temperature from 23 to 32°C or above. Both cause acidification (Weitzel et al., 1985). Whether dinitrophenol and arsenite elicit similar heat-shock or other stress proteins remains to be ascertained. Parenthetically, to our knowledge, uncouplers of oxidative phosphorylation have never been tried as tools for inhibition analysis as a way to rank oxidative substrates-a lack brought to mind by the report that hamster ovary fibroblasts are much more sensitive to heat shock in energy-starved media (Calderwood et al., 1985). Hyperosmolarity induces stress proteins in chick embryo proteins; how they relate to other hsp’s is unknown (Petronini et al., 1985). Several viruses induce stress proteins (Schlesinger, 1985)-an area awaiting development.

OF STRESS PROTEINS c . POSSIBLE FUNCTIONS

Stress-protein production correlates with sweeping redeployment of microtubules and intermediate filaments. They aggregate to perinuclear cup, as in heat-stressed rat embryo fibroblasts (Welch et al., 1985), which accords with other evidence of conservation of these components of eukaryotic architecture. Protozoologists have learned that morphoIogical transformations in the life cycle of protozoa reflect changing patterns of microtubules, conspicuous in Leishmania and Trypanosoma (Section IV). Conservation of this cytoarchitectural system is evident from cross-immunity between a high-molecular-weight actinfilament-gelation protein from Acanthamoeba with similar fractions from the slime mold Physarum, the mollusk, the fish brain, and human platelets (Pollard,

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1985). Transient ischemia in the brain of the gerbil increased synthesis of the 70kD protein (Nowak, 1985). If heat-shock proteins do protect against oxidative stresses, this might seem at odds with their extraordinary conservation. As generally accepted, 0, is accumulated in the atmosphere only after advent of oxygenic photosynthesis. Was there a preexisting threat? Demonstration of cross-protection between heat stress and radiation stress (Shimada, 1985; Shimada et al., 1985) dissolves that seeming anomaly: a primordial function of heat-stress proteins might have been to protect against the then greater risk of radiation injury, the ultraviolet (UV)absorbing ozone layer not yet formed. This idea could be put to a test of sorts by ascertaining whether the aforementioned Dienococcus pink tetracocci are exceptionally adept at forming stress proteins. There are, too, the red archaebacterial halophiles which carry out an anoxygenic retinal-dependent photosynthesis in direct tropical sunlight. Easy-to-grow strains of both groups are in the American Type Culture Collection (ATCC). The most-studied strain, Deinococcus (formerly Micrococcus) radiodurans, grows well in rather simple defined media (Shapiro et al., 1977). As seen, stress-protein work with protozoa should not suffer from scarcity of interesting prokaryote controls. A possibly specific interaction between polyamines and the cytoskeleton is the observation that aggregation of a cold solubilized microtubule fraction from brain is facilitated by polyamines at physiological concentrations (Anderson et al., 1985).

IV. Hemoflagellates (Trypanosomatids): Biopterin, Heme and Fe, Oxidative Stress, Polyamines A. GENERAL The trypanosomatids ( ‘ ‘ h e m o ~ ~ g e ~ l a ~are e s ”common ) in blood-sucking insects, e.g., mosquitoes and biting flies, and leeches. They rival Tetrahymena as research objects for elucidating growth-regulating and genetic mechanisms. Their advantages are: 1. Intrinsically stressful transitions figure in the life cycles of hemoflagellate species of enormous medical and veterinary importance in passing between insect vector and mammalian host. 2. A bizarre mitochondrion contains a uniquely DNA-rich kinetoplast whose double-stranded DNA is deployed in catenated mini- and maxicircles, with some linear strands. The mitochondrion almost totally disappears in bloodstream forms of salivarian trypanosomes, only to be regenerated in the insect vector, and there functions as a rather conventional cytochrome system with 0, the terminal electron acceptor.

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3. The pathogens are obviously related to species infesting a wide variety of insects and some other invertebrates, but are not known to have a vertebrate host in the life cycle; hence these are nonpathogenic and are so handled. The pathogens cause such scourges as African sleeping sickness, Chagas' disease (South American trypanosomiasis), and leishmaniasis. 4. Heme is an absolute requirement of the family Trypanosomatidae, which includes the nonpathogenic insect forms. 5 . All trypanosomatids examined require members of the biopterin series of pteridines. Biopterin is replaceable by a 100-fold or higher concentration of folic acid. Biopterin, or rather its coenzyme form, tetrahydrobiopterin, mediates the hydroxylations which convert phenylalanine to tyrosine and tyrosine to 3,4dihydroxyphenylalanine (dopa), precursor of dopamine, norepinephrine, and epinephrine. Therefore the relation of trypanosomatid catecholamine metabolism to that of mammals may illuminate the role of catecholamines in shock and later cell repair and cell replacement. The connection of biopterin to forms of phenylketonuria whose genetic defect is on the biopterin rather than the apoenzyme side of phenylalanine hydroxylase is attracting interest among pediatricians; recent availability of tetrahydrobiopterin has permitted maintenance of such children (see below). 6. Many insect trypanosomatids and a nonpathogenic trypanosome (Trypanosoma mega of the toad) grow luxuriantly in autoclavable defined media, all of whose components are commercially available, none expensive. 7. Leishmania lives intracellularly in the macrophage. Its insect stage is almost indistinguishable from an insect species, Leptomonas seymouri, which has no vertebrate host; the host is not hematophagous. As immunity to leishmania1 infection is predominantly cellular, and trypanosome infections are strongly immunosuppressive and, moreover, involve a unique mechanism in the T . brucei species complex, they attract many immunologists. Missing is a manipulable gene-recombination procedure. However, as outlined later, there are signs of DNA recombination in the most studied insect trypanosomatid, Crithidia fasciculata. and there is indirect evidence from others.

A landmark paper (Van der Ploeg et a l . , 1985) shows that for Leishmania major to adjust to the amastigote (nearly flagellaless condition) intracellular stage in the macrophage at mammalian skin temperatures, and for T . brucei to adjust to mammalian bloodstream and tissue temperatures, heat-shock proteins are brought into play. These morphological differentiations can be brought about in vitro by raising the temperature and are correlated in T . brucei with an at least 25-fold increase in hsp messenger RNA. But, unlike in other systems, the intracellularlike forms produced in vitro by raising the temperature from 25 to 37" C and the culture-form procyclic trypanosomes produced by an increase from 27 to 34" C continue to proliferate rather than entering a quiescent stage which, if prolonged in other cells, would be fatal. It was therefore concluded that heat-

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shock genes may control the differentiation. Independently, Hunter et al. (1984) and Lawrence and Robert-Gero ( 1985) describe heat-shock proteins for Leishmania and likewise ascribe to them a role in differentiation to the amastigote form. Granted that stress proteins are a response to life-threatening anaerobiosis and membrane damage with resultant diminishing ATP supply and to free radicals generated and loosed by a breach in the defenses against O,, then the digenetic (i.e., two-host) Leishmania and Trypanosoma live precariously. Yet, unfortunately, theirs are among the great successes of evolution: able to pass to the mammal from the austerities of the insect gut at ambient temperatures, there eking out a living by glycolyzing what little hexose escapes a competing glycolyzing microflora, employing a cytochrome-mediated oxidation of the end products of fermentation, and making do with the little 0, left. Trypanosomes, inserted into the bloodstream and tissue spaces at 37" C or so, with no competition for that lush smorgasbord, including ample glucose, of the bloodstream, exhaust the immune apparatus by slipping over themselves set after set of suits of variable-antigen glycoproteins, evading one antibody after another. Leishmania, cunning in another way, seduces the keystone of the defense, the macrophage, allows itself to be engulfed, and there multiplies. A Circean wile may then be exerted: Leishmania perhaps secretes a factor stimulating proliferation of macrophages. A hamster moribund with visceral leishmaniasis (L. donovani) mimics malignant histiocystosis, every resource having been diverted to macrophage production with otherwise normal phagocytic function, yet a macrophage may harbor as many as 200 viable leishmania bodies (Matzner et al., 1979). Mucocutaneous leishmaniasis (L. braziliensis braziliensis) in the human elicits macrophage-rich facially and pharyngeally destructive lepromalike swellings. Many strains defy standard chemotherapy and are difficult to grow-a desperate therapeutic situation in regions of the Amazon basin (Marsden, 1985). Metabolism of hemoflagellates is therefore investigated for many motives. It is unclear how Leishmania withstands the respiratory burst of the activated macrophage. The metabolic armor of Trypanosoma brucei-group bloodstream forms has a chink. Various trypansomatids had proven much more susceptible to antipolyamine toxicity than were rodents; 2-difluoromethylornithine (DFMO), which irreversibly inhibits ornithine decarboxylase, stood out. This enzyme mediates one of the two rate-limiting reactions in initial synthesis of polyamines: formation of putrescine. This assault (Bacchi et al., 1980) led to cures by DFMO of West African sleeping sickness patients (Van Nieuwenhove et al., 1985). An important chemotherapeutic target may be unique for Trypanosomatidae (and perhaps euglenoids): their glutathione reductase needs a cofactor, closely resembling glutathione itself, trypanothione, containing two moieties of the polyamine spermidine (Fairlamb et al., 1985). It was discovered through investigating glutathione reductase in Crithidia.

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A difficulty of salivarian bloodstream trypanosomes in satisfying their intense need for 0, for their unique terminal oxidase system, with resultant vulnerability to O,, is surmisable from their glutathione-centered defense in depth. They had been grown at 37" C with a feeder layer of macrophages, replaceable by mercaptoethanol in the presence of pymvate (Baltz et al., 1985). Mercaptoethanol turned out to be merely a means of keeping their essential cysteine from oxidizing to cystine (Duszenko et a/., 1986). In surmounting those perils, the pathogens (almost certainly including T. cruzi, the agent of Chagas' disease, which has an intracellular stage in the mammal) deploy, as noted above, stress proteins. But, as seen, enough survive to initiate and maintain infection. Work with pathogens obviously poses technical and administrative problems. The remarkable conservation of stress proteins renders it likely that they will be well represented in extant lower trypanosomatids, sharing some attributes which enabled the invasions of homeothermic hosts by the pathogens. A crucial role of temperature in invasion is conspicuous in L. enriettii. At room temperature it elicits only a small sore on the nose and paws of the guinea pig. Heightened ambient temperature cures (Pereira et al., 1958); it does not grow at 35" C in vitro (Fish et al., 1983). Leishmania enriettii is related to the Leishmania species causing cutaneous leishmaniasis in man. Local heat was curative for three patients with diffuse cutaneous leishmaniasis (Neva et al., 1984). Similarly, T . cruzi penetrated bovine embryo skeletal muscle cells in vitro (as it does in human infections) at 29-35" C, but penetration decreased sharply at 35-38" C (Dvorak and Poore, 1974). Trypanosoma cruzi, unlike Leishmania, after intracellular multiplication in ganglion and muscle cells, breaks out in the trypanosome (trypomastigote) form which is nonmultiplicative for T . cruzi. Crithidia lucillae var. thermophilu and C. hutneri are easily grown at 37" C in rich defined media (Roitman et al., 1977); several nutrients become critical near the upper temperature limit. Guttman (1963), using the standard strain of Crithidia fasciculata, whose upper limit in rich peptone media is ca. 34" C , showed the following increments in growth requirements with temperature: 2227" C, minimal defined medium; 27" C, stimulation by increased leucine, isoleucine, phenylalanine, and tyrosine; 28-30" C, glutamate, succinate, lactate, and additional Ca, Fe, and Cu now stimulatory; 32.4" C, additional substrate carbohydrate required; 33.6-34" C, lecithin, choline, and inositol stimulatory; better, dried egg yolk, replaceable by a combination of lecithin, choline, inositol, and cholesterol. These findings may not be clear-cut because an osmotic requirement became evident at 33" C; with this taken into account, growth at 35" C was obtained by a combination of a-glycerophosphate and isethionate, glycine, arginine, sorbitol, mannitol, and various inorganics (Ellenbogen et al., 1972). Easy-to-assemble media for the pathogens is not quite assured. Trypanosoma cruzi is dangerous to handle; no satisfactory drug is available for many strains. It therefore is mentioned that it may not be difficult to devise a defined

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medium for Leishmania enriettii as a representative intracellar form in the mammal. Some observations may align stress factors with invasion of the mammal. Prolonged survival of lower Trypanosomatidae in the peritoneal cavity of mice make this probable. Leptomonas costoris could be recovered up to 6 hours and C. luciliae and Herpetomonas muscarium to 24 hours, and, after some serial passages, C. harmosa, H . samuelpessoai, and H . megaseliae survived up to 24 hours (Daggett et a l . , 1978). There are many recent reviews on hemoflagellates and the flagellate line to which they belong, the Kinetoplastida. Some provide many references, diagrams, and illustrations of various features: nutrition of Kinetoplastida, Hutner et al. (1979); Leishmania, especially its intracellular phase, Chang (1983); leishmania1 invasion of the macrophage, Bray (1982); life cycles of trypanosomes infesting man, Vickerman (1985); life cycle of T . cruzi, De Souza (1984); oxidative response of macrophages to intracellular parasites generally, (Klebanoff et a l . , 1983) and to T . cruzi specifically, Nogueira (1983); morphological and biochemical differentiation during life cycles of Leishmania and Trypansoma, Williams ( 1985); cultivation systems for mammalian pathogenic trypanosomes, Brun and Jenni (1985). Docampo and Morano (1984) have exhaustively reviewed the role of oxygen-free radicals in normal physiology and of antihemoflagellate agents whose mode of action may depend on free-radical formation. B. HEME

To our knowledge heme status has never been examined in stress although heme has been used with acceptable risk for porphyria, a group of diseases with overproduction of the photosensitizing protoporphyrin (the Fe-free stage preceding synthesis of heme) (McColl et al., 1981; Bloomer and Pierach, 1982). In rats whose phenobarbital-induced cytochrome P-450 had been 48% destroyed by allylisopropylacetamide, heme infusion alleviated the impaired function (Farrell and Schmid, 1979). The short shelf-life of heme-containing culture media drew attention to a likely cause: oxidative decomposition of heme by microsome heme oxidase (Kikuchi et a l . , 1982) superimposed on accumulation of peroxides and oxygen-free radicals generated by heme’s catalytic activity. (A fairly satisfactory way of storing heme solutions is to dissolve hemin in a viscous alkali.) If ATP deficiency is one of the direr consequences of mitochondrial damage, repair of mitochondria should have high priority. Several mitochondrial components might be considered separately: membranes, DNA and RNA, cytochrome heme, and proteins. Lipids might give the most trouble. One could supply cholesterol or perhaps as utilizable water-

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soluble esters and the lipid precursor mevalonic acid (as lactone). Other likely permeant membrane precursors might include ethanolamine [a growth essential for human fibroblasts (Witte et al., 1985) and well taken up by Tetrahymena (Wilton, 1983)l. Ethanolamine is readily metabolized by the intact rat and heart slices; over 50% of the compound injected intraperitoneally is converted to lipid and 11% is oxidized to CO, (Taylor and Richardson, 1967). It may be assumed that a serine supplement may also help. Lower trypanosomatids are obviously uniquely suitable for such experiments. To employ heme in enzymes and as 0, carrier, with its heightened productivity for ATP, a price was paid to be taken into account if and when exogenous heme is tried in stress repair. Hemin in the presence of 0, and mercaptoethanol degrades isolated DNA (Aft and Mueller, 1983). Whether the equivalent could significantly happen in vivo is unknown. At 0.17 pM, hemin killed lymphocytes from patients with chronic lymphocytic leukemia (Malik and Djaldetti, 1980). Favorable signs: stimulated heme promotes growth of rat glioma cells (Zwiller et al., 1982) and neurite outgrowth in mouse neuroblastoma cells (Ishi and Maniatis, 1978). Transferrin, the protein which is the main Fe transporter in blood (Hanover and Dickson, 1985), is a common ingredient in tissue-cell media. Few papers have investigated whether it is replaceable by Fe, hemin, or the two together. Minute quantities of transferrin, hemoglobin, myoglobin, or hemin stimulated growth of Walker carcinoma cells, and Fe substituted for embryo juice as a stimulating factor (Neuman and Tytell, 1961). Heme in intact mammals has a transport mechanism distinct from that for inorganic Fe; heme is better absorbed than inorganic Fe (Bjorn-Rasmussen et al., 1974). Counterpart experiments with Crithidia fasciculata revealed that its ostensibly high heme requirement could be lowered at least 100-fold by supplying extra Fe plus such potent fairly stable chelating ligands as 1-(2-pyridylaz0)-2-naphthol (Shapiro e f al., 1978). The chelator 2-pyridinecarboxaldehyde-2-pyridylhydrazonecured mice infected with an acutely lethal strain of T. b. brucei (Shapiro e f al., 1982) in a medium with minimal heme. These experiments may be convergent with studies on the treatment of genetic and accidental poisoning by Fe, a promising compound to remove Fe being pyridoxal isonicotinylhydrazone (Huang and Ponka, 1983; Ponka et al., 1984). Crithidia experiments were flawed by the media being sterilized by autoclaving, hence first thoroughly de-aerated and aerated on cooling, with consequent alterations. Recent advances with broad-spectrum antibacterial antibiotics and fungicides may make it practical to dispense with autoclaving altogether except for media for stock cultures and inocula. The desirability of stabilizing these media is enhanced by the report that heme may affect glutathione metabolism: two forms of glutathione transferase in liver have peroxidase activity and high affinity for heme binding (Jagt et al., 1985).

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C. BIOFTERIN The popular Crithidia assay for biopterin is based on its response to biopterin being ca. 500-fold more sensitive than its response to folate, so that in many biological samples simple dilution eliminates interference from folates. To eliminate any folate interference, a preliminary autoclaving with acid inactivates folates by hydrolysis of the p-aminoglutamyl side chain (Baker et al., 1980). Better estimation of tetrahydrobiopterin, often the dominant form in tissues, may be obtained by preliminary treatment with a mild oxidant, which converts this coenzyme to the much stabler biopterin (Milstien, 1983)-a step similar to converting coenzyme tetrahydrofolate to the stabler folic acid. Crithidia can make biopterin, however inefficiently, from folic acid by a reaction having no counterpart in metazoa (Kidder et a l . , 1967), which explains the biopterin-folate relation in Trypanosomatidae. Tetrahymena has an absolute folic requirement, insensitive to biopterin (H. Baker, unpublished) spared by thymidine or thymine (Dewey and Kidder, 1953). In a symbiont-free strain of Paramecium aurelia, which is fairly close to Tetrahymena, the Crithidia situation oddly repeats itself: a very high ostensible folate requirement was brought down by biopterin to a physiologically realistic level (Soldo and Goday, 1974). Because of multiple lipid requirements, Paramecium is tricky to handle in defined media. It would be interesting to know the intracellular relation of folate to biopterin in Tetrahymena and chrysomonads (Section V) under stress conditions. Do either or neither conform to the only preliminarily explored mammalian pattern? Would they imitate the trypanosomatid pattern, or would their patterns be unique? This situation illustrates that developing a physiologically minimal medium (Section 11) is not simple when multiple interactions among metabolites are to be taken into account. That biopterin functions in Crithidia conventionally in mediating synthesis of catecholamines and indoleamines was shown by catecholamines (Janakidevi et a l . , 1966a) and serotonin (Janakidevi et al., 1966b), respectively, sparing the biopterin requirement. Other sparers were found but have not been followed up, perhaps because of the instability of the amines in the slightly alkaline growth medium and the need for aseptic additions of the amines to avoid destruction on steam sterilization. Melatonin stimulates growth of rodent brown adipose tissue (Heldmaier and Hoffmann , 1974). Biopterin and close intermediates in its synthesis appear intimately bound to cell proliferation. Thus activated lymphocytes excrete neopterin [6-(1' ,2' , 3 ' - ~ erythro-trihydroxypropylpterin)] (Rokos et al., 1984), an intermediate which is active for Crithidia but much less so than the related biopterins. Dihydrobiopterin and tetrahydrobiopterin stimulate proliferation of lymphocytes activated by concanavalin (Ziegler et a f . , 1983). Whether the body's capacity is impaired to synthesize or retain biopterin or keep it reduced in prolonged stress is unknown.

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Oral tetrahydrobiopterin is now a treatment for inborn defects of tetrahydrobiopterin synthesis, compensating for the defective neurotransmitter production (Niederwieser, 1984; Watts, 1985).

D. CHEMOTHERAPY AND POLYAMINES Drugs (low-molecular-weight or highly lipid-soluble ones excepted) enter by deceiving preexisting receptors and transport mechanisms, else they would not be drugs, aside from compounds directly affecting the plasma membrane. Internalization or not of leishmanicides or trypanocides spells life or death for the host and parasite. Trypanocides have contributed to cytology. Ethidium, a standard reagent for histochemical demonstration of double-stranded DNA, was synthesized and employed successfully for prophylaxis or cure of the cattle pathogen Trypanosoma congolense. Antipolyamines An immense cytological literature depicts polyamines as tissue-cell proliferation factors and antipolyamines as antiproliferation agents with applications to cancer increasing. That trypanosomatids are exceptionally favorable experimental material for polyamine research receives support from the aforementioned chemotherapeutic success in the field. Many reviews and symposia on polyamines have been published in the past decade; at least three symposium volumes are announced as near publication or at the advanced planning stage. For workers seeking comprehensive critical reviews with medical orientation, Janne et al. (1983) and Heby et al. (1984) are recommended. Those desiring more background information on hemoflagellates and related flagellates are referred to an outline by Lee and Hutner (1985). Current hemoflagellate literature presents a busy biochemical-cytological scene.

E. POSSIBLE DNA RECOMBINATION Alignment of stress proteins with (1) stress-enhanced nutritional requirements and (2) genetic analysis should assist arriving at stress-protein functions in eukaryotes. Findings bear on the likelihood of developing a procedure for effecting gene recombination. That some sort of recombination does occur in trypanosomatids is a near certainty for the lower trypanosomatid Crifhidiafasciculata; 24 markers used were for drug resistance, one for cysteine auxotrophy, and three for colony morphology. Stable recombinant phenotypes were isolated (Glassberg et al., 1985). The picture is less clear for the symbiont-bearing Crifhidia oncopelti, where mixing two strains, one resistant to cycloheximide, the other to chloramphenicol, yielded double-resistance strains (Krylov et al., 1985). Tait (1985), reasoning from the variation with respect to protein variation (enzyme

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zymodemes), concluded that some species of Leishmania and Trypanosoma probably have a nonobligatory sexual cycle, and others lacking it are “asexual.” Mating is well known in other flagellate groups.

V. Chrysomonads, Vitamin BIZ, Carnitine, Branched-Chain Amino Acids A. GENERAL Data from protozoa, especially those used to assay B vitamins (Baker and Frank, 1968), might yield hints on how branched-chain amino acids (BCAAs) and other essential amino acids might promote stress repair and, as by-product, provide clues to the identity of primordial fuels of the ur-phagotrophic cell. That biochemical micro-workhorse, Crithidia fasciculata (Section IV), needs the same assortment of amino acids as birds and young mammals. Could conditions be devised where amino acids collectively could constitute important fuels, with little or no sparking from Krebs or near-Krebs cycle intermediates? That experiment has not to our knowledge been carried out with any mammal despite much information on catabolism pathways of the individual amino acids. Presumably technical obstacles intervened, such as the problem of avoiding upset by the animal having to dispose of unusually large amounts of urea and uric acid. There might also have been problems with the cost of a ration based on crystalline amino acids. A further discouragement might be fear that the experiment would be artificial: what natural diet for mammal or chick would be so high in protein? Another fear: what if nothing new was learned-that the results were no more than expected from the summation of the individual catabolic pathways? That is, there might be little important left to learn about the pathways and their interactions. Whether this latter assumption holds for normal conditions is questionable; a B,,-dependent step is reported for leucine (Poston, 1984) and a hitherto neglected pathway for methionine catabolism (Benevenga, 1984). In stress, as mentioned, lean body mass-largely protein-is cannibalized (Section I). Might protein be the critical emergency fuel? Are the protein digests for parenteral or enteral nutrition adequately accompanied by cofactors and minerals? Is “hyperalimentation” actually hypoalimentation if one goes by actual benefit, not biochemical indices? Another caveat: preparations made from eukaryotic crudes are likely to have a disturbing concentration of histamine. Therefore one has to know whether an amino acid digest has been prepared from a fairly pure protein and hydrolyzing agent. If acid or a purified enzyme was used, important cofactors and inorganics might have been overlooked entirely. The trypanosomatid and Tetrahymena systems may serve as models to test these possibilities. Crithidia excretes excess purine as the soluble hypoxanthine

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(Kidder et al., 1977) and amino acid N as ammonia, urea, or both (Yoshida and Camargo, 1978). These end products may be a sign that nucleic acids and protein can be degraded by serving as fuels. We conclude that whether all or most of the amino acids of complete proteins, especially muscle proteins, can serve as important fuels for unstressed mammals or by the protozoa well accepted as laboratory animals is an open issue. As we emphasize, the protozoa proposed here for such studies are so diverse that a positive answer with only one might not justify trials with rodents, but a consistent positive pattern with several protozoa would be compelling. By way of sketching how patchily information from biochemistry has been applied to stress repair we have concocted a scenario in which literature, some very recent, is assembled to formulate a comprehensive inquiry as to whether amino acids are preferred fuels in stressed cells. It retraces much textbook material on individual amino acids but-the point of this exercise-synthesis may have been deficient. Three preliminary steps may be listed: (1) glucose (or hexose) had generally been thought necessary for trypanosomatids. Glucose proved replaceable for Crithidia fasciculata by glycerol + Krebs cycle intermediates several so-called nonessential amino acids, i.e., those readily entering the Krebs cycle (Tamburro and Hutner, 1971). (2) Much the same as (1) held for Tetrahymena (Cox et al., 1968); and (3) the nutritional supplements for growth at supraoptimal temperatures of the weakly photosynthetic, unmistakably phagotrophic chrysomonad Poterioochromonas (Ochromonas) malhamensis comprised an intriguing parallel to factors proposed as favoring wound healing or favoring tissue repair after other insults (Hutner et al., 1957).

+

B. TEMPERATURE FACTORSFOR Poterioochromonas malhamensis: A MODELFOR MAMMALS UNDER STRESS? Chrysomonad nutritional temperature factors were studied before stress proteins were discovered and as trials of BCAAs and other hypothetically desirable supplements in parenteral and enteral nutrition were barely begun. The spur (Hutner et al., 1957) was a puzzle: Poterioochromonas malhamensis, biochemically prominent because of its mammalianlike B , 2 requirement, came from an upland acid moorland pond whose temperature almost certainly never rose ' C. In the laboratory in crude or defined media its optimum was about above 8 28-30' C. Curiosity about its temperature responses was whetted by the common use of thyroactive materials, e.g., thyroxin or iodinated casein (Frost et at., 1949), to clarify the B requirement of laboratory (and experimental farm animals) to rapidly deplete B , 2 stores. Would heightened environmental temperature similarly heighten the chrysomonad's B , 2 requirement? It did, and other factors entered. Thus, in going from 31 to 36.3" C, growth inhibition was complete and completely restored by a Zn supplement; it was restored less

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effectively by Ca, with some restoration by B,,, especially when joined with folic acid, thiamin, BCAAs, and threonine. Growth above 36.3” C required complex supplements, e.g., autoclaved suspensions of cells grown at the baseline temperature (probably supplying lipids as well as water-soluble metabolites). Parallels with the sparse data on wound healing were noticed. Some experiments showed growth enhancement by thyroactive compounds (Baker et al., 1961). Several laboratories later investigated the relation between its B ,* requirement and methylations, e.g., Lust and Daniel (1966). Those observations today hold more interest. First, there is the relation to catabolism of BCAAs. Poston (1984) described an unexpected B ,,-dependent catabolic pathway for leucine: the P-keto pathway leading to p-leucine; it accounts for 1-2% of total flux and in the rat testis it accounts for one-third of the flux. 5‘-Methyladenosine (MTA) is produced stoichiometrically from S-adenosylmethionine in synthesis of polyamines (Kubota el al., 1985) and is conserved in resynthesis of methionine, salvaging the methylthio group (Parks et al., 1983). As shown in children deficient in enzymes for purine uptake, MTA is an important source of endogenous adenine as adenosine (Sahuta et al., 1983). 5‘Methyladenosine may be a regulator of polyamine synthesis (Section IV): it is a strong inhibitor (Iizasa and Carson, 1985) and is probably itself controlled by a phosphorylase (Kubota et al., 1985). It inhibits various other enzymes as well as growth and proliferation of some mammalian cells, especially lymphocytes (Di Padova et al., 1985). The startling report that MTA, though at much higher concentration, totally replaces B,, for P . malhamensis (Sugimoto et al., 1976) has been confirmed by H. Baker and 0. Frank (personal communication), ruling out contamination with cobalamins of the commercial MTA used, by Euglena, E . coli, and Lactobacillus leichmannii assays (each respond to cobalamins inactive for mammals as well as to B,,). Would MTA likewise replace B for chick or mammal? Poterioochromonas rnalhamensis so closely parallels the mammalian response to B,, (Baker et al., 1986b) that the problem might be posed as “are humans ochromonoid, B ,,-wise?” Vitamin B,, and folates are intimately concerned with regulation of methionine synthesis and catabolism (Chanarin et al., 1985; Shane and Stokstad, 1986) and have links with other regulatory systems. Certain DNA sites are modulated by methylation, notably cytosine. Methylation suppresses gene expression; hypomethylation favors expression and increases accessibility of DNA to oncoviruses (Feinberg, 1985). Most lines of cancer cells show DNA hypomethylation as well as inability to synthesize methionine from homocysteine plus B,, and folate (Hoffman, 1984, 1985); 23 human colon neoplasms were consistently hypomethylated (Goetz et al., 1985). Reexamination of B,, metabolism in P . malhamensis might assist in aligning its stress proteins with intersections here of regulators of one-carbon metabolism, methylation, and BCAAs, as well as interactions of folate and B,, cofactors with

,,

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still other regulatory systems. Interestingly, the closest mammalian model for the neurological as well as hematological consequences of B deficiency is the African fruit bat, Rousettus aegyptiacus. When its B,, is inactivated by wholebody exposure to N,O, the resultant picture is that seen in pernicious anemia (Chanarin et al., 1985; Van der Westhuyzen et al., 1985). Induction of an Nmethyltransferase by long-term heat stress (Leland and Hanson, 1985) encourages that hope that, whether by applying short-term or long-term heat stress, as in the aforementioned supplement-permitted growth at supraoptimal temperatures, useful information on transmethylations is to be gained from chrysomonads. The high temperature-enhanced Zn requirement of P . malhamensis merits more attention: at least two reports specifically relate Zn to stress. Mice stressed by implantation of Ehrlich ascites tumor cells developed depleted plasma Zn and shift of Zn to the liver (Kraker and Petering, 1983). Zinc is mobilized into injured tissue in surgical trauma and to the liver in burn-stressed rats, where uptake is several-fold higher than intake in wounds (Van Rij et al., 1981). Copper competes with Mo for transport, and therefore Mo can buffer Cu (Hutner, 1972). Perhaps buffering of cheltaable metals-a form, in pharmacological parlance, of controlled release-may prove as important in this area as H buffering. A strategic role for Zn in all cell and tissue repair is implicit in Kornberg’s (1980) support of an earlier suggestion by B. L. Vallee that Zn may cofactor all nucleotidyl transfers, as it is for all RNA and DNA polymerases examined. By implication, here may reside a shortcoming in supplying essential trace elements for parenteral nutrition; trace-metal buffering of media (standard practice in microbiology) has been ignored. Effective use of Fe, also competitive with Zn, requires ample but nontoxic availability of Cu. To meet this situation in microbiology, essential transition elements are not supplied nakedly but buffered by chelation, e.g., with citrate, EDTA, and the like; for Ca, ethyleneglycol bis(B-aminoethyl ether)-N,N,N’ ,N’-tetracetic acid (EGTA) is in wide use; for Cu there is the fairly specific 2,9-dimethyl-4,7-diphenyl1,lO-phenanthroline sulfonic acid, as used to sequester Cu to minimize the Cu-catalyzed autoxidation of cysteine, an absolute requirement of a mutant of mouse leukemia L1210 defective in cystine transport (Ishi and Bannai, 1985). The possibly more specific triethylenetetramine is given to drain away pathological accumulation of Cu in Wilson’s disease (Walshe, 198l), replacing the erratically toxic penicillamine.

,*

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C. A CARNITINE, B,,, BRANCHED-CHAIN SCENARIO

Carnitine transports long-chain fatty acids into the mitochondrion, where they are oxidized. Carnitine likewise functions for medium- and short-chain fatty acids (Bieber et al., 1982). Carnitine availability might govern the effectiveness as fuels of acetate produced by lipolysis and short-chain fatty acids produced in

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the metabolism of BCAAs and methionine. Perhaps it can help restore mitochondria function to patients in shock and aftercare; Corbucci et al. (1985) administer a 4.0-g bolus. Carnitine is given to patients with errors of organic-acid metabolism, For example, three patients with increased acylcarnitines and depressed free camitine were given carnitine. The object was to transfer acyl groups out of the mitochondria where they had accumulated as acyl CoA compounds, thus freeing CoA for other uses, e.g., relieving a pileup of CoA in the mitochondrion. There was some benefit (Chalmers et al., 1984). An infant with glutaric acidemia had symptoms alleviated by a combination of carnitine, riboflavin, and insulin (Mooy et al., 1984). Rationale for trial of carnitine in stress was, then, favorable. Additional indications: although in rats mobilization of fatty acids from adipose tissue occurs 24 hours after bum injury, the increase in plasma carnitine at 6 hours may be a response to the then-decreased oxidation of fatty acid rather than secondary to deficiency of plasma-free fatty acid (Van Alstyne et al., 1977). Skeletal. and cardiac muscle may be particularly vulnerable to carnitine malfunction; carnitine is not synthesized there (Cederblad er al., 1983). Shug et al. (1980) described loss of carnitine from various tissues, especially heart muscle, in ischemic rats, dogs, and sheep. Snoswell and Henderson (1980) reported dramatic increase in liver carnitine in various metabolic stress conditions, e.g., fasting; also, severely diabetic sheep had a 28-fold increase in carnitine synthesis, which presumably severely drained methionine methyl (a step in carnitine synthesis is trimethylation of lysine). They adduced that diabetic sheep had diminished creatine synthesis, thereby sparing methyl. In liver mitochondria exposed to stresses and damaging concentrations of Ca2 ,as in aging in the presence of r-butylperoxide, a cause of injury was perhaps loss of membrane-bound carnitine, impairing longchain acyl CoA metabolism (Di Lisa et al., 1985). Despite the immense investigative investment in BCAAs and the clear role of carnitine in BCAA metabolism, it is not yet known how important carnitine’s role is in stress. How well can the body take up exogenous carnitine, i.e., by the salvage pathway, and deliver it to the mitochondrion? What limits carnitine synthesis aside from product feedback somewhere along the biosynthesis chain? Does degradation of muscle in stress increase availability of lysine for carnitine synthesis? Carnitine methyls come from S-adenosylmethionine; can this compound be in short supply? If so, is it its adenine or its methyl? If methyl is limiting under stress, what is the best methyl source-methionine, choline, betaine, or synthesis of methyl de now by reduction of methylenetetrahydrofolate? Does stress impair folate uptake or synthesis? Carnitine synthesis requires ascorbic acid (Borum, 1983; Dunn et al., 1984): another potential shortage? Methionine is the most toxic amino acid. Its catabolism generates propionic acid, whose metabolism may be partly carnitine dependent. Catabolism of propionate (from valine, isoleucine, and threonine) proceeds by biotin-mediated carboxyla+

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tion to methylmalonate, which in excess is toxic owing to its propensity to be taken up to yield abnormal methyl-branched, long-chain fatty acids. Methylmalonate is isomerized to succinate by a B ,,-mediated reaction succinate (ignoring CoA acyl esters), which then is oxidized by the Krebs cycle or used for gluconeogenesis. Several steps are B, dependent. Does B,, biotin, or B,, thereby become limiting in stress? Or, for that matter, do any of the cofactors in the Krebs cycle or in various reductive steps in the above train of reactionsnicotinamide, riboflavin, and the aforementioned members of the keto acid dehydrogenase complex: lipoamide, thiamin, and pantothenate? Aspects of impaired BCAA catabolism are discussed at length in the literature on inborn defects, which describes the havoc wrought by pileup of catabolic intermediates, e.g., isovaleric and methylmalonic acids (Nyhan, 1984). There seems to be little information on damage from other acids, e.g., the series of dicarboxylic acids with a methyl sidechain seen in neonates with propionic and methylmalonic acidemias (Jacobs et al., 1985); are they incorporated into abnormal, poorly functional long-chain fatty acids much as low-molecular-weight isoacids may be? Lund and Williamson (1985) point out that injured extrahepatic tissue develops large fluxes of glycine and serine as well as the much-studied fluxes of BCAAs and that the metabolism of glycine (“something of an enigma”) is concerned, with alanine, with transport of carbon and nitrogen between the major mammalian tissues. Intriguing, because the folate-dependent glycine-splitting reaction in kidney provides NH, to correct acidosis: renal uptake of glycine increases five-fold in man during prolonged starvation (Lowry et al., 1985). Is glycine neglected as a fundamental fuel because glycine’s splitting reaction and conversion to serine (and thereby entry to the Krebs cycle) require, between them, more folate than usually available, perhaps especially in stress? This scenario could be spun out further. Thus, folic acid might be drawn upon for several repair syntheses, e.g., how best is purine available for repair? Lipoic acid (as lipoamide), as mentioned, is essential in BCAA catabolism. What governs how much lipoic acid derives from food and how much from endogenous synthesis? Is this ratio set awry in the stressed mammal? If synthesis gains importance in stress, should lipoic synthesis precursors be administered? If one is thus confronted with a regulatory network which under stress might be reduced to tom tangles, sorting out what is salvageable from what must be replaced could imply that rodent models would have to be employed on a scale matching that of industrial screening for antibiotics or the government screening for anticancer drugs and the famed World War I1 U.S. screen for antimalarials. At the risk of unconscionably piling up hypothetical experiments, another possibility is considered in the next section: that the best fuels have not yet been used for stressed protozoa, rodents, or patients. Hemoflagellate metabolism and chemotherapy (Section IV) provide a closing

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fillip to this discussion: carnitine stimulates ATP synthesis in Trypanusuma b. brucei (Gilbert and Klein, 1982); bromacetyl-L-carnitine interfers with carnitine uptake and prolongs the life of infected mice (Gilbert et al., 1983). When this antimetabolite becomes generally available it might serve for exploring ramifications of carnitine metabolism.

D. POSSIBLE ALTERNATIVE FUELSI N THE RUMINANT PATTERN:BIOTIN Ruminants are the most successful mammals, judging by diversity of species and number of harsh environments colonized. Nevertheless ruminants, oblivious of textbook dogma for proper mammals, flourish, and with their predominant fuels lower fatty acids. In nearly all circumstances 90-100% of their circulating glucose derives from gluconeogenesis (Lindsay, 1978). The microflora of the rumen intercepts nearly all soluble carbohydrate as fast as it is released on digestion. Since the rumen is anaerobic, the resultant fatty acids plus those from protein remain behind for the ruminant to dispose of. For the sheep, propionate is the main fatty acid, then lactate (Lindsay, 1978). Other low-molecular-weight fatty acids are important, including those originating from catabolism of methionine and BCAAs (valine, isobutyric; leucine, isovaleric; isoleucine, 2-methylbutyric). Their utilization was demonstrated in the rat: a diet whose energy except for 5% corn oil came from acetate, propionate, or butyrate served them as well, as did the glucose-based control diet; perhaps significantly the diet was “heavily fortified with B,,” (McAtee et al., 1968). A mixture of isobutyrate, isovalerate, n-valerate, and 2-methylbutyrate administered to lactating cows improved cow milk production and maintained body weight (Felix et al., 1980), and was essentially confirmed for dairy cows on full lactation fed the acids as ammonium salts; also agents, which shifted the rumen fermentation toward propionic and succinic acids, e.g., the antibiotic monesin, markedly improved feed utilization (for review see Van Niekerk, 1985). Is ruminant nutrition an atavistic survival of an ancient pattern of the chordate cell or a later adaptation to the ruminant’s life as ambulatory fermentation tank? Be that as it may, it may provide clues as to why clinical use of BCAAs, after high hopes and expenditures for use mainly for parenteral nutrition after grave injury and for a diversity of pathological chronic metabolic disorders, e.g., liver cirrhosis and hepatic encephalopathy, is thought to be of no clear benefit (Walser, 1984a,b). The rationale for attributing special virtue to BCCAs is that they have a regulatory role in protein synthesis and are unique in serving directly as fuels for skeletal muscle (Harper and Zapalowska, 198I ) , avoiding their destruction in the liver (Krebs, 1981; Fagan and Goldberg, 1985). There may be a clue here as to why benefits of BCAAs or their derived keto acids remain equivocal. An untested assumption may have contributed to his dismay: that the body in stress retains an ample working supply of cofactors for efficient catabolism of the

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BCAAs. Since a description of BCAA cofactors has been outlined above and is given in recent biochemical texts, only a few aspects will be further discussed, especially biotin and B ,. If a ruminant-style substrate cocktail deserves trial, how then to render those low-molecular-weight odoriferous acids acceptable to rodents and humans? One possibility is neutralization with arginine base: Barbul et al. (1985) found that high parenteral arginine given to rats helped heal skin incisions an improved blastogenesis in peripheral blood lymphocytes. Deficiency in one or all of the three biotin-dependent mitochondria1 carboxylases usually leads to excretion of low-molecular-weight fatty acids. Where correctible by administration of biotin, oral dosage may go as high as 10 mg/kg-a “pharmacological” dosage (Baker, 1985; Packman et al., 1985). There is no information on biotin in stress, although breakdown of muscle and increased metabolism of BCAAs might deplete biotin stores. Curiously, support for investigating biotin in stress is the sudden-death syndrome in chicks, marked by fatty liver and kidney in birds on a low-fat, low-protein, low-biotin diet. Birds stressed in this manner have a sudden onset of hypoglycemia and soon die, presumably from irreversible damage to the central nervous system (Whitehead, 1985). It is unclear exactly to which forms of bound as well as free biotin the bioassay reagent Ochromonas danica (Baker, 1985) is responding. Nevertheless, correlation of the assay with clinical status has held up. This situation is reminiscent of early use of Lactobacillus casei to measure folate activity in serum. The assay was clinically valid. Lactobacillus casei, besides responding to folate, was also responding to methylfolate, the hitherto unknown predominant form of folate in blood. Biotin-deficient HeLa cells do not incorporate thymidine into DNA, hence biotin may have functions besides carboxylation. A biotin carrier-protein occurs in serum (Dakshinamurti et al., 1985); it might be, rather than biocytin, the indicator form of bound biotin. The safety of biotin in mega-doses for inborn deficiencies of the fetus, administered orally via the mother and then orally to the infant indefinitely with complete alleviation of symptoms (Baker, 1985), implies that examining biotin status in stress is safe. Vitamin B,, figures in BCAA catabolism, especially that of valine, at the methylmalonate isomerase step (Section V). Ochromonas danica does not have a B requirement, nor is it known whether B,, is taken up and enters its metabolic stream. A specific anti-B,, is now available: the awkward N,O, as mentioned. A Shive inhibition analysis applied to both chrysomonads might yield surprises about functions of B,, and folate. Various rat tissues, as mentioned, metabolize leucine by an additional pathway involving an initial B ,-dependent conversion of leucine to p-leucine, the “P-keto” pathway. In the testis 4 the flux is by this route. It is of obvious interest to ascertain whether this pathway is affected by stress and, if its activity is

,

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enhanced, whether it correspondingly accounts for some of the enhanced demand for B,, in temperature stress in a chrysomonad (Section V,B). E. OTHERPOSSIBLEFUELS Overlooked or neglected effective fuels or spare-part metabolites may exist as judged from literature on intact animals or cells. Some are listed below. 1. Nucleosides as Fuel, Purine, and Pentose Sources Many, perhaps most, mammalian cells have a high-capacity nucleoside transport system. Potentialities of nucleosides as glycolytic substrates and emergency fuels have only recently attracted much general attention. Emphasis has been on whether their uptake detracts from activity of drugs designed as antipurines or antipyrimidines. Dipyridamole is a popular inhibitor of nucleoside transport (Parks et al., 1985). The efficiency of nucleoside transport is implied by how gout attacks are soon triggered after ingestion of nucleic acid-rich foods, e.g., pancreas and liver. Inosine may be the factor in liver perfusates maintaining ATP in dwarf pig blood cells which are impermeable to glucose and, if the cell membrane is broken, they can also use adenosine, ribose, and deoxyribose besides dihydroxyacetone and glyceraldehyde (Jarvis et al., 1980), with restoration of glycogen stores (Aussedat et al., 1985). Ribose is glycolyzed (Young et al., 1985). Ehrlich ascites cells die in ca. 12 hours when starved; glucose permits 48-hour survival. Glutamine inosine + uridine matched glucose (Zaporowska et al., 1985). Uridine also replaced glucose as source for 90% of the ATP (Linker et al., 1985). Various vertebrate cells grew indefinitely on a sugar-free medium if given uridine or cytidine (Wice and Kennel, 1983). Presumably a ribose kinase sweeps ribose into metabolism. Ribose in the rat was protective in myocardial ischemia and during reperfusion (Chatham et al., 1984). The ribosome as emergency source of ribose may be widespread: ribosomes break down during starvation in Tetrahymena and in eukaryotes generally (Klemperer and Pilley, 1985). That the erythrocyte may at least partly depend on nucleoside ribose, perhaps as a source of ATP by glycolysis, is suggested by the abundant literature on how prolongation of its functional effectiveness correlates with maintenance of ATP and 2,3-diphosphoglycerate by nucleosides in combination with adenine, especially by inosine or, somewhat less effectively, adenosine (Nishiguchi, 1981; Sasakawa et al., 1981). Ribose and nucleosides may protect the myocardium (Zimmer and Ibel, 1984). The myocardium is slow to replenish its adenine nucleotide pool after brief regional ischemia; ribose markedly enhanced adenine nucleotide synthesis and the ATP pool within 12 hours; without intervention normalization of the ATP pool took 72 hours. Xylitol in parenteral nutrition may partly resemble ribose (Georgieff et al., 1984, 1985). Georgieff et al. contend that excess carbohydrate in parenteral

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nutrition may injure the liver; glucose increases catecholamines and cortisol. Xylitol utilization does not require insulin and, entering the pentose shunt directly, might provide ribose and deoxyribose for synthesis of nucleic acids. On evolutionary and ecological grounds nucleosides should be prime substrates for phagotrophs. Many bacteria are rich in nucleic acids. Yeast cells, the commercial source of RNA, may have as much 20% RNA by weight. Such prey is packaged with a full complement of the cofactors, inorganic and organic, necessary for nucleic acid metabolism. But, in the laboratory, surveys of carbon sources or fuels, oxidative or fermentative, for an organism have, at least for phagotrophs, including cells of the monocyte-macrophage line, usually been carried out in defined or otherwise simplified media, perhaps resulting in misleading negative results. When the basal medium contains natural crudes, e.g., peptones and yeast extracts, their content of nucleic acid and degradation products may mask the activity of added nucleosides and nucleotides. 2 . Glutamine Instability of glutamine, a common stimulant or requirement for many, perhaps most, cell cultures, is vexatious. Glutamine content in human muscle exceeds that of all the other amino acids combined (in rat muscle glycine is the main intracellular amino acid) (Furst, 1985). In rats depletion of intracellular muscle glutamine, the typical quantitative response to trauma, resists dietary supplementation (Furst, 1985). Depletion is also produced by starvation (Hannaford et al., 1982) and inactivity (Gil et al., 1985). Glutamine (oxidized to CO,) and glycolysis yielding lactate are the primary energy sources of cultured mammalian cells (Zielke et al., 1984), e.g., glutamine is the major energy source for HeLa cells (Reitzer et al., 1979). Hamster cells exposed to 40" C survived for 2 hours or more in Eagle's minimal essential medium + 10% undialyzed fetal-calf serum, but not if glucose and glutamine were withdrawn during the treatment; the cells were then nearly all killed in as little as 20 minutes (Gomes et al., 1985). Glutamine readily permeates cells. Our interest was attracted to a patent claiming that glutamine for cell culture was replaceable by the autoclavable N-acetyl-L-glutamine ( S . Yamane et al., 1972). In a review on nutritional requirements of cultured cells (Yamane, 1978) the acetyl derivative went unmentioned. In any event tests of acetyl glutamine and of glutamine esters and other low-molecular-weight, moderately lipophilic derivatives are needed. Proliferation of fibroblasts, markedly decreased by glutamine depletion, was restored by adenine or adenosine, hypoxanthine or inosine (Engstrom and Zetterberg, 1984).

3. Glycerol Perhaps preoccupation with rapid utilization of glucose had led to trial of glycerol at unduly high concentrations; untoward osmotic effects resulted. As noted, glycerol permitted growth of two protozoa in hexose-free media. Follow-

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up of these experiments appears desirable in view of the rat's excellent tolerance of monobutyrin (glyceryl monobutyrate) (Birkhahn and Border. 1978). 4. Ethanolamine As noted, ethanolamine is easily metabolized by the intact rat. Given intraperitoneally, over 50% was incorporated into the lipid fraction and 1 1% oxidized to CO, (Taylor and Richardson, 1967). Rats fed ethanolamine 10 mg/day for 30 days had increased liver and muscle protein; acetylethanolamine was even better (Vartanyan and Kamalyan, 1968).

F. CHRYSOMONAD LITERATURE Chrysomonads and related flagellates make up a vast diversity of organisms (Lee et al., 1985) from many habitats. Only 0. danica besides P. malhamensis has been much studied by biochemists; it uniquely combines vigorous photosynthesis and voracious phagotrophy. Ochromonas danica is a reagent for thiamin as well as for biotin (Baker and Frank, 1968). It does not require exogenous B , 2 . Its behavior under stress is unknown. Chrysomonad physiology has been reviewed (Aaronson, 1980; Ishida and Kimura, 1986).

VI. Tetrahymena The bizarre DNA organization and unique reciprocal mating behavior in some Tetrahymena species and other ciliates are well known (e.g., Jeon, 1986). Nanney (1980) provided an incisive account of the biology of ciliates. Introductory material in the amply illustrated account of ciliate systematics in Small and Lynn's (1985) mind-boggling depiction of fantastic ciliate diversity provides some updating. Tetrahymena should be exceptionally good for learning how stress proteins act. Cells of T. thermophila moved to 40 from 37" C show the typical heat-shock messenger RNAs and brief survival at 43" C (Hallberg et al., 1985). The heatshock proteins appear to protect the ribosome against irreparable degradation (Hallberg et al., 1984). With one of the nonmating species group, T. pyriformis (some T. thermophila isolates were "T. pyriformis" in old literature), the effective shift was from 28 to 34" C (Galego and Rodriques-Pousada, 1985). Deciliating agents, e.g., dibucaine (Satir et al., 1976), also induce heat-shock proteins (Guttman et al., 1980). Many Tetrahymena species are easily grown in defined media, exemplifying a surprising genotypic diversity beneath phenotypic similarity. Most species except T. pyriformis (by definition nonmating, lacking a micronucleus) are available as mating types; they vary in upper temperature limit from 32.7" C (T. tropicalis) to 40.7" C for T. thermophila (Nyberg, 1981).

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The drastic reprogramming of biochemical priorities and architecture manifested by switching on stress proteins might parallel drastic alterations in morphology and behavior-an expectation is heightened by several striking transformations. Starvation-induced dispersal form. Starved T. thermophila transforms from a food vacuole-containing, plumpish form (its usual laboratory-pampered aspect) to an elongate, fast-swimming dispersal or hunting form, the ordinarily wide near-apical buccal cavity moves to the underside and becomes a slit; cilia form grown basal granules not previously so provided, and a long caudal cilium appears-all within 5 hours (Nelsen and Debault, 1978). Microstome to macrostome in T. vorax. Offered live ciliates as food, T. vorax changes from a small saprozoic form with a modest buccal cavity to one with a monstrous cytopharynx. After some hours without prey the cytopharynx is pinched off as a giant food vacuole and it reverts to the meek microstome form. The macrostome form is inducible by heat shock plus phylloquinone or coenzyme Q3 (Ryals and Smith-Somerville, 1985). This axenic strain is in the ATCC . Cell-diminution in starvation. In starving Tetrahymena autophagy is continuous. In 48 hours the cell may shrink to one-fourth the original volume. Such cells had first been grown in a rich medium (Nilsson, 1984), raising the question of how this reversible shrinkage depends on nutrients in the growth medium. Resorption of buccal organelles in conjugation. As demonstrated above, druginduced deciliation induces stress proteins. Are the same stress proteins induced during conjugation where, on apposition of their oral regions, the buccal membranelles, consisting of fused cilia, are resolved while intracellularly mobile gametic nuclei are exchanged (Elliot, 1973)? Heat-shock proteins (HSPs) are expressed at one time or another during normal development of Drosophila; the low-molecular-weight HSPs can be induced by the molting hormone ecdysterone (Tanguay, 1985). Is indulgence in DNA recombination a stressor in eukaryotes? If so, stress proteins might be evoked in Tetrahymena, with its much-studied disintegration of a ca. 45-ploid giant macronucleus, later regenerated from a zygotic (diploid) micronucleus. Another stress is preliminarily superimposed on the postulated conjugation stress: a period of starvation is necessary to induce conjugation (Elliott, 1973). Dividing tetrahymenas have no food vacuoles (Nilsson, 1979). Is this a starvation stress or does pinocytotic nutrition suffice, unstressfully, under these circumstances'? Which nutrients efficiently favor resumption of normal life after the aforementioned established and putative stressful episodes? Tetrahymena species need a postconjugation recovery or maturation period before sexual maturity, i.e., before being able to conjugate again. For most species it is about 50-60 mitoses (Elliott, 1973; Goodenough, 1980). No nutritional intervention has succeeded in lowering that number of divisions. Whether a critical mating promoter accumulates or an inhibitor is slowly diluted down by divisions is unknown.

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Severe injury in mammals is attended with membrane damage with consequent impairment of phagocytic internalization of nutrients or by ostensibly lessdemanding micropinocytosis. In injured leukocytes damage is manifest as immunosuppression centered on macrophage function (Lundy and Ford, 1983), the lynch-pin of cellular immunity. The outpouring of phagocytes at the site of injury, above all in the acute inflammatory response (Ward, 1985), is being studied intensely now that cell-mediated immunity is in the forefront as a determinant of resistance to eukaryotic parasites (see Section IV on hemoflagellates) and cancer cells. How dependent is Tetrahymena on phagocytosis, a process involving food receptors and recycling of food vacuole (phagolysosome) membranes and of some receptors (Pastan and Willingham, 1985)? Since phagocytosis and pinocytosis in Tetrahymena have been reviewed (Nilsson, 1979), only a few points are mentioned. It had been established that Tetrahymena does not form food vacuoles in rigorously particle-free ordinary media; multiplication was now slow. Addition of high concentrations of nucleosides and glucose brought up the rate of multiplication to match that in particle-containing media. Subsequent work with mutant NPl of T . thermophila, which formed vacuoles at 2830" C but not at 37" C, showed that a supplement of Fe, Cu, and folinic acid allowed good growth. Presumably these enter by pinocytotic surface uptake (Orias and Rasmussen, 1977). Before concluding that these nutrients are linked, it should be recalled that the mutant had been induced by chemical mutagenesis. Fortunately, the readily available dichloroisoproterenol, a P-adrenergic blocker, reversibly inhibits food vacuole formation (Ricketts, 1983). Perhaps Tetrahymena's capacity for pinocytic uptake of nutrients is underestimated. Thus uptake of ferritin and the lipid-soluble chelate complex trisacetonato-Fe(II1) was independent of food vacuole formation (Rasmussen et al., 1985). In charting which essential nutrients are significantly taken up by phagocytosis, if a similar pattern emerges in the other test protozoa, therapeutic possibilities would come to mind. Nanney (1982) placed the origin of eukaryotes at 1.5-2 billion years ago and thinks that ciliates were in the first wave to exploit eukaryotic organization. Did absorption of nutrients through the body surface precede phagocytosis; hence, was this the earliest mode of nutrition of the eukaryotes? Hormonal resemblances in Tetrahymena to vertebrate hormones are implied by occurrences there as revealed by immunochemical probes of amino-acid sequences characteristic of corticotrophin and P-endorphin-like material (LeRoith er al., 1985), somatostatin-like material (shown also by biological activity) (Berelowitz et al. , 1982), and, curiously, relaxin, the hormone mediating softening of the ligaments of the pubic symphysis in the pregnant sow and rabbit and relaxation of the cervix (Schwabe et al., 1983). Catecholamines occur in Tetrahymena. Reserpine inhibited growth and reduced the epinephrine and norepinephrine content (Blum e f al., 1966)-an inhi-

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bition potentiated by triiodothyronine, from which, along with other evidence, it was postulated that Tetruhymenu had an adrenergic system (Blum, 1967). Gundersen and Thompson ( 1 985) cautioned that much dopamine accumulated in T . pyriformis 01 and was secreted into the growth medium but originated from an extracellular nonenzymatic conversion of tyrosine to dopa. On autoclaving, catalyzed by Fe-EDTA in the medium, dopa was converted to dopamine; even 500 FM had no effect on cell motility, phagotrophy, or transformation to the hypermotile dispersal form. The organism does have, however, an L-aromatic aminoacid oxidase capable of producing dopamine from dopa; dopamine receptors on the cell surface were not found. A different criterion yielded positive results: in T . thermophilu addition of glucose or millimolar dopa, dopamine, norepinephrine, or epinephrine repressed galactokinase (Ness and Morse, 1985). There may be a zone where the temperature may be in the range for induction of stress proteins and concomitant transient thermotolerance, yet where the otherwise inevitable lethality, if exposure is prolonged, may be obviated by a nutritional supplement-a familiar situation with temperature mutants of bacteria and fungi but seldom investigated for phagotrophs, protozoan or mammalian. A striking instance was described by Rosenbaum et ul. (1966), who noted grotesque cell shapes in T . thermophilu grown at 40" C (optimal temperature ca. 35" C) in a defined medium. Formation of these abnormalities was eliminated by crude soybean phospholipid. The biochemical lesion was in fatty-acid desaturation (Erwin, 1970). This system might be superb for elucidating participation of stress proteins in synthesis of membrane lipids. Composition of Tetrahymenu lipids as affected by temperature has been much studied (Nozawa and Thompson, 1979). Tetruhymena's requirements for amino acids so closely resembles those of humans that Tetrahymena nicely measures the biological value of proteins; one hardly has to do more than solubilize the sample with the cathepsin-like pineapple enzyme bromelain to make the proteins adequately accessible (Baker et u l . , 1978)-a feature bearing on the question of whether proteins can serve as a significant or even predominant fuel for phagotrophs under normal and stressrecovery conditions. Csaba (1985) reviewed his extensive work on responses of Tetruhymena to hormones. The main positive index was stimulation of phagotrophy. The issue addressed is the evolutionary import of the responsitivity to environmental change of the receptors of a unicellular organism as compared with the circumscribed activities imposed by the multicellularity of the intracellular receptors of a metazoan. Since the ciliates were grown in peptone media, interpretations of those observations are not altogether clear. Whatever the outcome of comparisons of the behavior of Tetruhymena in defined media with those in crude natural media, Csaba and co-workers will have stimulated development of the comparative cytoendocrinology of phagotrophy, from protozoan to macrophage.

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VII. Hypoxia In temporary anaerobiosis, many invertebrates exploit glycolytic mechanisms yielding more ATP than usual glycolysis. In mammalian cells in culture, glycolysis of hexoses is traditionally regarded as stereotyped, yielding lactate. In mollusks and some other invertebrates extra ATP is generated by condensation of pyruvate with one of several amino acids, forming compounds such as octopine, where arginine is the amino acid (Fields, 1983; Hammen and Ellington, 1984; Hochacha and Somero, 1984) with oxidation of NADH to NAD and consumption of one H+ . An unexpected example was detected in bloodstream Trypanosoma b. brucei. It appeared to be wholly dependent on 0, to sustain glycolysis by regenerating NAD from NADH. Hopes of chemotherapy were inspired when salicylhydroxamic acid and similar Fe(II1)-sequesteringligands were found to be powerful inhibitors of this system. But the flagellates remained anaerobically motile and viable, apparently by reversal of the a-glycerophosphate kinase reaction, i.e., a-glycerophosphate + glycerol ATP (Hammond and Bowman, 1980). If hypoxia is the prime initial damaging agent in shock and the initial damage sets in train further impairments in defenses against oxygen, a dilemma is created: aerobiosis has to be restored without unacceptable formation of oxygen free radicals (reviewed by Clark et al., 1985; Jones, 1985). One solution may be to find a temporary substitute for 0,itself, i.e., an alternative electron acceptorpermeant, nontoxic, readily metabolizable or inert, and safely disposable. One candidate is dihydroxyacetone, already employed to maintain 2,3-diphosphoglycerate and ATP in the erythrocyte, with extension of useful life (Dawson et al., 1981) or maintenance of ATP as index of functionality (Moore et al., 1981). High concentrations of electron acceptor may not be necessary if important cells behave like cultivated heart cells, apparently undamaged with only 1% 0, in the gas phase (Vemuri et al., 1985). Another defense might be to prevent formation of oxygen free radicals or, that failing, to find free-radical scavengers, especially antioxidants, not themselves apt to produce free radicals on oxidation. Blood is rich in uric acid. We have not encountered reports of urate oxidation yielding free radicals. Ames et al. (1981) reported that it protected erythrocyte ghosts against peroxidation, only to be challenged by Koster et al. (1984), using as index of activity suppression of malondialdehyde formation. The contradiction might be resolved if uric acid is active only in the aqueous phase (Niki et al., 1985). Perhaps uric acid is best effective conjoined with a Iipid-soluble antioxidant. The test oxidant might well imitate free radicals formed by oxygen or include compounds themselves giving rise to toxic free radicals which deplete glutathione. Hydroperoxides, especially t-butylhydroperoxide and cumene hydroperoxide, are standard peroxides. As glutathione-depleter diamide (diazendicarboxylic acid bis-N,N-diethylamide)

+

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(Sies, 1984) is much employed, so also is diethylmaleate (Freeman et al., 1985). Naphthoquinones are exceedingly toxic and highly efficient in generating free radicals (Doherty et al., 1984). Menadione-a vitamin K-shares this toxicity; tested on hepatocytes, it induces oxidation of glutathione, membrane lesions, and perturbation of Ca homeostasis, which may be the proximate cause of cell death (Orrenius et al., 1984; Orrenius, 1985; Smith et al., 1985). That abnormal entry of Ca2+ can be very damaging is lent credence by the protection against hypoxic brain damage afforded by agents blocking Ca entry (Meldrum et al., 1983). If, as is likely, production of superoxide by macrophages is the first line of defense, especially in the lung (Tritsch and Niswanda, 1985), then interest attends compounds which imitate superoxide dismutase. They might be twoedged swords: possibly interfering with macrophage function, yet blocking formation of the more toxic free radicals originating from superoxide. One suck compound, the Cu(1I) complex with 3,5-diisopropylsalicylate, attenuated the severity of streptozotocin diabetes in the rat (Gandy et al., 1983). The diabetogenic alloxan resembles a monocyclic counterpart of a toxic naphthoquinone. Does the extreme toxicity of this series to protozoa signify a vulnerable site analogous, if not homologous, to p-cells of the pancreas? Outstanding in pathogenesis is the action of xanthine oxidase on hypoxanthine liberated in stress from nucleotide and coenzyme breakdowns during reperfusion of tissues and organs. An abundance of free radicals may be generated, especially damaging in reperfusion after intestinal ischemia (Schoenberg et al., 1984). Allopurinol, used in cancer chemotherapy to minimize the surge of uric acid from mass cell death with danger of uric acid overload of the kidneys, is a potent inhibitor of xanthine oxidase. As pretreatment to skeletal muscle subjected to 4- to 6-hour ischemia, it attenuated to increase in vascular permeability (Korthuis et al., 1985). Polyamine catabolism yields products highly antiproliferative for mammalian cells, the most toxic among them probably amino alcohols (Seiler et al., 1981); H202 is also formed (Gaugas et al., 1981). The diamine-oxidase inhibitor aminoguanidine, low toxicity, may achieve wide use as antipolyamine cancer chemotherapy develops (Janne et al., 1983; Seiler et al., 1985). Polyamine intoxication in rodents given spermidine and spermine intraperitoneally may be a variable, nonspecific component of trauma (Campbell and McGrath, 1985). Tierney et al. (1985) emphasize the toxicity resulting from the sensitivity of polyamines to oxygen, hence their likely role in various clinical disorders. Serum is notoriously high in diamine oxidase, which thereby contributes nonspecific toxicity in trials of 2-difluoromethylornithine in cancer chemotherapy (Heston et al., 1981). Polyamines abound in all proliferating tissues. As set forth in many reviews, essentiality of polyamines had surfaced from their constituting an absolute growth factor for a Neisseria and, later, of some mutant mammalian cells. A tantalizing report has it that infusion of putrescine or ethylamine into the ileal

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lumen of rats stimulated growth of the mucosa around the tip of the catheter with concomitant increase in polyamine biosynthetic enzymes (Seidel et al., 1985). An association of polyamines with stress is induction in liver by glucocorticoids of ornithine decarboxylase, the more prominent of the two rate-limiting enzymes of polyamine synthesis (Corti et al., 1985). Supporting the classification by Whelan and Hightower ( 1985) that an oxidizing intracellular environment elicits heat-shock proteins (Section 111) is the induction by H,O, of heat-shock proteins in Drosophila (Ashburner and Bonner, 1979). In a report on Salmonella, oxidative stress overlapped heat stress (Christman et al., 1985).

VIII. Concluding Reflections A. Crypthecodinium; Dictyostelium Inspection of the panorama of protozoa (Lee et al., 1985) will suffice to show how small is the sampling of protozoa pressed into biomedical research services. The protozoal panel might later include the colorless marine dinoflagellate Crypthecodinium cohnii: hardy, fast-growing, and marine. Some isolates grow well in simple defined media; some have marked phagotrophic proclivities. Mating behavior of many strains has been worked out (Beam and Himes, 1984); many are in the ATCC. Fresh perspectives on metabolism under stress thus are likely from their study, aided by two multiauthor volumes (Spector, 1985; Taylor, 1986). The extraordinarily dense growth of one isolate in simple media has been illustrated (Keller et al., 1968). No metabolic pathway yet elucidated in C . cohnii seems uniquely relevant to the stress problem. Dinoflagellates are nominated by some workers, on cytological grounds, as outstandingly archaic. Among the protozoa described here as investigative tools, only the ciliates and the cellular slime molds, e.g., Dictyostelium, offer no clear clues to any algal affinity. Infusion into human volunteers of cortisone, glucagon, and epinephrine elicited some characteristics of the shock phase: increased serum glucose and free fatty acids, glucose uptake by muscle, nitrogen, excretion, and 0, consumption, basal metabolic heat production, and endogenous glucose production not completely suppressed by insulin and hormones as in controls. It was concluded that the response resembled that seen in critically ill patients (Bessey et al., 1985). In this light, and given the occurrence of epinephrine, norepinephrine, and serotonin in Crithidia (Section IV) and dopamine in Tetrahymena (Gunderson and Thompson, 1985), besides mammalian hormone sequences detected in Tetrahymena (Section VI), relevance of protozoological studies to at least the initial phase of stress is plausible on endocrinological grounds. Lack of steroid

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hormones in protozoa obviously would detract from their value as models for the mammalian cell. As in this instance. negative data are not conclusive. The joke E . coli = E. lephartt has lost some appositeness as the gulf between prokaryotes and eukaryotes is inferred to go back deep in evolution. It is logical, seeing the enormous diversity of metabolic pathways among prokaryotes which were not retained by eukaryotes, to think of the model protozoa as an assemblage of microorganisms enriched in mammal-relevant mechanisms. That is not to overlook that protozoa invented regulatory mechanisms absent in chordates; work with protozoa would permit a higher metabolic signal-to-noise ratio than work with prokaryotes if understanding mechanisms peculiar to the metazoan cell is the goal. Protozoa in pure culture in the ATCC provide abundant subjects for comparative studies within the groups singled out here (Daggett and Nerad, 1985). We wish neither to encroach further on Corliss’s chapter in this volume nor expound more natural history of our protozoan elect than a biochemist or clinician may wish to know. Still, some orientation may not be amiss. We have given short shrift to several popular protozoa such as the cellular slime mold Dictyostelium on the practical ground that, by present technology, it would be difficult to grow rapidly and tridimensionally on an industrial scale, even though its complex, manipulable life cycle has been shown to be under hormonal control at several morphogenetic decision points and much is known about the molecular biology of its stress proteins (Rosen et al., 1985). The same applies to the slime mold Physarum. With few exceptions, for identification of vitamins and other growth factors, large-scale cultivation guided by a reliable bioassay becomes necessary. If the metabolite sought abounds in that estimable eukaryote, yeast, tonnages are not difficultly procurable. Biochemical history abounds in instances where success depended on large-scale fractionation of a biological crude guided by a microbiological assay. Had the focus here been on parallels between hormones affecting the life cycles of Dictyostelium and mammals, Dictyostelium would have been center stage. Dictyostelium, as do many other acrasians (cellular slime molds with a fascinating diversity of life cycles, most of them biochemically unexplored), provide valuable insights. It comes down to a question of tempo and technical hindrances, e.g., Dictyostelium’s need of a solid substratum for clear expression of all stages of its life cycle, some of them quasi-metazoan. For those desiring better acquaintance with Dictyostelium there are good monographs and a profuse flow of research reports. The cellular slime molds, like the ciliates, are thought to have diverged very early from the main eukaryotic trunk as judged from the nucleotide sequences of genes for small-subunit rRNA (McCarroll et al., 1983). Despite their huge literature, little about polyamines has been said here. A clichC in polyamine papers is that the essential function of polyamines is unknown. The sperrnidine moieties in trypanothione, despite their probable impor-

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tance as target for chemotherapy, may be no more than an idiosyncrasy of the hemoflagellate-kinetoplastid (and euglenoid?) line, obscuring the essential function of polyamines. Many polyamines, some simple, some highly substituted, are known. Which-if any-will emerge as signpost(s) to polyamine quintessentiality remains conjectural. Trypanothione may at least serve phylogeneticists as marker to explore the origin of the Kinetoplastida-euglenoid line.

B. SOMELONG-RANGEPROBLEMS The aforementioned advantage of physiologically minimal, defined media in using antimetabolites to probe the biochemical boundaries of their targets makes us try to formulate media by the guidelines proposed by McKeehan et al. (1977 and Ham, 1984): an objective concentration of a nutrient is the midpoint of the plateau obtained on plotting growth in linear units (ordinate) versus log concentration of nutrient. These authors confess that it is a “grueling” procedure. We are heartened (1) by recalling that Shive (1950), as noted, employing a minimal defined medium, deciphered virtually the entire hierarchy of folate metabolites by systematically testing sparers of p-aminobenzoic acid in annulling inhibition of growth of E . coli by sulfanilamide, culminating in his entirely independent isolation of cyanocobalamin; (2) Spiegel (1984) stated, “. . . any chemical or biological agent that is essential for cell function and survival is a potential target for a cytotoxic drug,” in pointing out that “most antineoplastic agents act by interfering with molecular processes to the cancer cell which are essential for the cell replication process. These tactics presumably are applicable to stress research. One might identify compounds that alleviate the growth inhibition induced by compounds that induce stress proteins, e.g., arsenite and 2,4-dinitrophenol; these uncouplers might afford a test for critical fuels by their permitting growth under stressful circumstances, as noted for a chrysomonad grown at supraoptimal temperatures (Section V,B) and a hemoflagellate (Section IV). The time scale for growth rules out explicit comparison with the minutes or hour or two for inducing stress proteins, except, as seen, for Leishmania and Trypanosoma adjusting to life in the mammalian host. Even so, one might look for compounds acting during a short interval such as that for inducing heat-shock proteins. Another issue is evaluation of thermotolerance as a factor in use of local hyperthermia in cancer therapy (Gerwick, 1985; Gibbs and Stewart, 1985). It exceeds the scope of this article to discuss advantages of enteral nutrition in the convalescent stage except to note that a rule applies there as well to media for protists: do not exceed the organism’s osmotic tolerance-an argument for unballasted media; osmotic excess is often manifest as persistent diarrhea (McArdle et al., 1981; Ann Quinn-Nathan, personal communication). Some cytologists may envision a time when the cytocosm will be essentially understood. An icy shock of present reality is the distance to be traversed before ”

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the cellular basis of stress repair is understood, the telling test of understanding being rational, effective regimens of therapeutic interventions. Thorny problems may be encountered. Repair might be hindered if, demanding many cell divisions, a Hayflick limit enters, i.e., normal diploid mammalian cells are programmed for finite divisions, averaging ca. 50 (Phillips and Cristofalo, 1985). Immortalization is a newly common term to denote establishment of cell lines which, by definition, escape the Hayflick limit upon transformation by an oncogenic factor or a mutagenic equivalent. Gene cloning to introduce genes into the body to confer a noncancerous immortality on cell lines temporarily valuable for injury repair is of course highly speculative. Some ciliates, especially paramecia, die out unless undergoing gene recombination by mating or other inducers of genic reorganization (Smith-Sonnebom, 1985), but it is uncertain whether these phenomena relate to the Hayflick effect. Vast data document deviations from normal biochemical values before, during, and after therapeutic interventions. As noted, the statistics of morbidity and mortality dismay. An intellectual debridement of unproductive assumptions has been sternly performed in symposia on which we have relied heavily (Barton, 1985; Bozzetti and Dionigi, 1985), as evidenced from the references. Simple objective criteria have been needed to judge whether an intervention does help a patient escape organizational collapse-that stark point of no return (Moore, 1985). Release of enzymes from damaged cells, notably certain transaminases, is routinely employed to detect cardiac infarction. A simple similar test is proposed by Miiller et al. (1985): irreversibility is imminent when serum proline (as representative free amino acid), exceeds 700 k*.Mper liter. Baker et al. (1986a) find in alcoholic hepatitis that plasma (B ,,>4 ng/ml) predicts 67% mortality within 30 days. Presumably certain B ,,-retaining proteins are representative of proteins whose integrity is crucial. Misgivings have been voiced (Bier and Young, 1984) about present understanding of intact-body protein metabolism; more detailed models are called for than those derived from single amino-acid probes, whose measurements are of plasma amino acids, not of intracellular amino acids. One could add a counsel of near-perfection: theoretically, rigorously valid tests should rest on gnotobiotic animals. But this exacerbates the kinds of difficulties noted earlier with tissue cell-culture models. The protozoa singled out in this article are, as practically taken for granted (sterility-test results favorable), gnotobiotic, hence free of the perturbations in ordinary rodent laboratory models contributed by fluctuations in intestinal syntheses of vitamins or other substances influencing growth. For example, vitamin-K deficiency is well known for some newborns before their microflora is established, as well as the presence in adult sera of substantial amounts of cobalamins, presumably inert as judged from microbiological assays and partly of intestinal origin and of unknown significance (Muir and Chanarin, 1983). There may be subtler perturbing factors in the background, e.g., queu-

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nine, a highly modified purine in tRNA, taken up by fibroblasts in culture, is of microbial origin in the diet or gut, and whose decrease in tRNA correlates with carcinogenesis as promoted by phorbol ester; its increase inhibits tumor growth (Elliott et al., 1984, 1985). If the brain indeed programs resource marshaling for repair, a new note of hope is sounded: the central nervous system has unexpected capacities to repair damage (Cotman and Nieto-Sampedro, 1985). Priorities for effective therapeutic intervention may thus become clear if, in serious injury, the brain’s own stock of spare parts or other resources might be dangerously depleted. The reparative agent should traverse the blood-brain barrier. A step in this direction is suggested by the remarkable effectiveness of oral thiamin propyl disulfide in replenishing thiamin stores in the cerebrospinal fluid of normal subjects and alcoholics; the compound is in wide use in Japan (Thompson et al., 1971). Well-nourished patients survive injury better. This common observation is, strictly speaking, anecdotal. The favorable nutrients can only be guessed at. Ethical considerations preclude trials with patients to identify these nutrients. It might prove to be an enormous multifactorial problem with multicellular laboratory animals. A new literature genre afflicts general biological and medical journals: defensive biology and medicine, cutting into research time. That situation, crisply summarized by Pocock (1983), may impart new impetus to using protozoa as biomedical research tools. One need not ask protozoa for informed consent before experiments, or assure an official veterinarian that the protozoa are comfortably housed, or explain one’s motives to a Friends of the Protozoa League. Past abuses have justified the aforementioned biomedical regulations. Still, experiments with protozoa need no approval from an ethics committee nor from a committee to decide whether the experiments conform with state of the art. Work with pathogenic protozoa presents obvious problems of containment, licensing, and is unsuitable for instructing microbiological tyros, hence emphasis here on nonpathogenic models. Laboratory infections have inspired a rich literature (Collins, 1983).

ACKNOWLEDGMENTS This chapter is dedicated to the memory of a long-time colleague, Dr. Paul A. Zahl, who introduced the senior author to stress research, and to the memory of Dorothy T. Krieger, M.D. In late full career her efforts were divided equally between clinical endocrinology and probings of protozoa for amino-acid sequences paralleling those diagnostic of mammalian polypeptide and protein hormones and neuroeffectors. Illness ended her collaboration with protozoologists at Haskins when about to start. We thank Herman Baker, Ph.D., and Daniel P. Petrylak, M.D., for enlightenments over many years. We thank Ann Quinn-Nathan, R.N., our consultant on patient care from a nurse’s standpoint.

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Dr. G. A. M. Cross kindly provided a paper before publication. The junior author thanks his clinical mentors, Peter H. Wernik, M.D., and Louis M. Sherwood, M.D., for encouragement and advice.

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Index

A Abdominal ganglion, electron microscopy and, 141 Acanthamoeba, mammalian stress repair and, 385 Acetabularia, nucleocytoplasmic interactions and, 249, 251-264, 270, 308, 309 Actin genes, cell reproduction and, 121 Actin mRNA, centrifuges and, 26 Actinomycin D cell reproduction and, 106 mitotic cycle and centrosome cycle, 70 chromosome cycle, 54 nucleocytoplasmic interactions and Acetabularia, 254, 257 cytoplasmic determinants, 298 sea urchin eggs, 266, 267 Xenopus, 277 Actinopods, protistan phylogeny and, 340 Activation cell reproduction and, 111 , 116 nucleocytoplasmic interactions and, 25 1, 292, 293 cytoplasmic determinants, 296 Adenylate cyclase, Xenopus and, 277 Adsorption, ionized groups and aldehyde fixation, 222 colloidal probes, 215, 216, 218, 220 polyions, 227, 232, 236, 239 Air-turbine ultracentrifuge, 19-21, 30 Aldehyde fixation, ionized groups and formaldehyde, 224 glutaraldehyde, 221-224 polyionic probes, 224, 225 427

Algae mammalian stress repair and, 376 nucleocytoplasmic interactions and. 252 protistan phylogeny and, 324, 329; 330, 334-338, 346, 348, 351, 355, 356, 358 a-Amanitin, cell reproduction and, 110 Amebae, cell reproduction and, 94, 97, 98 Amino acids cell reproduction and, 93 Go state, 118 G,-less cells, 116 G I switch, 104, 105 ionized groups and glycoproteins, 206 interstitial tissues, 206 lipids, 205 polyions, 239 mammalian stress repair and, 372, 394, 398, 413 branched-chain, 394, 395, 397-401 glutamine, 403 hypoxia, 408 stress proteins, 382-384 nucleocytoplasmic interactions and, 273, 304 protistan phylogeny and, 327 Amino groups, cytochernical detection of aldehyde fixation, 222, 224 cationic dyes, 209 colloid titration, 21 1 ionic dyes, 208 ionization, 212 Amoeba, protistan phytogeny and, 324, 329 Amoeba proteus, cell reproduction and, 94, 97, 98

428

INDEX

Amplification, nucleocytoplasmic interactions and, 272, 293 Anabaena, centrifuges and, 42 Anaerobiosis, mammalian stress repair and, 385, 388, 408 Anaphase centrifuges and, 37 centrosome cycle, 70, 72-74 chromosome cycle, 56, 60 mi@tic apparatus, 85 nucleocytoplasmic interactions and cleavage, 287 Xenopus, 281 Aneuploidy mitotis and, 80 nucleocytoplasmic interactions and, 301 Anionic colloid particles, ionized groups and, 229-23 1 Anionic ferric hydroxide colloid, ionized groups and, 220, 221, 224, 229, 233, 240 Anionic groups, cytochemical detection of, 203 aldehyde fixation, 221, 222, 224, 225 blood vessels, 232, 233 cationic dyes, 209 colloidal prQbes, 214-219 ionization, 212 macromolecules, 210 peritoneal cavity, 229, 230 Anionic macromolecules, ionized groups and, 240 blood vessels, 236 colloid titration, 210, 21 I ionization, 212 peritoneal cavity, 229 Anionic molecules, cytochemical detection of, 209 Anionic polymers, ionized groups and, 240 Anionic sites, cytochemical detection of aldehyde fixation, 222 cationic dyes, 208, 209 colloidal probes, 215, 218, 220 polyions, 235 Antibiotics, mammalian stress repair and, 382 Anticentrosome serum, centrosome cycle and, 65, 67, 68, 71 Antigens, centrosome cycle and, 65, 67, 77 Aphidicolin, centrosome cycle and, 70 Apicomplexans, protistan phylogeny and, 342, 343

Araldite, electron microscopy and, 156, 170 Arbacia centrifuges and, 23-25, 28 nucleocytoplasmic interactions and cleavage, 290 sea urchin eggs, 267-270 Arsenite, mammalian stress repair and, 385, 412 Ascaris centrifuges and, 22, 28 mitosis and, 81 Ascaris megalocephala, centrosome cycle and, 63 Ascetosporans, protistan phylogeny and, 354 Ascidian eggs, nucleocytoplasmic interactions and, 298 Asters centrosome cycle and, 74, 75, 77, 78 mitotic apparatus and, 80, 83 nucleocytoplasmic interactions and, 25 1 early embryonic development, 305 sea urchin eggs, 265, 269 Xenopus, 282, 285, 286 Asymmetry, electron microscopy and freeze-fracture etch technique, 189, 194 unit membrane, 161, 171 ATP mammalian stress repair and, 373, 376 carnitine, 400 hemoflagellates, 388, 390 hypoxia, 408 nucleosides, 402 stress protein, 382, 384 Xenopus and, 280 Autoantibodies, centrosome cycle and, 65 Autonomously replicating sequences, cell reproduction and, 120-122 Axon membrane, electron microscopy and, 174

B Bacteria cell reproduction and, 94, 119, 120 centrifuges and, 42 mammalian stress repair and, 371, 376, 378, 379 protistan phylogeny and, 3 19

INDEX Basement membranes, ionized groups and, 209, 234, 235, 238, 239 Biopterin, mammalian stress repair and, 372, 373, 38 I , 386 hemoflagellates and, 392, 293 Biotin, mammalian stress repair and, 38 I , 398-402, 404 Bipolarization, mitosis and, 52, 53 centrosome cycle, 74, 78 mitotic apparatus, 87 Blastula, nucleocytoplasmic interactions and cleavage, 287, 289 early embryonic development, 301, 302, 305, 306 sea urchin eggs, 265-267 Xenopus. 276, 282, 283 Blockage, cell reproduction and cell cycle, 119 G I period, 117 G I switch, 104-106, 108, 109 nuclear structure, 122 Blood coagulation, ionized groups and, 210, 21 1 Blood vessels, ionized groups and, 232-238 Blue-green algae, centrifuges and, 42 Bond energy, ionized groups and, 203, 204 Boveri’s rules, mitotic apparatus and, 79, 80 Branched-chain amino acids, mammalian stress repair and, 394, 395, 397-401 Budding yeast, cell reproduction and GI switch, 102-104 G I transit, 100 nuclear structure, 120

C C fibers, electron microscopy and, 145 Cacodylate iron colloid, ionized groups and, 215 Calcium mammalian stress repair and, 378, 396-398 nucleocytoplasmic interactions and sea urchin eggs, 265 Xenopus, 277, 285 Calmodulin, mitosis and centrosome cycle, 70 mitotic apparatus, 85 Cancer cells, centrifuges and, 37, 39

429

Carbohydrate electron microscopy and freeze-fracture etch technique, 190 unit membrane, 163 ionized groups and glycoproteins, 206 lipids, 204 mammalian stress repair and, 389, 400 protistan phylogeny and, 351 Carboxyl group, cytochemical detection of, 206 aldehyde fixation, 225 colloidal probes, 214-216, 218 ionization, 212 Carcinogenicity, mammalian stress repair and, 380 Carnitine, mammalian stress repair and, 397, 398, 400 Cartesian diver balance, cell reproduction and, 94 Catalase, ionized groups and, 219 Catecholamines, mammalian stress repair and, 373, 379, 387, 392, 403, 406 Cationic dyes, ionized groups and, 208, 209 Cell cycle, cell reproduction and, 93-95 four part, 100 Go state, 118, 119 G1-less cells, 113, 116, 117 GI/S border, 119, 120 G I switch, 102-104 higher eukaryotes, 99, 100 with three sections, 96-98 Cell division, cell reproduction and, 93,94, 123 cell growth, 95, 96, 98-100 G I transit, 100, 104, 108, 112, 113, 117 Cell fission, GI switch and, 102 Cell growth, 123 cell cycle and four-part, 100 three-section, 96-98 DNA replication, 96 Gl-less cells, 116 GI transit, 101, 108 in higher eukaryotes, 99, 100 Cell reproduction, 93-95, 123 cell growth and, 95-100 Go state, 118, 119 G,-less cells of early embryos, 109-1 13 mammalian cells in culture, 114-1 17

430

INDEX

G,/S border, 119, 120

GIswitch in animal cells, 105-107 in budding yeast, 102-104 in cultured animal cells, 104, 105 operation, 107-109 GI transit, 100, 101 G2 period, 122, 123 nuclear structure, 120-122 shortening, 117, 118 Cell surface, ionized groups on, see Ionized groups Central nervous synapses, electron microscopy and, 144 Central nervous system, mammalian stress repair and, 373, 401, 414 Centrifuges, 15-17, 43, 44 animal eggs and, 22, 23 Arbacia, 23-25 germ cell determinants, 28 mosaic eggs, 25-27 polarity, 28-30 symmetry, 28-30 bacteria, 42 blue-green algae, 42 cancer cells, 37, 39 developmental history, 17, 18 air-turbine ultracentrifuge, 19-2 1 microscope centrifuge, 2 1 suspension media, 21 Svedberg oil turbine ultracentrifuge, 18, 19 ground substance, 42, 43 protozoa, 39-42 somatic cells, 30-37 viscosity, 30 Centrioles centrosome cycle and, 63, 64, 66, 70, 71, 73-75, 78 protistan phylogeny and, 349 sea urchin eggs and, 26, 27 Centrosome cycle, mitosis and, 52, 63, 78, 79, 88, 89 embodiment, 63-65 identification, 65 mitotic apparatus, 79 model, 74-78 natural history, 65-70 pole separation, 70-74

Cen trosomes mitosis and, 49-5 1, 62 mitotic apparatus, 87 Xenopus, 285, 286 Chaetopterus centrifuges and, 26, 28 nucleocytoplasmic interactions and, 290, 299 Chagas’ disease, mammalian stress repair and, 387, 389 Charge related endocytosis, ionized groups and, 227, 229, 230, 240 Chemotherapy, mammalian stress repair and, 376, 380, 399, 412 hypoxia, 408, 409 polyamines, 393 stress proteins, 382, 383 Chloramphenicol, Acetabularia and, 254, 257, 258 Chlorarachniophytes, protistan phylogeny and, 346, 350, 35 1, 356 Chlorobionts, protistan phylogeny and, 335 Chlorophylls, protistan phylogeny and, 347, 349-351 Chlorophyte series, protistan phylogeny and, 335, 336 Chloroplast envelope, protistan phylogeny and, 349 Chloroplasts centrifuges and animal eggs, 29 Euglena. 41 somatic cells, 35 mammalian stress repair and, 376 nucleocytoplasmic interactions and, 252, 254, 257-259, 262, 264 protistan phylogeny and, 331, 335, 336, 346, 350 Chloroprotists, protist phylogeny and, 335, 336, 346, 351 CHO cells, cell reproduction and, 105, 114, 115, 117 Choanoflagellates, protistan phylogeny and, 351 Chondroitin sulfate, ionized groups and, 206 colloidal probes, 215 ionization, 212 Chondroitin sulfate ferric hydroxide colloid, ionized groups and, 220, 229, 230, 233, 236

INDEX Chromatin cell reproduction and, 112 centrifuges and protozoa, 39, 41 somatic cells, 35 mitosis and, 49, 5 1 chromosome cycle, 54-56, 58-60, 62 mitotic apparatus, 80 nucleocytoplasmic interactions and cleavage, 289 early embryonic development, 304 later stages, 293 Xenopus, 282, 285 Chromobionts, protistan phylogeny and, 336, 337, 339, 341, 346, 351 Chromomeres Acetabularia and, 259 Xenopus and, 273 Chromophyte series, protistan phylogeny and, 336, 337 Chromosome condensation cell reproduction and cell growth, 97 GI-less cells, 110, 11 1, 114 nuclear structure, 123 mitosis and, 56, 61 nucleocytoplasmic interactions and sea urchin eggs, 270 Xenopus 277, 279, 282, 285 protistan phylogeny and, 347 Chromosome cycle, mitosis and, 52, 54-57, 62, 63, 73 condensation, 59, 60 factors, 60-62 interphase, 53, 54 mitotic apparatus, 79 structure, 58 Chromosome decondensation, cell growth and, 197 Chromosomes cell reproduction and, 120- 122 centrifuges and cancer cells, 37 somatic cells, 37 nucleocytoplasmic interactions and Acetabularia, 259, 262, 264 early embryonic development and, 301, 303, 305, 306 sea urchin eggs, 269

43 1

Xenopus, 273, 279, 284 protistan phylogeny and, 327, 348 Chronic lymphocytic leukemia, mammalian stress repair and, 391 Chrysomonads, mammalian stress repair and, 376, 377, 379, 392, 395, 397, 401, 402, 404 Chytrids, protistan phylogeny and, 339 Ciliates mammalian stress repair and, 381, 404, 405, 407, 410, 411, 413 potistan phylogeny and, 329, 331, 332, 343, 344, 346, 347, 351, 352 Ciliophorans, protistan phylogeny and, 343 Circadian rhythm, Acetabularia and, 254, 255 Citrate, mammalian stress repair and, 378, 397 Citrate ferric hydroxide colloid, ionized groups and, 221, 229, 230 Cleavage cell reproduction and, 93 cell growth, 99 G1-less cells, 109-1 13, 117 nuclear structure, 122 nucleocytoplasmic interactions and, 249, 25 1 Acetabularia, 261 cytoplasmic determinants, 293, 296, 298, 299 early embryonic development, 301, 303, 305, 307 fertilized eggs, 286-290 sea urchin eggs, 265-267, 269 Xenopus, 279, 282 Coated pits, ionized groups and, 227, 230 Coiling, mitosis and, 88 centrosome cycle, 76 chromosome cycle, 58 Colcemid cell reproduction and, 113 Colchicine cell reproduction and, 11 1 chromosome cycle and, 56 Collagen fiber, electron microscopy and, 134 Collagen fibrils, electron microscopy and, 136 Collar flagellates, protistan phylogeny and, 35 1 Colloid titration, ionized groups and, 210212, 216 aldehyde fixation, 222, 225

432

INDEX

Colloidal probes, ionized groups and, 209, Acetabularia, 254 219-221 cytoplasmic determinants, 298, 299 cationic cacodylate ferric hydroxide colloid, Cytochemical detection, ionized groups and, 215-218 see Ionized groups ferric hydroxide colloid, 214, 215 Cytochrome c, protistan phylogeny and, 347 ferritin molecules, 218, 219 Cytocosm, mammalian stress repair and, 373, ionization, 212 374, 382 Condensation, mitosis and, 52, 53, 62 Cytokinesis centrosome cycle, 73 cell reproduction and, 101, 102 chromosome cycle, 54, 55, 57-59, 61 centrifuges and, 28 mitotic apparatus, 85. 88 protistan phylogeny and, 335 Contact inhibition, cell reproduction and, 104 Cytoplasm Cortex, centrifuges and, 25, 42 cell reproduction and, 93, 94, 98 Cortical cytoskeletal domain, centrifuges and, Go state, 118 26 GI-less cells, 110, 113, 114 Corticosteroids, mammalian stress repair and, G I switch, 102, 107, 108 375 centrifuges and Cortisol, mammalian stress repair and, 403 animal eggs, 22, 23, 28 Cortisone, mammalian stress repair and, 410 bacteria, 42 Craspedomonads, protistan phylogeny and, blue-green algae, 42 351 protozoa, 39 Crayfish synapse, electron microscopy and, somatic cells, 31 173-177 ionized groups and, 203 Crepidula, centrifuges and, 28 aldehyde fixation, 225 Criegee reaction, electron microscopy and, 168 lipids, 204 Crichidia, mammalian stress repair and, 377, polyions, 226, 227, 230, 238, 239 379, 388, 391, 392, 394, 410 mammalian stress repair and, 384 Crithidia fasciculara, mammalian stress repair mitosis and, 52 and, 373, 387, 389, 391, 393-395 centrosome cycle, 64,69, 70 Cryprhecodiniurn, mammalian stress repair chromosome cycle, 56, 61, 62 and, 410 mitotic apparatus, 80, 83 Cryptomonads, protistan phylogeny and, 346, nucleocytoplasmic interactions and, 249349-35 1 252, 293, 294, 307, 309 Cryptophytes, protistan phylogeny and, 349, Acetabularia, 254-256, 259 350 asidian eggs, 298, 299 Cyclic AMP cleavage, 286, 287, 289, 290 cell reproduction and, 105, 106 early embryonic development, 301-307 nucleocytoplasmic interactions and, 277 gray crescent, amphibian eggs and, 295 Cyclin Ilyanassa eggs, 296-298 cell reproduction and, 110, 112, 113 insect, 296 chromosome cycle and, 61 later stages, 290, 292 Cycloheximide sea urchin eggs, 266, 267, 269, 270 cell reproduction and, 106, 108, 109, 11 1, Xenopus, 271, 273, 275, 279, 280, 282, 115, 116 285 mammalian stress repair and, 393 protistan phylogeny and, 349, 354 nucleocytoplasmic interactions and, 254, rhizopods, 337 258 serial endosymbiosis theory, 325 Cytobiosis, 326 Cytoplasts, nucleocytoplasmic interactions and, Cytochalasin B, nucleocytoplasmic interactions 249, 264 and Cytoskeleton, centrifuges and, 42, 43

INDEX Cytosol centrifuges and, 44 mammalian stress repair and, 373 Xenopus and, 285 Cytospectrophotometry. cell reproduction and, 93 Cytsokeleton, mitosis and, 80 Cytostatic factor, nucleocytoplasmic interactions and cleavage, 289, 290 Xenopus, 279-281, 286 Cytostome, protistan phylogeny and, 347 Cytotoxicity, mammalian stress repair and, 376, 382, 412

D D period, cell reproduction and cell growth, 97 G I transit, 101, 104, 113 Decondensation, mitosis and, 52, 53, 57, 62, 88 chromosome cycle, 54, 55 factors, 61 Degradation, mammalian stress repair and, 404 Deinococcus, mammalian stress repair and, 385, 386 Deletion, centrifuges and, 26, 28 Dendraster, centrifuges and, 29 Density-dependent inhibition, cell reproduction and, 104, 115, 118 Deoxycytidine, cell reproduction and, 103 Deoxycytidine monophosphate deaminase, Acetabularia and, 258, 259 Deoxynucleoside diphosphates, cell reproduction and, 103 Deoxynucleoside triphosphates, cell reproduction and, 119 Diadenosine tetraphosphate, cell reproduction and, 116 Diaminobenzidine, ionized groups and, 220 Dictyostelium cell reproduction and, 97, 98 mammalian stress repair and, 41 1 Differentiation cell growth and, 99, 121 centrifuges and, 43 animal eggs, 22, 23, 25 somatic cells, 31

433

mammalian stress repair and, 388 mitosis and, 49 nucleocytoplasmic interactions and, 25 1, 290, 291, 293 cytoplasmic determinants, 294, 295, 298 later stages, 292 sea urchin eggs, 266 Xenopus, 283, 284 protistan phylogeny and, 330 Dihydrofolate reductase, cell reproduction and, 119, 121, 122 Dinoflagellate mammalian stress repair and, 410 protistan phylogeny and, 346-350 Dinozoa, protistan phylogeny and, 347, 348 Diploidy cell reproduction and, 103, 109, 120 mammalian stress repair and, 405, 413 nucleocytoplasmic interactions and, 301 protistan phylogeny and, 330, 343 Displacement, centrifuges and, 30, 31, 35 DNA ionized groups and, 207 mammalian stress repair and, 386, 387, 390, 391, 393, 394, 396 Tetrahymena. 404, 405 mitosis and, 49, 50, 88 centrosome cycle and, 68 chromosome cycle and, 54, 5 5 , 58, 59, 62 nucleocytoplasmic interactions and, 307, 308 Acetabularia, 257-259, 262, 264 cleavage, 287, 289, 290 cytoplasmic determinants, 297, 299 early embryonic development, 302, 304 later stages, 293 sea urchin eggs, 265, 266, 269, 270 Xenopus, 273, 215, 279, 284, 285 protistan phylogeny and, 325, 332 DNA lipase, cell reproduction and, 107 DNA polymerase cell reproduction and, 107, 116, 120 mammalian stress repair and, 397 DNA primase, cell reproduction and, 107 DNA replication, cell reproduction and, 93, 94, 123 cell growth, 96-100 Go state, 118, 119 Gl/S border, 119

434

INDEX

G I transit, 100, 101 GI-less cells, 109, 110, 112-1 14, 116 GI switch, 102-109 shortening, 117 G2 period, 122, 123 nuclear structure and, 120-122 DNA synthesis cell reproduction and, 93, 94 four-part cell cycles, 100 GI-less cells, 114 GI switch, 102, 103, 105, 107, 108 nuclear structure, 120 chromosome cycle, 54, 56 DNA topoisomerase, cell reproduction and, 107 DNase chromosome cycle and, 54, 55 nucleocytoplasmic interactions and, 293 Drosophila cell reproduction and cell growth, 99 G1-less cells, 109, 113 nuclear structure, 122 protistan phylogeny and, 371, 379 hypoxia, 410 stress repair, 383 Tetrahymena, 405 mitosis and, 81 nucleocytoplasmic interactions and, 250, 308 cleavage, 287 cytoplasmic determinants, 296 early embryonic development, 303-305 late stages, 293 Drosophila melanogaster, centrifuges and, 28 Dynein, mitosis and centrosome cycle, 70 mitotic apparatus, 85

E Echinochrome, sea urchin eggs and, 268 EDTA, mammalian stress repair and, 378, 397 Eggs, centrifugal force and, 22, 23 Arbacia. 23-25 germ-cell determinants, 28 mosaic eggs, 25-27 polarity, 28-30 symmetry, 28-30

Ehrlich ascites tumor cells ionized groups and, 218 mammalian stress repair and, 397, 402 Electron microscopy centrifuges and, 19, 44 ground substance, 43 somatic cells, 31 ionized groups and, 240 anionic colloid particles, 229 cationic dyes, 208, 209 colloidal probes, 215-218, 220 ferric hydroxide colloid, 226, 227, 240 ionization, 212 macromolecules, 229 polyions, 234, 238, 239 mitosis and centrosome cycle, 65, 69, 75, 79 chromosome cycle, 55, 56, 58 mitotic apparatus, 81 nerve tissue membranes and, 134-142 crayfish synapse, 173-177 freeze-fracture etch technique, 186-195 lanthanum tracer technique, 184, 185 motor-end plate, 143-145 muscle, 148-154 myelin sheath, 155 spiral myelin, 145- 148 synaptic disk, 177- 184 unit membrane, 155-172, 186 nucleocytoplasmic interactions and, 259, 26 1 protistan phylogeny and, 327, 343, 350, 356 Elodea, centrifuges and, 29 Embodiment, centrosome cycle and, 63-65 Embryogenesis centrifuges and, 26 nucleocytoplasmic interactions and, 297, 303 Embryo1ogy cell reproduction and, 93 cell growth, 99, 100 GI-less cells, 109-1 13, 117 nuclear structure, 122 nucleocytoplasmic interactions and, 24925 1 cleavage, 287, 289 cytoplasmic determinants, 293-295, 298, 299 early development. 301 hybiids, lethal, 301-303

435

INDEX later stages, 292, 293 molecular embryology, 307-309 mutations, lethal, 303-307 sea urchin eggs, 266, 267 Xenopus, 271, 282 Endocytobiology, 326 Endocytosis ionized groups and, 240 charge-related, 227, 229, 230, 240 polyions, 227-229 Xenopus and, 271 Endoplasmic reticulum centrifuges and, 35 protistan phylogeny and, 349 Xenopus and, 285 Endosymbiosis, protistan phylogeny and, 338, 341, 345, 346 Endosymbiosis theory, serial, see Serial endosymbiosis theory Endothelium, ionized groups and, 203 colloidal probes, 217, 218 polyions, 227, 232-239 Endotoxin, mammalian stress repair and, 377, 378 Enzyme histochemistry, 5 , 6 Enzyme synthesis, Acetabularia and, 256, 259 Ephapses, electron microscopy and, 141 Epinephrine, mammalian stress repair and, 378, 387, 406, 407, 410 Epithelial cells, electron microscopy and, 194 crayfish synapse, 176 lanthanum tracer technique, 184 Epithelial growth factor, cell reproduction and, 107, 118 Epithelium, ionized groups and, 217, 235, 238-240 Erythrocyte membrane, electron microscopy and, 156, 189, 190, 194 Erythrocytes centrifuges and, 3 1, 33 mammalian stress repair and, 402, 408 nucleocytoplasmic interactions and, 276, 293 Escherichia coli cell reproduction and, 97 mammalian stress repair and, 383, 384, 396, 411, 412 nucleocytoplasmic interactions and, 257, 308 Ethanol, mammalian stress repair and, 384

Ethanolamine, mammalian stress repair and, 404 Euchromatin, cell reproduction and, 121 Euglena centrifuges and, 41, 42 mammalian stress repair and, 376, 378, 396 nucleocytoplasmic interactions and, 259 protistan phylogeny and, 356 Euglenophytes, protistan phylogeny, 346, 347 Euglenozoa, protistan phylogeny and, 346, 347 Eukaryogenesis, protistan phylogeny and, see Protistan phylogeny, eukaryogenesis and Eukaryotes cell reproduction and cell growth, 98-100 G,-less cells, 110, 113 GI/S border, 119 G I switch, 102 nuclear structure, 120, 121 centrifuges and, 42, 50-52 centrosome cycle, 64,71, 78 chromosome cycle, 60 mammalian stress repair and, 371, 379 hemoflagellates, 393 nucleosides, 402 stress proteins, 383, 385 Tetrahymena, 405, 406 nucleocytoplasmic interactions and, 27 1, 284, 308

F Factors, chromosome cycle and, 60-63 Fatty acids, mammalian stress repair and, 397-401, 410 Ferric hydroxide colloidal probes, ionized groups and, 212, 214, 215, 221 Ferric hydroxide colloids, ionized groups and, 225-230, 232, 234, 236, 239, 240 Ferritin ionized groups and, 218-221 aldehyde fixation, 222 polyions, 227, 230, 233, 235 mammalian stress repair and, 406 Fever, mammalian stress repair and, 374 Fibril, nucleocytoplasmic interactions and, 262, 291, 303 Fibrin, ionized groups and, 232, 236, 238, 240

436

INDEX

Fibrinogen, ionized groups and, 212, 220, 225 Fibroblasts cell reproduction and, 115, 116, 119 mammalian stress repair and, 385, 391, 403, 414 Fibronectin, nucleocytoplasmic interactions and, 291, 303 Fission yeast, cell reproduction and, 97, 98, 104 Flagella mammalian stress repair and, 373 protistan phylogeny and, 331, 336, 337, 339, 340, 343, 345-347, 350, 352 Flow cytophotometry, chromosome cycle and, 55

Flow microfluorimetry, cell reproduction and, 106 Fluid mosaic model electron microscopy and, 188-190 ionized groups and, 205 Fluorescence microscopy, centrosome cycle and, 65 Folic acid, mammalian stress repair and, 379, 396 hemoflagellates, 387, 392 stress proteins, 384 Formaldehyde electron microscopy and, 178 ionized groups and, 222, 224, 225 Fractionation, cell reproduction and, 1 1 1 Freeze-fracture, protistan phylogeny and, 327 Freeze-fracture etch technique, electron microscopy and, 186-195 Fucus furcatus, centrifuges and, 29 Fungi mammalian stress repair and, 378 protistan phylogeny and, 300, 322, 329, 332, 338 evolutionary lines, 337, 352 Fusion cell reproduction and Go state, 118 G1-less cells, 111, 112, 114 G I switch, 108 ionized groups and, 229, 230 mammalian stress repair and, 405 nucleocytoplasmic interactions and, 252, 265 protistan phylogeny and, 352

G

Go period, cell reproduction and, 105, 118, 119 GI-lesscells, cell reproduction and early embryos, 109-1 13 mammalian cells in culture, 114-1 17 GI period, cell reproduction and, 122 four-part cell cycles, 100 in higher eukaryotes, 99, 100 transit, 100, 101 early embryos, 109-1 13 mammalian cells, 114-1 17 shortening, 117, 118 switch, 102-109 G I phase, nucleocytoplasmic interactions and, 287, 289 G,/S border, cell reproduction and, 119, 120 G Iswitch, cell reproduction and, 100, 101 animal cells, 105-107 cultured, 104, 105 budding yeast, 102-104 G1-less cells, 113, 115, 116 operation, 107- 109 Gz period, cell reproduction and, 122, 123 cell growth, 97-99 early embryos, 109-1 13 G I transit, 101, 104, 108 G2 phase, nucleocytoplasmic interacting and, 287, 289 GA, see Glutaraldehyde Gastrulation, nucleocytoplasmic interactions and, 249, 290-292 cytoplasmic determinants, 295, 298, 299 early embryonic development, 301-305 Xenopus, 276 Gene activation, centrifuges and, 26 Genes, chromosome cycle and, 63, 79 Geotropism, centrifuges and, 15, 22 Germ-cell determinants, centrifuges and, 28 Germinal localizations, nucleocytoplasmic interactions and, 293-299, 309 Germinal vesicle, nucleocytoplasmic interactions and, 25 I , 252 Acetabularia, 261 early embryonic development, 306 Xenopus. 273, 275, 277, 281, 282 Gleocapsa, centrifuges and, 42 Glucagon, mammalian stress repair and, 410

INDEX Glucocorticoids, mammalian stress repair and, 373, 410 Glucose, mammalian stress repair and, 375, 378, 395, 410 biotin, 400 nucleosides, 402, 403 stress proteins, 384 Tetrahymena, 406, 407 Glutamine, mammalian stress repair and, 402, 403 Glutaraldehyde electronic microscopy and, 139, 140 freeze-fracture etch technique, 194 lanthanum tracer technique, 184 muscle, 154 unit membrane, 156 ionized groups and cationic dyes, 208, 209 colloidal probes, 217 fixation, 221-225 polyions, 234, 236 Glycerol ionized groups and, 204, 205 mammalian stress repair and, 403, 404 Glycine, mammalian stress repair and, 399, 403 Glycogen mammalian stress repair and, 402 nucleocytoplasmic interactions and, 27 1 Glycolipids electron microscopy and, 194 ionized groups and, 205, 238 Glycolysis, mammalian stress repair and, 376 glutamine, 403 hemoflagellates, 388 hypoxia, 408 nucleosides, 402 stress protein, 382 Glycophorin, ionized groups and, 206 Glycoproteins ionized groups and, 203, 205-207 polyions, 232, 236 nucleocytoplasmic interactions and, 29 1, 302, 303 Glycosylation, nucleocytoplasrnic interactions and, 291 Golgi apparatus centrifuges and, 31 protistan phylogeny and, 341, 353

437

Golgi bodies, centrifuges and ground substance, 43 somatic cells, 35 Golgi membranes, electron microscopy and, 163 Gravity, centrifuges and, 17, 44 animal eggs, 22 development history, 18 somatic cells, 31 Gray crescent, mitosis and, 249, 250, 294 amphibian eggs, 294-296 Griflrhsia, centrifuges and, 29 GVBD, see Hugh germinal vesicles

H Hale’s ferric hydroxide colloidal probe, see Femc hydroxide colloidal probe Haploidy cell reproduction and, 102, 103 nucleocytoplasmic interactions and, 252, 261, 301 protistan phylogeny and, 330 Haplosporidia, protistan phylogeny and, 354 Heat-shock proteins mammalian stress repair and, 385-388, 405, 410, 412 nucleocytoplasmic interactions and, 28 1, 282 Heat-stress proteins, mammalian stress repair and, 384-386 Heliozo, protistan phylogeny and, 338 Helix, centrifuges and, 3 I Helodea densa, centrifuges and, 29 Hemaglobin, centrifuges and, 31 Heme, mammalian stress repair and, 377, 387, 390, 391 Hemeundecapeptide, ionized groups and, 2 19, 220 Hemoflagellates, mammalian stress repair and, 386-389, 399 biopterin, 392, 393 DNA recombination, 393, 394 heme, 390, 391 polyamines, 393 Heparan sulfate, ionized groups and colloidal probes, 2 15 glycoproteins, 206 interstitial tissues, 206

438

INDEX

Heterochromatin, cell reproduction and, 121 Heterokaryotes, protistan phylogeny and, 343 Heterokonts, protistan phylogeny and, 336, 337, 339, 353 Hexose transport, cell reproduction and, 106 Hexoses, mammalian stress repair and, 388, 395, 403, 408 Histamine ionized groups and, 235, 236 mammalian stress repair and, 394 Histidine, ionized groups and, 222 Histone genes, cell reproduction and, 119, 122 Histone mRNA, centrifuges and, 26 Histones cell reproduction and, 110 ionized groups and, 224, 225 mitosis and, 58-60 nucleocytoplasmic interactions and cleavage, 287, 289 cytoplasmic determinants, 298 early embryonic development, 302 sea urchin eggs, 269 Xenopus, 279, 281 Hodgkin’s lymphoma, mammalian stress repair and, 379 Homeo box, nucleocytoplasmic interactions and, 304, 305 Homogenization, centrifuges and, 33 Hormones, mammalian stress repair and, 372, 376, 381, 411 stress proteins, 382 Teirahymena, 406, 407 Horseradish peroxidase, ionized groups and, 219, 220, 227, 233, 235, 239 Huge germinal vesicle, Xenopus and, 277, 279, 281, 282 Hyaluronic acid, ionized groups and, 206, 207, 212 Hybridization cell reproduction and, 114, 115 centrifuges and, 26 mitosis and cytoplasmic determinants, 299 early embryonic development, 301-303, 305 sea urchin eggs, 269 Xenopus, 275 protistan phylogeny and, 327 Hydra, cell growth and, 97

Hydrogenosomes, protistan phylogeny and, 34 1 Hydrophilic groups, cytochemical detection of, 203 Hydrophobic cell membrane, ionized groups and, 203 Hydroxyl groups, cytochemical detection of, 205 Hydroxyurea cell reproduction and G I transit, 103, 117, 118 nuclear structure, 122 nucleocytoplasmic interactions and, 259 Hyperglycemia, mammalian stress repair and, 373 Hypoglycemia, mammalian stress repair and, 40 1 Hypoxia, mammalian stress repair and, 373, 408- 10

I Ilyanassa eggs, nucleocytoplasmic interactions and, 296-298 Ilyanassa obsoleta, centrifuges and, 26 Immunocytochemistry centrifuges and, 44 centrosome cycles and, 65, 71, 77 Immunofluorescence microscopy centrifuges and, 43 Indoleamines, mammalian stress repair and, 373, 392 Inflammatory cells, ionized groups and, 207 Inflammatory changes, ionized groups and, 240 polyions, 225, 235, 236 Inflammatory response, mammalian stress repair and, 406 Inhibition, cell reproduction and cell growth, 98 Go state, 118 Gl/S border, 119 G I transit GI-less cells, 110, 111, 116 GIswitch, 103, 104, 106-108 mammalian cells, 117 G2 period, 122 nuclear structure, 122

INDEX Initiation cell reproduction and, 93 cell growth, 96 GI/S border, 119 G I switch, 102, 103, 109 nuclear structure, 120-122 nucleocytoplasmic interactions and, 256, 265 Insulin cell reproduction and, 107 mammalian stress repair and, 375, 398 nucleocytoplasmic interactions and, 27 1, 277 Interphase cell reproduction and, 93 G1-less cells, 109-111, 114, 115 nuclear structure, 121 mitosis and, 53 centrosome cycle, 71 chromosome cycle, 53-57, 59 nucleocytoplasmic interactions and, 285 Interstitial tissues, ionized groups and, 204, 206, 207, 240 anionic colloid particles, 229 colloid titration, 2 11 ferric hydroxide colloid, 214 ferritin molecules, 21 8 ionization, 21 1 polyions, 233 Intestine, ionized groups and, 239 Ionic bonds, ionized groups and, 203 colloidal probes, 215 ionic dyes, 208 macromolecules, 210 polyions, 227, 228 Ionic dyes, light microscopy and, 207, 208 Ionization, ionized groups and, 21 1-213 aldehyde fixation, 222, 224 colloidal probes, 216 polyions, 225 Ionized groups aldehyde fixation, 221-225 cationic dyes, 208, 209 colloid titration, 210, 21 1 colloidal probes, 219-222 cationic cacodylate femc hydroxide colloid, 215-218 ferric hydroxide colloid, 214, 215 in eukaryotic cell surfaces, 204-206 in interstitial tissues, 204, 206, 207

439

ionic dyes, 207, 208 macromolecules, 209, 210 polyions anionic colloid particles, 229-23 1 into blood vessels, 232-238 ferric hydroxide colloids, 225-229 into intestine, 239 into lungs, 238, 239 Isoleucine cell reproduction and GI-less cells, 115, 116 G I switch, 104, 106 mammalian stress repair and, 389, 398, 400

K Karyokinesis, mitosis and, 50, 56 Karyophilic proteins, nucleocytoplasmic interactions and, 273, 275, 306 Karyoplasts, nucleocytoplasmic interactions and, 285 Karyotype, chromosome cycle and, 60 Kinesin, mitosis and centrosome cycle, 70 mitotic apparatus, 85 K inetochores centrifuges and, 35 mitosis and, 50, 51, 88, 89 centrosome cycle, 69, 76 chromosome cycle, 56 mitotic apparatus, 79-87 Kinetoplast mammalian stress repair and, 385 protistan phylogeny and, 347 Kinetoplastideans, protistan phylogeny and, 346, 347 Kinetosomes, protistan phylogeny and, 332, 339

L Labyrinthomorphs, protistan phylogeny and, 352, 353 Labyrinthuleans, protistan phylogeny and, 352, 353 Lactobacillus, mammalian stress repair and, 378 Lamellae, centrifuges and, 42

440

INDEX

Lanthanum tracer technique, electron microscopy and, 184, 185 Lateral dendrite electron microscopy and, 177, I78 Leishmania, mammalian stress repair and, 376, 379, 385, 387-390, 394, 412 Leucine, mammalian stress repair and, 389, 394, 396, 400, 401 Light microscopy, 139, 141 ionized groups and, 240 cationic cacodylate ferric hydroxide colloid, 216 colloidal probes, 220 femc hydroxide colloid, 214, 215 ferritin molecules, 218 formaldehyde fixation, 224 ionic dyes, 207, 208 ionization, 212 macromolecules, 209 polyions, 225, 227, 229, 234 protistan phylogeny and, 327 Lipid bodies, centrifuges and, 35 Lipids electron microscopy and, 134 crayfish synapse, 176, 177 freeze-fracture etch technique, 187- 190, 193, 194 synaptic disk, 179, 183 unit membrane, 157, 159, 161, 163, 164, 166, 167, 169, 171, 172 ionized groups and, 204, 205, 234, 236 mammalian stress repair and, 372, 390, 391, 396, 407, 408 nucleocytoplasmic interactions and, 265, 27 1 Lipoid, electron microscopy and, 172 Liposomes, ionized groups and, 21 1 Liver, centrifuges and, 21, 3.5 Lungs, ionized groups and, 238, 239 Lymphocytes, mammalian stress repair and, 391, 392, 396, 401 Lysine, ionized groups and, 222 Lysosomes, ionized groups and, 204, 229, 230, 240 Lysozyme, ionized groups and, 219, 220

M Macromolecular probes, ionized groups and, 214, 224

Macrophages ionized groups and, 240 aldehyde fixation, 224 colloidal probes, 221 peritoneal cavity, 226, 229, 230, 232 polyions, 233, 236, 237, 239 mammalian stress repair and, 374, 377, 387-390, 403, 406, 407, 409 Mammalian stress repair, protozoological approaches to, 371-381, 412-414 biopterin, 379 biotin, 400-402 chrysomonads, 404 Crypthecodinium, 410 Dictyostelium, 410, 41 1 ethanolamine, 404 glutamine, 403, 404 glycerol, 403, 404 hypoxia, 408, 410 nucleosides, 402, 403 Poterioochromonas malhamensis, 395-397 stress proteins, 382-386 Tetrahymena, 404-407 tissue cell culture, 381, 382 trypanosomatids, 386-395 Mastigomycetes, protistan phylogeny and, 339, 340 Mating hormone, cell reproduction and, 102 Maturation-promoting factor cell reproduction and, 110-1 13, 123 chromosome cycle and, 61-63 nucleocytoplasmic interactions and cleavage, 289, 290 Xenopus, 279-282 Mauthner cell, electron microscopy and, 177 Meiosis cell reproduction and, 103, 110, I 1 1, 1 I3 mitosis and, 53 centrosome cycle, 7.5 chromosome cycle, 61 mitotic apparatus, 81, 86 nucleocytoplasmic interactions and Acetabularia, 261 Xenopus, 276, 277, 280, 281 protistan phylogeny and, 341, 343, 345, 349 Membranous organelles, electron microscopy and, 157, 170 Merotomy , nucleocytoplasmic interactions and, 249, 252 Mesaxon, electron microscopy and, 146, 147

INDEX crayfish synapse, 173, 174 unit membrane, 157 Metaphase cell reproduction and, 110, 1 1 1 mitosis and, 53, 88 centrosome cycle, 72, 74 chromosome cycle, 57, 58, 60 mitotic apparatus, 81, 82, 86 nucleocytoplasmic interactions and cleavage, 290 Xenopus, 280, 281, 285 Metaphyta, mammalian stress repair and, 383 Metaphytan tissues, protistan phylogeny and, 319, 355 Metatropic sheath, electron microscopy and, 173, 174 Metazoa, mammalian stress repair and, 376, 377, 411 biopterin, 392 stress protein, 383 Tetruhymena, 407 Metazoan tissues, protistan phylogeny and, 319, 355 Methionine, mammalian stress repair and, 394 398, 400 Methotrexate, mammalian stress repair and, 379 5’-Methyladenosine, mammalian stress repair and, 396 Micelles, electron microscopy and, 166, 179 Microcephaly , nucleocytoplasmic interactions and, 294, 295, 301 Micronucleus cell growth and, 97 mammalian stress repair and, 404, 405 Micropinocytosis, mammalian stress repair and, 406 Microscope centrifuge, 2 1 Microsomes mammalian stress repair and, 384 protistan phylogeny and, 332 Microsporans, protistan phylogeny and, 353 Microsporidia, protistan phylogeny and, 353, 354 Microtome, electron microscopy and, 186, 187

Microtubules centrifuges and, 43 mammalian stress repair and, 385 mitosis and, 50, 89

44 1

centrosome cycle, 64,70, 71, 74-79 mitotic apparatus, 81-85 nucleocytoplasmic interactions and, 214 early embryonic development, 305 Xenopus. 281, 282, 285 protistan phylogeny and, 331, 332, 340, 343, 351, 352 Microtubule-organizing centers mitosis and, 65, 69, 71, 75, 88, 89 nucleocytoplasmic interactions and, 269 Mitochondria centrifuges and Euglena, 41 ground substances, 43 somatic cells, 3 1, 35 electron microscopy and, 163 mammalian stress repair and, 376 carnitine, 397, 398 hemoflagellates, 386, 390 stress protein, 382, 384 mitosis and, 69 nucleocytoplasmic interactions and, 265, 270 protistan phylogeny and, 330, 331 chlorophyte series, 335 evolutionary lines, 337, 339, 340, 343, 344, 347, 349-354 lower eukaryotes, 325, 326 Mitosis cell reproduction and, 93, 95 in budding yeast, 102, 104 cell growth, 97, 99 G , transit, 109-1 17 G2 period, 122, 123 centrifuges and, 35, 37 mammalian stress repair and, 405 nucleocytoplasmic interactions and, 309 in Acetabuluria, 254 cleavage, 287, 289, 290 cytoplasmic determinants, 299 later stages, 292 sea urchin eggs, 267 Xenopus, 280, 281, 284, 285 protistan phylogeny and, 33 I Mitotic apparatus cell reproduction and, 95 mitotic cycle and, 74, 79, 89 Boveri’s rules, 79, 80 monopolar, 8 1-83 new model, 83-87

442

INDEX

puppet show, 80, 81 nucleocytoplasmic interactions and, 289 Mitotic cycle, 49-51, 62, 63 centrosome cycle, 63, 78, 79 embodiment, 63-65 identification, 65 model, 74-78 natural history, 65-70 pole separation, 70-74 chromosome cycle, 54-57 condensation, 59, 60 factors, 60-62 interphase, 53, 54 structure, 58 mitotic apparatus, 79-87 whole cell cycle, 51-53 Mitotic poles centrosome cycle, 67, 69 mitotic apparatus, 81, 85 Monoclonal antibodies, centrosome cycle and, 65 Monophyly, protistan phylogeny and, 329, 334, 336, 340, 342, 345 Morphogenesis, nucleocytoplasmic interactions in, see Nucleocytoplasmic interactions, morphogenesis and Morphology mammalian stress repair and, 385, 393 nucleocytoplasmic interactions and, 249, 250

later stages, 291 sea urchin eggs, 255 Xenopus, 275, 276 protistan phylogeny and, 331, 337, 346 Morula, nucleocytoplasmic interactions and, 266, 306, 307 Mosaic eggs, centrifuges and, 25-27 Motor-end plate, electron microscopy and, 143-147 Mouse teratocarcinoma cell, cell reproduction and, 99, 114 MPF, see Maturation-promoting factor mRNA

cell reproduction and, 106, 110, I19 centrifuges and, 26 nucleocytoplasmic interactions and, 308, 309 in Acetabularia. 257, 259, 262, 264 cleavage, 287

cytoplasmic determinations, 294, 296299 early embryonic development, 304, 305 sea urchin eggs, 266-270 Xenopus, 273, 275, 276, 281, 282 Mucopolysaccharides, ionized groups and, 206, 207, 21 1 Muscle electron microscopy and capacitance, 172 synaptic disk, 184 transverse membranes of, 152- 154 transverse-tubule system of, 148-151 mammalian stress repair and, 389 nucleocytoplasmic interactions and, 294296, 298, 299 Mutagenesis, cell reproduction and, 115, 116 Mutants cell reproduction and cell growth, 98 GI-less cells, 115, 116 GI switch, 102, 103, 106 mammalian stress repair and, 406 mitotic cycle and, 74, 80 nucleocytoplasmic interactions and, 250, 308 early embryonic development, 301, 303307 Xenopus, 272 Mycoplasrna, mammalian stress repair and, 38 1 Myelin, electron microscopy and, 137 crayfish synapse, 176 spiral, see Spiral myelin unit membrane, 159, 163 Myelin lamellae, electron microscopy and, 137, 155, 157, 176 Myelin sheath, electron microscopy and, 137, 139, 146, 155 crayfish synapse, 176 unit membrane, 157, 163 Myofibrils, electron microscopy and, 148, 152, 153 Myxosporans, protistan phylogeny and, 354, 355 Myxosporidia protistan phylogeny and, 354, 355 Myxozoa, protistan phylogeny and, 354, 355

443

INDEX N Necturus, centrifuges and, 33 Negative charges, ionized groups and, 205, 222, 224, 230 Nereis, centrifuges and, 25 Nerve fiber, electron microscopy and, 134136, 141 crayfish synapse, 173, 174 myelin sheath, 155 spiral myelin, 146 unit membrane, 156, 157, 167 Neuroblastoma. centrosome cycle and, 75 Neuroblasts, cell growth and, 99 Neurospora, centrifuges and, 33 Neurotubules, electron microscopy and, 136 Nexus, electron microscopy and, 184, 185 Niiella flexilis, centrifuges and, 30 Norepinephrine, mammalian stress repair and, 387, 406, 407, 410 Nuclear division, cell growth and, 97 Nuclear envelope cell reproduction and, 11 1, 120 mitosis and, 51, 61, 71 nucleocytoplasmic interactions and, 279, 284, 290 Nuclear structure, DNA replication and, 120122 Nucleic acid mammalian stress repair and, 402, 403 nucleocytoplasmic interactions and, 256, 257, 296, 307 Nucleocytoplasmic interactions, morphogenesis and, 249-252 Aceiabularia, 252-264 cytoplasmic determinants, 293-299 early embryonic development, 301-307 fertilized eggs, 286-290 later stages, 290-293 molecular embryology, 307-309 sea urchin eggs, 264-270 Xenopus maturation, 276-282 oogenesis, 271-276 unfertilized eggs, 283-286 Nucleolus centrifuges and, 35, 37 nucleocytoplasmic interactions and, 259, 273,277,305

xi,

Nucleoplasm, centrifuges and, 37, 41, 42 Nucleoprotein, chromosome cycle and, 59 Nucleosides, mammalian stress repair and, 402, 403, 406 Nucleosomes chromosome cycle and, 58, 60 nucleocytoplasmic interactions and, 279, 281, 287, 289 Nucleotides, mammalian stress repair and, 403, 411 Nucleus cell reproduction and cell growth, 97 Go state, 119 GI-less cells, 112, 113 G I switch, 102, 107, 108 centrifuges and, 35. 37 mitotic cycle and, 49 centrosome cycle, 73 chromosome cycle, 55, 57-62 nucleocytoplasmic interactions and, 24925 1 Acetabularia, 252, 254-259, 261, 262, 264 cleavage, 286, 289, 289 cytoplasmic determinants, 295 early embryonic development, 301-303 sea urchin eggs, 265, 266, 269, 270 Xenopus, 273, 276, 282-284, 286 protistan phylogeny and, 330, 331, 347, 353 Nutrient deprivation, cell reproduction and cell growth, 97-99 G I switch, 102-104, 106 G I transit, 101

0 Oligosaccharides, ionized groups and, 205, 206 Oncogene, cell reproduction and, I18 Oocyte cell reproduction and, 110, 11I nucleocytoplasmic interactions and, 25 1 Acetabularia, 259, 261, 262 cleavage, 286 early embryonic development, 304, 306 later stages, - 293 sea urchin eggs, 267

444

INDEX

Xenopus, 271-273, 275-277, 279-282,

285 Oogenesis, nucleocytoplasmic interactions and, 250, 252 early embryonic development, 304, 307 sea urchin eggs, 267, 269 Xenopus, 271-276 Organelles centrifuges and, 26, 30, 31, 43, 44 mammalian stress repair and, 405 mitosis and, 65, 79 nucleocytoplasmic interactions and, 257 protistan phylogeny and, 331, 334, 340, 341, 344, 345, 350 lower eukaryotes, 325, 326 Organogenesis, nucleocytoplasmic interaction and, 251, 290, 292 Xenopus, 272, 284 Oscillation cell reproduction and, 1 I 1 nucleocytoplasmic interactions and, 28 1 , 289 Osmiophilia, chromosome cycle and, 65, 71

P Paracentrotus, nucleocytoplasmic interactions

and, 270 Paraflagellates, protistan phylogeny and, 35 1, 352 Paramecium, centrifuges and, 39-41 Parthenogenesis centrifuges and, 25 centrosome cycle and, 66, 67 nucleocytoplasmic interactions and, 25 1 early embryonic development, 301 sea urchin eggs, 264-267 Xenopus, 282 FCC see Premature chromosome condensation Pelomyx illinoisenesis, centrifuges and, 4 1 Perideneans, protistan phylogeny and, 347, 348 Peritoneal cavity, polyionic colloid particles and, 225-232 Peroxisomes, centrifuges and, 35 Phagocytosis, mammalian stress repair and, 376, 378, 406 Phagotrophy, mammalian stress repair and, 374, 376, 377, 395, 403, 407, 410

Phenylanine, mammalian stress repair and, 387, 389 Phosphatases, nucleocytoplasmic interactions and, 256, 298 Phosphate group, 204, 205, 212 Phosphoglycerides, ionized groups and, 204, 205 Phospholipids electron microscopy and, 164, 190 mammalian stress repair and, 407 Phosphorylation chromosome cycle and, 60 mammalian stress repair and, 385 nucleocytoplasmic interactions and, 277, 279, 282 Photosynthesis mammalian stress repair and, 376, 386 nucleocytoplasmic interactions and, 254, 255 protistan phylogeny and, 331, 332, 334, 339, 344 Phylum, protistan phylogeny and, 330, 336, 340, 342, 343, 345-353, 355 Phymocytes, protistan phylogeny and, 339, 340 Physarum cell reproduction and, 97, 98, 116, 117, 119, 121 chromosome cycle and, 60 mammalian stress repair and, 385, 41 1 Physicochemical studies, 4, 5 Pinocytosis, mammalian stress repair and, 374, 405, 406 Plasma membranes, electron microscopy and, 163, 170 Platelet derived growth factor, cell reproduction and, 107, 118 Pleurodeles, nucleocytoplasmic interactions and, 276 Polarity centrifuges and, 22, 25, 28-30, 43 nucleocytoplasmic interactions and, 250 Acetabularia, 254 cleavage, 286 early embryonic development, 304 later stages, 292 Xenopus, 272, 273 Polarization microscopy, I34 Pole separation, centrosome cycle and, 70-74

INDEX Polyadenylation, nucleocytoplasmic interactions and, 266, 269 Polyamines chromosome cycle and, 59 mammalian stress repair and, 372, 386, 388, 396, 41 I , 412 chemotherapy, 393 hypoxia, 408, 410 nucleocytoplasmic interactions and, 264 Polyanionic macromolecules, ionized groups and, 209, 210, 240 Polyanions, ionized groups and, 240 blood vessels, 236-238 cationic dyes, 209 colloid titration, 2 1 1 ferric hydroxide colloid, 214, 215 interstitial tissues, 207 peritoneal cavity, 230 Polyarchal formation, centrosome cycle and, 75 Polycationic macromolecules, ionized groups and, 209, 210, 240 Polycations ionized groups and cationic probes, 220 colloid titration, 2 10, 2 1 1 ferric hydroxide colloid, 214 interstitial tissues, 207 polyions, 233, 234, 236-238 mitotic cycle and, 59, 60 Polyethyleneimine, ionized groups and, 219, 220, 233 Polyglutaraldehye-embedding methods, electron microscopy and, 140, 156, 197, 194 Polyion complexes, ionized groups and, 207, 210 Polyionic macromolecules, ionized groups and, 240 Polyionic probes, ionized groups and, 224, 225 Polyionic tissue, cytochemical detection of, 208 Polyions, ionized groups and, 209 anionic colloid particles, 229-231 into blood vessels anionic ferric hydroxide colloids, 233 cationic probe, 232, 233 polyanions, 236-238 polycations, 234-238

445

colloidal probes, 220 ferric hydroxide colloids, 225-229 into intestine, 239 into lungs, 238, 239 Poly-L-lysine, ionized groups and, 222, 233, 236 Polymastigotes, protistan phylogeny and, 34 1 , 342 Polymonads, protistan phylogeny and, 341, 342 Polypeptide chains electron microscopy and, 163 ionized groups and, 205, 206, 236 Polyphyly, protistan phylogeny and, 338 Polyploidy nucleocytoplasmic interactions and, 259 protistan phylogeny and, 330, 343 Polysaccharides, ionized groups and, 203, 240 aldehyde fixation, 225 cationic dyes, 208 colloid titration, 210 colloidal probes, 225 glycoproteins, 206 interstitial tissues, 206 ionic dyes, 208 ionization, 2 1 1, 2 12 Polyvinyl sulfate ferric hydroxide colloid, ionized groups and, 221, 229, 230, 233 Postsynaptic membranes, electron microscopy and, 141, 142 crayfish synapse, 173, 174 synaptic disk, 177, 178 Potassium polyvinyl sulfate, ionized groups and aldehyde fixation, 222, 225 colloid titration, 21 1 colloidal probes, 221 Poterioochromonas malhamensis, mammalian stress repair and, 379, 395-397 Premature chromosome condensation mitosis and, 55-58, 61 nucleocytoplasmic interactions and, 270 Presynaptic membranes, electron microscopy and, 141, 142 crayfish synapse, 173, 174 synaptic disk, 177, 178 Progesterone, nucleocytoplasmic interactions and, 276, 277, 279, 281, 282, 285

446

INDEX

Prokaryotes mammalian stress repair and, 371, 372, 374, 376, 379, 411 stress proteins, 383, 385, 386 protistan phylogeny and, 319, 321, 322, 330, 345, 346, 349, 356 eukaryote distinction, 323, 324 serial endosymbiosis theory, 325 Proliferating cell nuclear antigen, cell reproduction and, 112, I16 Proliferation, cell reproduction and, 123 cell growth, 96, 99 Go state, 118 G1-less cells, 112, 114 G I switch, 104 G I transit, 100 Prometaphase, mitosis and, 86-88 Pronuclei, nucleocytoplasmic interactions and, 307 early embryonic development, 301 sea urchin eggs, 265, 266, 268-270 Xenopus, 285 Prophase cell reproduction and, 93, 110 mitosis and, 57, 62, 72 nucleocytoplasmic interactions and, 261, 280, 285 Protamine, ionized groups and, 21 1, 235 Protein, see also Ribosomal proteins; Stress proteins cell reproduction and cell growth, 97 Go state, 118 GI-less cells, 110, 112 G I switch, 103, 104, 106-109 G2 period, 123 nuclear structure, 120 centrifuges and, 18 electron microscopy and, 135, 195 crayfish synapse, 176, 177 freeze-fracture etch technique, 187-191, 193, 194 synaptic disk, 183 unit membrane, 157, 159, 161, 163, 167, 186 ionized groups and, 204, 240 aldehyde fixation, 222, 224, 225 colloidal probes, 220 interstitial tissues, 207 ionic dyes, 207

ionization, 211, 212 polyions, 232-236, 238, 239 mammalian stress repair and, 372, 375, 381, 384, 390, 394, 39.5, 400, 401, 407, 413 mitosis and, 59-61, 70, 89 nucleocytoplasmic interactions and, 307, 308 Acetabufaria, 264 cleavage, 287 cytoplasmic determinants, 294, 297 early embryonic development, 303 later stages, 291, 293 sea urchin eggs, 266 Xenopus, 271, 273, 277, 282 protistan phylogeny and, 332 Protein-kinase activity, cell reproduction and, 107, 118

Protein synthesis cell reproduction and, 97, 117 G1-less cells, 110, 111, 115, 116 GI switch, 103, 104, 106-109 nuclear structure, 122, 123 mammalian stress repair and, 371, 382, 383, 386, 401 nucleocytoplasmic interactions and, 307, 308 Acetabularia, 254-258 cytoplasmic determinants, 296-298 sea urchin eggs, 265, 268-270 Xenopus, 373, 375, 376, 379, 381, 382 Proteoglycans, ionized groups and, 206, 234, 238, 239 Protistan phylogeny, eukaryogenesis and, 319323, 330, 331, 355-358 diversity, 228-330 evolutionary lines, 333-345 isolated groups, 345-355 lower eukaryotes, 323-325 new techniques, 326-328 prokaryote distinction, 323, 324 serial endosymbiosis theory, 325, 326 Protooncogenes, cell reproduction and, 102, 118 Protoplasm, centrifuges and, 43, 44 animal eggs, 22, 23 bacteria, 42 viscosity, 30 Protozoa cell reproduction and, 95

INDEX centrifuges and, 22, 30, 39-41 nucleocytoplasmic interactions and, 249 protistan phylogeny and, 319, 324, 329, 332, 356 evolutionary lines, 334, 337, 338, 353 Protozoology mammalian stress repair and, see Mammalian stress repair, protozoology and protistan phylogeny and, 320, 321, 333, 356 evolutionary lines, 333, 334, 337, 340, 342, 346, 352 lower eukaryotes, 327 Pseudopodia, protistan phylogeny and, 330, 33 1, 337, 340, 350 hromycin, nucleocytoplasmic interactions and, 254, 257, 298 Pyrimidine ribonucleoside diphosphates, cell reproduction and, 347 Pymhophytes, protistan phylogeny and, 347

R Radioautography cell reproduction and, 93, 100- 102, 114, 120 chromosome cycle and, 56 nucleocytoplasmic interactions and, 56 protistan phylogeny and, 327 Radioimmunoassays, mammalian stress repair and, 372 Rana pipzens, nucleocytoplasmic interactions and, 283, 285 Rana temporaria, nucleocytoplasmic interactions and, 249 rDNA cell reproduction and, 121 nucleocytoplasmic interactions and, 272 Red blood cells electron microscopy and, 172 ionized groups and, 218, 222 Red cell surface, ionized groups and, 206, 217, 218, 238 Reducing agents, 2, 3 Regeneration, nucleocytoplasmic interactions and, 257, 262, 210 Replication mitosis and, 52, 88 centrosome cycle, 68, 69, 75, 78

447

chromosome cycle, 53-56, 59, 62 nucleocytoplasmic interactions and, 299 Acetabularia, 259 cleavage, 287, 289, 290 sea urchin eggs, 265, 269 Xenopus, 279 Reticulocytes, centrifuges and, 31, 33 Rhizoids, Acetabularia and, 252, 254, 259, 262 Rhizopods, protistan phylogeny and, 337, 338, 340 Rhodophytes, protistan phylogeny and, 346, 348, 349 Riboflavin, mammalian stress repair and, 398, 399 Ribonucleotide reductase, cell reproduction and, 119 Ribosomal proteins, cell reproduction and, 103 Ribosomes centrifuges and, 31 mammalian stress repair and, 402, 404 nucleocytoplasmic interactions and Acetabularia, 255, 258, 259, 261 early embryonic development, 305 sea urchin eggs, 265, 268, 270 Xenopus, 271, 272 Rifampicin, nucleocytoplasmic interactions and, 254 RNA cell reproduction and, 102, 103 centrifuges and, 26 electron microscopy and, 170, 190, 191 ionized groups and, 207, 208 mammalian stress repair and, 387, 390, 403, 404 mitosis and, 68, 69 nucleocytoplasmic interactions and, 307, 308 Acetabularia, 254, 256, 257, 259, 262, 264 cleavage, 289 cytoplasmic determinants, 297-299 early embryonic development, 304-306 sea urchin eggs, 266, 269, 270 Xenopus, 271, 273, 276, 277 protistan phylogeny and, 332 RNA polymerase, mammalian stress repair and, 397 RNA polymerase I cell reproduction and, 107

448

INDEX

nucleocytoplasmic interactions and, 262 RNA polymerase I1 cell reproduction and, 107, I10 nucleocytoplasmic interactions and, 262, 270 RNA polymerase 111, nucleocytoplasmic interactions and, 110 RNA synthesis, cell reproduction and cell growth, 97 G I transit, 106, 107, 117 G2 period, 122, 123 RNase, nucleocytoplasmic interactions and, 257, 296 rRNA cell reproduction and, 121 mammalian stress repair and, 374, 41 1 nucleocytoplasmic interactions, 305 Acetabularia, 262, 264 sea urchin eggs, 270 Xenopus, 27 1, 273, 279 protistan phylogeny and, 327, 347

S S period, cell reproduction and cell growth, 96, 97 GI-less cells, 109, 112, 113 G I switch, 102-105, 108, 109 GI transit, 100, 110 G2 period, 123 nuclear structure, 121, 122 S phase, nucleocytoplasmic interactions and, 287, 290 Saccharomyces cerevisiae mammalian stress repair and, 385 mitosis and, 50 Sarcodinids, protistan phylogeny and, 338, 340, 341 Sarcolemma, electron microscopy and, 152, 153 Sarcoplasmic reticulum, electron microscopy and, 153, 154 Scaffold, mitosis and, 58, 59 Schwann cell, electron microscopy and, 146, 157, 161, 173, 174 Sciatic nerve fibers, electron microscopy and, 157 Sea urchin eggs, nucleocytoplasmic interactions and, 249, 251, 264-270, 289, 307

Sedimentation, centrifuges and, 17, 19 Sepsis, mammalian stress repair and, 371, 373, 380 Septum, electron microscopy and, 141 Serial endosymbiosis theory, protistan phylogeny and, 325, 326 Serotonin ionized groups and, 236 mammalian stress repair and, 392, 410 Serum deprivation, cell reproduction and, 105, 106, 115, 116, 118 Serum growth factors, cell reproduction and, 104-106, 118 Sialic acid, ionized groups and, 206, 212, 214, 215 Skeletal muscle fiber, electron microscopy and, 134 Small nuclear RNA, nucleocytoplasmic interactions and, 273, 275, 289 Solenoids, mitotic cycle and, 56-58, 60, 88 Somatic cells cell reproduction and, 1 I I , 122 centrifuges and, 22, 30-32, 35 blood, 31-34 cell division, 35-37 organelles, 3 1 plant, 33 ionized groups and, 203, 206, 240 nucleocytoplasmic interactions and, 271 Somites, nucleocytoplasmic interactions and, 295, 296, 302 Spectrin, electron microscopy and, 189 Spermidine, mitosis and, 59 Spindle, mitosis and, 49, 51 centrosome cycle, 63, 64, 67, 69, 70, 76, 76 mitotic apparatus, 79-81, 83-87 pole separation, 71-73 Spindle fibers centrifuges and, 35 mitosis and, 50 Spiral myelin, electron microscopy and, 145148 Spirillum volutans, centrifuges and, 42 Spirogyra, centrifuges and, 29 Sporozoa, protistan phylogeny and, 342, 343, 353 Starvation, cell reproduction and, 97, 102, 103

449

INDEX Stephanopogan, protistan phylogeny and, 344, 346, 347 Stratification, centrifuges and, 43 Amoeba, 4 I animal eggs, 26 ground substance, 42, 43 somatic cells, 30, 33, 35 Streptococcus, mammalian stress repair and, 377 Stress proteins, mammalian stress repair and, 371, 374, 382, 411, 412 functions, 385, 386 hemoflagellates, 388, 389, 393 inducers, 382-385 Poterioochromonas malhamensis, 395 Tetrahymena, 405, 407 Stress repair, mammalian, see Mammalian stress repair Stronglyocentroius, nucleocytoplasmic interactions and, 269 Subcutaneous tissue, ionized groups and, 225, 226, 240 Sulfanilamide, mammalian stress repair and, 381, 412 Sulfate groups, cytochemical detection of, 211, 215, 225 Sulfonate groups, cytochemical detection of, 215 Sulfonic groups, cytochemical detection of, 218 Suspension media, centrifuges and, 21 Svedberg oil turbine ultracentrifuge, 18 Symbiosis, protistan phylogeny and, 325, 329, 332, 346 Symmetry, centrifuges and, 22, 25, 28-30 Synapses, electron microscopy and, 140, 141, I78 Synaptic disk, electron microscopy and, 177185 Synaptic vesicles, electron microscopy and, 144, 145, 178 Synchrony cell reproduction and GI-less cells, 110, 112-1 14 G I switch, 107 nuclear structure, 120, 121 electron microscopy and, 148, 153 nucleocytoplasmic interactions and, 286, 289

protistan phylogeny and, 330 Syndineans, protistan phylogeny and, 347, 348 Systemic lupus erythematosus, cell reproduction and. 112

T 3T3 cells cell reproduction and G,-less cells, 112, 1I6 GI switch, 104, 105, 107, 108 ionized groups and, 227 Telophase cell reproduction and, 97 centrifuges and, 37 mitosis and, 54. 62 Tetrahydrobioptenn, mammalian stress repair and, 373, 379, 387, 392, 393 Terrahymena cell reproduction and, 97 mammalian cell repair and, 376, 378, 386, 391, 392, 394, 395, 402, 404-407 Tetraploid cells, cell reproduction and, 109, 113 Thraustochytriaceans, protistan phylogeny and, 352, 353 Thylakoids, protistan phylogeny and, 335, 336, 339 [3H]Thymidine, cell reproduction and, 93 cell growth, 99 GI-less cells, 114, 115 G l switch, 102, 105 [14C]Thymidine, cell reproduction and, 93, 99 Thymidine kinase cell reproduction and, 102, 113, 119 nucleocytoplasmic interactions and, 258, 279 Thymidine triphosphates, cell reproduction and, 103 Thymidylate kinase, nucleocytoplasmic interactions and, 256, 259 Tobacco mosaic viruses, electron microscopy and, 190 Toluene, electron microscopy and, 137 Transcription cell reproduction and, 110, 1 1 1, 113, 119, 121, 122

450

INDEX

mitosis and, 79 nucleocytoplasmic interactions and, 292, 298 Acetabularia, 257, 258, 262, 264 cleavage, 287, 289, 290 early embryonic development, 302, 304, 305 sea urchin eggs, 267 Xenopus, 271, 273, 275. 276, 279, 282 Transcytosis, ionized groups and, 235, 239 Transfemn, mammalian stress repair and, 391 Transverse membranes, electron microscopy and, 152-154 Trauma, mammalian stress repair and, 373 Trirurus, cell reproduction and, 122 tRNA mammalian stress repair and, 414 nucleocytoplasmic interactions and, 275, 289 protistan phylogeny and, 327 Trypanosoma, mammalian stress repair and, 376, 379, 385, 388, 390, 394, 412 Trypanosoma cruzi, mitosis and, 51 Trypanasomatids, mammalian stress repair and, 373, 381, 382, 386-389, 394 biopterin, 392, 393 DNA recombination, 393, 394 heme, 390, 391 polyamines, 393 Trypanosomes mammalian stress repair and, 384, 387-390 protistan phylogeny and, 341 Tubulin mitosis and, 65, 70, 76, 77, 83 nucleocytoplasmic interactions and, 269, 305 Tumor cells cell reproduction and, 109 ionized groups and, 21 1, 234 Tumorigenicity, cell reproduction and, 104 Tyrosine ionized groups and, 222 mammalian stress repair and, 387, 389

U Ubiquitin, mitosis and, 60 Ultracentrifueation. 17. 43. 44

animal eggs and, 29 cancer cells and, 37 ground substance and, 43 somatic cells and, 31, 33, 35 Ultra-high voltage, protistan phylogeny and, 327 Unit membranes, electron microscopy and, 137, 155-172, 186 crayfish synapse, 173 freeze-fracture etch technique, 188, 189, 194 [3H]Uracil, cell reproduction and, 102 Uranium, centrifuges and, 19 UV-irradiation, nucleocytoplasmic interactions and, 295, 296, 301

v Viruses cell reproduction and, 120 centrifuges and, 17 ionized groups and, 205 mitosis and, 79 Viscosity, centrifuges and, 30, 33, 37, 42, 43 Vitamin B12, mammalian stress repair and, 378, 395-397, 400-402, 404, 413 Vitamins, mammalian stress repair and, 372, 378, 394, 411, 412 stress proteins, 384 tissue-cell culture, 381, 382 Vitellogenin, nucleocytoplasmic interactions and, 271, 272, 275, 276

W Withdrawal, cell reproduction and, 105 Wound healing, 3, 4

X Xenopus cell reproduction and, 99, 110, 11 1 , 113 nucleocytoplasmic interactions and, 252 Acetabularia, 262 cleavage, 290 cytoplasmic determinants, 299 early embryonic development, 303-305

45 1

INDEX maturation, 276-282 oogenesis, 271-276 sea urchin eggs, 267, 270 unfertilized eggs, 283-286 Xenosomes, protistan phylogeny and, 326, 327, 346, 349 X-Rays electron microscopy and, 134, 135, 139, 140 synaptic disk, 183 unit membrane, 157, 159, 161, 163, 164, 166, 186 mammalian stress repair and, 377 nucleocytoplasmic interactions and, 249 protistan phylogeny and, 327

Xylitol, mammalian stress repair and, 402, 403

Y Yeast, cell reproduction and, 95 G I transit, 106, 107, 115, 117 nuclear structure, 121

Z Zooflagellates, protistan phylogeny and, 341, 346 Zwitterions, ionized groups and, 205

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