International Review of Cytology, Volume 61

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME61

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN GARY G. BORISY ROBERT W. BRIGGS STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO CHARLES J. FLICKINGER M. NELLY GOLARZ DE BOURNE K. KUROSUMI MARIAN0 LA VIA GUISEPPE MILLONIG ARNOLD MITTLEMAN DONALD G. MURPHY

ROBERT G. E. MURRAY ANDREAS OKSCHE VLADIMIR R. PANTIC W. J. PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL WILFRED STEIN ELTON STUBBLEFIELD HEWSON SWIFT DENNIS L. TAYLOR TADASHI UTAKOJI ROY WIDDUS ALEXANDER L. YUDIN

INTERNATIONAL

Review of Cytology EDITED BY

G . H. BOURNE

J . F. DANIELLI

St. George's University School of Medicine

Worcester Polytechnic Institute Worcester, Massachusetts

St. George's, Grenada Wesr lndies

ASSISTANT EDITOR K . W. JEON Department of Zoology University of Tennessee Knoxville, Tennessee

VOLUME61

ACADEMIC PRESS New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich, Publishers

1979

COPYRIGHT @ 1919, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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ISBN 0-12-364461 -5 PRINTED IN THE UNITED STATES OF AMERICA

79 80 81 82

9 8 7 6 5 4 3 2 1

Contents LISTOF CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . .

ix

The Association of DNA and RNA with Membranes MARYPATMOYER I . Introduction . . . . . . . I1 . Membrane-Associated DNA 111. Membrane-Associated RNA IV . Some Additional Theoretical V . Conclusions . . . . . . . References . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2 21

34 39 40

Electron Cytochemical Stains Based on Metal Chelation DAVIDE . ALLENA N D DOUGLAS D . PERRIN

I. I1 . 111. IV . V. VI .

Introduction . . . . . . . . . . . . . . . . . Incipient Instability . . . . . . . . . . . . . . Postchelation . . . . . . . . . . . . . . . . "Robust" Complex Formation . . . . . . . . . Immunocytochemical Methods . . . . . . . . . Some Possible Developments . . . . . . . . . . References . . . . . . . . . . . . . . . . .

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63

64 65 71

74 76 80

Cell Electrophoresis

.

THOMAS G . PRETLOWII AND THERESA P . PRETLOW

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Medium for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of Electrophoresis to Specific Cell Separations . . . . . . . . . Viability and Function . . . . . . . . . . . . . . . . . . . . . . Effects of Lytic Enzymes on Electrophoretic Mob es of Cells . . . . . . . Adsorbable and Other Materials . . . . . . . . . . . . . . . . . . . . . Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isoelectric Focusing . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I1 . I11 . IV . V. VI . VII . VIII . IX . X. XI .

V

85 86 88 89 96 112 113 117 118

119 121 121

vi

CONTENTS

The Wall of the Growing Plant Cell: Its Three-Dimensional Organization JEAN-CLAUDE ROLANDA N D BRIGITTEVIAN

.

I 11. I11. IV . V. VI .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Some Characteristics of Plant Cell Wall Growth . . . . . . . . . . . . . . Organization of the Expanding Cell Wall . . . . . . . . . . . . . . . . . Modalities of Cell Wall Expansion . . . . . . . . . . . . . . . . . . . Possible Factors in Cell Wall Morphogenesis . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

129 130 132 149 154 160 161

Biochemistry and Metabolism of Basement Membranes NICHOLAS A . KEFALIDES. ROBERTALPER.A N D CHARLES C . CLARK I. I1. 111. IV . V. VI . VII . VIII . IX .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Morphology and Distribution . . . . . . . . . . . . . . . . . . . . . . Functions of Basement Membranes . . . . . . . . . . . . . . . . . . . Structural Chemistry of Basement Membranes . . . . . . . . . . . . . . . Basement Membrane Metabolism . . . . . . . . . . . . . . . . . . . . Immunochemistry of Basement Membranes . . . . . . . . . . . . . . . . Basement Membrane Changes in Disease . . . . . . . . . . . . . . . . . Supramolecular Organization of Basement Membranes . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . .

167 170 177 179 197 209 212 218 220 221 228

The Effects of Chemicals and Radiations within the Cell: An Ultrastructural and Micrurgical Study Using Amoeba proteus as a Single-Cell Model M . I . ORD I. I1. I11. IV . V. VI .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Micrurgy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Site and Mode of Action of Toxic Agents within the Cell . . . . . . . . Chemicals as Probes into Cellular Activities . . . . . . . . . . . . . . . . General Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of Amoeba proteus as a Model in Cell Biology and Toxicology . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

229 231 233 248 273 274 277

Growth. Reproduction. and Differentiation in Acanthamoeba THOMAS J . BYERS I . Introduction . . . . . . I1. Growth and Reproduction

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

283 286

vii

CONTENTS

I11. Molecular Biology and Differentiation . . IV . Beginnings of Acanrharnoeba Genetics . . V . Conclusions . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

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SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF PREVIOUS VOLUMES . . . . . . . . . . . . . . . . . . . . .

310 330 332 333 339 343

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List of Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

DAVID E. ALLEN(63), Anatomical Pathology Department, Prince Henry’s Hospital, Melbourne, Victoria 3004, Australia ROBERTALPER (1671, Departments of Medicine and Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania I9104 THOMAS J . BYERS(283), Department of Microbiology and Developmental Biology Program, The Ohio State University, Columbus, Ohio 43210 CHARLES C. CLARK(167), Departments of Medicine and Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania I9104 NICHOLAS A. KEFALIDES(167), Departments of Medicine and Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 MARYPATMOYER ( l ) , Thorman Cancer Research Laboratory, Trinity University, San Antonio, Texas 78284 M. J. ORD (229), Department of Biology, The University of Southampton, Southampton, and Medical Research Council Toxicology Research Unit, Carshalton, Surrey, England DOUGLAS D. PERRIN (63), Medical Chemistry Group, Australian National University, Canberra, Australian Capital Territory 2601, Australia

T H E R E SP.A PRETLOW ( 8 5 ) , Department of Pathology, University ofAlabama in Birmingham, Birmingham, Alabama 35294

THOMAS G . PRETLOW, I1 ( 8 5 ) , Departments of Pathology and Biochemistry, University of Alabama in Birmingham, Birmingham, Alabama 35294 JEAN-CLAUDE ROLAND (129), Universitt?de Paris, Ecole Normale Supkrieure, 75005 Paris, France BRIGITTE VIAN(129), Universitk de Paris, Ecole Normale Supkrieure, 75005 Paris, France

ix

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INTERNATIONAL REVIEW OF CYTOLOOY, VOL. 61

The Association of DNA and RNA with Membranes MARYPATMOYER Thorman Cancer Research Laboratory, Trinity University, San Antonio, Texas

. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . hkaryotes . . . . . . . . . . . . . . . . . . .

I. Introduction

11. Membrane-AssociatedDNA

A. B. Eukaryotes . . . . . . . . . . . C. Tmnsformation and Transfection . . . III. Membrane-Associated RNA . . . . . . A. Rokaryotes . . . . . . . . . . . B. Eukaryotes . . . . . . . . . . . IV. Some Additional Theoretical Considerations V. Conclusions . . . . . . . . . . . . References . . . . . . . . . . . .

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

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1 2 2 8 11

21 22 25 34 39 40

I. Introduction All models of membrane structure use the Danielli-Davson (1935) proteinlipid bilayer membrane as a baseline structure. A fluid mosaic (Singer and Nicolson, 1972) of proteins in a lipoprotein structural bilayer is the current conceptual model of organization of the multifunctional, nonrigid membrane. Noncovalent weak interactions (e.g., hydrophobic and electrostatic) provide membrane continuity. Biochemical and morphological studies have suggested that both RNA and DNA are attached to, or associated with, membranes. This appears to be a phenomenon ubiquitously seen in the biological kingdoms. Certain conformational and functional properties would be provided by the association of nucleic acids with membranes, but in many cases specific functions of this relationship remain to be elucidated. Disruption of the membrane by chemical means (e.g., detergent or high salt) or physical methods (e.g.. sonication) and subsequent association of nucleic acids with the resultant membranous fractions suggest membrane attachment but are not definitive. Except for the attachment of ribosome ribonucleoprotein particles to membranes, the electron microscope observations of nucleic acid-membrane association are open to question with regard to fixation artifacts (e.g., see Silva et al., 1976) and nucleic acid proximity, rather than attachment, to membranes. This article is a survey of the literature pertinent to the membrane association of nucleic acids. Supportive data are discussed, as are speculative aspects of the Copyright @ 1979 by Academic Press, Inc. All rights of rcproductlon in any form reserved ISBN 0-12-364461-5

2

MARY PAT MOYER

association of DNA and RNA with cell membranes. A more detailed account of many features of nucleic acid-membrane association is given in a recent monograph (Moyer and Moyer, 1980). 11. Membrane-Associated DNA

The membrane association of chromosomal DNAs appears to be a universal phenomenon which has been observed in prokaryotes, eukaryotes, viruses, and extrachromosomal DNAs. A variety of techniques have been employed to study this DNA-membrane association. Electron microscopy (EM) and autoradiography have provided morphological data. Further support has come from a variety of biochemical procedures including the preferential association of DNA with lipoprotein-rich extracts, selective salt extraction, density gradient centrifugation, incorporation of thymidine analogs (e.g., bromodeoxyuridine), and labeling with radioactive DNA precursors. A. PROKARYOTES 1. Chromosomal DNA

Biochemical extraction procedures and electron microscopy of whole cells have shown the circular bacterial chromosome associated with portions of the cell membrane (Table I). Similar observations have been made with other prokaryotes and prokaryote-like organelles such as mitochondria and plastids (Table I). This DNA-membrane interaction in Escherichia cofi involves the replication origin (Fielding and Fox, 1970; Parker and Glaser, 1974), which may remain bound even after the termination of chromosomal replication (Craine and Rupert, 1978). In some bacteria, a specific association with a membranous component, the mesosome [see Reusch and Burger (1973) for review], has been described. Whether or not the mesosome structure and, indeed, the DNA-membrane association is artifactual has been recently open to question. Silva et af. (1976) showed that the presence or absence of mesosomes varied with the fixation technique used, although chromosomal DNA appeared to remain attached to a membrane structure under the variety of conditions employed. Leibowitz and Schaechter (1975) have reviewed both the morphological and biochemical evidence supporting and refuting the association of the bacterial chromosome with the mesosomes or cell membrane. Attachment of the prokaryote chromosome to the cell membrane may provide a structural basis for DNA replication and chromosome segregation in vivo (Table I; Leibowitz and Schaechter, 1975; also see Lark, 1966, 1972; Pato,

MEMBRANE DNA AND RNA

3

1972; Pardee et al., 1973; Salton, 1967). Details of DNA replication in E. coli have been recently reviewed (Alberts and Stemglanz, 1977; Wickner, 1978). Based on phage and plasmid replication, at least three distinct DNA replication systems have been described in E. coli (Lewin, 1977; Schekman et al., 1974), and multiple modes of DNA synthesis may operate in Lactobacillus acidophilus (Soska et al., 1976). Although the association of DNA with membrane appears to be a prerequisite for in vivo DNA replication, the nature of the multienzyme-membrane complex is not known. The DNA-synthesizing proteins of E. coli have been isolated and are functional in a soluble form in vitro (Kornberg et al., 1974; Weiner et al., 1974). DNA synthesis is primed by (membrane-associated?)primer RNA (p’RNA), and both RNA and protein may be involved in binding the DNA to the membrane and in the initiation of DNA synthesis (Alberts and Sternglanz, 1977; Chargaff, 1976; Leibowitz and Schaechter, 1975; Schekman et al., 1974; Wickner, 1978). Membrane alterations are associated with certain DNA synthesis mutations in E . coli. Synthesis initiation (e.g., dna A, dna B ? ) and elongation of primed DNA (e.g., dnu B ) may be affected (Lewin, 1977). The DNA-membrane attachment varies during the log and stationary phases (Niveleau, 1974; Ryder and Smith, 1974; Ryter, 1968; Worcel and Burgi, 1972, 1974), although the replicative origin may remain membrane-bound throughout the cell cycle (Dworsky and Schaechter, 1973; Dworsky, 1976; Harmon and Taber, 1975). It should be noted, however, that criticisms of the interpretation of DNA replication associated with nuclear membranes in eukaryotes (Huberman, 1977) are applicable to membrane-associated replication of prokaryote DNA. Nonreplicating, essentially membrane-free nucleoid DNA is condensed into compact structures by folding and supercoiling (Petitjohn and Hecht, 1973; Worcel and Burgi, 1972, 1974). A continual synthesis of RNA, which binds to the folded bacterial genome to stabilize DNA folds and segregate domains of supercoiling, maintains the condensed state of the DNA (Petitjohn and Hecht, 1973; Worcel and Burgi, 1972, 1974). A variety of factors can affect the membrane association of the folded chromosome (Korch et al., 1976; Meyer et al., 1976a, b). The membrane-associated DNA (mbrDNA) of other prokaryotes and prokaryote-like organelles and plastids is replicated in a manner similar to that reported for eubacteria. Circular, double-stranded DNA (dsDNA) of mycoplasmas (Morowitz et al., 1967; Maniloff and Morowitz, 1972) is replicated at specific initiation sites that involve p’RNA (Maniloff and Quinlan, 1973, 1974; Quinlan and Maniloff, 1972, 1973; Smith, 1969). In blue-green algae, photosynthetic mechanisms, which are closely allied with membrane components, are also closely related to DNA synthesis (Leach and Herdman, 1973; Van Baalen, 1973), which proceeds by mechanisms similar to those observed in eubacteria (Allen, 1972).

STUDIES O N THE

TABLE I MEMBRANE ASWCIATION O F ~ O K A R Y O T EDNA

Organism

Observations

References

Esckrichia cofi

DNA bound at replication origin and/or during replication

Earhart er a f . (1968); FElding and Fox (1970); Fuchs and Hanawalt (1970); Gomez-Eichelmann and Bastarrach (1975); Kuippers and Stratling (1970); Kochiyama er al. (1966); Marsh and Worcel (1977); Parker and Glaser (1974); Smith and Hanawalt (1967); Stratling and Knippers (1971); Tremblay et al. (1%9) Abe d d.(1977); Ballesta er al. (1972)

ssDNA bound to membrane at replication origin; membrane isolation, nuclease sensitivity EM autoradiography; DNA synthesis enriched in membrane fractions RNA andor RNA polymerase may be involved in attachment of chromosome to the membrane and in nucleoid conformation

DNA and replication origin associated with outer membrane

Niveleau (1974) Dankberg and Cumrmn ’ gs (1973); Delius and Worcel (1974); Dworsky and Schaechter (1973); Dworsky (1976); Earha~lera!. (1973); Hanna and Carl (1975); Hecht and Petitjohn (1973); Petitjohn and Hecht (1973);Porter aad Fraser (1968); Stonington and Petitjohn (1971) Gudas et al. (1976); Nicolaidis and Holland (1978)

DNA attachcd at inner-uter membrane junction Synthesis of a special membrane region signals DNA replication initiation Bacillus subrilis

Heidtich and Olsen (1975);Olscn er al. (1974);Portalier and Worcel ( 1976) Helmstetter (1974a,bJ

Direct EM observation

Ryterand Jacob(1964,1966);KyterandLandman(1964)

EM, isolated membranes

Van llerson and G m n (1971)

DNA bound at replication origin andor during replication

Ganesan and Lederberg (1965);Hanawalt and Ray (1964);Harmon and Taber (1975);lvarie and Pene (1970); Smith and Hanawalt (1967):Sueoka and Hammers (1974);Sueoka and Quinn (1968); Yamaguchi and Yoshikawa (1975) lvarie and Peno (1970);Sueuka and Quinn (I 968);Snyder and Young (1969) Ivarie and Pene (1973) Cundliffe (1 970)

DNA bound at rephcakion terminus DNA bound at many regions of chromosome RNA involved in membrane attachment and nucleoid conformation

Pneumococcus

rnbrDNA replication complex

Firshein (1972);Firshein and Gilmore (1970)

Mycoplamia

EM morphology Unique attachment site; membrwe-associated replication complex

Anderson and Barile (1965) Morowitz et al. (1967);Quinlan and Manilo€f (1972, 1973);Smith (1969)

Blue-green algae

mbrDNA and DNA synthesis

Mann and Cam (1974)

6

MARY PAT MOYER

2. DNA Phage and Plasmids

Direct observation of the attachment of extrachromosomal DNAs of bacteriophages and plasmids to their host cell membranes is difficult. Membrane association of these DNAs (Table 11) during virus replication and maturation is inferred from experiments involving labeling, selective extraction techniques, or electron microscopy (which is generally preceded by labeling and/or selective extraction). Generally, the replicative form of the DNA and the enzymes for DNA replication appear to be membrane-associated (Clowes, 1972; Helinski, 1976; Korn and Thomas, 1971; Sakaki et a f . , 1971; Siege1 and Schaechter, TABLE I1 STUDIES SUGGESTING MEMBRANE ASSOCIATION OF PHAGE A N D PLASMID DNA AND DNA SYNTHESIS Host

Phage

References

Escherichia coli

A

Klaus eral. (1972a,b); Kolber and Sly (1971); Korn and Thomas (1971); Nishimoto and Matsubara (1972); Sakaki et al. (1971); Sakakibara and Tomizawa (1971) Brutlag eral. (1971); Forsheit and Ray (1971); Jazwinski er al. (1973); Wickner et al. (1972, 1978) Linial and Malamy (1970) Sinsheimer er al. (1968) Simon (1972) Buckley et al. (1972); Frankel (1966); Kozinski and Lin (1965); Simon (1972); Speyer er al. (1972); Tomich er al. (1974) Rosenkranz (1973) Center (1972); Hiebsch and Center (1977); Pacumbaba and Center (1973) Chattoraj (1978); Ljungquist (1973, 1976)

M13

4 4x174 T2 T4

T5 T7 P2 Salmonella

€22

Botstein (1968); Botstein and Levine (1968a.b); Darlington and Levine (1971); Levine et al. (1970)

Pseudomonas

PM2

Datta er al. (1971); Datta and Franklin (1969)

Mycoplasma

Mycoplasma virus

Host

Das and Maniloff (1976); Liss and Maniloff (1973)

Plasmid

References ~~

Escherichia coli

Mycoplasma

Col El Col E2 R factors A mutants Miscellaneous plasmids Plasmid

Blair er al. (1972); Inselberg (1974) Beppu and Arima (1972) Helinski (1973, 1976) Lieb (1970) Levy (1971); Shull er al. (1971) Zouzias er al. (1973)

7

MEMBRANE DNA AND RNA

1973). Phage or plasmid DNA may be attached at its origin of replication (Helinski, 1976; Siege1 and Schaechter, 1973), and DNA synthesis is usually primed by RNA (Table III;Helinski, 1976; Schekman et al., 1974; Westergaard et al., 1973; Williams ef al., 1973), as it is in the host bacterium (Keller, 1972; Klein and Powling, 1972; Kornberg, 1974). DNA synthesis in the uracilcontaining PBS 2 DNA phage of Bacillus subtilis is not primed by host RNA polymerase (Price et al., 1974) and may proceed by a novel mechanism. The membrane provides a site for the attachment of infectious viral DNA and for compartmentalization during virus assembly and maturation. Upon infection, some phages, e.g., M13 and 4x174, inject a protein which facilitates attachment of their DNA to the host cell membrane (Jazwinski et al., 1973; Schekman, personal communication). Newly synthesized T7 DNA associates with the host membrane (Center, 1972; Pacumbaba and Center, 1973), and an endonuclease essential for molecular recombination (Lee et al., 1976) is formed in association with the T7 DNA-membrane complex (Pacumbaba and Center, 1974). DNA TABLE 111 RNA PRIMING OF PHAGEA N D PLASMKI DNA SYNTHESIS Host Escherichia coli

Phage

References

A

T5 T7

Davies er al. (1972); Hayes and Szybalski (1973a,b, 1974) Geider et al. (1978); Gray et al. (1978) Blutlag er al. (1971); Dasgupta and Mitra (1978); Kessler-Liebscher and Staudenbauer (1 976); Olsen et al. (1972); Schneck et aZ. (1976); Wicker et aZ. (1972) Godson (1974) Alikhanian et al. (1976); Black and Gold (1971); Buckley et al. (1972); Speyer er al. (1972) Rosenkranz (1973) Miller (1972)

Plasmid

References

fd M13

.$x174 T4

Host

Escherichia coli

Episome Col El

R factor plasmid

Bazzicalup and Tocchini-Valentini (1972) Blair et al. (1972); Clewell and Evenchik (1973); Clewell et al. (1972); Sugino et al. (1975); Williams et al. (1973) Helinski (1973, 1976)

Staphylococcus aureus

Penicillinase plasmids

Johnston and Richmond (1970)

Mycoplasma

Plasmid

Zouzias er al. (1973)

8

MARY PAT MOYER

lesions in colicin-treated DNA may involve membrane association of the colicin and/or DNA (Beppu and Arima, 1972; Maeda et al., 1977; Saxe, 1975). Morphogenesis and maturation of mycoplasma viruses (Das and Maniloff, 1976; Liss and Maniloff, 1973; Maniloff et al., 1977), the enveloped phage PM2 (Braunstein and Franklin, 1971; Camerini-Otero and Franklin, 1972; CotaRobles et al., 1968; Dahlberg and Franklin, 1970; Datta etal., 1971; Espejo and Canelo, 1968; Franklin et al., 1969), and filamentous phages (Hsu, 1968; Ohnishe and Kuwano, 1971; Smilowitz et al., 1971, 1972; Trenkner, 1970; Wickner and Killick, 1977) are intimately associated with the host cell membrane (Siege1 and Schaechter, 1973). The membrane localization of phage coat proteins during maturation and assembly may involve endoproteolytic cleavage of coat protein by host proteases in the membrane (Chang et al., 1978). Alternatively, some proteins may be refolded as they enter the membrane (Wickner et al., 1978). Although most phages appear to replicate in association with the membrane, host membrane lipid synthesis (NUM and Groman, 1976; Thilo and Vielmetter, 1976) and membrane fluidity may not be required (Thilo and Vielmetter, 1976). It is interesting to speculate that interference phenomena observed in hosts containing certain viruses or plasmids (cf Fujimura, 1966) may be effected by competition to bind to cell membrane-associated polymerases either nonspecifically, because of physical structure, or specifically, as a result of base sequence homology. B. EUKARYOTES 1 . Nuclear DNA In eukaryotes, multiple chromosomes and the complexity of the nucleus and nucleosome structures preclude a direct comparison to the prokaryote nucleoid. However, many workers (Table IV), using a variety of cell types from numerous species, have reported an association of chromosomal DNA with the nuclear membrane. Most of the experimental evidence from these studies has been extensively reviewed by Franke (1974; also see Edenberg and Huberman, 1975; Huberman, 1977). The general consensus seems to be that the chromatin is anchored at multiple nuclear membrane sites, but its location relative to nuclear pore structures remains debatable (Ashley, 1974; Dyson, 1975; Franke, 1974). In most cells this attachment is disrupted during the prometaphase to late anaphase stages of mitosis, and in late meiotic prophase. In some protozoa, the chromosome material is membrane-associated throughout mitosis (cf. Hollande and Valentin, 1968; Leadbeater and Dodge, 1967; Kubai and Ris, 1969), possibly via a centromere or centromere-like region. The variety of observations supporting the special nature of this association have been reviewed (Franke,

MEMBRANE DNA AND RNA

9

1974). No overall general conclusions on structure-function relationships can be drawn from these observations, but the results suggest that the mbrDNA is predominantly constitutive heterochromatin, which may be preferentially attached at pericentromeric regions, and seems to be enriched in late-replicating DNA. This nuclear membrane-associated chromatin synthesizes little, if any, RNA. During meiotic prophase either telomeric or centromeric segments of the chromosomes may preferentially attach to the nuclear envelope at the termini of synaptonemal complexes, apparently to align and pair the homologous chromosomes. The DNA-membrane association is stable under a variety of conditions, including ultracentrifugation, high and low salt concentrations, and treatment with urea, detergents, or chelating agents (Franke, 1974). Although the majority of the DNA is susceptible to DNase degradation, some of it is protected by its close association with the membrane (Franke et al., 1973a; Franke, 1974). The functional role of the DNA-membrane association during DNA replication, which involves a number of enzymes (Wintersberger, 1977) and proceeds by standard RNA priming and discontinuous synthesis mechanisms (reviewed by Chargaff, 1976; Edenberg and Huberman, 1975; Huberman, 1977; Shimada and Terayama, 1976; Spadari and Weissbach, 1975; Werner and Maier, 1975), is also controversial. It is unclear as to how much DNA synthesis is membraneassociated, since DNA replication occurs at sites distributed throughout the nucleus (Table V). Initiation and/or termination may be membrane-associated (Kolodny, 1974; Spadari and Weissbach, 1975; Stavianopoulos et al., 1971), while continued DNA synthesis occurs in the internal nuclear regions. Cell cycle dependence with periodic changes at various stages reflects altered membrane association (Franke, 1974; Hildebrand and Tobey, 1973; Infante et al., 1973, 1976; Yamada and Hanaoka, 1973). The nature of a purported DNA-enzyme complex remains elusive (Fansler, 1974). Although a nuclear mbrDNA complex from cultured mouse cells can synthesize DNA in vitro (Infante et al., 1976), a soluble DNA-synthesizing system can be isolated from HeLa cells (Fraser and Huberman, 1977). A variety of DNA-synthesizing activities have been described. A DNA polymerase-like activity (Yoshikawa-Fukada and Ebert, 1971; Yoshida et al., 1971), an endogenous RNase-sensitive DNA-polymerizing activity (Cavalieri and Carroll, 1975; Cavalieri et al., 1975), a y-like DNA polymerase (It0 et al., 1976), and a DNA swivel (nicking-rejoining) enzyme activity (Yoshida et al., 1977) have been found in association with eukaryote nuclear membranes. However, other workers (Deumling and Franke, 1972) have been unable to detect deoxyribonucleotide triphosphate-incorporating activity in purified mammalian liver nuclear membranes. Table IV compiles results supporting a role of the membrane in DNA synthesis. Similar techniques have been employed in labeling studies suggesting membrane association of nascent DNA. Cells are labeled with t3H]thymidinefor a short

10

MARY PAT MOYER

TABLE IV DURING DNA SYNTHESIS STUDIES SUPPORTING EUKARYOTE CHROMOSOME-MEMBRANE ASSOCIATION ~~

Cell source

Observations and techniques used

References

I n vitro cell culture

Human Amnion HeLa

Lymphoid cells Various cell types where heterochromatin is lacking Chinese hamster Mouse 3T3 fibroblast cells

Lymphoma cells

DNA associated with nuclear periphery; EM, autoradiography M band contains nascent DNA

Isotope-labeled, newly replicated DNA associated with phenolaqueous interface upon extraction EM, autoradiography, chase of pulse-labeled DNA from the nuclear membrane to internal regions Antigen-stimulated; gradient purification, labeling EM autoradiography , morphology

Comings and Kakefuda (1968) Hanaoka and Yamada (1971); Yamada and Hanaoka (1973); Cabradilla and Toliver (1975) Levis et al. (1967); Friedman and Mueller (1969)

O’Brien et al. (1972)

Gottlieb er al. (1970) Milner (1969)

M band contains nascent DNA

Hildebrand and Tobey (1973)

M band and gradient fraction isolation of mbrDNA capable of in v i m synthesis Sucrose gradients; cesium chloride banding; decreased association of DNA with membrane

Infante et al. (1976)

Ormerod and Lehmann (1971)

(continued)

period (e.g., several minutes), and then the cells are lysed or the nuclei are isolated. The lysate or nuclear material is fractionated by various techniques, and the location of bulk nonreplicating DNA relative to labeled DNA is determined. In these types of studies the rH]DNA acts as if it were attached to a large, low-density,possibly hydrophobic cell component, which is presumably nuclear membrane. The M-band technique (Tremblay et al., 1969) is a selective salt extraction method by which a membrane-nucleic acid fraction can be preferentially isolated. The presence of labeled nascent DNA in this fraction has been taken as evidence for the association of newly replicated DNA with membrane.

11

MEMBRANE DNA AND RNA TABLE IV (continued)

Cell source Bovine Liver cells

Observations and techniques used

Gently extracted DNA-membrane complexes had newly replicated (labeled) DNA tightly bound to membrane (>DNase resistance) EM, sucrose gradient purification; small amountofDNAassociated with isolated nuclear membrane Organs or whole organisms Rat Liver High-salt membrane extract contains nascent DNA and mbrDNA enriched in satellite DNA but no preferential localization of DNA replication points or initiation Isotope-labeled, newly replicated Regenerating liver DNA at interface upon phenol or chloroform isoamyl extraction M band contains labeled, nascent Sea urchin DNA EM observation, Kleinschmidt Honeybee embryos (1962) technique In vivo labeling, DNA gently Guinea pig extracted from adrenal glands, gradient centrifugation; nascent DNA associated with membrane material EM morphology Fungi

References

Wanka et al. (1977)

Berezney er al. (1972)

Franke et al. (1970, 1973)

Franke et al. (1970, 1973)

Infante et al. (1973) Dupraw (1965) Barrieux er al. (1973)

Burnett (1968)

Huberman (1977) has presented arguments which suggest that previous interpretations of DNA-membrane association during DNA synthesis may be incorrect. This conclusion is based primarily on EM autoradiographs such as those listed in Table V. When cells were pulse-labeled in early S phase and then prepared by EM autoradiography after 2- 15 minutes, label was preferentially located at the nuclear periphery. These data suggest that the heterochromatin is late-replicating DNA, and that replication is not initiated on the nuclear membrane. Huberman (1977) attempted to explain the apparently contradictory results for membrane and nonmembrane association of DNA synthesis. A 30second pulse time was used, since it had been calculated that only 1.25 pm of

12

MARY PAT MOYER

TABLE V STUDIESINDICATING THAT DNA SYNTHESIS Is NOT PREFERENTIALLY ASSOCIATED WITH NUCLEAR MEMBRANE BUT MAYBE DISTRIBUTED THROUGHOUT THE NUCLEUS

In v i m cell culture Human KB cells HeLa cells Stimulated lymphocytes, embryo lung fibroblasts, epithelial cells from laryngeal carcinoma Chinese hamster

Rat liver

Physarum

Technique

THE

References

EM autoradiography EM autoradiography EM morphology and autoradiography

Blonde1 (1968) Erlandson and deHarven (1971) Milner (1969)

EM autoradiography

O’Brien etal. (1973);Williams andockey (1970); Fakan et al. (1972); Comings and Okada (1973); Huberman ef a[. (1973); Huberman (1977); Wise and Prescott (1973) Berezney and Coffey (1975a,b, 1977); Franke er al. (1973); Kashnig and Kasper (1969); Monneron et at. (1972)

EM autoradiography, biochemical extraction of nuclear matrix, labeled DNA EM autoradiography

Kuroiwa (1973)

DNA could be synthesized in 30 seconds (Huberman and Riggs, 1968). Synchronized Chinese hamster cells were pulsed, trypsinized, fixed, and sectioned for EM. Since grains were distributed throughout the nucleus in early S phase at sites too distant from the nuclear membrane to have allowed these DNAs to have been initiated there (as they were > 1.25 pm away), Huberman (1977) concluded that the nuclear membrane was not required for DNA replication. Franke (1974) and Huberman (1977) have similarly interpreted some of the biochemical experiments which suggest that the apparent membrane association of newly synthesized DNA may simply represent preferential adventitious adsorption of single-stranded DNA (ssDNA) (Fakan et d ,1972), which is present in eukaryotic DNA (Henson, 1978), and DNA-synthesizing enzymes (including nascent DNA) to membrane fractions (e.g., the M band). The “sticking” of newly replicated DNA to the hydrophilic-hydrophobic interface of a bufferphenol or buffer-chloroform-isoamyl alcohol extract (Ben-Porat et al., 1962; Fakan et al., 1972; Friedman and Mueller, 1969; Levis et al., 1967; Mizuno et al., 1971b) may be adventitious and be a result of the DNA conformation (Franke, 1974). The EM autoradiography data of O’Brien et al. (1972), indicating that label could be chased from the nuclear membrane to the internal regions,

MEMBRANEDNAANDRNA

13

may be explained by insufficient exposure time of the autoradiographs (Huberman, 1977). Many interpretations of the studies on DNA synthesis do not consider the skeletal structure of the nuclear matrix. The nuclear matrix is a flexible, dynamic structure which is closely coupled to nuclear function (Berezney and Coffey, 1977). At least some of the integral proteins of the nuclear membrane are associated with the internal nuclear matrix, with which newly replicated DNA of regenerating rat liver is closely associated (Berezney and Coffey, 1975a, b; 1977). Perhaps some of the variety of results on DNA-membrane binding and mbrDNA synthesis can be explained by the recent observation (Daskal et al., 1978) of membranous patches on isolated chromosomes. Although this could be an adventitious association, these patches may contain the mbrDNA synthesis enzymes actually located within the nuclear matrix but which are readily isolated with nuclear membrane fractions. Virus-specific gene products, the DNA-binding proteins, are involved in viral (and often cellular) DNA synthesis [e.g., simian virus 40 (SV40)T antigen, Butel and Soule, 1978; D’Alisa and Gershey, 1978; Martin and Oppenheim, 1977; Weinberg, 19771. Since the antigens are located within the nuclear matrix (in association with membranous patches?), as well as closely associated with the nuclear membrane, it is not unreasonable to assume that perhaps both sites are involved in DNA synthesis. The replication of different chromosome regions during characteristic times of the S phase may reflect more than one replication mechanism (Mattern and Painter, 1978) and/or a means of temporal regulation (Cabradilla and Toliver, 1975). It has also been suggested (Lark et al., 1966; Sved, 1966) that nuclear membrane association of sister chromatids may be involved in their segregation.

2. Cytoplasmic DNA Mitochondrial DNA (mtDNA) and plastid DNA exist as closed circular duplex molecules attached to, and replicated in association with, their membranes (Borst, 1972; Dyson, 1974; Kuroiwa et al., 1977; Manning and Richards, 1972; Manning et al., 1971, 1972; Newton and Burnett, 1972; Newton et al., 1973; Sprey and Gietz, 1973). The mtDNA-membrane association may be altered in transformed and tumor cells (White et al., 1975). Ribonucleotides, which may be p’RNA, have been detected in all mtDNAs thus far tested (Borst, 1972; Grossman et al., 1973; Miyaki et al., 1973; Wong-Staal et al., 1973). Although a variety of plasmidlike cytoplasmic DNAs have been detected in eukaryotes (Table VI), only those described by Guerineau et al. (1974) have a known function. These yeast plasmids, like basterial plasmids, appear to confer antibiotic resistance. It has been proposed (Guerineau et al., 1974) that they also play an informational role in the function and/or biogenesis of membranes. In a variety of studies, cytoplasmic DNA and DNA-binding proteins (Vaughan and

14

MARY PAT MOYER

TABLE VI EUKARYOTES I N WHICHSMALL, CIRCULAR DNAs, POSSIBLY PLASMIDS, HAVEBEENDESCRIBED Group

Organism

References

Fungi

Yeast Neurospora Saccharomyces

D. E. Griffith et al. (1975); Guerineau et al. (1974) Agsteribbe et al. (1972); Mishra (1976) Billheimer and Avers (1969)

Protozoa

Euglena Paramecium Trypanosomes

Nass and Ben-Shaul (1972) Dilts (1977) Ono et al. (1971)

Algae

Acetabularia

Green (1976)

Plants

Tobacco

Wong and Wildman (1972)

Insects

Drosophila

Stanfield and Helinski (1976)

Amphibians

Xenopus

Buongiomo-Nardelli er a f . (1976)

Mammals

Human Monkey Mouse

Smith and Vinograd (1972) Smith and Vinograd (1972) Rush (1973)

Comings, 1973) have been detected in close association with cytoplasmic memBell (1969, 1971) has termed the DNA, which he observed in branes (Table W). cultured embryonic cells, informational DNA. He has proposed that this DNA associates with membranes and serves as a template for RNA transcription during differentiation (Bell, 1969, 1971). The 15 and 25s dsDNA particles seen in the nucleus of developing tissues and cells (David et al., 1970) may later be involved in cytoplasmic translation control, as described by Felicitti and Urbani (1977). The role of these DNAs, if any, remains controversial, as some studies suggest that the small extranuclear plasmidlike DNA may be a result of nuclear breakdown from random sets of genes (Williamson, 1970; Williamson et al., 1976). Although DNA-synthesizing enzymes have been detected in association with isolated cytoplasmic membranes from a number of sources (Table VIII; also see Wintersberger, 1977), their actual location and role during the cell cycle are not known. Most of these are DNA polymerases which require p’RNA but have different cofactor requirements than most viral RNA-dependent DNA polyrnerases. Surface membrane association of DNA appears to involve membranous components and neuraminic acid (Aggarwal er al., 1974, 1975; Rosenberg, personal communication; Strothkamp and Lippard, 1976). Characterizations of the DNA have not been done, but it has been suggested (Schjeide and DeVellis, 1970) that

15

MEMBRANEDNAANDRNA

DNA as an integral membrane component would exist as a low-molecular-weight form (possibly s105 daltons). DNA may be released from the surface membrane in v i m and in vivo. Minute, ring-shaped nucleoprotein particles can be detected in conditioned cell culture medium (Narayan and Hendren, 1976) and in medium of stimulated lymphocytes (Rogers el al., 1972). DNA is present in normal sera (Cox and Gokcen, 1976) and may become elevated during certain disease states (e.g., systematic lupus erythomatosis). This information exchange may be augmented in virro by certain viruses (Ayad and Delinassios, 1975). 3. DNA Viruses Cellular membranes play an important role in eukaryote virus replication (Fenner, et al., 1974; Klenk, 1973; Russell and Winters, 1975). Attachment and penetration of an infecting virus or viral DNA occurs at the plasma membrane. Subsequent synthesis of viral DNA, RNA, and proteins is often membraneassociated, as are assembly and maturation of virions. Although some of the same arguments presented against mbrDNA synthesis of eukaryote chromosomal DNA (Huberman, 1977) are applicable to viral DNA synthesis, a number of results suggest that at least some replication is membrane-associated. The prototype viruses have been the papovaviruses, principally SV40, which assume a nucleosome configuration in productively infected TABLE VII CYTOPLASMIC DNAs OF EUKARYOTES Cell source

Observations

Embryonic cells in virro Human lymphocyte cell line (in virro)

DNA in discrete particles in cytoplasm, membrane-associated Cytoplasmic mbrDNA; synthesized in nucleus and then transported to cytoplasm; linear dsDNA (ca. 4.2 X lo6 daltons) In virro DNA synthesis on smooth membranes detected by fluorescence Unique, 15s DNA-containing particles may play a role in the initiation of protein synthesis High amounts of cytoplasmic DNA present during formation of secondary nuclei

Rabbit reticulocyte lysates Acetabularia

References Bell (1969, 1971) Hall er al. (1971); Kuo er al. (1975); Lemer er al. (1971); Meinke and Goldstein (1974); Meinke er al. (1973) Cavalieri el al. (1975)

Felicitti and Urbani (1977)

Boloukhere (1970)

16

MARY PAT MOYER TABLE VIII DNA-SYNTHESIZING ENZYMES ASSOCIATED WITH CYTOPLASMIC MEMBRANES Source

Human Normal diploid skin cells Skin cells from leukemic patients Normal lymphocytes

References

Scolnick er al. (1971) Scolnick et al. (1971) Bobrow ef al. (1972); Cavalieri et al. (1975); Jachertz (1973); Robert et al. (1972); Scolnick et al. (1971); Smith and Gallo (1972)

Human leukemic blood lymphocytes KB cells African green monkey Transformed kidney cells Bovine Calf thymus Rabbit Bone marrow cells Rat Regenerating liver Cell cultures Transformed cells Mouse BALB/3T3 normal and transformed cells Cultured mouse embryo fibroblasts Chicken Embryo cells

Gallo et al. (1972); Scolnick et al. (1971); Samgadharan et al. (1972) Sedwick et al. (1974) Scolnick et al. (1971) Goodman and Spiegelman (1971) Byrnes et al. (1973) de Recondo and Abadiedebat (1976) Coffin and Temin (1971) Bandyopadhyay (1975) Scolnick et al. (1971) J. Speckmiear and R. Moyer (unpublished) observations) Kang and Temin (1972)

cells and are replicated as part of the host chromosome in transformed cells (Fareed and Davoli, 1977). The DNA-binding protein (T antigen; Butel and Soule, 1978; D’Alisa and Gershey, 1978; Martin and Oppenheim, 1977; Weinberg, 1977) is involved in both viral and cellular DNA synthesis, probably at the site of initiation (J. Griffith et al., 1975; Jessel et al., 1975; Oppenheim and Martin, 1978; Reed et al., 1975). T antigen exists as both a membrane-bound form and an intranuclear matrix entity (Anderson er al., 1977; Deppert, 1978; Martin and Oppenheim, 1977). A T antigen-membrane-nascent SV40 DNA complex (LeBlanc and Singer, 1974) suggests that SV40 DNA synthesis is membrane-associated. The T antigen or a portion of the T-antigen molecule with cellular membranes may be the tumor-specific transplantation antigen (TSTA), in addition to its other functions (C. Chang et al., 1977; Martin and Oppenheim, 1977; Tevethia and Tevethia, 1976).

MEMBRANEDNAANDRNA

17

Replicating adenovirus DNA appears to involve membrane attachment (Doerfler el al., 1972; Pearson and Hanawalt, 1971), and adenovirus DNA-binding protein may function in DNA synthesis initiation and elongation (Van der Vliet et al., 1977). DNA synthesis as well as virion maturation and assembly of herpes viruses are closely associated with the nuclear membrane (Fenner et al., 1974). Cytoplasmic membranes are the site of poxvirus DNA replication and virion assembly (Fenner et al., 1974; Morgan, 1975). As in other DNA replication systems, p‘RNA seems to be involved in the synthesis of papovavirus DNA (Champoux and McConaughy, 1975; Hunter and Francke, 1974) and herpesvirus DNA (Hirsch and Vonka, 1974). For more details on general aspects of eukaryote cell membrane-DNA virus interactions, the reader is referred to Fenner (1974) and Russell and Winters ( 1975). C. TRANSFORMATION A N D TRANSFECTION 1. Prokaryotes Genetics and other aspects of bacterial transformation (cf. Lewin, 1977, Tomasz, 1973) are beyond the scope of this article which only considers nucleic acid-membrane relationships. During genetic transformation of bacteria (Table IX),the initial step is the irreversible binding of DNA to the cell surface (Dooley and Nester, 1973; Dubnau and Cirigliano, 1972a,b). After binding, host endonucleolytic activity may reduce the molecular weight of the bound DNA as much as 10-fold (Dubnau and Cirigliano, 1972b). Competence factor is a protein produced by competent bacteria (bacteria in a physiological state which allows them to incorporate extracellular DNA). This protein can induce a DNA-binding (and genetic transformation) capability in cells which are not competent (Tomasz, 1972, 1973); that is, it can “recruit” noncompetent cells to competency. Membrane-associated competence-specific

TABLE IX SOMEREPRESENTATIVE BACTERIAL TRANSFORMATION SYSTEMS Bacteria Bacillus subtilis Pneumococcus Streptococcus Hemophilus

References Dooley and Nester (1973); Dubnau and Cirigliano (1972a.b); Hams and Ban (1969); Levine and Strauss (1965); Wolstenholme er al. (1966) Set0 er al. (1975); Tomasz (1972, 1973); Tomasz and Mosser (1966) Tomasz (1972, 1973) Tomasz (1972, 1973)

SOME

TABLE X STUDIES O N TRANSFECTION OF EXOGENOUS h R 3 a M A T I O N INTO EUKARYOTIC CELLS*

Donor material

Defined SV40 DNA fragments

Metaphase c h o s o m e SV40transformed Chinese hamster celk SV40 DNA recombinants

Herpes simplex virus DNA fragments Herpes simpkx virus DNA or herpesvirus-transformed cell

Changes in recipient ceUs

References

Rat cells BSC-I monkey cells

Altered cell phenotype, expression of sume virus-specific antigens, persistence of viral subgenoms Transformation Infectivity of proviral DNA

Moyer and Moyer (1976b); Moyer er al. (1977, 1978); Hurtado, Moyer, and Moyer (in preparation) Atrrahams et a/. (1975) Shani et al. (1976)

Rat cells Monkey cells TI- mouse cells

Genome uptake and maintenance Fareed (1977) Genome uptake and maintenance Upcroft et al. (1977, 1978) Conversion to TK+ cells Maitland andMcDougall(l977)

TK- mouse cells

Conversion to TK' cells

Miuson er al. (1978)

Human cells TI- mouse cells

Infectivity of viral DNA

TK+,also galactokinase

ChinnadUrai er al. (1978) McBride et al. (1978)

Recipient cells Monkey cells

DNA Adenovinrs-2 DNA ChiDese hamster metaphase chromosomes

expression

Phosphotipid vesicles containing metaphase chromosomes of human-mouse hybrids A9MRBC2, containing the human X chromosome and HGPRT gene Rat metaphase chromosomes

Mouse HGPRT- A9 cells

HGPRT’

Mouse cells

T7 DNA bacteriophage

Hamster cells

Chinese hamster cell metaphase chromosomes Cellular DNA, DNA virustransformed cells

Permissive and nonpermissive host cells

Incorporation an, eplication of rat chromosomes None assessed, but phage DNA attached and penetrated DNA synthesis inhibition Transcription of A DNA Incorporation of exogenous DNA RecoveIyfrom senescence,i.e., transformation Roductive infection andor transformation

Cellular DNA, retrovirustransformed cells

Permissive and nonpermissive host cells

Productive infection andor transformation

Human lymphocytes Human fibroblasts Indian barking deer cells Senescent hamster lung c e l l s

=TK, Thymidine kinase; HGPRT, hypoxanthoguanine ribosyltransferase

Mukhejee er al. (1978)

iguc ii et al. (1973) Leavitt et al. (1976) Wenger et al. (1978) Geier and Merril (1972) Petriciani and Patterson (1974) Spandios and Siminovitch (1978) Boyd and Butel (1972);Graham (1977); Lipotich, Moyer, and Moyer (in preparation); Moyer and Moyer ( 1979) Hill and Hillova (1971); Hill cr al. (1974); Nicolson et al. (1976 and references therein)

20

MARY PAT MOYER

antigens, which bind transforming DNA (Seto et a f . , 1975; Tomasz, 1973), are present on competent bacteria, including pneumococci, streptococci, and Hemophilus. In B. subtilis, transforming DNA may preferentially associate with mesosomes (Harris and Barr, 1969), which are present in greater numbers in competent cultures than in noncompetent cultures (Harris and Barr, 1969; Wolstenholme et al., 1966). The transforming DNA probably stays in close association with the membrane until the time of integration (Dooley and Nester, 1973; Tomasz, 1973; Williams and Green, 1972). Since pairing and recombination events appear to occur on, or very close to, the membrane (Dooley and Nester, 1973; Tomasz, 1973), it is interesting that competent B. subtilis cultures have a higher incidence of single-strandednicks in their DNA than noncompetent cultures (Harris and Barr, 1969; Wolstenholme et al., 1966). Conjugal transfer of DNA to E. coli minicells (Shull et a f . , 1971) and genetic transformation of Pseudomonas (Khan and Sen, 1974), which appears to involve an RNA primer, are also membrane-associated. The efficiency of transformation in organisms which are poorly transformable can be increased upon membrane modification by creating spheroplasts and/or by treatment with salts, sugars, or heat (Molholt and Doskocil, 1978, and references therein). Transfection of prophage DNAs across bacterial membranes is facilitated by similar techniques (cf. Benzinger et al., 1978 and references therein). Eukaryote DNAs transfected into prokaryote recipients via plasmids or other cloning vehicles may be expressed in some instances (cf. Polisky et al., 1976). Although attempts to exchange genetic information of Mycopfasma species by transformation and conjugation were unsuccessful (Folsome, 1968; Maniloff and Morowitz, 1972; Smith, 197 1), DNase-resistant membrane binding of donor DNA occurred. Blue-green algae undergo genetic recombination (Van Baalen, 1973), although the role of membranes in the attachment and replication of transforming nucleic acid has not been defined. However, membrane alterations are commonly associated with mutagenesis, which may be mediated by a RNA-DNA complex excreted into the medium of growing Anacystis nidulans cultures (Herdman and Carr, 1971).

2. Eukaryotes Attachment, subsequent uptake, and possibly genetic expression of transfected nucleic acid involves membranes. Transfection of exogenous nucleic acids into eukaryotic cells has been reviewed (Bhargava and Shanmugan, 1971; Graham, 1977). Some additional studies employing this technique are listed in Table X. The plasma membrane generally provides a barrier to exogenous nucleic acids. However, calcium chloride and DEAE-dextran treatment of recipient cells enhances nucleic acid membrane attachment and subsequent uptake. The

MEMBRANE DNA AND RNA

21

mechanism of action is unknown, but perhaps the nucleic acid is protected from nuclease degradation by both its membrane attachment and association with the transfection-facilitating agent. Although the potential biological applications of genetically engineering eukaryotic cells with exogenous DNAs are numerous and exciting, only one eukaryotic system is known in which foreign nonviral DNA is naturally transferred in vivo and confers permanent phenotypic changes in the host. Crown gall tumors induced by Agrobacterium in plants appear to result from the transfer of plasmid DNA from the bacterium to the host (Drummond et al., 1977; Chilton et al., 1977). Some studies of xenogeneic tumor cells suggest that DNA may be acquired from the host (Fenyo etal., 1973; Goldenberg et al., 1974; Weiner e t a l . , 1972; Moyer et al., unpublished observations). The initial step in binding the DNA to the surface membrane might protect the tumor cells from being rejected (Rosenberg, personal communication). The DNA surface membrane association might be augmented by the presence of DNA-binding proteins on the surfaces (e.g., the TSTA form of SV40 T antigen), or by chemical and physical factors, which might act as carcinogens and mutagens (Kubinski et a l . , 1976). A number of studies have shown that DNA (particularly viral DNA) inoculated into animals is biologically active (cf. Graham, 1977 and references therein; Moyer et al., unpublished observations). No data, however, are available indicating whether or not a membrane-DNA binding step is involved. It is probable that the injected DNA becomes attached to surface membranes of the cells in the inoculated animal, but it cannot be ruled out that the DNA is phagocytized by macrophages or other cells, thus precluding a membrane-associatedattachment and penetration step.

111. Membrane-Associated RNA

It is difficult to separate adventitiously adsorbed RNA from cellular membrane components, but extensive biochemical and EM studies have shown that RNA is present as an integral membrane component and as a substructural part of membrane-bound ribosomes (Emster et a l . , 1962; Leive, 1973; Scherrer and Darnell, 1962; Shapot and Davidova, 1971). Membrane RNA is relatively insensitive to RNase, is soluble in organic solvents, and is most readily extracted with acidified water or hot (60°C) phenol or other standard solvents following treatment of the membrane with sodium dodecyl sulfate (Emster et al., 1962; Leive, 1973; Scherrer and Damell, 1962; Shapot and Davidova, 1971.) This membrane-associated RNA (mbrRNA) may play a variety of roles, including ribosome binding via rRNA-mbrRNA associa-

22

MARY PAT MOYER

tion, stabilization of mRNA by membranes, translation of mRNA on membrane-bound ribosomes, membrane association of p’RNAs for DNA synthesis, compartmentalization of template RNA for RNA-dependent RNA or DNA synthesis, and membrane-associated functions of tRNA. Many interesting and mysterious biological processes, particularly those concerned with differentiation, may involve mbrRNA. A. PROKARYOTES 1. Cellular RNA Certain prokaryote RNAs appear to be membrane-associated.The requirement for mbrRNA and protein to initiate DNA synthesis (see Table I) suggests that p‘RNA is membrane-associated; and isolation of a membrane fraction containing newly synthesized DNA, RNA, and protein suggests that other RNAs are also associated with membranes in bacteria (Knippers and Stratling, 1970; Miller et al., 1970; Rouviere et al., 1969; Tremblay et al., 1969; Van Knippenberg and Duijts, 1971; Van Knippenberg et al., 1971; Varrichio, 1972). Similar observations have been made in studies of mycoplasmas (Tourtellote, 1969) and bluegreen algae (Mann and Carr, 1974; Wolk, 1973). Three classes of ribosomes have been described in E. coli (Varrichio, 1972); Walsh and Cohen, 1974), whose RNA synthesis requires some protein component(s) integrated in the cell membrane (Kimura, 1976). The predominant class, which comprises over 50% of the total cell ribosomes, is cytoplasmic and not associated with membranes. The other two classes are membrane-associated,one presumably via the DNA (Cundliffe, 1970; Varrichio, 1972; Walsh and Cohen, 1974). Thus polysomes may be formed at the membrane as mRNA is removed from the DNA by ribosomes, as suggested by Bremer and Konrad (1964). A similar conclusion might be drawn from the results of Ishida et al. (1970), who observed that labeled ribosomes of blue-green algae were initially membraneassociated and that free ribosomes became labeled after prolonged incubation. However, other workers (Smith et al., 1978) have recent evidence which suggests that bacterial polysomes may only be attached to membranes via their nascent peptides. The interaction of a “signal peptide” region with the membrane may be involved in intramembrane localization or transmembrane transfer of certain proteins (Chang et al., 1978). The potential membrane association of initiator proteins and RNA during translation is beyond the scope of this article, although RNA conformation (which can be altered by a variety of factors, including membrane association) may effect a differential translation, that is, may modulate the frequency or start time of translation (Berissi et af., 1971; Lodish, 1971). In addition, it is of interest that ribosomes can stabilize mRNA (Walsh and Cohen, 1978).

23

MEMBRANEDNAANDRNA

The best candidates for bacterial mbrRNA are the stable mRNA species associated with membranes in sporulating bacteria and stationary-phase cells (Aronson, 1965a, b; Aronson and del Valle, 1964; del Valle and Aronson, 1962; Doi and Igarashi, 1964a, b; Petit-Glatron and Rapport, 1975; Scott and Bell, 1964). In Bacillus species, induced penicillinase formation and secretion correlate with mesosome formation and the presence of a mbrRNA (Ghosh et al., 1969). RNA from penicillinase-positive cells can be used to transform penicillinase-negative recipient cultures phenotypically (Kirtikar and Duerksen, 1968a, b). The transforming RNA may be dsRNA or a RNA-DNA hybrid (Spizizen et al., 1966). Other stable bacterial RNA species have been described (Table XI). Many of the ascribed functions of these RNAs involve membranes, but there are no definitive data for this association. The greater stability of these RNAs may be conferred by membrane association, as occurs in the sporulating bacteria. Since bacterial sporulation has been considered a primitive form of differentiation (Szulmajster, 1973), studies of this system may have applications to other differentiation systems (Hanson et al., 1970). The mRNA of sporulating bacteria may be synthesized by a RNA polymerase modified from the vegetative bacterial polymerase in the p subunit (Donachie et al., 1973). This might explain the insensitivity of the sporulation-phasepolymerase to actinomycin D (Aronson and del Valle, 1964; Hanson et al., 1970). A third form of RNA polymerase may be involved in transcription during germination of the spore to a vegetative cell (Kennet and Sueoka, 1971). Selective translation of the stable mRNA species (Legault-Demare and Chambliss, 1975) may be involved in bacterial differentiation processes (e.g., sporulation, germination, or morphogenesis of Caulobacter or other species). Enzymes such as ribonucleotide phosphorylase (Kinscherf and Apirion, 1975) or RNases (e.g., RNase III, DUMand Studier, 1973) may also exert transcriptional and translational control. Many of the transcription studies showing differences in RNA polymerases in vegetative and sporulating cells have utilized phage +e TABLE XI PROKARYOTES IN WHICH STABLE mRNA HASBEENDESCRIBED BUT ASSOCIATION WITH MEMBRANE Is NOTKNOWN Associated function

References

Clostridium perfringens Caulobacter

Sporulation, entemtoxin synthesis Morphogenesis

Escherichia coli minicells Bacillus subtilis

Outer membrane synthesis Flagellin synthesis

Labbe and Duncan (1977) Donachie er al. (1973) Newton (1972) Levy (1975) Martinez (1966)

Organism

24

MARY PAT MOYER

infection of B. subtilis. The altered host RNA polymerase, which is present during sporulation, prevents 4 e replication (Sonenshein and Roscoe, 1969). The DNA, change in the RNA polymerase disables transcription of the phage though it is an excellent template in vegetative cells (Losick and Sonenschein, 1969). No stable mbrRNA has been reported in mycoplasmas, but cell-free protein synthesis is stimulated 20-fold upon addition of a purified cell membrane fraction (Tourtellotte, 1969; Tourtellotte et al., 1967). Ribosome-membrane association, addition of membrane-associated factors or enzymes, or stable RNA templates could explain this increased efficiency of protein synthesis. In addition to mRNA, qualitative and quantitative differences in tRNA have been detected in dormant and germinating bacterial spores (Sussman and Douthit, 1973). Although these may exert modified translational control, they may also represent tRNAs involved in nonribosomal polypeptide syntheses (Table XII). Similar biosyntheses may operate in blue-green algae, since the chemical composition and formation of the cell envelope are similar to those of the eubacteria envelope (Drews, 1973; Wolk, 1973). The tRNA in the nonribosomal polypeptide syntheses is probably transiently associated with the membrane, as the enzymes involved in peptidoglycan chain assembly as well as the other syntheses shown in Table XI1 are membrane-associated (Glaser, 1973). Some of these tRNAs may be inactive in ribosomal polypeptide synthesis (Bumsted et al., 1968; Glaser, 1973). The tRNAs may also function as amino acid donors during the membraneassociated enzymatic syntheses of small peptides such as gramicidin S and tyrocidin (Kurahashi, 1974; Lipmann et al., 1971). It is of interest that unique, apparently modification-deficient forms of tRNAs are formed when E. coli is cultured under conditions which lead to imbalanced growth (Kitchingman et al., 1976).

+

TABLE XI1 FUNCTIONS OF PROKARYOTE tRNA NOT ASSOCIATED WITH RIBOSOMAL POLYPEETIDE SYNTHESIS Function

References

Aminoacyl-tRNA transfer of activated amino acids to cell wall peptidoglycan Aminoacyl-tRNA addition of terminal amino acids Aminoacyl phosphatidyl glycerol synthesis

cf. Bumsted et af. (1968); Ghuysen and Shockman (1973); Littauer and Inouye (1973) Littauer and Inouye (1973)

Synthesis of glycyl lipopolysaccharide

Gould et al. (1968); Lennarz et af. (1966); Nesbitt and Lennan (1968) Gentner and Berg (1971)

MEMBRANE DNA AND RNA

25

2. RNA Phage The various stages of replication of a RNA phage, including attachment, penetration, uncoating, and RNA replication, are closely associated with cellular membranes (Allison, 1971; Calendar, 1970; Haywood et at., 1969; Siegel and Schaechter, 1973; Strauss and Sinsheimer, 1963; Thach and Thach, 1973). Membrane-associated ‘‘replication complexes” synthesize RNA, and translation of the viral proteins principally occurs on membrane-associated polyribosomes. Two classes of membrane-binding replicative RNA have been observed in bacteriophage MS2 (Haywood, 1973), but their relative functions are obscure. It has been suggested (Haywood, 1971) that MS2 and rRNA compete for membrane synthesis sites. This may also be true of R23, whose RNA replication results in the inhibition of host rRNA synthesis and depends on the E. coli “RNA control” locus (Emberg and Skold, 1976). Conformational alterations of membrane-associated phage genomes and gene products are related to multiple gene function and regulation of RNA phage gene expression (Allison, 1971; Godson, 1968; Haywood et al., 1969; Kozak and Nathans, 1972; Price et al., 1974; Stavis and August, 1970; Sugiyama et al., 1972). An interesting example is Qp phage whose membrane-associated replicase functions can be changed by host translation factors (Haselkorn and Rothman-Denes, 1973; Zubay, 1973). Mutations in the replicase gene can essentially convert Qp to a RNA plasmid (Valentine et al., 1969). The importance of RNA phage in the study of prokaryote transcriptional and translational control mechanisms has been considered in reviews on bacteriophages (Allison, 1971; Calendar, 1970; Siegel and Schaechter, 1973) and protein synthesis (Haselkom and Rothman-Denes, 1973; Zubay, 1973).

B. EUKARYOTES

1 . Nuclear RNA RNA may comprise 1.4-9% of the total weight of isolated nuclear membrane fractions (Franke, 1974; Shapot and Davidova, 1971), but its role is a mystery. Since DNA synthesis involves p’RNA (Brewin, 1972; Chargaff, 1976; Edenberg and Huberman, 1975; Fansler, 1974), this RNA may be membrane-associated during the initiation of DNA synthesis. Some of the nuclear mbrRNA may be present as the dense material seen within the pores of the nuclear membrane (Franke, 1974). Active nuclear metabolism and RNA synthesis increase with pore frequency, and the ribonucleoprotein may stabilize the pore complex (Franke, 1974), possibly by acting as a core pivot structure (Shapot and Davidova, 197 1).

26

MARY PAT MOYER

Ribosomes tightly bound to the nuclear membrane (Franke et al., 1970; Kirschner et af., 1977) may reflect transport of completed ribosome structures from the nucleus (Sat0 et af., 1977a, b) or membrane-associated mRNAribosome complexes (Branes and Pogo, 1975). Direct translational control of certain nuclear membrane and intranuclear proteins might be provided by this association. Although it may vary during the cell cycle (Fakan and Nobis, 1978), RNA synthesis is principally associated with the euchromatin in the internal nuclear regions and not with the peripheral heterochromatin (Franke, 1974). However, some RNA may be synthesized in association with a DNA-nuclear membrane complex or the chromosome-associatedmembrane patches described by Daskal et al. (1978). The ADP ribosylation of nuclear proteins (reviewed by Hayaishi and Ueda, 1977) may regulate DNA synthesis and allow selective transcription during the cell cycle (Hayaishi and Ueda, 1977; Lehmann et al., 1974; Smulson et al., 1975; Stein et af., 1974; Tanigawa et al., 1978), but specific membrane association has yet to be defined. It has been proposed (Apirion et af., 1977; Dickson and Robertson, 1976) that small, stable RNAs in the nucleus may mediate gene expression at the transcriptional and/or translational level. Some small RNAs are transcribed in the nucleus, released into the cytoplasm, and then returned to the nucleus, where they are long-lived (Elicieri and Gurney, 1978). The small RNAs may provide binding sites for proteins involved in heterogeneous nuclear RNA (hnRNA) processing (Apirion et af., 1977; Calvet and Pederson, 1977; Fakan and Nobis, 1978). They may also represent the intervening “intron” sequence transcripts (Gilbert, 1978) which are removed from the hnRNA to generate the active RNA (Table XIII). The double-strandedness and other features of regions of the hnRNA and some of the nuclear RNAs (Calvet and Pederson, 1977; Stollar et al., 1978) would provide them with the potential to serve a regulatory function in translation (cf. Carter and deClerq, 1974; Sarma et af., 1978; Torelli et al., 1975) and also to serve as substrate for, or activator of, endoribonucleases (Clemens and Vaquero, 1978; Marcus et al., 1975; Rech e t a f . , 1976; Robertson and Mathews, 1973). There is no direct evidence demonstrating a membrane-associated regulatory role for the small nuclear W A S . They could associate with the nuclear membrane during or after hnRNA processing, upon transmembrane transport, or possibly with the endoplasmic reticulum and/or ribosomes to effect translational control. Other hypothetical membrane-associated phenomena might include stereochemical effects of membrane binding, functioning as a core pivot (Shapot and Davidova, 1971) or receptor for certain membrane or other proteins, or intercellular exchange upon release from the plasma membrane. The relationship of small nuclear RNAs, if any, to small cytoplasmic mbrRNAs (Elicieri, 1976) whose function is obscure, is unknown.

27

MEMBRANEDNAANDRNA

TABLE XIII STUDIES SUGGESTING THATEUKARYOTE DNA CONTAINS INTERVENING NONCODING (INTRON) SEQUENCES BETWEEN CODING (EXON)DNA Description of genetic source and described RNA or protein Mouse Globin Immunoglobin (light-chain) Rabbit Globin Ovalbumin Immunoglobulin Yeast tRNA viruses Papovaviruses (primarily SV40)

Adenoviruses

References

Tilghman et al. (1978); Gilmore-Herbert and Wall (1978) Jeffreys and Flavell (1977) Breathnach et al. (1977); Dugaiczuk et al. (1978); Weinstock et al. (1978) Brack and Tonegawa (1977) Goodman etal. (1977);O’Farrell etal. (1978); Valenzuela et al. (1978) Aloni et al. (1977); Berk and Sharp (1978); Haegemann and Fiers (1978); Hsu and Ford (1977); Kitchingman et al. (1977); Lavi and Groner (1977); Paucha et af. (1978) Berget et al. (1977); Chow et al. (1977); Klessig (1977)

An interesting type of RNA processing is observed in Amoeba proteus, in which the nuclear membrane-associated RNA helices may represent RNA synthesis complexes which subsequently break down into ribonucleoprotein (Stevens and Prescott, 1965; Wise et al., 1972).

2. Cytoplasmic RNA RNA is an integral membrane component of endoplasmic reticulum (ER) and plasma membranes (Shapot and Davidova, 1971). It is biochemically detected using methods modified from those of Emster et al. (1962) and Schemer and Damell (1962). EM morphology and autoradiographyprovide further support for this association. When considering the association of cytoplasmic RNA with the ER, the membrane association of ribosomes is important. Both free and membrane-associated ribosomes are found in the cytoplasm (Murthy, 1972; Noll and Burger, 1974; Ojakian et al., 1977; Sabatini et al., 1966; Shires and Pitot, 1972). No obvious differences in the rRNA size or base composition can be detected, but different turnover rates have been reported (Bourguignon and Katz, 1978). The membrane-associated ribosomes may be loosely or tightly bound (Rosbash and Penman, 1971a, b; Van Venrooij et al., 1975). Although ER membranes appear

28

MARY PAT MOYER

to have specific binding sites (Shires et al., 1971, 1975), the nascent polypeptide may exert additional binding effects (Adelman et al., 1973; Blobel and Dobberstein, 1975; Rosbash and Penman, 1971a, b; Van Venrooij el al., 1975; Wirth et al., 1977), possibly to generate more tightly bound ribosomes. Ribosomes may also be bound to membranes by some degree of complementary base pairing between mbrRNA and rRNA (Shires et al., 1971), or by specific recognition of mRNA by a class of membrane ribosomes (Borgese et al., 1973). Certain factors may specifically detach ribosomes from the membranes (Blobel, 1976; Rez et al., 1976). As in bacteria, the membrane association of ribosomes may be a function of the age of the culture. In the fungus Aspergillus niger, for example, ribosomes appear to be free in young hyphae and membrane-associated in old hyphae (Moyer and Storck, 1964). Some recently proposed models for the ribosome-membrane mRNA complex are interesting. Biogenesis of polysomes and transport of mRNA have been envisioned as coupled events (Branes and Pogo, 1975). Following transmembrane transport of the ribosome-mRNA complex from the nuclear membrane, the complex associates with the endoplasmic reticulum. Depending upon the characteristics of the individual complex and possibly upon the protein to be synthesized, the complex either remains associated with the membrane or is released as a free entity. Intermediate functional complexes that begin protein synthesis in association with the ER, but complete synthesis on free polysomes, would also be present. Alternatively, ribosomes may initially exist free and then become membrane-bound upon association of the nascent polypeptide with the membrane (Wirth et al., 1977), possibly via hydrophobic amino-terminal sequences (Palmiter et al., 1978). Neither of these models is mutually exclusive. Although most synthesis of secretory proteins occurs on membrane-bound polysomes (Yap et al., 1977), membrane, secretory, and nonsecretory proteins can be synthesized on both free and membrane-bound polysomes (Floor et al., 1976; Morrison and Lodish, 1975; Okuyama et al., 1977). These observations suggest that previous concepts of secretory and membrane protein synthesis occurring solely on membrane-bound ribosomes, with other protein synthesis occurring on free polysomes (Takagi et al., 1970), should be critically reviewed. Cytoplasmic membranes, alone or in combination with other factors (Table XIV), may stabilize certain populations of mRNA (Table XV). A variety of stable mRNAs have been described, whose definitive association with membranes is not known (Table XVI) but suggestive. The entire subject of mRNA structure and function has been recently reviewed by Cohn and Volkin (1977). mRNA may be bound to membranes indirectly by association with rRNA or protein components of the attached ribosome (Chang et al., 1977; Kabat, 1975; Kaempfer et al., 1978; Warren and Dobberstein, 1978). Alternatively the mRNA may be directly bound to the ER. Membrane attachment to ER may occur at the mRNA poly-A 3’-end (Cardelli et al., 1976; Lande et al., 1974, 1975;

29

MEMBRANE DNA AND R N A TABLE XIV FACTORS WHICHALONEOR IN COMBINATION CANSTABILIZE OR DESTABILIZE mRNA TEMPLATES Associated structures or molecules Membranes rRNA tRNA dsRNA Ribosomal or other proteins Structure of the mRNA S’cap “Leader” sequences Untranslated regions 3 ’ - ~ l y - Atail dsRNA or hairpin regions

Milcarek and Penman, 1974) and/or the “cap” 5’-end (Busch, 1976; Naora and Whitelam, 1975; Naora et al., 1974). It is of interest that both the 3’- and 5’-ends of the mRNA contain doublestranded RNA (dsRNA) regions (Naora and Whitelam, 1975), which may actually be hairpin noncoding regions (cf. Chang et al., 1977). These dsFWA regions might serve in membrane or ribosomal attachment and/or in functions attributed to dsRNA. Such functions include endoribonuclease activation (Clemens and Vaquero, 1978) and interaction with translation initiation factors (Carter and deClerq, 1974; Kaempfer and Kaufman, 1973; Levin and London, 1978; Safer et TABLE XV DIRECTASSOCIATION OF mRNAs WITH ER

OR

MICROSOMAL MEMBRANES

~~

Cells with membrane-associated mRNA Human diploid lung Rat liver cells Rat liver tumor cells Rat lung cells Mouse reticulocytes Chironornonas salivary gland Yeast Euglena Entamoeba Dictyosrelium Blastocladiella

References Adesnik et al. (1976); Lande er al. (1974, 1975) Cardelli etal. (1976);Pitot (1969);Pitot etal. (1969, 1974); Pitot and Shires (1973) Pitot (1969) Johannesen et al. (1977) Morrison and Lingrel (1975) Lonn (1977) Branes and Pogo (1975) Mandel (1967) Serrano er al. (1975) Bourguignon and Katz (1978) Lovett and Wilt (1975)

30

MARY PAT MOYER TABLE XVI EURKARYOTES IN WHICH STABLEmRNAs MAYBE MEMBRANE-ASSOCIATED

0r ganism

Stable mRNA associated function

Acetabularia

Morphogenesis

Paramecium aureliu

Protein synthesis in amacronucleate cells Spore germination

Allomyces

References Brachet (1967, 1968, 1970); Brachet et al. (1964); Dillard (1970); Hammerling (1963); Janowski and Bonotto (1970); Zetsch (1967); Zetsch et al. (1970) Kimball and Prescott (1964) Smith and Burke (1975)

al., 1976). Differential degradation and, consequently, stability of mRNAs, as well as mRNA-ribosome-membrane binding, may be partly a function of their 5’-end cap structure (Busch, 1976; Perry and Kelley, 1976; Rose, 1977; Sonenberg and Shatkin, 1977; Stiles et al., 1976). Posttranslational processing of proteins may involve membrane-associated enzyme activities (e. g., signal peptidase, Jackson and Blobel, 1977). Some cytoplasmic mRNA and other RNA may be amplified by membraneassociated RNA-dependent RNA or DNA polymerases (Table XVII). Subpopulations of mbrRNA in the cytoplasm could be true eukaryotic RNA plasmids TABLE XVII CYTOPLASMIC mbrRNAs A N D RNAs WHICHMAYBE SYNTHESIZED BY CYTOPLASMIC RNA- OR DNA-DEPENDENT RNA POLYMERASE Cell description Acetabularia Allomyces Blasrocladiella Euglena HeLa cells Lymphocytes

Rabbit reticulocytes Tobacco plants

Observations RNA synthesis in absence of nucleus, DNA in cytoplasm Stable mRNA during mitospore germination Stable mRNA during zoospore germination RNA-dependent RNA polymerase Small mbrRNAs in cytoplasm Cytoplasmic mbrRNA and DNA synthesis mbrRNA-dependent RNA polymerase mbrRNA-dependent RNA polymerase

References Dillard (1970); JanowskiandBonotto (1970); Werz (1974) Smith and Burke (1975) Lovett and Wilt (1975) Mandel (1967) Elicieri (1976) Jachertz (1973) Boyd and Fitschen (1975, 1977); Downey et al. (1973) Ikegami and Fraenkel-Conrat (1978)

31

MEMBRANE DNA AND RNA

(Table XVIU). The RNA detected on the surface membrane (Shapot and Davidova, 1971) may have a role in cell recognition, information exchange, or cell wall biosyntheses (Table XIX) . tRNA plays a key role in membrane and nonmembrane-associated protein synthesis, in addition to other biological activities, many membrane-associated. For reviews of tRNA and its multiple functions in the cell and during membrane biosynthesis, see Rich and RajBhandary (1976) and Soffer (1974).

3. RNA Viruses Smooth, membrane-bound replication complexes serve as the site of RNA synthesis for many RNA viruses. These complexes contain the viral RNA polymerase and replicative intermediate molecules. Viral proteins are synthesized elsewhere, principally on rough ER but also on free polysomes (Table XX; also see Fenner et al., 1974; Klenk, 1973; Russell and Winters, 1975). The genome-linked protein, VPg, described for poliovirus mRNA, appears to be cleaved from the mRNA prior to membrane-associated translation, suggesting that the enzyme which removes the polypeptide is on the membrane (Nomoto et al., 1977). Virus-infected mammalian cells have been used as model systems to study the synthesis of membrane and nonmembrane proteins. Vesicular stomatitus virus (VSV) and Sindbis virus have been used primarily, since each has a membrane envelope structure with associated proteins, in addition to their nonmembrane proteins. Glycoprotein G of VSV is synthesized from a membrane-associated mRNA, and the nascent polypeptide is inserted transmembranally into the membrane during synthesis (David, 1977; Morrison and Lodish, 1975; Morrison and McQuain, 1978; Toneguzzo and Ghosh, 1978). Another VSV membrane protein, M, which appears to be synthesized on both free and membrane-bound ribosomes (David, 1977), associates with host cell membranes after synthesis in TABLE XVIIl EUKARYOTIC CYTOPLASMIC RNAs WHICHMAYBE PLASMIDS Source

Associated characteristics ~~

~~

References

~

Saccharomyces cerevisiae

dsRNA plasmid in killer yeast strains; secretes a toxin that kills strains not carrying the plasmid; chromosomal genes affect maintenance and expression of the killer phenotype

Herring and Bevan (1974); Leibowitz and Wickner (1976); Vodkin and Fink (1973); Wickner (1976); Wickner and Leibowitz (1976)

Lymphocytes

Immune RNA; can transfer both humoral and cellular immunity

Cohen (1976); Fink (1976)

32

MARY PAT MOYER

TABLE XIX SOURCEA N D POSSIBLE ROLESOF RNA ASSOCIATED WITH EUKARYOTIC PLASMA MEMBRA.NES Source

Possible role

References

Rabbit ovarian follicles

Information exchange from plasma membrane

Brain synaptosomes or synaptic vesicles

Information exchange from plasma membrane

Albertini and Anderson (1974); Espey and Stutts (1972); Merk et al. (1973) Bondy and Roberts (1968); Ovscharoff (1976); Shashoua

Human lymphoid cells

Cell recognition; information exchange from plasma membrane Cell recognition; information exchange from plasma membrane Cell wall biosynthesis involving dictyosome vesicles Isoaccepting membrane tRNAs involved in enhancement of glycosyl transferase ectoenzyme systems during gametic contact Deposition of membrane vesicles during growth

(1973)

Ehrlich ascites tumor cells Acetabularia Chlamydomonas

Fungal hyphae

Weiss and Mayhew (1966, 1967); Weiss and Sinks (1970) Weiss and Mayhew (1969) Werz (1974) McClean and Bosmann (1975)

Bartnicki-Garcia (1973); Syrop (1973)

preparation for virion assembly and budding (Morrison and McQuain, 1978). Thus an as yet unknown mechanism must be used for the insertion of membrane proteins which have been synthesized on free polysomes. In a study on the three structural proteins of Sindbis virus, Wirth et al. (1977) found that the mRNA which codes for all three proteins was translated primarily in association with membrane-bound polysomes. The nascent polypeptide is important in ribosome-membrane binding, and the virus structural proteins result from proteolytic cleavage of the longer protein molecule (Wirth et al., 1977). The membrane glycoproteins associate with the lipid bilayer ER membrane, whereas core protein localizes on the cytoplasmic side of the ER. The retroviruses have a characteristic mbrRNA-directed DNA polymerase (Green and Gerard, 1974; Leis and Hurwitz, 1972; Temin, 1974; Temin and Baltimore, 1972; Verma et al., 1971), and their entire replication cycle is closely associated with host cell membranes (Dalton, 1972; Dalton and Hagenau, 1973; Dalton ef al., 1975; DeHarven, 1974; Fenner et al., 1974). Virus-specific DNA synthesized on cytoplasmic membranes from the viral RNA is a precursor of the covalently closed circular viral DNA which integrates into the host chromosome (Shank and Varmus, 1978; Varmus ef al., 1974). Infectious virus particles bud from the surface membrane after the viral nucleic acid-containing nucleoid aligns

MEMBRANE DNA AND RNA

33

itself at surface membrane sites where viral envelope proteins have been inserted (Dalton, 1972; DeHarven, 1974; Dubois-Dalcq et al., 1976). Viruslike particles are also seen in close association with ER membranes, but it is not known if they are infectious. Rough ER is the main site of viral protein synthesis, and the firm adherence between viral structural proteins and membrane proteins may provide some compartmentalization (Lueders and Kuff, 1975; Lueders, 1976; Van Zaane et al., 1975). It is of interest that the morphogenesis of intramitochondrial viruslike particles resembles that of retroviruses (reviewed by Lunger and Clark, 1976), and these mitochondrial particles may simply represent normal constituents which have gone awry. It is perhaps the membrane-associated nature of the viroids (Table XXI) which results in their physicochemical properties as well as their pathological effects. Pathogenesis of some viroids may also be explained by cell fusion induction (Kidson et al., 1978). Most viroids are sensitive to agents which destroy membranes but are resistant to nucleases, proteases, heating, and irradiation (reviewed by Diener, 1972a, b; Hunter ef al., 1973; also see references in Table XXI). A model of viroid secondary structure has been proposed (Henco et al., 1977). It may be that viroid RNA serves a regulatory function in the infected organism (Grill and Semancik, 1978; Semancik et al., 1977), but the infecting viroid determines its own nucleotide sequences (Dickson et al., 1978). The

TABLE XX SOMERNA VIRUSESWHOSEREPLICATION A N D MATURATION ARE MEMBRANE-ASSOCIATED Virus ~~

References

~

Cytoplasmic membrane Kungin flavivirus Poliovirus Newcastle disease virus Encephalomyocarditis virus Foot-and-mouth disease virus Lactic dehydrogenase virus Sindbis virus Tobacco mosaic virus St. Louis encephalitis Vesicular stomatitis virus Nuclear membrane Japanese encephalitis virus

Boulton and Westaway (1976) Caliguiri and Tamm ( 1 969); Girard et al. (1 967); Penman (1964); Wiegers et al. (1976) Marcus and Zuckerbraun (1970) Dalgamo and Martin (1965); Dalgamo er at. (1966); Hortoner al. (1964) Polatnick and Arlinghaus (1967) Darnell and Plagemann (1972); Fenner et al. (1974) Sreevalsan (1970); Wirth et al. (1977) Scotnicki ef al. (1976); White and Murakishi (1977) Qureshi and Trent (1972) Atkinson er al. (1976); Brown and Riedel (1977); Momson and McQuain (1978); Orenstein et al. (1975) Kos etal. (1975);Takedaetal. (1977, 1978);Zebowitzetal. (1 974)

Pichinde virus

Banejee et al. (1976); Farber and Rawls (1975)

34

MARY PAT MOYER TABLE XXI rnbrRNA VIROIDS Disease

Natural host

Scrapie

Sheep

KUN, Creutzfeldt-Jacob disease

Human

Spindle tuber disease

Potato

Exocortis disease

Citrus plants

References Clarke and Millson (1976); Diener (1972a); Hunter (1972); Hunter etal. (1973);Kimberlin et al. (1971); Kirnberlin and Walker (1978); Lampert er al. (1971); Semancik et al. (1976a); Siakaras et al. (1976) Gajdusek (1973, 1977); Gajdusek and Gibbs (1973) Diener (1972b); Diener (1973); Hadidi and Diener (1978); Owens et al. (1977) Semancik and McGeelan (1975); Sernancik etal. (1976b, 1977); Semancik and Vandenvaude (1976); Grill and Semancik (1978)

regulation might be exerted directly, or via a DNA intermediate (Semancik and McGeelan, 1975). Hepatitis is an unusual virus which has a variety of membranous and nonmembranous structural forms (Fenner et al., 1974). Although one of the hepatitisassociated particles may represent a viroidlike mbrRNA, the other is a DNAcontaining entity (Hruska et al., 1977; Joswiak e? al., 1971, 1975).

IV. Some Additional Theoretical Considerations The membrane association of DNA or RNA may be involved in, or explain some of the mysteries of, the following areas of active research inquiry. These are discussed in more detail elsewhere (Moyer and Moyer, 1980). A variety of immune phenomena can result from the transfer of immune RNA (Table XXII; also see Cohen, 1976; Gottlieb, 1973; Fink, 1976). The hot-phenol extraction techniques used to extract the RNA are essentially modifications of the original Schemer and Darnel1 (1962) method, which readily extracts mbrRNA (Shapot and Davidova, 1971). It has been suggested (Jachertz, 1973) that immune RNA is produced in macrophages and then transferred to immunocompetent lymphocytes via intercellular membrane bridges. Immune RNA is a rather heterogeneous population upon isolation. Some of the functional RNA appears to be mRNA, while the other RNAs belong to no specific class (i.e., mRNA, rRNA, or tRNA). The immune RNAs may serve a core pivot structural role (Shapot and Davidova, 1971), be p’RNA(s) (Chargaff, 1976), act as viruslike templates for endogenous RNA-dependent DNA andor RNA-dependent RNA

MEMBRANEDNAANDRNA

35

polymerase (Jachertz, 1973), or have a control function as yet undefined. The immunity induced by RNA of ribosomal preparations from bacteria (Table XXIII) are also very likely membrane-associated and may initiate immune responsiveness by mechanisms similar to those suggested for immune RNA. For further discussion of the involvement of membrane-associated nucleic acids in the immune response see Moyer and Moyer (1976a, 1980). Changes in eukaryote cellular growth patterns, resulting in indefinite growth transformation (Ponten, 1971) and tumorigenesis, may reflect changes in the membrane association of DNA or RNA. Proviral DNAs may code for membrane-associated DNA-binding proteins (e.g., SV40 T, U, or TSTA antigens; Anderson et al., 1977; Oppenheim and Martin, 1978; Weinberg, 1977) responsible for cell transformation. The DNA-binding protein could alter the membrane association of host cell chromatin, possibly resulting in increasing the frequency of DNA synthesis initiation (Oppenheim and Martin, 1978). The membrane-associated transfer and subsequent integration of plasmid DNA from Agrobacterium tumefaciens into plant cells results in tumor formation (Kerr et al., 1977; Lippincott, 1977; Matthyse and Stump, 1976; Van Larabeke et al., 1974; Watson et al., 1975; Zaenen et al., 1974). In addition, Beljanski et al. (1974) have reported that the tumorigenicity can be transferred by A . tumefaciens RNA, which may have an associated RNA-directed DNA polymerase. The binding of chemical carcinogens to nucleic acids and membranes may explain their ability to induce tumors (Brookes, 1975; Heidelberger, 1975; Irving, 1973; Mekler, 1972; Yoshida and Holoubek, 1976). A variety of modifications in RNA structure, function, and synthesis (Table XXIV) may result from or cause cell transformation or tumor cell formation. Abnormal processing and a concomitant increase in membrane association of RNA (Gillespie and Gallo, 1975; Shearer, 1974) may be related to the decreased RNase activity seen in transformed and tumor cells (Daoust and de Lamirande, 1975). Changes in both membrane and nonmembrane-associatedribonucleoprotein particles, which accompany the decreased RNase activity, could ultimately result in abnormal growth regulation (Daoust and de Lamirande, 1975). As suggested by Pitot and co-workers (Pitot, 1969; Pitot et a l . , 1969) direct alterations in the membrane association of mRNA may result in phenotypic transformation, which might be a manifestation of translational control (Pitot, 1976; Pitot et al., 1974; Shires and Pitot, 1972; Sharma et al., 1976). The altered stability of the mRNA template may be responsible for the activation of developmental genes in neoplastic tran:formation (Fishman, 1976). Translational control by membrane attachment of ribosomes (cf. No11 and Burger, 1974), either directly or by nascent polypeptides, could effect cell transformation. The significance of the membranous RNA-containing inclusions seen in transformed and malignant cells (Hulanicka et al., 1977; Woessner and Roman, 1976) is unknown.

TABLE X X I I INDUCTIONOF IMMUNEPHENOMENA BY NUCLEIC ACIDS Type of nucleic acid RNA

Immunity (cell type) Humoral (B-cell)

Immune phenomenon induced by nucleic acid Antibody synthesis

Cell-mediated (T-cell)

8-12S, RNase (+),

lsograft rejection

ss-RNA? dsRNA? ssRNA? dsRNA, ssRNA? Antigen-RNA complex 8-12S, RNm (+), SSRNA? dsRNA? dsRNA?

Homograft protection

dsRNA?

Resistance to tuberculosis or other “intraceIlular” pathogens

dsRNA ribosomal fractions: sensitized cells or pathogenic organism

Allotype transfer Autoantibody production Increased antigenicity of autigen Transplantation immunity

RNA

Characteristics of the nucleic acid

Notes Possibly mRNA

May act as adjuvant

Microsomal RNA from sensitized lymphocytes of allogenic individual RNA from recipient percolated through donor graft Probably membrane RNA

Tumor immunity

dsRNA?

Cytoxicity Schwartzman reaction Lymphokine induction

dsRNA dsRNA dsRNA, ssRNA?

Macrophage activation

dsRNA, microsumal ILVA

Humoral and/or cell-mediated

Fewer stem cells Ocular toxicity Fewer stem cells Increased tumor incidence

dsRNA dsRNA dsRNA dsRNA, ssRNA?

Unknown

Strung immunosuppmsiveeffect Unknown

dsRNA Linear? dsDNA

Fmm tumor immune donor protection in nonimmune recipient; regression of primary tumors; no effect on established tumors

RNase (+I, cross-species baniers Direct rnacmphage-activating factor induction, indirect via interferon

RNA extract from immunized cells; m a y be for blocking anti-

M Y mbrDNA

May be nonspecific 2% of total cellular DNA (30%of which is unique)

38

MARY PAT MOYER

FROM BACTERIA

WHICH

TABLE XXIII EFFECTIVE RIEIOSOMAL VACCINES HAVEBEENPREPARED

Organism

References

Mycobacterium tuberculosis Salmoneila typhimurium

cf. Youmans and Youmans (1974 and references therein) Venneman(l972);VennemanandBigley(1969);Venneman eral. (1970) Winston and Berry (1970a,b) Winston and Berry (1970a,b) Thompson and Snyder (1971) Thomas and Weiss (1972)

Sraphylococcus aureus Pseudomonas aeruginosa Diplococcus pneumoniae Neisseria meningir idis

The increased glycolysis in malignant cells (Warburg, 1956) might reflect altered membrane association of mtDNA (D’Agostino and Nass, 1976; Paoletti and Riou, 1973; White et al., 1975) mitochondrial RNA (mtRNA) (Lunger and Clark, 1976), or mitochondria and ER (Shore and Tata, 1977). The presence of retrovirus-like particles in mitochondria (Lunger and Clark, 1976) may reflect changes in mtRNA. The characteristics of RNA tumor viruses (retroviruses) are described in Section III, B, 3. Although some viruses of this group can induce leukemias or sarcomas in their hosts, no pathogenicity or oncogenicity has been demonstrated

TABLE XXIV CHANGES IN RNA POPULATIONS IN TRANSFORMED A N D TUMOR CELLS ~

Observations rRNA mRNA

tRNA

mRNA, rRNA, tRNA, others?

Increase in membrane-bound ribosomes in transformed cells Membrane associationof mRNA from transformed and tumorcells; differs from that of normal cells Qualitative, quantitative and functional differences in the tRNA populations and tRNA-associated enzymes

Altered RNA transpolt from nucleus to cytoplasm Release of RNAs normally restricted to nucleus into the cytoplasm

References No11 and Burger (1974) Pitot (1969, 1976); Pitot and Shires (1973); Pitot er al. (1969, 1974); Shires and Pitot (1976) Agris et al. (1974); Borek and Kerr (1972); Itoh et al. (1975); Kuchino and Borek (1978); Ouellette and Taylor (1973); Randerath er al. (1974); Sharma (1973); Sharma et al. (1975);YangandNovelli (1968) Schumm er al. (1973) Shearer (1974); Shearer and Smuckler (1971, 1972)

39

MEMBRANE DNA AND RNA

TABLE XXV OBSERVED VIRUSLIKE PARTICLES WHICHAREAPPARENTLY NONINFECTIOUS AND MAYSIMPLY REPRESENT ABERRANT mbrRNA Source and description of organ, organelle, or cell type Murine pituitary tumor Thraustochytrium ER Neurospora mitochondria Mitochondria of transformed and tumor cells Human malignancies Placentas

Embryos: pre- and postimplantation

References Petrea and Gardner (1973) Pollack (1975) Schatz and Mason (1974) Lunger and Clark (1 976) Gillespie and Gallo (1975); Kufe etal. (1973a.b); Steinke et al. (1976) Dalton et a / . (1974); Feldman (1975); Feldman et al. (1976); Kalter et al. (1973a,b); Schidlovsky and Ahmed (1973) Church and Schultz (1974); Yang et al. (1975)

for the majority of nonrodent viruses. Many of the retrovirus-like particles may actually be abnormal mbrRNAs whose appearance is viruslike when seen in the electron microscope (Dalton, 1972; Dalton and Hagenau, 1973; Dalton et al., 1974, 1975; DeHarven, 1974). Morphogenetic or cytodifferentiation events in which retrovirus-like particles have been detected are exemplified by the studies shown in Table XXV. Some of these viruslike particles may actually reflect abnormal RNA processing (Gillespie and Gallo, 1975) or normal RNA processing and not be viruses at all.

V. Conclusions The membrane association of DNA and RNA provides a means for structurefunction compartmentalization. At least some DNA replication of both prokaryotes and eukaryotes, and their viruses, is membrane-associated. In prokaryotes, the initiation and/or termination of DNA synthesis may occur at the membrane. The role of the membrane in eukaryote DNA replication is more controversial, but membranous patches rather than the limiting nuclear membrane may provide chromosome replication-attachment sites. The association of DNA with membranes may also provide a transcriptional control. RNA may be membrane-associatedin a variety of ways: the rRNA constituent of membrane-bound ribosomes; mRNA providing a template for translation; tRNA serving a role in protein, membrane, or other syntheses; RNA serving a control function in transcription or translation; RNA providing a structural basis for protein assembly (i.e., a core pivot); p’RNA during mbrDNA synthesis; or,

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in eukaryotes, an RNA template for cytoplasmic RNA-dependent DNA or RNA synthesis. Many unexplained differentiation processes may correlate with the association of DNA andor RNA with membranes. The future holds many exciting discoveries in this area.

ACKNOWLEDGMENTS My sincerest gratitude goes to my husband, Rex Moyer, for his critical reading of the manuscript, stimulating discussions, and encouragement throughout this endeavor. Special thanks are extended to Cindy Williams, Debbie Davis, and our other student secretaries, whose talents in convening scribbles and hieroglyphics into readable material were invaluable. The help of Katia Pierson and Jack and Cindy Egan in preparing parts of the manuscript is also appreciated.

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Weinstock, R., Sweet, R., Weiss, M., Cedar, H., and Axel, R. (1978). Proc. Narl. Acud. Sci. U.S.A. 75, 1299. Weiss, L., and Mayhew, E .(1966). J. Cell Physiol. 68, 345. Weiss, L., and Mayhew, E. (1967). J. Cefl Physiof. 69, 281. Weiss, L., and Mayhew, E. (1969). Int. J . Cancer. 4, 626. Weiss, L., and Sinks, L. F. (1970). Cancer Res. 30,90. Wenger, S. L., Turner, J. H., and Petricianni, J. C. (1978). In Virro 14, 543. Werner, D., and Maier, G.(1975). Eur. J . Biochem. 54, 351. W e n , G. (1970). In “Biology of Acerubulariu” (J. Brachet and S. Bonotto, eds.), p. 125. Academic Press, New York. Werz, G. (1974). Int. Rev. Cyrol. 38, 319. Westergaard, O., Brutlag, D., and Kornberg, A. (1973). J . Biof. Chem. 248, 1361. Wettenhall, R. E. H., and Slobbe, A. (1976). Exp. Cefl Res. 99, 189. White, J . L., and Murakishi, H. H. (1977). J . Virol. 21, 484. White, M. T.. Wagner, E. K., and Tewari, K. K. (1975). Cancer Res. 35, 873. Wickner, R. B. (1976). Bacreriof. Rev. 40,757. Wickner, R. B., and Leibowitz, M. J. (1976). J . Mol. Biof. 105, 427. Wickner, S. H. (1978). Annu. Rev. Biochem. 47, 1163. Wickner, W . , and Killick, T. (1977). Proc. Narl. Acud. Sci. U.S.A. 74, 504. Wickner, W., and Kornberg, A. (1973). Proc. Narf. Acud. Sci. US.70, 3679. Wickner, W., Brutlag, D., Schekman, R., and Komberg, A. (1972). Proc. Nurl. Acud. Sci. U.S.A. 69, 965. Wickner, W., Mandel, G.,Zwizinski, C., Bates, M., and Killick, T. (1978). Proc. Nurl. Acud. Sci. U.S.A. 75, 1754. Wiegers, K. J., Yamaguchi-Koll, U., and Drzeniek, R. (1976). Biochem. Biophys. Res. Commun. 71, 1308. Williams, C . A., and Ockey, C. H. (1970). Exp. Cefl Res. 63, 365. Williams, G.L., and Green, D. M. (1972). Proc. Narl. Acud. Sci. U.S.A. 69, 1545. Williams, P. H., Boyer, H. W., and Helinski, D. R. (1973). Proc. Nurl. Acud. Sci. U.S.A. 70, 3744. Williamson, R. (1970). J . Mof. Biol. 51, 157. Williamson, R., Young, R. D., and McShane, T. (1976). Biochem. Biophys. Res. Commun. 68,29. Wilson, T., Papabadjopoulos, D., andTaber, R. (1977). Proc. Nutl. Acud. Sci. U.S.A. 74,3741. Winston, S . H., and Berry, L. J. (1970a). J . Rericuloendorhul. SOC. 8, 13. Winston, S. H . , and Beny, L. J. (1970b). J . Reticufoendothul. SOC.8, 66. Wintersberger, E. (1977). Trends Biochem. Sci. March, 58. Wirth, D. F., Katz, F., Small, B., and Lodish, H. F. (1977). Cell 10, 253. Wise, G.E., and Goldstein, L. (1972). Chromosoma 36, 176. Wise, G. E., andPrescott, D. M. (1973a). Proc. Nutl. Acud. Sci. U.S.A. 56, 1571. Wise, G. E., and Prescott, D. M. (1973b). Proc. Nurl. Sci. Acad. U.S.A. 70, 714. Wise, G.E., Stevens, A. R., and Prescott, D. M. (1972). Exp. Cefl Res. 75, 347. Woessner, S . , and Rozman, C. (1976). Blut 33, 23. W o k , C. P. (1973). Bacreriol. Rev. 37, 32. Wolstenholme, D. R., Vermeulen, C. A., and Venema, G.(1966). J. Bacreriol. 92, 1 1 1 1 . Wong, F. Y., and Wildman, S. G.(1972). Eiochim. Biophys. Acra 259, 5 . Wong-Staal, F., Mendelsohn, J., and Goulian, M. (1973). Biochem. Biophys. Res. Commun. 53, 140. Woodcock, C. L. F. (1971). J . Cell. Sci. 8, 611. Woodcock, C. L. F., and Bell, P. R. (1968). J. Ulrrusrrucr. Res. 22, 546. Woodcock, C. L.,Maguire, D. L., and Stauchfield, J. E. (1974). J . Cell Biol. 63, 377a.

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INTERNATIONAL REVIEW OF CYTOMGY, VOL. 61

Electron Cytochemical Stains Based on Metal Chelation DAVIDE. ALLEN Anatomical Pathology Department, Prince Henry’s Hospital, Melbourne, Victoria, Australia

DOUGLAS D. PERRIN Medical Chemistry Group, Australian National University, Canberra, Australian Capital Territory, Australia I. Introduction

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

11. Incipient Instability

III. Postchelation . . . . . . . . . . . . . . . . . . . A. Nonosmiophilic Methods . . . . . . . . . . . . . . B. Osmiophilic Methods . . . . . . . . . . . . . . . IV. “Robust” Complex Formation . . . . . . . . . . . . . V . Immunocytochemical Methods . . . . . . . . . . . . . VI. Some Possible Developments . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

63 64 65 65 65 71 14

76 80

I. Introduction Despite marked growth in the use of electron microscopy over the last decade, available staining methods have remained relatively few, and of these only a small number are in common use. A review of the principles involved in producing suitable metal chelate stains is timely, partly because of the need to gather proposed methods together and partly because such a review can in tum suggest directions in which new stains might be developed. The precise physical property which makes a substance useful as an electron stain has been a matter of controversy (Omstein, 1957; Valentine, 1958). The problem is made difficult by the fact that quantitative measurements of contrast in the electron image are not easy to obtain; however, subjective opinions are unreliable. Valentine (1958, 1961) concluded that the effectiveness ’of a stain varied with the manner of its use. However, where molecules of stain are bound by tissue or where a deposit of material is formed at a tissue site (conditions met by the stains considered in this article), an effective electron stain is a material of high molecular weight containing atoms of high atomic number. This results in a 63 Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-364461-5

64

DAVID E. ALLEN AND DOUGLAS D. PERRIN

maximum change in the density at the site where the stain is bound, hence in maximum contrast. Heavy-metal ions fulfil these requirements and are in common use as staining agents. An important property in staining with metal ions is that the resulting tissuemetal complex be sufficiently stable to withstand subsequent manipulations without reversible dissociation. This stability may be either thermodynamic (i.e., the complex has a sufficiently high formation constant) or kinetic (i.e., the rate of dissociation is very slow as compared to the time for tissue manipulation). Where metal chelates are sufficiently stable in either of these senses they offer the advantage of much “cleaner” staining, because better washing procedures can be used and nonspecific deposition is diminished. One type of metal-containing stain excluded from the present discussion relies on the simple precipitation of inorganic species, usually as a result of enzyme activity on an appropriate substrate. Thus phosphatases can be visualized by the liberation of phosphate, with subsequent precipitation of lead phosphate following the addition of lead ions. Such methods, which have been tabulated elsewhere (Geyer, 1973), do not involve metal chelation.

II. Incipient Instability One type of method commonly used for electron staining of tissues with heavy-metal complexes depends on the adjustment of conditions so that the metal complex in the solution is near the limit of its stability. Immersion of tissue sections in the solution allows binding groups on the tissue to compete successfully for some of the metal ion present, hence to undergo staining. The dynamic character of the metal complex equilibria provides a reservoir of metal ions, but ligand concentration, solution pH, and metal ion concentration must be carefully adjusted because a slight variation in conditions might lead to the precipitation of hydrolyzed metal species or, alternatively, to a significant decrease in the activity of free metal ion, hence to a decrease in staining ability. Direct competition between ligands in the solution and binding sites in the tissue leads to selective staining. Examples include the use of alkaline lead citrate (Reynolds, 1963), uranyl acetate (Watson, 1958), and silver complex preparations (Movat, 1961; Marinozzi, 1963). This type of method is prone to random deposition of metal, causing contamination of tissue sections. Nevertheless, this technique is used in such cytochemical methods as the staining of nucleic acids (Albersheim and Killias, 1963), intensification of ferritin labels with alkaline bismuth reagent (Ainsworth and Karnovsky, 1972), and staining of elastic tissue with a mixture of tannic acid, uranyl acetate, and lead acetate (Kajikawa et al., 1975). Synaptic junctions have been stained with a bismuth-iodide complex (Pfenninger et at., 1969), and

ELECTRON CYTOCHEMICAL STAINS

65

periodate-engendered aldehyde groups have been localized with silver methenamine (Rambourg et al., 1969) and also with alkaline bismuth reagents (Ainsworth et al., 1972).

111. Postchelation

When cytochemical reagents that also have metal-complexing ability are reacted with tissue, they allow subsequent selective chelation of heavy-metal ions at their binding sites. This technique, postchelation, has been of great importance in electron cytochemistry. A. NONOSMIOPHILIC METHODS Early developments of postchelation procedures were based on modifications of optical histochemical methods to produce electron-opaque reaction products. Several optical procedures based on metal chelation were found to be directly applicable to electron microscopy. An example is the chelation of formazans with metal ions to improve the localization of dehydrogenases (Pearse and Scarpelli, 1959). In some cases, methods for use in electron microscopy have been designed a priori. Thus Kendall and Mercer (1958) proposed that proteins could be stained by forming bromoacetate derivatives which subsequently could be rendered electron-opaque by chelation with lead salts. These and other examples are listed in Table I.

B. OSMIOPHILIC METHODS The most successful applications of the postchelation principle to electron staining have been in the development of osmiophilic methods. Originally introduced by Seligman and his co-workers (Hanker et al., 1964), these methods are based on established histochemical procedures, except that reagents are used which contain organic functional groups, usually nitrogen or sulfur moieties, which are capable of reacting with osmium tetroxide. The reaction products of these methods are very stable substances termed osmium blacks. They are believed to be coordination polymers of osmium and the organic ligands (Hanker et al., 1967), though in some cases reduction of osmium tetroxide by the ligand may also result in the deposition of lower oxides of osmium, their hydrates, and possibly osmium metal (Holt, 1974). In many respects these products are ideally suited to electron cytochemistry. They are insoluble in organic solvents, lipids, and epoxy resins, and their fine, amorphous character permits them to be precisely localized in the electron microscope. However, in many cases, osmium tetroxide may also react nonspecifically with tissue components (Hayat, 1978a).

66

DAVID E. ALLEN AND DOUGLAS D. PERRIN TABLE I NONOSMIOPHILIC POSTCHELATION METHODS FOR ELECTRON MICROSCOPY"

Tissue components localized (as stated in method) Nucleic acids

Protein

Periodate-reactive substances

Complex carbohydrates

Polyanions

Reagent and reaction Aromatic sulfonic acid derivatives; chelation of uranyl acetate Schiff's reagent; chelation of thallium ions 1-Fluoro-2:4-dinitmbenzene derivatives; chelation of lead or silver salts Bmmoacetae derivatives; chelation of lead salts N-Acetylhomocysteine thiolactone derivatives; chelation of silver salts p-Chloromercuriphenylsulfonic acid derivatives; chelation of uranyl acetate Thiocarbohydrazide used to label periodateengendered aldehyde groups; chelation of silver from silver proteinate Tannic acid; chelation of ferric chloride, uranyl acetate, or gold chloride Cetylpyridinium chloride; chelation of ferric ion

Reference Beer and Moudrianakis (1962); Beer (1966) Moyne (1973) Lamb et al. (1953)

Kendall and Mercer (1958) Kendall and Barnard (1963)

&be1 and Roe (1967)

Thikry (1967)

Sannes et al. (1978)

Courtoy er al. (1974)

(continued)

A wide range of such osmiophilic reagents have now been synthesized. They provide the basis of a considerable number of methods, including those listed in Table 11, for the cytochemical demonstration of enzymes and functional groups of macromolecules by both light and electron microscopy. This concept of osmiophilia has been extended, through the use of multidentate ligands, to methods by which osmium blacks can be bound to metal compounds localized at tissue sites. Such deposits may arise through the selective affinity of a metal compound for a particular macromolecule or as the result of cytochemical reactions carried out to demonstrate enzyme activity. Thiocarbohydrazide has an affinity for many metal ions and retains the ability to react further with osmium by bridging. By this means, osmium has been bridged to

67

ELECTRON CYTOCHEMICAL STAINS TABLE I (continued)

Tissue components localized (as stated in method) Esterases

Cholinesterases and thiocholinesterases

Dehydrogenases

Cytochrome oxidase

Peroxidase

Reagent and reaction Enzyme-released quinoline derivatives; chelation of bismuth(II1) ions Enzyme-released p-nitrothiopheny1derivatives;chelation of aurous gold Enzyme-released thiocholine; chelation of silver(1) ions Enzyme-released thiocholine; chelation of copper(I1) ions Enzyme-released thiocholine or thiolacetic acid; chelation of aurous gold Enzyme-engendered formazans; chelation with cobalt ions Enzyme-engendered formazans; chelation with alkaline lead Oxidation product of N-phenyl-pphenylenediamine; chelation of copper and reaction with ferrocyanide Homovanillic acid; chelation of lead ions by an enzymecatalyzed dimer

Reference Biicking er al. (1976)

Vatter et al. (1968)

Birks and Brown (1960) Lewis and Shute (1969) Brzin and Pucihar (1976) Davis and Koelle (1967) Koelle et al. (1968) Pearse and Scarpelli (1959) Franchiotti et al. (1971) Kerpel-Fronius and Hajos (1967)

Papadimitriou et al. (1976)

“To avoid repetition, chemically related methods are not referenced separately. For details of these techniques the cited papers should be consulted.

palladium, osmium, uranium, lead, mercury, iron, chromium, copper, calcium, zinc, and tin cations on tissue (Hanker et al., 1966a). Alternatively, traces of transition-metal complexes may be used to catalyze the oxidative generation of an osmiophilic polymer from organic monomers such as 3,3’-diaminobenzidine, p-phenylenediamine, catechol, and biogenic amines (Hanker et al., 1972a). Both these techniques have found application in enzyme cytochemistry (Table 11), and thiocarbohydrazide can also be used to enhance the contrast of tissue generally impregnated with metal ions (e.g., following fixation with osmium tetroxide) to the point where it offers an alternative to lead and uranium staining (Hanker et al., 1966a; Seligman, 1966; Seligman et al., 1966). The development of osmiophilic methods for transmission electron microscopy, particularly

TABLE I1 OSMIOPHILIC METHODSFOR ELECTRON MICROSCOPY Tissue components localized (as stated in method) General morphological and lipid staining following osmium tetroxide fixation

Organelle staining Periodate-reactive substances

Sulfated mucopoly. saccharides Acid phosphatase, P-glucuronidase Acid and alkaline phosphatase

Acid phosphatase, nonspecific esterase

Nonspecific esterase, cholinesterases

Cholinesterases

Reaction sequence"**

Reference

Osmium bridging with TCH

Hanker et al. (1966a); Seligman et al. (1966)

p -Phenylenediamine binding to osmium residues DAB oxidation to osmiophilic polymer Reaction of periodate-engendered aldehyde groups with TCH Carbonyl reaction products with pentafluorophenylhydrazine and p-fluorophenylhydrazine rendered osmiophilic with ammonium sulfide Reaction of periodate-engendered aldehyde groups with thiosemicarbazide Oxidation and osmium binding by DAB attached to sulfate groupings Osmium bridging to mercury by TCH

Ledingham and Simpson (1972) Novikoff (1970) Hanker et al. (1964)

Osmiophilic diazothioether formation by coupling enzyme hydrolysis product by di(dicyclohexy1ammonium)-2-napthylthiolphosphate with fast blue BN Hydrolysis product of di(dicyclohexy1ammonium)-2-naphthylthiolphosphate or 2-thiolacetoxybenzanilide used to form Hatchett's brown, followed by osmium bridging with TCH or catalytic generation of osmiophilic polymer from DAB Osmiophilic a m dyes formed by coupling tetrazotized N , N ' bis(4-aminopheny1)-1,3-xyIenediamine with enzyme-released indoxyl Osmiophilic diazothioethers formed by coupling enzyme hydrolysis products of thiocholine esters with fast blue BN

Seligman er al. (1970a)

Bradbury and Stoward (1967) Stoward and Bradbury (1968) Stastna and Travnik (1971)

Monga

et

al. (1972)

Smith and Fishman (1969)

Hanker er al. (1972b)

Kawashima and Murata (1969)

Bergman

et

al. (1967)

(continued)

68

TABLE I1 (continued)

Tissue components localized (as stated in method)

Reaction sequence".* Osmiophilic polymer generation from

S-acetylthio-3-toluenediazonium ion, 3-acetoxyl-5-indolediazonium ion, or p-acetylthiobenzenediazonium ion Thiocholine substrates used to form Hatchett's brown, followed by osmium bridging with TCH or catalytic generation of osmiophilic polymer from DAB Osmiophilic indigos produced by enzymic hydrolysis of a mixture of 5 '-(5-iodo-3-indolyl)-5-fluorodeoxyuridine phosphodiester and 5'-(5nitro-3-indolyl)-5-fluorodeoxyuridine phosphodiester Osmiophilic a m dyes formed by coupling hexamtized pararosaniline with enzyme-released indoxyls Osmiophilic diazothioethers formed by coupling enzyme hydrolysis products of various thioesters with fast blue BN Osmiophilic diazothioethers formed by coupling enzyme hydrolysis products of 2-naphthylthiolnonanoate or 2thiolnonanoxybenzanilide with fast blue BN Osmiophilic azo dye formed from diazotized 4-aminophthalhydrazide and enzyme hydrolysis product of L-alanyl or y-glutamyl-4-methoxy-2napthylamide Osmiophilic polymer generation at the sites of copper capture following enzyme hydrolysis of 4-nitro-] ,2benzenediolmono-(hydrogensulfate) Nonosmiophilic tetrazolium salt yielding an osmiophilic formazan 2-(2'benzothiazolyl)-5-styryl-3-(4'phthalhydrazidyl)tetramlium chloride

5'-Nucleotide phosphodiesterase

Esterase

Esterase, lipase

Lipase

Aminopeptidase, y-glutamyl transpeptidase

Aryl sulfatase

Dehydrogenases

Reference Mednick et at. (1971) Davis et al. (1972)

Hanker et al. (1973b)

Tsou er al. (1974)

Holt and Hicks (1966)

Hanker et al. (1966b)

Seligman et al. (1965)

Seligman et al. ( 1 9 7 0 ~ )

Hanker et al. (1975)

Kalina et al. (1972)

~

(continued)

69

70

DAVID E. ALLEN AND DOUGLAS D. PERRIN TABLE I1 (continued)

Tissue components localized (as stated in method) Dehydrogenases, monoamine oxidase

Monamine oxidase

Cytochrome oxidase

Peroxidase Polyviny lpyrrolidone and dextrans used as tracers

Reaction sequencea,b Fem-ferncyanide reduction used to form Hatchett's brown, followed by catalytic generation of osmiophilic polymer from DAB Nonosmiophilic tetrazolium salt yielding an osmiophilic formazan 2-(2'benzothiazolyl)-5-styryl-3-(4'phthalhydrazidy1)tetrazolium chloride Osmiophilic indoaniline formed from N-benzyl-p-phenylenediamineand substituted naphthols (NADI reaction) Osmiophilic polymer generation from three bisphenylenediamines: N , N ' bis(4-aminopheny1)-1.3-xylenediamine, N,N'-dimethylethylenediamine, and DAB Osmiophilic polymer generation from DAB Osmiophilic polymer generation from DAB Visualization by fixation with osmium tetroxide partially reduced with potassium ferrocyanide

Reference Hanker et al. (1973a)

Shannon et al. (1974) Shannon et al. (1977a,b)

Seligman et al. (1967)

Seligman et al. (1970b)

Anderson et al. (1975) Graham and Kamovsky (1966) Ainsworth (1977)

"To avoid repetition, chemically related methods are not referenced separately. For details of these techniques the cited papers should be consulted. *TCH,Thiocarbohydrazide; DAB, 3,3'-diaminobenzidine; fast blue BN is the stabilized diazotate of N-(4-amino-2,5-diethoxyphenylbenzamide); Hatchett 's brown is copper ferncyanide.

those relating to the localization of enzyme activity, has been reviewed by Holt ( 1974). Thiocarbohydrazide-mediatedosmium binding was applied to scanning electron microscopy by Kelley et al. (1973). By impregnating the specimen with osmium following treatment with thiocarbohydrazide a conductive coating is produced. This coating is sufficient to prevent charging effects when the specimen is exposed to the electron beam, and the method has advantages over other metal-coating methods in that sputtering and evaporation steps are avoided. The

ELECTRON CYTOCHEMICAL STAINS

71

coating is continuous over the entire surface of the specimen and extends into its interior (Woods and Ledbetter, 1976). Modifications of the original method which reduce the incubation times and the quantity of osmium used have been described by Postek and Tucker (1977) and by Hayat (1978b).

IV. “Robust” Complex Formation Another approach to staining for electron microscopy is to use a robust metal complex which has on its periphery a cytochemically active group. The osmium-containingtetrazolium salt developed by Tsou et al. (1968) is an example. Robust complexes are kinetically inert toward dissociation into their components, thereby greatly reducing the risk of random metal deposition that can arise when labile metal species are used as electron stains. This approach has the advantages of retaining the selectivity of the postchelation procedures and of decreasing the number of reaction steps. Examples of this type of reagent and the tissues for which they are used are listed in Table HI. By eliminating the postchelation step it is possible to avoid metal binding by native tissue groups, which might lead to false localizations. Several approaches can be recognized in the development of robust complex methods. Thus complexes used in optical microscopy have been tested for their ability to produce contrast in the electron microscope, and noncomplex optical stains have been rendered electron-opaque by the formation of metal chelate derivatives. In some cases, metal complexes incorporating biologically active ligands have been synthesized in attempts to design specific electron stains. As might be expected, the metals incorporated into complexes for electron staining have generally been heavy transition metals such as osmium, gold, silver, and platinum. These metals are characterized by a suitably reactive chemistry, high electron scattering power, and the formation of stable complexes. Lead and mercury have also proven useful, but in general the use of lead compounds is hindered by low water solubility, and some mercury derivatives have demonstrated instability under the conditions existing in the electron microscope (Pepe and Finck, 1961; Dobb et al., 1972; Allen and Perrin, 1974). Vaporization of the mercury label may occur, leading to loss of contrast. Stains of this type, in which a single metal atom is responsible for producing electron opacity, cannot provide the degree of contrast obtainable with polynuclear complexes such as alkaline lead preparations, or which follows from methods that result in the formation of osmiophilic polymers. However, many staining situations involve labeling of localized deposits within tissues or labeling of selected tissue components where a comparatively large number of staining sites are available. For example, nucleic acids after Feulgen hydrolysis, and

72

DAVID E. ALLEN AND DOUGLAS D. PERRIN TABLE ILI ROBUSTMETAL-CONTAINING REAGENTS USEDAS ELECTRON STAINS Tissue components localized

Reagent

Reference

General stain

Dipotassium tetramethyl osmate

Hmckley and Murphy

Membranes and products of diaminobenzidine cytochemistry Carbonyl groups, DNA following acid hydrolysis

Potassium-osmium-cyanide complex

Hoshino etal. (1976,1977)

Silver complex of thiosemicarbazide

Woods and Livingston

(1975)

Nucleic acids

Acidophilic or basophilic tissue stmctures Acid mucopolysaccharides

(1966)

Ferrocenylcarboxyhydrazide “Osmium ammine” Acriflavine-phosphotungstic acid complex Osmium tetroxide-pyridine complex Iron-hematoxylin complex Platinum-pyrimidine complexes Platinum-uracil complex cis-Dichlorodiammine platinum(I1) Osmium coordination compounds with multiple acidic or basic groups Alcian blue, a cationic copper phthalocyanin derivative Ruthenium red, a cationic ammoniated ruthenium oxychloride [Ru~O,(NHJ&~.~H~O High iron diamine, an iron-N,Ndimethylphenylenediamine complex

Allen and Perrin (1974) Gautier (1976) Chan-Curtis et al. (1970) Subbaraman (1971) Brissie et al. (1974) Agganval (1976) Babitch et al. (1976) Heinen (1977) Seligman et al. (1968)

Rothman (1969) Schofield et al. (1975) Ruggeri et al. (1975) Luft (1971)

Spicer et al. (1978) (continued)

polysaccharides after periodate oxidation, have locally high concentrations of aldehyde groups available for reaction with a stain. The success of the reagents listed in Table I11 illustrates that under these conditions single metal ion complexes can lead to satisfactory increases in electron opacity while at the same time providing simplified staining procedures, improved selectivity, and decreased contamination. Considerable effort has been devoted to the production of a metal-containing diazonium salt (Tice and Barrnet, 1965; Livingston et al., 1970; Beadle et al., 1971). Such a reagent could serve as an electron-opaque marker for a range of hydrolytic enzymes by acting as a capture agent for naphthols released from commercially available enzyme substrates. Such substrates are available for es-

73

ELECTRON CYTOCHEMICAL STAINS TABLE I11 (continued)

Tissue components localized Elastic tissue

Sulfhydryl groups

Acid phosphatase (also staining due to a diamnium moiety) Acid phosphatase, P-glucuronidase Acid phosphatase, P-glucuronidase, nonspecific esterase Dehydrogenases

Cytochrome c

Reagent

Reference

Anionic gold and silver chelates of tetraphenylporphine sulfonate Iron-hematoxylin complex Organomercurials: mercury orange 1-(4-chloromercuriphenylam)-2naphthol 4-Hydroxy-1 -naphthylmercuric acetate p-Chloromercuribenzoate Methylmercuric halides

Albert and Fleischer (1970) Albert (1973) Brissie et al. (1974) Mundkur (1964)

Chloromercuriferrocene Metal (Mg, Cu, Pb) phthalocyanin diazonium salts

Barmett et al. (1958) Dobb et al. (1972) Dobb et al. (1972) Gersch er al. (1960) Allen and Pemn (1974) Tice and Bannett (1965)

Lead phthalocyanin diazonium salt

Livingston et al. (1969)

Diazotized triphenyl lead derivatives: triphenyl-p-aminophenethyllead and triphenyl-p-aminophenyl lead Osmium-containing tetrazolium salt: osmate of 2,2‘,5,5‘-tetra-p-nitrophenyl-3,3’-stilbene ditetrazolium chloride

Livingston et al. (1970)

Bis-(4,4’-diamino-2.2’-bipyridyl)

Beadle et al. (1971) Tsou et al. (1968)

Tsou et al. (1976)

ferrous chloride

terases, phosphatases, peptidases, and glucuronidase (Sigma, Polysciences). If a diazonium function is to be attached to a metal complex, it is necessary to select a complex of considerable chemical stability if it is to survive the harsh conditions of diazotization. While the diazonium salt should be very soluble in water, the dyes formed with the enzyme-released naphthols should be insoluble if precise enzyme localizations are to be obtained. Further, the dyes must also be insoluble in organic solvents used for dehydration and in resins used for embedding the tissue. These requirements are severe, particularly because metal complexes are generally more soluble in organic solvents than metal ions. None of the metal-containing diazonium salts so far described in the literature meet all these requirements, though some of them have yielded useful results in electron microscopy and solubility problems have been lessened by using special processing procedures (Livingston et al., 1969, 1970; Beadle el al., 1974). A

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DAVID E. ALLEN AND DOUGLAS D. PERRW

FMC

CMF

Diferrocenylmercury

FIG.1 . Ferrocene derivatives as electron stains.

further problem with lead-containing diazotates is their nonspecific affinity for lysosumal material, reported by Beadle et al. (1974). In an extension of this staining principle a robust metal compound, after appropriate substitution with various functional groups, can be used as an electron-dense label for a number of cytochemical procedures. We have previously illustrated this concept (Allen and Perrin, 1974) by preparing ferrocenylmethylcarboxyhydrazide (FMC) and chloromercuriferrocene (CMF). In these compounds (Fig. 1) the electron-opaque ferrocene core can serve as a stain for aldehyde groups (FMC) or sulfhydryl groups (CMF).Clearly, other ferrocene derivatives could be prepared as stains for other tissue components, and the same principle could be applied to other robust complexes. Possible future developments along this line are considered in Section VI.

V. Immunocytochemical Methods Antigen-antibody combinations require labeling with an electron-opaque marker before they can be visualized in the electron microscope. Metal complex formation has been used for this purpose. Thus Singer (1959) introduced the metalloprotein ferritin as a reagent. This stable iron-protein complex was attached to antibodies by the use of difunctional coupling agents (Williams and Gregory, 1967). More recently, Farrant et al. (1974) separated “heavy” and ‘‘light” ferritins for the simultaneous localization of two antigenic sites.

ELECTRON CYTOCHEMICAL STAINS

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A number of organomercurials have been employed to mark antibodies through mercaptide formation (Pepe, 1961; Pepe and Finck, 1961; Zhadanov et af.,1965, 1966). Yasuda and Yamamoto (1975, Yamamoto, 1977) developed an indirect antibody-labeling method by conjugating sera with a thiolated egg albumin coupled with CMF. Electron scattering due to the CMF can be amplified by staining thin sections with silver nitrate. These authors claim that this method retains the advantages of direct metal labeling (low molecular eight of the label, sharp electron images) and overcomes the disadvantage that relatively few metal atoms can be attached to an antibody without reducing its activity. Gill and Mann (1966) suggested that ferrocene itself might be useful as a marker for proteins, and Franz (1 968, 1971) demonstrated the application of ferrocene derivatives in immunoelectron microscopy. Franz has synthesized 3carboxy-4-ferrocenyl isothiocyanate (CFPI) in which the isothiocyanate group allows combination with protein amino groups, the carboxyl promotes solubility in basic aqueous solutions, and the iron provides the electron opacity. CFPI gave excellent results when used to demonstrate bacterial capsular antigens (Wildfuhr and Franz, 1971). Osmiophilic methods can also be used to enhance the electron opacity of immunoglobulins (Sternberger et af., 1966a,b). An indirect osmiophilic method is currently the most frequently used procedure for visualizing in the electron microscope the sites of antigen-antibody binding. In this method antibodies conjugated with horseradish peroxidase (HRP) (Avrameus, 1969) are subsequently localized through the enzyme-catalyzed formation of an osmiophilic polymer from diaminobenzidine in the presence of hydrogen peroxide (Graham and Kamovsky, 1966). More recently, Papadimitriou et af. (1976) have suggested an alternative reaction sequence for demonstration of the HRP label in which the enzyme-catalyzeddimerization of homovanillic acid is followed by the chelation of lead ions. Schurer et al. (1977) have proposed the use of an ethyleneimine polymer for marking immunoglobulins. This polymer meets specifications for such an application because it has free amino groups for conjugation to antibodies, polar groups to confer solubility, and complexing groups which allow postchelation with osmium tetroxide or phosphotungstic acid to confer electron opacity. The use of polyethyleneimine of molecular weight 40,000 could avoid transport difficulties that arise in the use of femtin (MW 600,000) and also permit more accurate localization than is possible with an HRP marker. A specificity resembling antigen-antibody combination is shown by certain plant lectins toward tissue saccharides. This permits highly selective staining of certain cellular carbohydrate components, based on prior conjugation of lectins with metal chelates. Thus concanavalin A, a lectin from the jack bean, selectively binds to a-D-glucopyranosyl, a-D-mannopyranosyl, and p-D-fructofuranosyl residues. This material was first used in ultrastructural cytochernistry by Bernhard and Avrameus (1971). Similarly, wheat germ agglutinin, specific for

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DAVID E. ALLEN AND DOUGLAS D. PERRIN

N-acetylglucosamineresidues, was employed by Franqois et al. (1972) and Huet and Ganido (1972). Visualization techniques employing metal chelates have included conjugation with an iron-dextran complex (Martinand Spicer, 1974), an iron-mannan complex (Roth and Franz, 1975), ferritin (Nicolson and Singer, 1972; Roth e f al., 1975), a gold complex (Roth and Binder, 1978), and the ironcontaining hemocyanin (Weller, 1974). Mercury labeling was used by Horisberger et al. (1971), and conjugation with microperoxidase by Temmink et al. (1975). The latter authors visualized the microperoxidase by formation of the osmiophilic polymer of oxidized diaminobenzidine and compared the effectiveness of this method with visualization by means of ferritin, hemocyanin, and HRP. The HRP method was introduced by Bernhard and Avrameus (1971), who found that the glycoprotein component of HRP would bind to concanavalin Asaccharide combinations. Again the HRP label was visualized by diaminobenzidine oxidative polymerization. Other plant lectins can be used as tracers and can also be rendered electron-dense with metal complexes. The interaction of lectins with animal cells has been reviewed by Nicolson (1974) and by Roth (1977). The egg white protein avidin has a very high association constant per mole) for biotin and can be rendered electron-opaque by conjugation with ferritin. Heitzmann and Richards (1974) introduced the avidin-biotin complex as a selective histochemical marker. Tissue components can be labeled with biotin through the use of biotinyl-N-hydroxysuccinimideester (which reacts with protein amino groups) or by reacting biotin hydrazide with aldehyde groups produced by a prior cytochemical reaction. Heitzmann and Richards conjugated avidin with ferritin by cross-linking with glutaraldehyde. Bayer et al. (1976) have used a reductive alkylation process which precludes the production of aggregates of ferritin polymers.

VI. Some Possible Developments A criticism of osmiophilic methods has been that the reagents are not readily available and are difficult to synthesize. Nevertheless, the success of these procedures in demonstrating a wide range of tissue components will no doubt lead to further stains which utilize this principle. As alternatives to using osmium tetroxide in the formation of osmium blacks, Hanker ef al. (1976) examined several compounds of osmium (VIII) including potassium osmiamate and coordination complexes with ammonia and nitrogen heterocycles. They found the most useful of these to be a molecular addition complex of hexamethylenetetramineand osmium tetroxide, osmeth, which is used as a dispersion in water. Advantages of osmeth cited by the authors are that it is safer and more convenient to use than osmium tetroxide and that it is as effective as equivalent concentrationsof the tetroxide in the formation of osmium blacks. The compound reacts only slowly with

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tissue and is therefore not as good a fixative as osmium tetroxide. This property might be used to advantage to decrease nonspecific osmium deposition in cytochemical studies. As well as serving as alternatives to osmium tetroxide in the formation of osmium blacks, robust complexes of osmium (and iridium) with nitrogen heterocyclic ligands could also be a source of electron stains provided the ligand already carries a suitable substituent or is capable of substitution. Several of the stains listed in Table In have demonstrated the applicability of osmium complexes in this field, and Taube (1975) has shown that a series of kinetically stable osmium complexes containing substituted pyridines can be formed. Taube and his coworkers have also described a series of binuclear mixed-valence complexes of ruthenium (Creutz and Taube, 1973) and osmium (Magnusson and Taube, 1972), which can also be prepared using substituted pyridines as ligands (Creutz and Taube, 1973). Such complexes suggest the possibility of synthesizing metal complex stains of increased effectiveness in which two metal atoms are present. In contrast to the development of osmiophilic reagents, the formation of robust complex stains by direct coordination of metal ions with existing ligands generally requires few chemical manipulations. This is an important factor where electron microscopists do not have chemical facilities. A special class of stains can be formed by incorporating a metal ion into a multidentate ligand which still retains groups able to bind to tissue components. As examples we have prepared robust platinum complexes of the histological dyes chrysoidine orange, Bismarck brown and Congo red (Allen, 1975). In these complexes the metal atom is chelated by ortho aminoazo groups present in the dye molecules, leaving free amino groups (chrysoidine orange and Bismarck brown) or free sulfonic acid groups (Congo red) available for reaction with tissue moieties. The properties of the metal complexes resemble those of the parent dyes apart from a decrease in solubility in water. Using these dyes we have enhanced the contrast of acidic and basic tissue components in the electron microscope. Similarly, a number of histological dyes, including chromotrope 2R, Sudan red B, and oil red 0, contain ortho hydroxyazo groupings and can also form platinum complexes. The possibility exists that the use of these dyes can be extended to electron microscopy by chelation with a heavy-metal ion. The platinum derivatives of chrysoidine orange and Bismarck brown (Fig. 2) can be diazotized with retention of the metal atom. These metal-containing diazotates may prove useful as capture reagents in enzyme cytochemistry. In the case of the complex from the diazo dye Bismarck brown, the products of coupling with enzyme-released naphthols, especially naphthols of the AS series (Burstone, 1958, 1962), are amorphous, insoluble compounds in which electron opacity can be increased by postchelation of other metal atoms such as copper and osmium. Procion dyes (Polysciences, 1974) comprise another series of dyes which may have potential applications in electron microscopy. These dyes are derivatives of

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DAVID E. ALLEN AND DOUGLAS D. PERRIN

c1

Chrysoidine orange

HaHp+=N

Pt -C1

t

wN=N&NH2

Bismarck brown

FIG.2 Platinum complexes of aminoazo and hydroxyazo dyes.

chloro-S-triazinyl coupled with a chromophore, and they have already found use in cytology, fixation of tissue (Levine et al., 1964; Goland and Grand, 1968), and neuron profile studies (Remler et al., 1968; Purves and McMahon, 1972; Kellerth, 1973). These dyes can react with hydroxyl or amino groups on tissues to produce covalently bonded colored adducts. Their reaction with proteins has been reviewed by Shore (1968, 1969). The chromophore group can be modified to contain a heavy-metal atom, thus rendering it electron-opaque. Procion brown contains chromium and was used in electron microscopy by Christensen (1973) to study neurons. Available Procion dyes contain copper, chromium, and cobalt and may offer further possibilities for electron microscopy. The concept of a metallic “tag” is not confined to electron cytochemistry. Since metal ions possess useful spectroscopic and radioactive properties, the preparation of robust complexes which interact with biological macromolecules may lead to new applications of metal ions as probes of biological systems (Mann, 1967; Kornicker and Vallee, 1969; Sundberg et al., 1974; see also references in Riley and Perham, 1973). Already, heavy-atom isomorphous derivatives of proteins have played a large part in the elucidation of protein function by x-ray crystallography. Conversely, these labeling studies may yield methods of relevance to electron cytochemistry. For example, Sundberg er al. (1974) synthesized a phenyl-substituted ethylenediaminetetraaceticacid (EDTA) carrying a diazonium function on the aromatic ring. Following the reaction of proteins with the diazonium moiety, the binding sites can be labeled with metal ions by means of the EDTA function. The use of this diazonium salt might well be extended to enzyme cytochemistry using a technique similar to that noted in

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Section IV. The method is potentially adaptable to the production of other stains by exchanging the diazo moiety for other functional groups. Thus the system is a further example of a single robust complex system furnishing several stains by substitution with various functional groups, as described for the ferrocene-based stains CMF and FMC. Groups that might be attached to a robust complex system to give electron stains include a chelating portion able to bind to osmium attached to tissue as a result of fixation with osmium tetroxide, acidic or basic groups for demonstrating acido- or basophilia, and a quaternary amonium group to localize polyanions. Substrates containing the complex for enzymic release might be prepared as a means of localizing hydrolytic enzyme activity, and the variable valency of many metal ions might be exploited to permit use of the complex as an electron acceptor for demonstrating oxidoreductase activity. As an example of the latter use we have tested a number of ferrocene compounds as indicators of succinic dehydrogenase activity (Allen, 1975). Conversion of the complexes to ferricinium ions produces water-soluble forms. Reduction of these ions leads to precipitation of the insoluble ferrocene compound at the sites of enzymic activity. Ferrocene itself is unsuitable for this reaction, because its ferrocinium ion is unstable in aqueous solution and it is too soluble in organic solvents to survive conventional dehydration and embedding processes. However, these problems can be avoided by using substituted ferrocenes, and been applied to demonstrate succinic one of these-diferrocenylmercury-has dehydrogenase activity in rat heart muscle. This compound has the additional advantage of containing three electron-opaque metal atoms, and in this case the mercury atom is not lost in the electron microscope. Franz (1967) pointed out that electron stains based on ferrocene could be amplified by treating the stained tissue with silver (I) ions. Oxidation of the ferrocene nucleus’by the silver ions results in the precipitation of silver metal at the reaction sites. We have previously shown that this reaction can be carried out on ultrathin sections of tissue treated with ferrocene compounds prior to embedding in epoxy resins. In some cases the silver deposit so formed is very fine and is suitable for high-resolution studies (Allen and Perrin, 1974). This reaction is potentially valuable because it could extend the use of ferrocene derivatives as electron stains, and it may also be applicable to other metal complex systems. In recent years considerable interest has centered on the preparation and investigation of synthetic macrocyclic compounds. Many macrocyclic polyethers, polythioethers, polyamines, and other related molecules have interesting and unusual binding properties (Christensen et al., 1971, 1974). Some of these complexes are intensely colored and exhibit thermal and chemical stability comparable to that of the related phthalocyanin complexes over which they have the advantage of a significant reduction in molecular size. Substituted macrocycles may thus prove to be a further source of electron stains. Macrocycles containing

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heavy-metal atoms (e .g., platinum) can be more easily prepared than some other types of heavy-metal complexes, and their hydrophobic exteriors which shield the hydrophilic center containing the metal ion could facilitate the penetration of tissue components. These properties might be advantageous for cytochemical purposes. Clearly, metal complexes play major roles in electron staining. Hopefully, this article, by illustrating principles used in designing stains, will lead electron microscopists to a wider use of existing methods and stimulate them to develop new methods. The possibilities inherent in metal chelation as a source of electron cytochemical stains are by no means exhausted.

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Riley, M., and Perham, R. N. (1973). Biochem. J . 131, 625. Roth, J. (1977). Exp. Purhol. (Suppl.) 3. Roth, J., and Binder, M. (1978). J . Hisrochem. Cyrochem. 26, 45. Roth, J., and Franz, H. (1975). Histochemie 41, 365. Roth, J., Thoss, K., Wagner, M., and Meyer, H. W. (1975). Histochemie 43, 275. Rothman, A. H. (1969). Exp. Cell Res. 58, 177. Ruggeri, A., Dell'orbo, C., and Quacci, D. (1975). Hisrochem. J. 7, 187. Sannes, P. J., Katsuyama, T., and Spicer, S. S. (1978). J. Hisrochem. Cyrochem. 26, 5 5 . Schofield, B. H., Williams, B. R., and Doty, S. B. (1975). Hisrochem. J. 7 , 139. Schurer, J. W., Hoedemaeker, P. J., and Molenaar, I. (1977). J . Hisrochem. Cytochem. 25, 384. Seligman, A. M. (1966). J. Histochem. Cyrochem. 14, 1975. Seligman, M. L., Ueno, H., Hanker, J. S., Kramer, S. P. Wasserkrug, H. L., and Seligman, A. M. (1965). J. Hisrochem. Cytochem. 13, 705. Seligman, A. M. Wasserkrug, H. L., and Hanker, J. S. (1966). J . Cell Biol. 30, 424. Seligman, A. M., Plapinger, R. E., Wasserkrug, H. L., Deb, C., and Hanker, J. S. (1967). J. Cell Biol. 34, 787. Seligman, A. M., Wasserkrug, H. L., Deb, C., and Hanker, J. S. (1968). J. Histochem. Cyrochem. 16, 87. Seligman, A. M., Kawashima, T., Ueno, H., Katzoff, L., and Hanker, J. S. (1970a). Acru Histochem. Cyrochem. 3, 29. Seligman, A. M., Wasserkrug, H. L., and Plapinger, R. E. (1970b). Histochemie 23, 63. Seligman, A. M., Wasserkrug, H.L., Plapinger, R. E., Seito, T., and Hanker, J. S. (1970~).J . Hisrochem. Cyrochem. 18, 542. Shannon, W. A., Wasserkrug, H.L., and Seligman, A. M. (1974). J. Hisrochem. Cyrochern. 22, 170. Shannon, W. A., Wasserkrug, H. L., Plapinger, R. E.,and Seligman, A. M. (1977a). J. Hisrochem. Cytochem. 24, 527. Shannon, W. A., Wasserkrug, H. L., and Seligman, A. M. (1977b). J. Hisrochem. Cyrochem. 24, 546. Shore, J. (1968). J . SOC. Dyers Colour. August, 408. Shore, J. (1969). J. SOC. Dyers Colour. January, 14. Singer, S. J. (1959). Nature (London) 183, 1523. Smith, R. F., and Fishman, W. H. (1969). J. Hisrochem. Cyrochem. 17, 1. Spicer, S. S . , Hardin. J. H., and Setser, M. E. (1978). Hisrochem. J . 10,435. Stastna, J., and Travnik, P.(1971). Hisrochemie 27, 63. Stemberger, L. A., Donati, E. J., Hanker, J . S., and Seligman, A. M. (1966b). Exp. Mol. Purhol. Suppl. 3, 36. Stemberger, L. A., Hanker, J. S., Donati, E. J., Petroli, J. P., and Seligman, A. M. (1966a). J . Hisrochem. Cytochem. 14, 71 1. Stoward, P. J., and Bradbury, S. (1968). Histochemie 15, 93. Subbaraman, L. R., Subbaraman, J., and Behrman, E. J. (1971). Bioinorg. Chem. 1, 35. Sundberg, M. W., Meares, C. F., Goodwin, D. A., and Diamanti, C. I. (1974). J. Med. Chem. 17, 1304. Taube, H. (1975). Proc. R. Ausr. Chem. Insr. 42, 139. Temmink, J. H. M., Collard, J. G., Spits, H., and Roos, E. (1975). Exp. Cell Res. 92, 307. Thiery, J. P. (1967). J. Micros. 6, 987. Tice, L. W., and Bannett, R. J. (1965). J. Cell Biol. 25, 23. Tsou, K. C., Goodwin, C. W., Seamond, B., and Lynm, D. (1968). J. Hisrochem. Cytochem. 16, 487. Tsou, K. C., Hendricks, J., Gupta, P. D., and La, K. W. (1974). Hisrochem. J. 6, 327.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 61

Cell Electrophoresis THOMAS G. PRETLOW, I1 A N D THERESA P. PRETLOW Departments of Pathology and Biochemistry, University of Alabama in Birmingham, Birmingham, Alabama I. Introduction . . . . . . . . . . . . . . . . . . . 11. Theory . . . . . . . . . . . . . . . . . . . . . I n . Medium for Electrophoresis . . . . . . . . . . . . . IV. Apparatus . . . . . . . . . . . . . . . . . . . . A. Cytopherometer . . . . . . . . . . . . . . . . B. Laser Doppler Spectroscopy . . . . . . . . . . . . C. Electrophoresis in Columns . . . . . . . . . . . . D. Endless-Belt Electrophoresis . . . . . . . . . . . . E. Free-Flow Electrophoresis . . . . . . . . . . . . V. Applications of Electrophoresis to Specific Cell Separations . A. Lymphoid, Blood, and Hemopoietic Cells . . . . . . . B. Neoplastic, Dividing, and Immature Cells . . . . . . . VI. Viability and Function . . . . . . . . . . . . . . . VII. Effects of Lytic Enzymes on Electrophoretic Mobilities of Cells VIII. Adsorbable and Other Materials . . . . . . . . . . . . IX. Miscellaneous . . . . . . . . . . . . . . . . . . X. Isoelectric Focusing . . . . . . . . . . . . . . . . XI. Concluding Remarks . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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I. Introduction In 1902, Ralph S. Lillie (1902) described the movement of isolated nuclei, leukocytes, muscle cells, and red blood cells in an electric field in an isotonic solution of cane sugar as viewed through a microscope. Among his several observations, he noted that “the resistance of the sugar-solution is very high and only a slight current passes. . . . Two years later, Girard-Mangin and Henri (1904) described somewhat similar experiments on the electrophoresis of cells in Europe. As discussed in greater detail below, more than half a century later, the development of electrophoretic buffers of low conductivity which are made isosmolar with saccharides has become an important innovation in the modem electrophoresis of cells. The long history of the electrophoresis of cells is reviewed in the symposium edited by Ambrose (1965). Despite its venerable antiquity, cell electrophoresis was not used extensively for the preparative purification of viable cells until the ”

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1970s. During the first half-century of cell electrophoresis, investigators were preoccupied with the design of apparatus, the construction of model systems, the electrophoresis of red blood cells, alterations of electrophoretic mobility following the exposure of cells to lytic enzymes, and so on. In the 1970s, the techniques, apparatus, and buffers of Hannig and Zeiller (Hannig, 1972) were applied to a variety of problems in cell separation and demonstrated the utility of the electrophoresis of cells for the separation of large numbers (lo8 to lo9 cellsflour) of viable cells for biochemical and biological investigations.

11. Theory

There have been many excellent reviews of the theory of electrophoresis as related to the electrophoresis of cells (Hannig, 1967, 1969, 1971, 1972; Bier, 1967; Ambrose, 1965; Shortman, 1972; Abramson, 1934; Overbeek and Wiersema, 1967; van Oss, 1975). The theory of electrophoresis has not been useful in permitting the precise prediction of conditions which will allow optimal cell separations. While many variables in experimental design are known to be important in influencing the electrophoretic migrations of mammalian cells, Shortman (1972) has summarized the state of our knowledge in the statement: ‘‘There is no completely satisfactory formulation directly relating electrophoretic mobility to the ionic composition of the medium and to the surface charge, surface shape, and size of the cell. ” In this article we confine our discussion of theory to a very superficial presentation of the equations which describe the electrophoretic migration of cells and to a brief description of selected experimental studies on some of the important experimental variables. As described below, many different types of apparatus have been used for the electrophoresis of cells. In fact, there are few other methods for the separation of cells in which the apparatus and design of equipment have assumed the importance given them in cell electrophoresis. In reviewing the literature relevant to the electrophoresis of cells, one is struck by the fact that the design of hardware is often a preoccupation which has distracted investigators from consideration of biologically important aspects of their investigations. Commonly, two physical systems are used for the electrophoresis of cells. In the first of these, cells are electrophoresed in a static liquid medium; and the electrophoretic migration of the cells is described by Eq. (1):

v = &E/6.rrr) (1) where v is the electrophoretic mobility of the cell, 5 is the electrokinetic potential of the cell, E is the dielectric constant, E is the field strength, and r ) is the viscosity of the medium. The second commonly employed physical system for the electrophoresis of cells, developed by Hannig (1972), is termed free-flow

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electrophoresis. Cells are introduced from a point (Fig. 1) source near the top of a vertical laminar flow of electrophoresis buffer which enters the top of the electrophoresis chamber, flows in a laminar fashion between two parallel electrodes, and leaves the chamber through multiple parallel exits at the bottom of the vertical chamber. Cells are exposed to the electrical field for 5-10 minutes. If we regard their sedimentation at unit gravity as negligible during this interval, their electrophoretic migration is described by Eq. (2): tan a = vi/qPw (2) where a is the angle between the diagonal course described by the migrating cell and the vertical course described by an uncharged particle, v is the electrophoretic mobility of the cell, i is the current, q is the cross section of the chamber, P is the specific conductivity of the medium, and w is the velocity of flow of the belt DIRECTION OF LAMINAR FLOW

uuuuuuuuuuuuuuuu

1

1

uuuu

innnr COLLECTION TUBES

FIG.1. In free-flow electrophoresis, cells are introduced from a point at the top of the apparatus and migrate laterally toward the electrode in the vertically flowing buffer.

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of fluid in the chamber. Effective separation of cells and the applicability of this equation to this system are dependent upon avoidance of thermoconvective disturbances with a sophisticated cooling system; Hannig has designed and tested extensively several such systems (Hannig, 1972). As noted by Hannig, the accuracy of the equation is dependent upon several assumptions which are not completely validated. Among these is the assumption that the cell is a spherical insulator. The several other assumptions required for the mathematical description, as well as several complicating practical considerations, are discussed in detail by Hannig (1972; Hannig et al., 1975). The actual observed migration of cells in free-flow electrophoresis is influenced by several practical considerations such as the thickness of the flowing curtain, the configuration of the sample inlet jet, the charge on the material used for construction of the chamber, and other relatively undefined variables.

111. Medium for Electrophoresis

Much has been written about the effect of the medium selected for electrophoresis on the electrophoretic mobilities of cells. The mobilities of cells are affected by many properties of the medium including ionic strength, dielectric constant, temperature, viscosity, pH, and the ability of the medium to maintain cell viability (Weiss, 1966; Zeiller et al., 1975a; Mehrishi and Thomson, 1968). Several investigators have emphasized changes in the electrophoretic mobilities of cells associated with the death of cells (Leise and LeSane, 1974; Carstensen et al., 1968; Weiss, 1966). Hannig (1971) reviewed both the properties desirable for media used for specific problems in electrophoresis and the several kinds of media employed for specific problems. Hannig (1972) has pointed out that “in the electrophoretic separation of particle suspensions (cells and cell organelles), the ionic strength of the particles plays a minor role owing to their relatively small surface charge . . . and that “a higher conductivity of the buffer solution is unfavorable, not only because of increased production of unwanted Joule heat but also because the absolute and relative electrophoretic mobility of the components to be separated drops . . . ” when buffers of higher conductivity and ionic strength are employed. This decrease in electrophoretic mobility with increase in conductivity of the suspending medium was known much earlier from work with bacterial cells (Brown and Broom, 1935); however, practical advantage of this knowledge was not taken before Hannig and his associates emphasized this important point. In our laboratory, we have found that the buffers of Zeiller and Hannig have been most satisfactory in permitting us to separate rat proximal tubule cells (Kreisberg et al., 1977b) and human tonsillar cells (Pretlow et al., in preparation). ”

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IV. Apparatus The first electrophoresis of cells was conducted in a cane sugar solution layered on top of a microscope slide (Lillie, 1902). Since that time, many different kinds of apparatus have been described. Often the apparatus has so absorbed the attention of the investigator as to prevent any serious consideration of the effect of the physical conditions on the biological integrity of the cells being electrophoresed. We briefly review some of the available apparatus. A. CYTOPHEROMETER

Most of the early work on the electrophoresis of cells was conducted with a cytopherometer. The technique was introduced by Ellis (1911), and many interesting data resulted from investigations with this instrument. The major limitations inherent in the use of this instrument resulted from the fact that measurements were made on only one cell at a time. The capacity of the instrument dictated that it could be used only for analytical investigations, and preparative separations of cells by electrophoresis had to wait until instruments with larger capacities could be developed. Even for analytical purposes, instruments with capacities larger than that of the cytopherometer facilitate a more adequate sampling of the cells to be studied and obviate the necessity for holding cells in solution while the electrophoretic mobilities of other cells are being measured one cell at a time. In some cases, investigators have been particularly interested in the electrophoretic mobilities of blood cells in the sera of specific donors; and for such studies (Seaman and Heard, 1961) the small volume of the cytopherometer appears to offer advantages. With various modifications the cytopherometer has continued to find applications in recent years (Bangham et at., 1958; Bert et al., 1971; Donald et al., 1974; Goldstone et al., 1974; Larcan et al., 1974; Rubinstein et al., 1974; van Oss et al., 1974; Chollet et al., 1975, 1976a,b, 1977; Gallin et a l . , 1975; Hanjan and Talwar, 1975; Jenkins, 1975; Brown et al., 1977; Donner et al., 1977; Hanjan et al., 1977; Chiu et al., 1977; Lampert et al., 1977; Sabolovic et al., 1977; Talwar et al., 1977).

B. LASERDOPPLER SPECTROSCOPY Laser Doppler spectroscopy is a technique used for work with cells primarily by one laboratory (Uzgiris and Kaplan, 1974a,b, 1976a,b; Kaplan and Uzgiris, 1975, 1976). This technique is clearly explained and applied to a problem in the detection of molecular heterogeneity and aggregation by Ware and Flygare (1972). A laser is used in conjunction with an optical scanning system to detect subpopulations of eIectrophoresed cells or molecules as they change in concen-

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tration at various locations in the apparatus during the course of electrophoresis. Because of its rapidity and convenience, the technique has been promoted as a means of avoiding other methods which require “the tedious microscopic observation of the time of migration of individual cells” (Uzgiris and Kaplan, 1974a). After reviewing the work done by Uzgiris and his associates in studying the electrophoreticmobilities of cells by laser Doppler spectroscopy, one is struck by the fact that the biological meaning of this work is uncertain. Most of the articles cited above simply report that one obtains electrical signals from the apparatus during the electrophoresis of cells in the apparatus. The meaning of these signals is limited to statements such as that found in the report of Kaplan and Uzgiris (1976) that during their study of “electrophoretic mobility distributions of human peripheral blood lymphocytes. . . three major subpopulations were spectrally resolved”; two of these subpopulations were stated to be T cells, and one was said to be B cells. There are several problems with this work (Kaplan and Uzgiris, 1976) which lead us to question the conclusions. We believe that it is important to point out that one of these subpopulations would disappear from their plot of the distribution of cells if one data point were deleted or altered in magnitude by approximately 25%; that is, one subpopulation (with an electrophoretic mobility of 2.35 p m sec-’ V-’ cm-* in their Fig. 1) is identified by a single data point. Deductions are made about the identities of these subpopulationsof cells by comparing the distributionsof cells by laser Doppler spectroscopy before and after depletion of the population by passage over nylon fiber columns, by “a one-step rosette procedure with sheep erythrocytes,” and by other commonly used cell separation procedures. Unfortunately, we are not told how pure the purified B or T cells were; we are not told what proportion of the cells subjected to the purification procedures was recovered; and the presence or absence of monocytes after the purification of cells with Ficoll-Hypaque and/or a nylon column was not mentioned. In fact, we are given only the statement: “The lymphocytes appeared to be virtually free of all other blood cell elements (98 to 100% lymphocytes as determined by Wright’s stain examination). ” After another purification procedure, we are told that “by these procedures, a cell preparation can be obtained comprising mostly B cells.” We believe that expressions such as “mostly B cells” and “virtually free of all other [cells]” fall short of the precise numerical expression more appropriate to the description of analytical separation procedures in the 1970s. These investigators (Kaplan and Uzgiris, 1976) found that, after separation only on Ficoll-Hypaque (their Table I), blood cells from six of their eight donors contained 10% or fewer “lymphocytes. . . which have surface immunoglobulins (Ig) as determined by immunofluorescence.” We are not told where the monocytes went or how they were accounted for; and one suspects that these

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authors are not aware that some excellent investigators have found more than 40% monocytes among cells separated on Ficoll-Hypaque (Zucker-Franklin, 1974). Starting with cells from Ficoll-Hypaque from donors whose cells contained 4 , 6 , 8 , or 9%cells with surface Ig, Kaplan and Uzgiris purified B cells by depleting the starting suspension of T cells; they then reported the laser Doppler patterns obtained with these purified B cells. It never seemed to occur to them to measure what proportion of these cells had surface Ig by direct irnmunofluorescence. The closest they came to making this measurement was their attempt to quantitate the B cells in the suspension of purified B cells when they “reacted them with polyvalent anti-human Ig serum and determined the fraction of these cells that were positive for surface IG [not by observing them directly with fluorescence microscopy but] by the resulting depletion of the low frequency peak. ” Like many of the other methods used in this research, this method for assaying the number of B cells was worse than it appears at first glance. To compare the proportion of B cells found in blood in their laboratory with that reported by others, the authors (Kaplan and Uzgiris, 1976) cite work ( h b o et al., 1975; Winchester et al., 1975) showing that purified anti-IgG may bind to cells that lack surface Ig but have surface Fc receptors. Unfortunately, it appears that Kaplan and Uzgiris missed the central points of these two reports since, both in their attempts to deplete cells of B cells and in their immunofluorescent staining of B cells, these authors used “polyvalent antiserum to human Ig.” Thus it is likely that, with the antiserum used to deplete cell preparations of B cells, they depleted the unseparated cell preparation of other cells in addition to B cells. Similarly, while they do not appear to have examined their purified B cells by immunofluorescence, their reported proportion of Ficoll-Hypaque purified cells with surface immunoglobulin probably includes cells other than B cells which have Fc receptors. Since only Wright stains were done to exclude monocytes, it is impossible to know what proportion of monocytes was present in the studied suspensions of cells (Zucker-Franklin, 1974). We have dwelt on this paper rather at length because it is among the few papers (Luner et al., 1977; Smith et al., 1976, 1978) we have encountered which attempt to demonstrate that laser Doppler spectroscopy is suitable for obtaining data that can be interpreted as biologically significant. We believe that the usefulness of this technique for the electrophoresis of cells has not been demonstrated. There are other problems associated with any technique for the separation of cells which is not followed by an attempt to recover, examine, and characterize the separated subpopulations after they are separated. These problems are discussed in Section IV,C. For the sake of completeness, a study of blood cells from normal subjects and patients with acute leukemia (Smith et al., 1976, 1978) is considered in our discussion of neoplastic cells in Section V,B.

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c. ELECTROPHORESIS IN COLUMNS In 1973, Fike and van Oss (1973) described their design of a system for preparative electrophoresis of cells in a column. They stated the view that “freeflow electrophoresis. . . ,until now the only feasible method for preparative cell electrophoresis, is both costly and complicated.” In their attempt to design a cheaper, effective system for preparative electrophoresis of cells, they investigated several alternative supports for the column of fluid in which electrophoresis was to be accomplished. In their investigation of soluble dextrans, methyl cellulose, and polyoxyethylene, they encountered ‘‘various drawbacks, mainly attributable to osmotic pressures,” which were not described in detail; and they therefore investigated the use of solid packing materials. When Sephadex was used as the supporting material, they found that cells adhered to the beads on the sides of the beads that faced the negative pole. They hypothesized that the paths of the electrophoresed cells “led through the porous beads, directly in line with the electric field.” They therefore used glass beads as a support for column electrophoresis in the belief that the electric field and the courses of the cells would bend around the glass beads. This interesting, original, and economical approach allowed them to separate 0.2 to 2.0 X 10” cells in a single experiment. The glass beads did not appear to retard the migration of the cells significantly, since the values they obtained for the electrophoretic mobilities of the cells were similar to those reported form other laboratories. Other investigators have electrophoresed cells in columns stabilized by density gradients. Boltz et al. (1973) electrophoresed cells in an isosmotic gradient of decreasing sucrose and increasing Ficoll concentration. The combination of these two solutes was necessary because, under the conditions used, increasing concentrations of Ficoll caused increased osmotic pressure. They found that 75% of Chinese hamster cells plated were able to form colonies after cell electrophoresis. Of particular interest (Boltz et al., 1973), “the plating efficiency of the cells was the same with and without the application of an electric field. ” It would not have been necessary to include sucrose with the Ficoll in the gradient if the Ficoll had been processed slightly differently, as described previously by Pretlow et al. (1975). The values obtained for the osmolarity of Ficoll by Boltz et al. are markedly different from those reported by Munthe-Kaas and Seglen (1974). The values obtained by Boltz were observed by us after Ficoll was autoclaved similarly to the Ficoll used by Boltz et al.; however, Ficoll has an osmotic pressure of less than 25 milliosmoles/liter when it is prepared as described by us (Pretlow et al., 1975). The data of Munthe-Kaas and Seglen are in agreement with out data for Ficoll at concentrations below 20%; it is our opinion that the data of Munthe-Kaas and Seglen for concentrations above 20% Ficoll are complicated by technical problems (which they do not discuss) in the performance of their experiments.

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Since the original electrophoresis of cells in columns (Boltz et al., 1973; Fike and van Oss, 1973), numerous investigators have employed this technique (Gillman er al., 1974; Griffith et al., 1975, 1976; Catsimpoolas et al., 1975; Ault et al., 1976; Platsoucas et al., 1976; Catsimpoolas and Griffith, 1977a,b,c). Before leaving our discussion of column electrophoresis, we should mention the recent development of a TRANS-analyzer by Catsimpoolas and his associates (Catsimpoolas, 1975; Catsimpoolas et al., 1975; Catsimpoolas and Griffith, 1977b,c). This instrument is designed for the rapid analysis of cell electrophoresis patterns during column electrophoresis. As described by Catsimpoolas et al. (1975), “the distribution of cells is monitored repetitively during migration by absorbance measurements at any wavelength in the 200-800 nm range. A computer program provides the statistical analysis of each peak (baseline correction, smoothing, area, mean, standard deviation, skewness, and kurtosis) which can be further utilized for computing additional parameters, such as resolution and heterogeneity. We wish to emphasize our opinion that this kind of analysis will be useful, if at all, only when the results obtained are corroborated closely and in detail by an independent method such as cell counts done by light microscopy. The TRANS-analyzer has the advantage over laser Doppler spectroscopy that fractions can be collected and characterized; however, if one does the necessary careful characterization by other means, it is not clear that the TRANS-analyzer adds much additional, valuable information. Cells are mutable. Small differences in the methods for preparing them may cause cell death or aggregation of cells. At the present time, optical scanning systems such as those used in laser Doppler spectroscopy and in the TRANS-analyzer cannot distinguish monomeric viable cells from dead cells, debris, organelles, aggregates of cells, and so on. With probably more experience than anyone else in the careful analysis of cells separated by electrophoresis, Hannig (1972) has emphasized that “the best way of deciding on suitable milieu conditions for the isolation of a particular cell fraction is to observe the morphological behavior in the optical or electron microscope. Aggregation is always a sign of damage.” Automated systems such as those described above do not discriminate between aggregated and nonaggregated or between viable and nonviable cells. ”

D. ENDLESS-BELT ELECTROPHORESIS In 1969, Kolin and Luner described a very cleverly designed device for the electrophoresis of cells in a continuously flowing endless belt of buffer. Stability of the system against thermoconvection and protection from artifacts secondary to sedimentation of cells at unit gravity were afforded by periodic reversal of the gravitational field. Descriptions of the device and preliminary cell separation

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experiments performed with the apparatus were published in 1969 (Kolin and Luner, 1969) and again in 1971 (Kolin and Luner, 1971); however, it appears that applications of this device to the separation of cells have been too limited to permit a thorough evaluation of its utility for this purpose. E. FREE-FLOW ELECTROPHORESIS As discussed above, cell electrophoresis has been useful for analytical purposes since 1902. The electrophoresis of cells in sufficient quantities (lo8 to lo0 cells/hour) to permit detailed characterization and biochemical studies became a possibility when Hannig, Zeiller, and their associates at the Max Planck Institute in Munich introduced two major deviations from the electrophoresis of cells as practiced during the preceding half-century . First, they employed Hannig 's (Hannig, 1964, 1967, 1969, 1971, 1972) continuous-flow apparatus to conduct a type of experiment in electrophoresis termed free-flow electrophoresis. Second, they developed a new series of buffers for electrophoresis which differed from the buffers in common use in that they were of low conductivity. These buffers were made isosmotic with solutes of low ionic strength such as glucose, sorbitol, and sucrose. Media of low ionic strength permitted Hannig and Zeiller to use sufficient voltage to obtain good separations while avoiding the thermoconvective problems commonly observed with salt solutions of higher conductivity. Hannig , Zeiller, and their associates have applied free-flow electrophoresis to a wide variety of problems in experimental immunology and hematology (Hannig, 1964; Hannig and Krusmann, 1968; Ganser et al., 1968; Hannig and Zeiller, 1969; Zeiller et al., 1970, 1971a,b, 1972a,b,c, 1974, 1975a,b, 1976;Zeiller and Hannig, 1971; Schubert et at., 1973;Zeiller and Pascher, 1973; Seiler etal., 1974; Mehrishi and Zeiller, 1974; Droege et al., 1974a,b; Hannig et al., 1975, 1977; Zeiller and Hansen, 1978). Probably because of the expense of the freeflow electrophoresis equipment and the difficulty of keeping it in good repair, other laboratories were slow to use the technique until after it had been proved several times by Hannig and Zeiller. In 1972, the first of a series of experiments from Nordling, Andersson, and Hayry (Nordling et al., 1972a) in which cells of the immune system were separated by free-flow electrophoresis appeared; and later articles in this series from their laboratory have appeared regularly (Nordling et al., 1972b; Hayry et al., 1973, 1975a,b; Andersson et al., 1973a,b, 1975; Hayry and Andersson, 1975). At almost the same time that Nordling et al. (1972a) published their first application of the technique of Hannig and Zeiller, Stein et at. (1973) published his first use of free-flow electrophoresis for the separation of human lymphocytes; and Stein has continued to employ free-flow electrophoresis for the separation of lymphoid cells (Stein, 1975a,b). A little later, Shortman, von Boehmer, Schlegel, and their associates began to report their applications of free-flow

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electrophoresis to the separation of lymphoid and hemopoietic cells (von Boehmer, 1974; von Boehrner et al., 1974; Schlegel et al., 1975a,b; Shortman et al., 1975; Melchers et al., 1975; Roelants et al., 1975; Osmond etal., 1975). Other laboratories have studied lymphoid, hemopoietic, and/or blood cells by free-flow electrophoresis (Herve et al., 1975; Just et al., 1975; Heuer et al., 1977; Levy et al., 1977, 1978; Schubert et al., 1977, Chollet et at., 1978). In 1977, free-flow electrophoresis was first applied to the separation of proximal tubule cells from rats in our laboratory (Kreisberg et al., 1977b) and from rabbits by Heidrich and Dew (1977). It is difficult to assess the degree of purification obtained by Heidrich and Dew. They state that “renin activity is found only in fractions 16-21 ”; however, we cannot determine if this is really evidence of purification, since no evidence was presented to establish that renin activity was more concentrated in the cells in fractions 16 to 21 than in the kidney cells prior to electrophoresis. In fact, no numerical expression of the degree of concentration or purity obtained is given. Unaware of a considerably earlier report of the purification of proximal tubule cells (Pretlow et al., 1974) and of an additional report on the purification of proximal tubule cells published only a few months before their report (Kreisberg et al., 1977a), Heidrich and Dew stated that ‘‘the successful isolation and separation of homogeneous cell populations from kidney cortex in preparative quantities has not yet been described. Unfortunately, in their report (Heidrich and Dew, 1977) we are not given numerical data which would permit us to evaluate either the quantity or the homogeneity of the purified cells. The details of many of the reported cell separations by free-flow electrophoresis are discussed below; however, before leaving our treatment of the technique we should comment briefly on several practical aspects of the procedure. Initially, we were guarded in our estimation of the breadth of applicability of this technique because we were skeptical about (1) possible injurious effects of the electrophoretic procedure on the cells to be studied, (2) possible injurious effects of the rather unusual electrophoretic buffers on the cells, and (3) the effects of the proteolytic enzymes which we have usually found necessary for work with human tissues on the separability of the cells to be electrophoresed. In order to examine these potential problems, we studied the separation of human tonsillar cells by electrophoresis. These cells were selected because we have had considerable experience in their separation, characterization, and culture (Willson et al., 1975, 1976a,b). To our surprise, the human tonsillar cells obtained in suspension with trypsin or collagenase responded to mitogens and excluded trypan blue equally as well before and after electrophoresis (Pretlow et al., in preparation). Of equal interest, while the electrophoretic mobilities of all cells were somewhat decreased as compared with those of human tonsillar cells obtained in suspension mechanically (without trypsin or collagenase), all cells were as well purified after being obtained in suspension with trypsin as after ”

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being obtained in suspension mechanically. At least this one group of cells in our laboratory has shown no evidence of injury or decreased separability after electrophoresis with the technique of Hannig and Zeiller and after exposure to their buffers for several minutes at 4.0"C. There are several practical aspects of the use of this technique which might be listed briefly. In this article, we do not consider the theoretical factors which influence the artifacts encountered during free-flow electrophoresis. These factors have been discussed with authority in the many reviews by Zeiller and Hannig, and we simply refer the reader to two of the more recent of these (Hannig et al., 1975; Zeiller et al., 1975a). Practically, Hannig et al. (1975) have found that coating the wall of the electrophoretic apparatus with various substances such as albumin has been useful in improving the operation of the apparatus. More recently, Zeiller (verbal communication) has preferred to use hemolyzed rat blood to coat the chamber. Zeiller sterilizes the chamber with 3.5% formaldehyde prior to experiments conducted under sterile conditions; others have used 2% acetic acid '(Stein, 1975b). After coating the chamber, Zeiller has found it useful to test the electrophoretic mobility of a standard batch of glutaraldehyde-fixed erythrocytes to be certain that the coating is satisfactory. Zeiller has found that aggregation of the cells in the apparatus can be minimized by treatment of the cells with DNase (Zeiller et al., 1975a); we did not find this to be necessary in our work with rat kidney cells (Kreisberg et al., 1977b) or human tonsillar cells (Pretlow er al., in preparation). We did find some slight variation in the locations of cells in different exit channels from the apparatus in different experiments; however, experiments were highly repeatable if the locations of cells were expressed relative to the modal location of red blood cells. Others have found this kind of adjustment of the data useful (Stein, 1975a).

V. Applications of Electrophoresis to Specific Cell Separations A. LYMPHOID, BLOOD,AND HEMOPOIETIC CELLS Because of the large volume of studies focused on the electrophoretic separation of T and B lymphocytes, we review their separation first and then consider other blood, lymphoid, and hemopoietic cells exclusive of T and B lymphocytes. 1. T and B Lymphocytes

Probably because of their ready availability as a suspension of human cells, blood cells were among the first subjects of cell electrophoresis (Lillie, 1902). Perhaps for the same reason and because of the growth of immunology in more recent years, lymphocytes have been the most frequently electrophoresed cells in the 1970s. The extensive investigations of blood cells prior to 1965 have been

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reviewed thoroughly by others (Arnold, 1965; Seaman and Cook, 1965; Ruhenstroth-Bauer, 1965; Rottino and Angers, 1965; Sachtleben, 1965; Straub, 1965). In 1965, blood lymphocytes were regarded by most investigators as a single population distinct in electrophoretic mobility from granulocytes and erythrocytes. In 1968, a report from Hannig and Krusmann (at the Max Planck-Institut fiir Eiweiss- und Lederforschung in Munich), interpreted retrospectively, gave the first clue that T and B lymphocytes might exhibit different electrophoretic mobilities (Hannig and Krusmann, 1968): “In addition to the normal lymphocyte fraction an electrophoretically slower moving fraction of larger lymphocytes is found, which stains differently from the normal one. We might consider this slower moving lymphocyte fraction as being enriched in antibody producing cells.” Also in 1968, from another Max Planck Institute in Munich with a very different type of instrument, Ruhenstroth-Bauer and Lucke-Huhle (1968) reported work which led them to hypothesize that there were two subpopulations of lymphocytes in peripheral blood. Based on their measurements of the electrophoretic mobilities of 308 rat thoracic duct lymphocytes, they plotted the frequency distribution of rat thoracic duct lymphocyte mobilities. In what appears to the casual observer to be a somewhat asymmetrical curve, they thought that they could identify two gaussian distributions and wrote (Ruhenstroth-Bauer and Lucke-Huhle, 1968): “If the two resultant overlapping distributions are plotted as integrated curves on probability paper, then each population gives rise to a straight line. ” In addition, their analysis of the volume distributions of these cells by a similar method led them again to the conclusion that they were studying two distinct subpopulations. Parenthetically, it has never been clear to us why anyone should assume that any single type of cell, not identified by another parameter independent of size or partition characteristics, should follow a gaussian distribution with respect to size or frequency distribution after electrophoresis, sedimentation, or other partitioning; however, this assumption is common in the published work on cell separation. Other indirect evidence reported by these investigators (1968) led them to state that “it may be said that our experiments are in agreement with the hypothesis that the so-called small lymphocytes of the rat in fact comprise two cell populations.” In 1971, Zeiller and Hannig published their observations that rat “thymus and bone marrow were comprised mainly from lymphocytes of low mobility, duct lymph mainly from high-mobility lymphocytes, whereas lymph node and spleen showed similar numbers of cells in both regions. In a companion paper (Zeiller et al., 1971a), they demonstrated that spleen cells active in an assay for graftversus-host reactivity were found among lymphocytes from the high mobility region (HMR)of their electrophoretic profile. They cited their previous report that antibody forming cells were concentrated in the low mobility region (LMR) and concluded that ‘‘a summary of these results suggests that two basic popula”

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tions of lymphocytes can be separated on the basis of different zeta potentials. One population encompasses cells dealing with the humoral immune response, the other population contains cells which are responsible for reactions of the delayed hypersensitivity type. ” They stated that their evidence supported the hypothesis that they had electrophoretically separated lymphocytes of thymic and bursal derivation. They also noted that they were preparing a paper which would describe similar results for lymphocytes from mice. Since these early papers by Zeiller, Hannig, and their associates, there have been numerous papers which have supported the general principle that, in most studied mammals, the electrophoretic mobilities of most T lymphocytes are different from those of most B lymphocytes (Zeiller et al., 1971b, 1972b, 1974, 1976; Nordling et al., 1972a,b; Zeiller and Pascher, 1973, 1978; Andersson et al., 1973b; Hayry et al., 1973; Stein et al., 1973; Wiig and Thunold, 1973; Donald et al., 1974; Platsoucas et al., 1976; von Boehmer et al., 1974; Mehrishi and Zeiller, 1974; von Boehmer, 1974; Goldstone et al., 1974; Seiler et al., 1974; Sabolovic et al., 1974; Gillman etal., 1974; Schlegel et al., 1975a,b; Shortman et al., 1975; Herve et al., 1975; Eckert, 1975; Chollet et al., 1975, 1976a,b, 1977, 1978; Durandy et al., 1975; Hayry et al., 1975a,b; Weiss et al., 1976; Dumont and Barrois, 1977; Dumont and Bischoff, 1977; Dumont and Robert, 1977a,b; Smith et al., 1978). Rather than review the extensive and often repetitive studies reported in these articles, we refer interested readers to the original sources for discussions of the responses of fractionated lymphocytes to mitogens, surface markers in fractionated lymphocytes, and so on. We emphasize that the completeness of the separation of T cells from B cells by electrophoresis has varied enormously as a function of different laboratories, different species, and different tissues. In an excellent critical article, Vassar et al. (1976) discuss the several variables which may contribute to the different experimental results reported; these authors observed marked differences in electrophoretic mobility and separability of T and B lymphocytes from the same individual donors at different times. They sound an important warning in their statement that “. . . pooling of electrophoretic data may be potentially misleading. ” Furthermore, this observed variability suggests that work attempting to correlate lymphocyte mobilities measured over a period of time with disease processes, clinical treatments, or immune rejection phenomena should be interpreted with caution. Two reports have suggested specific chemical differences which may help to account for the observed differences in the electrophoretic mobilities of T and B lymphocytes (Mehrishi and Zeiller, 1974; Nordling et al., 1972b). Before leaving our discussion of the electrophoretic separation of T and B cells, we should mention the observation of Stein et al. (1973) in their study on human blood lymphocytes that “no difference in stimulation by PHA in vitro or in the percentage of cells with immunoglobulin determinants was found between unseparated cells and separated cells when all fractions were pooled after separa-

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tion. ” The functional characterization of cells from pooled fractions of all recovered cells is a particularly valuable kind of control which is most commonly not included in reports on cell separations. Our laboratory has observed a similar maintenance of response to mitogens by pooled fractions of human tonsillar cells after free-flow electrophoresis (Pretlow et al., in preparation). 2. Other Cellsfrom Peripheral Blood

a. Red Blood Cells. Since early in the history of cell electrophoresis (Lillie, 1902; Kozawa, 1914; Coulter, 1921; Eagle, 1930; Abramson, 1930; Anderson and Mackie, 1939), red blood cells have been widely employed because of their

ready availability in suspension. Most often, studies employing red blood cells have not been directed primarily toward their investigation, rather erythrocytes have been employed to test new physical systems for cell electrophoresis, new buffers, the effect of pH on electrophoretic mobility, and so on. Most applications involving erythrocytes are reviewed in other sections which describe the purposes for which they were employed, for example, Sections IV, VII, and X. In addition to their ready availability in suspension, erythrocytes offer other advantages as prototypic mammalian cells for use in testing new systems for cell electrophoresis. Erythrocytes from single donors are relatively uniform in size and electrophoretic mobility as compared with other kinds of cells from single donors. Erythrocytes from different species have different, characteristic electrophoretic mobilities (Abramson, 1929; Howitt, 1934). The different mobilities of erythrocytes from different species have provided investigators with widely used model systems in which erythrocytes from two or more species are mixed and separated in various electrophoretic systems into distinct zones. For those primarily interested in demonstrating that a particular apparatus works, the red color of erythrocytes has made them easily visible as pink bands or zones; and some investigations have relied totally on the ability of the unaided eye to see erythrocytes in electrophoresis chambers. We note that for this purpose the unaided eye is not only quantitatively insensitive but also unable to distinguish erythrocytes from hemoglobin crystals which result from the isoelectric focusing of lysed erythrocytes at high concentrations. Numerous studies of the electrophoresis of red blood cells have been reviewed in the symposium edited by Ambrose (1965). Most of the experiments reported in this symposium were conducted with a cytopherometer. Interestingly, erythrocytes were among the first cells to be obtained in a high degree of purity by free-flow electrophoresis (Hannig, 1964); and work by Hannig and his associates (Gamer et al., 1968) demonstrated the separation of red blood cells from other blood cells even before the development of modem buffer systems which are better suited for this purpose. Before leaving our discussion of red blood cells, we note that there have been a few studies which suggest that measurement of the electrophoretic mobilities of

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red blood cells may find practical applications in the study of disease. In mice, study of several inbred species has resulted in the demonstration that the electrophoretic mobility of erythrocytes is inherited as an autosomal codominant trait which correlates with certain other properties of erythrocytes and which segregates independently of coat color and the H-2 genes. Study of a large number of patients led Rottino and Angers (1961) to the conclusion that “barring the concurrent presence of inflammatory disease, microelectrophoreticdata will enable differentiation of benign from malignant neoplasm, peptic ulcer of the stomach from carcinoma of the stomach. ” The electrophoretic mobilities of erythrocytes have not been studied extensively for the purpose of characterizing genetic or pathological conditions. b. Miscellaneous Separation of Nucleated Blood Cells. Despite the extensive study of the electrophoresis of red blood cells and lymphocytes, there have been comparatively few studies on the electrophoresis of granulocytes and monocytes. As reviewed in the symposium edited by Ambrose (1965), work with a cytopherometer established that erythrocytes, granulocytes, and lymphocytes were separable electrophoretically in laboratory rodents (Arnold, 1965) and humans (Ruhenstroth-Bauer, 1965). Generally, all these cell types were described as exhibiting relatively limited but distinctive ranges of electrophoretic mobility. Monocytes, now known to constitute a much larger proportion of nucleated blood cells than was recognized in the 1960s (Zucker-Franklin, 1974), were largely ignored in the work with the cytopherometer; and one can only speculate that they were present among the morphologically somewhat similar lymphocytes. With free-flow electrophoresis, Hannig and his associates (Hannig and Krusmann, 1968) observed that “the distribution of granulocytes and also that of the lymphocytes show two peaks.” As discussed above, he recognized that one of the peaks of lymphocytes contained concentrated antibody-producing cells. There have been a few electrophoretic characterizationsof blood lymphocytes, which differ either in purpose or scope from the numerous separations of T and B lymphocytes described above; these are reviewed here. Several investigators have described more than two populations of lymphocytes, which can be distinguished wholly or in part by their electrophoretic mobilities. Chollet et al. (1975, 1977) thought that they could distinguish a third population of lymphocytes with electrophoretic mobilities intermediate between those of T and B lymphocytes; this third population lacked the conventional markers for both T and B lymphocytes. Since they do not discuss any approach to either eliminating or accounting for monocytes, these papers are difficult to interpret. Ault et al. (1976) reported the concentration of several classes of lymphocytes by electrophoresis. Of particular interest, one fraction of separated cells was obtained which contained 65% cells with surface IgM or IgD (B cells in the terminology of Ault ef al.). Unfortunately, we are not told what proportion of all recovered IgM- and IgD-bearing cells were contained in this fraction, and we do

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not know how representative the cells in the purified fractions were of all such cells. Further, we are not told what proportions of all IgM- and IgD-bearing cells in the starting sample were recovered after electrophoresis. We are not given much cause for optimism by the statement (Ault et al., 1976) that “the overall recovery of cells after electrophoresis ranged from 30 to 50%.” There are other features of this report which lead us to wonder how generally applicable the reported data are; for example, all experiments were carried out with cells from a single donor. The purity of the separated cells is given without any estimate of the confidence interval, standard deviation, or range observed. It is stated that ‘‘. . . in some experiments the T cells recovered from electrophoresis did not form good rosettes and thus T cells are reported here as those lymphocytes which were Ig negative.” We wonder if cells which were Ig-negative were null cells, cells which had lost surface Ig, T cells which had lost surface receptors for sheep erythrocytes, or T cells with intact receptors; certainly, the absence of a marker is not a very reliable means for the positive identification of cells as T cells, injured cells, or any other particular kind of cells. In the introduction to the paper, the authors state: “In the course of these studies we have also observed that a newly described class of lymphocytes shows electrophoretic behavior that is distinct from both monocytes and IgM and IgD bearing B lymphocytes. These lymphocytes have IgG on their surface but lose it rapidly at room temperature or at 37°C. . . . Unfortunately, we are not given evidence in the paper that these claims are justified. The only presented data show the distribution of cells which react with anti-IgG, and we are referred to thus far unreported data for the identification of these cells (Ault et al., 1976): “In other experiments we have shown that greater than 90% of cells thus labeled by whole anti-IgG antibody are the same cells with labile IgG that have been described by others. . . .” We must wonder if these other experiments were performed with just one blood donor like those in the report under consideration. If so, was it the same donor? Were the cells in the other experiments treated with the same electrophoretic procedures which resulted in the loss of 50-70% of all cells, the loss of an unspecified proportion of IgG-bearing cells, and “T cells recovered from electrophoresis. . . which. . . did not form good rosettes. . .”? We are told that “there was no consistent preferential loss of any one cell type noted. Unfortunately, from the meager data presented, there is obviously an enormously preferential loss of particular cell types in particular experiments; and it appears that the preferential losses, like many other aspects of this report, were inconsistent. Stein ( 1975a) has employed free-flow electrophoresis in combination with other cell separation techniques for the purification of individual kinds of cells from peripheral blood. He has observed that consistent separations are obtained when the peaks are located in relation to “. . . the peak of an erythrocyte distribution for the standardization of results. . . . In our laboratory, we too have found that this practice is essential, since the peaks of specific kinds of cells may be ”

”

”

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found in different fractions in different experiments despite the fact that peaks of specific kinds of cells are located a constant distance from the modal population of red blood cells. Electrophoresis has been suggested as a means to study the effects of drugs on blood cells. Sauvezie et al. (1974) observed that the administration of chlorambucil to rheumatic patients resulted in a lymphopenia which affected primarily a reduction in circulating lymphocytes of high electrophoretic mobility. Larcan et al. (1974) incubated lymphocytes from normal individuals for 20 minutes in various concentrations of a wide range of immunosuppressive and cytotoxic drugs. Except for periwinkle alkaloids which had no effect on electrophoretic mobility, all the drugs studied caused decreased electrophoretic mobility; the mechanism responsible for this effect has not been defined. The electrophoresis of blood cells has found limited clinical application in the past. Work prior to 1965 was reviewed by Ruhenstroth-Bauer (1965) and by Arnold (1965). Wiig (1975) described the electrophoresis of mononuclear cells purified by Ficoll-Hypaque from single patients with the Bruton type of agammaglobulinemia, thymic dysplasia, and chronic lymphatic leukemia. Plagne et al. (1975) reported a decrease in the number of circulating lymphocytes of high electrophoretic mobility in the blood of patients with cancer. This decrease was more marked in patients with disseminated disease than in patients with localized disease; and Plagne et al. interpreted their observations as consistent with the observation of others that some cancer patients have decreased numbers of circulating T lymphocytes. Unfortunately, Plagne et al. (1975) did not distinguish between lymphocytes and monocytes among cells separated on Ficoll-Angiocontrix (sodium iothalamate), and the observed differences among normal patients, patients with localized cancer, and patients with disseminated cancer are subject to many different possible interpretations. 3. Macrophage Electrophoretic Mobility Test

In 1970, Field and Caspary described their observation that lymphocytes from cancer patients could be stimulated with an extract of human central nervous system to produce a substance having the capacity to retard the electrophoretic migration of macrophages. In their original preliminary communication, this effect was observed when lymphocytes were obtained from cancer patients but was not observed when lymphocytes were obtained from the blood of donors without cancer. In a subsequent communication, Caspary and Field (1971) reported that a similar extract of human tumor, termed “tumour antigens” by them, could be substituted for the extract from the nervous system. Since 1970, there has been a deluge of letters to editors, brief communications, and occasional articles from many laboratories around the world (Tognella et al., 1974; Iwaguchi and Sakurai, 1974; Pritchard et al., 1973; Rawlins et al.,

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1976; Chiu et al., 1977; Lampert et al., 1977), which confirm most of the observations of Field and Caspary. Because of the enormity of the literature in this area of investigation, we shall not attempt a comprehensive review of this topic but confine ourselves to a brief description of the current state of this area of investigation. The method has been modified often in different laboratories (Carnegie et d . , 1973; Pritchard et d . , 1973; Wass et al., 1977; Iwaguchi and Sakurai, 1974; Shenton and Field, 1975, Shenton et al., 1977), and the diverse nature of the modifications makes meaningful comparison of the results obtained difficult or impossible. Many of the problems encountered with the macrophage electrophoretic mobility (MEM) test were discussed at a workshop in England (Bagshawe, 1977). In general, there appears to be little doubt that a somewhat similar phenomenon has been observed in many different laboratories. The substances used to stimulate the lymphocytes to be tested have included extracts of human brain termed encephalitogenic factor (Field and Caspary, 1970), a myelin basic protein (Rawlins et al., 1976), substances extracted from human tumors (Caspary and Field, 197l), and, more recently, human chorionic gonadotropin (Wass et al., 1977). With discontinuous Ficoll gradients, Minderhoud and Smith (1972) found that the blood lymptxytes most effective in this test were separated in a density fraction between l .075 and l .090 g d m l . Shenton et al. (1973) found that the suitability of macrophages for this test varied with cell size. There have been reports of cancer patients with normal MEM tests (Crozier et al., 1976; Lewkonia et al., 1974); however, these have been infrequent, and it appears that the more common problem is positive tests among patients not known to have cancer. Abnormal macrophage electrophoretic mobility tests have been observed when lymphocytes are obtained from patients with a variety of other serious illnesses including asthma, sarcoidosis, Crohn ’s disease, and several kinds of neurological illnesses (Pritchard et al., 1976). It has been suggested that many false positive tests may be attributed to the sensitivity of the test in picking up very early cancers; and, in the face of this argument, one is confronted with the difficult problem of proving that a patient is free of subdetectable cancer. It has been suggested that the test may be valid if one postulates that it (Pritchard et al., 1976) “detects cancer about 16 years before the clinical appearance of the disease.” This hypothesis cannot be tested easily, if at all. In summary, it appears that the MEM test may merit investigation as a screening procedure for the detection of a variety of diseases including cancer. A normal test result is reassuring; an abnormal test result cannot be interpreted conclusively. The frequent modification of the test in different laboratories, often without extensive clinical correlations, complicates the kind of sharing and comparison of long-term, clinical data required for the definitive evaluation of this test. The mechanisms by which the test works at the cellular and molecular levels are thus far undefined.

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4. Thymus and Thymus-Dependent Cells In addition to the electrophoretic separations of T and B lymphocytes reviewed above, there have been several studies of thymocytes or thymus-derived cells. In 1971, Zeiller et al. (1971a) tested the graft-versus-host reactivity of electrophoretically separated rat spleen cells with the spleen weight assay. Cells were divided into groups with high, intermediate, and low electrophoretic mobility. Cells of low electrophoretic mobility showed little or no activity; cells of intermediate electrophoretic mobility, low activity; unseparated cells, somewhat greater activity; and cells of high electrophoretic mobility, the greatest activity. When present, activity was related to the approximate number of cells injected. Subsequently, Zeiller and his colleagues (Zeiller et al., 1974) characterized cells electrophoreticallyseparated from spleen, lymph nodes, blood, and thymus with many different markers including theta antigen, graft-versus-host reactivity, size, sensitivity to hydrocortisone, and morphology. In further studies from the same laboratory (Zeiller et al., 1975b; Zeiller and Pascher, 1978), these cells were characterized with transmission and scanning electron microscopy, antibodies to surface antigens, and reactivity with cationized fenitin before and after treatment with Vibrio cholerue neuraminidase. Droege et al. (1974b) characterized chicken thymocytes with respect to electrophoretic mobility and size. Droege et al. (1974a) also characterized thymocytes from mice of various ages with respect to electrophoretic mobility, density in continuous albumin gradients, and size. Dumont and Sabolovic (Dumont and Sabolovic, 1973; Sabolovic and Dumont, 1973) studied the distributions of cortisone- and hydrocortisone-sensitiveand phytohemagglutinin (PHA)-responsive mouse thymocytes with respect to electrophoretic mobility and distribution in discontinuous albumin density gradients. Subsequently, Dumont and his associates studied the effect of age (Dumont, 1974; Dumont and Bischoff, 1977; Dumont and Robert, 1977a) and various doses of hydrocortisone (Dumont and Robert, 1976; Dumont and Barrois, 1977) on subpopulations of electrophoretically different mouse lymphocytes. Many studies of subpopulations of thymocytes recognized by their electrophoretic mobilities must be interpreted with caution because of the failure of several investigators to report the proportions of cells recovered. When they examined recovery, most investigators found that they had lost one-third to one-half of their cells during the process of electrophoresis. Losses like these are common with other methods for cell separation. When losses of this magnitude occur, it is difficult to conclude that attrition results from factors other than the technique used for separation, that is, that cells are killed by treatment with a pharmacological agent, for example, hydrocortisone. In fact, cells may have been made more susceptible to destruction by experimental manipulation. Neither the procedure used to obtain the cells in suspension nor the technique for the separation of cells is without hazard; and the

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loss of a proportion of cells with high electrophoretic mobility, for example, could occur in the tissue during the process of obtaining the treated cells in suspension or during the procedure for cell separation. Interpretation is further complicated in many reports by the omission of data which would enable one to evaluate the range of variability observed, that is, standard deviations, ranges, confidence intervals, or similar statistical data. Before leaving our review of the electrophoresis of thymocytes, we should mention the very interesting report by Jenkins (1975) that the transplantation of tumor cells into mice markedly altered the frequency of subpopulations of cells recognized by their electrophoretic mobilities. In work with a cytopherometer, Jenkins observed two modal populations of mouse thymocytes which had electrophoretic mobilities of 0.77 ? 0.023 and 0.99 & 0.015 p m sec-I V-’ cm-’ respectively. After transplantation of certain tumors into the mice, the ratio of slow thymocytes to fast thymocytes changed from 9: 1 to 2: 1 for the remainder of the lives of the mice. Similar transplantation of syngeneic spleen cells produced an alteration of this ratio which was smaller and of briefer duration. 5. Antibody-Forming Cells and Their Precursors At approximately the same period during which they demonstrated that T and B lymphocytes had different electrophoretic mobilities (see above), Zeiller, Hannig, and their associates began a series of studies to determine changes in electrophoretic mobility during the course of differentiation of B lymphocytes and their precursors. Following the intraperitoneal injection of sheep blood cells into rats, Zeiller et al. (1970) demonstrated the sequential appearance of different peaks of plaque-forming cells defined by their ability to form plaques and their electrophoretic mobilities. Different peaks were felt to represent plaqueforming blasts and immature plasma cells. In a subsequent investigation, Zeiller et al. (1976) recognized “three distinct progenitor cells of direct PFC plaque forming cells, showing high, medium, and low electrophoretic mobility. ” These three electrophoretically different subpopulations of mouse spleen cells were biologically different. In light of the general relationship between electrophoretic mobility and maturity of cells (see below), it is interesting to note that Zeiller et al. (1976) found that the PFC with highest electrophoretic mobility were “members of a rapidly cycling compartment whereas . . . ” those of lesser electrophoretic mobility showed “low cycling. ” Other workers (Dumont, 1975) have combined centrifugation in discontinuous albumin gradients with electrophoresis in order to study the effects of treatment with cyclophosphamide and other manipulations on subpopulations of mouse spleen cells. They found that (Dumont, 1975) “both B and T cells were recovered throughout the gradient, but in different portions. ” B cells were stated to be “enriched in the light density fractions. . .” while T cells were concentrated in

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the heavy fractions. The addition of the discontinuous density gradient seems to have added very little of value to electrophoresis for the characterizationof T and B lymphocytes. The average concentrations of cells with surface immunoglobulins in the fractions from the gradients were always less than 50% different from those observed among spleen cells prior to centrifugation in the discontinuous density gradients, There is little evidence to suggest that isopycnic centrifugation even in continuous gradients is a potent method for the separation of T and B lymphocytes; and, as discussed below, it is probable that the insertion of interfaces into density gradients does not result in greater purity than can be achieved in continuous gradients. H.von Boehmer and his associates also attempted to use cell electrophoresis for study of the maturation of B lymphocytes. In an early study (Osmond et al., 1975) they reported both sedimentation at unit gravity and electrophoresis of mouse bone marrow cells reacted with radioiodine-labeled antiglobulin. They noted that labeled and unlabeled small lymphocytes were not appreciably separated by sedimentation at unit gravity, “except for 5% of the labeled small lymphocytes” which sedimented more rapidly than the majority of lymphocytes. T and B cells were separated electrophoretically much as expected based on the extensive previous work from other laboratories, despite the fact that the B cells had been reacted with the labeled antibody before electrophoresis. With respect to the 5% of B lymphocytes which sedimented more rapidly, they state: “The possibility is raised that the rapidly sedimenting immunoglobulin-bearing small lymphocytes in bone marrow are a functionally distinct group of potential precursor cells.” This is of course a testable hypothesis; however, no evidence for or against it was presented. We believe that a more likely possibility is that the 5% of rapidly sedimenting labeled lymphocytes were lymphocytes which aggregated during the 5 hours while marrow cells were sedimenting at unit gravity. Many kinds of viable cells which are allowed to associate at high concentrations for several hours form aggregates. Aggregated cells sediment more rapidly than monomers; and we believe that the possibility that subpopulationsof cells, which are defined by more rapid sedimentation than morphologically similar cells at unit gravity, are simply aggregates deserves more widespread consideration in the immunological literature. In later collaborative studies, von Boehmer and his associates characterized mouse lymphoid cells with various surface markers (Roelants et al., 1975), as well as (Melchers et al., 1975) “subpopulations of B cells from spleen and bone marrow separated by free flow electrophoresis. ”

6 . Lymph Node Cells We have discussed separations of some lymph node cells elsewhere in this article; however, there are a few remaining separations of lymph node cells which do not fit easily into the more specific discussions in other sections. In

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1968, Hartveit et al. noted that a new population of lymph node cells of rapid electrophoretic mobility appeared after immunization of mice with ascites tumor cells in Freund’s complete adjuvant. Jenssen et al. (1975) described characteristic changes in rabbit lymph node cells after the immunization of rabbits with Candida albicans. Hannig and Zeiller’s studies of changes in lymph node cells during the course of immunization have been described above (Hannig and Zeiller, 1969; Zeiller ef al., 1970).

B. NEOPLASTIC, DIVIDING,AND IMMATURE CELLS Subject to the limitations and exceptions inherent in the process of generalizing about cells, there is a large body of evidence which suggests that immature and proliferating cells have higher electrophoretic mobilities than mature and resting cells. In general, malignant, neoplastic cells have been found to have electrophoretic mobilities similar to or more rapid than those of dividing and immature normal cells. 1. Hepafoma Cells

In 1956, Ambrose et al. measured the electrophoretic mobilities of rat cells from normal liver, normal kidney, and tumors from liver and kidney induced with butter yellow and stilbesterol, respectively. Cells were obtained in suspension from all tissues by perfusion of the tissues with culture medium containing various chelating agents. Under these conditions, kidney tumor cells showed higher electrophoretic mobilities than cells from normal kidneys; and liver tumor cells showed higher electrophoretic mobilities than normal liver cells. Similar data were published with greater detail together with the characterization of cells from several ascites tumors (Lowick ef al., 1961). Normal, fetal, regenerating, and neoplastic liver have provided an interesting series of comparisons. Ben-Or ef al. (1960) observed that regenerating liver cells had higher electrophoretic mobilities than normal liver cells. Eisenberg et al. (1962) demonstrated that the electrophoretic mobility of rat liver cells decreased from birth to 21 days of age. In younger animals, these investigators observed “numerous, small, round cells . . . ” which they believed to be hemopoietic cells and excluded from their investigations. In addition, they expanded their earlier observations (Ben-Or et al., 1960) that cells from regenerating liver had a higher electrophoretic mobility than normal liver cells. The highest electrophoretic mobility was observed 48 hours after partial hepatectomy . Extending these studies of liver cells, Fuhrmann (1965) observed that the electrophoretic mobility of cells from regenerating liver increased “. . . parallel to that of the mitotic index taken in the morning. . . ”; however, they noted a diurnal variation in mitotic rate which made it necessary that this correlation be

108

THOMAS G. PRETLOW. II AND THERESA P. PRETLOW

observed in the morning. They also reported that the electrophoretic mobilities of regenerating liver cells were little affected by treatment with neuraminidase, while the electrophoretic mobilities of malignant cells from an ascitic hepatoma were markedly decreased by treatment with neuraminidase. When the experiments were conducted in vivo by injection of neuraminidase intraperitoneally into animals with ascitic tumors, the mobilities of the ascites tumor cells decreased rapidly and returned to pretreatment levels over the following 1-3 days. Similar decreased electrophoretic mobility of HeLa cells was observed after treatment with neuraminidase. The transplantability of the ascitic hepatoma cells and culturability of the HeLa cells were not altered by treatment with neuraminidase. Subsequently, Woo and Cater (1972) confirmed the observation that the mobilities of hepatoma cells were decreased by treatment with neuraminidase and noted that fetal liver cells were affected similarly. 2. Ascites Tumors

In addition to the studies on ascites tumors mentioned above (Lowick et al., 1961), there have been several others. Like blood cells, ascites tumor cells have the advantage that they are obtained already in suspension. Cook et al. (1962) used Ehrlich cells to study alterations of electrophoretic mobility according to altered pH and treatments with trypsin, neuraminidase, and aldehyde fixatives. Mayhew (1968) reported that the electrophoretic mobilities of Ehrlich cells varied with the age of the tumor; he observed that treatment of Ehrlich ascites tumor cells with neuraminidase and RNase alone and in combination caused decreased electrophoretic mobility. Hartveit et al. (1968) noticed that the electrophoretic mobility of Bf 8 and Ehrlich ascites tumor cells changed on successive days following transplantation. Cook et al. (1963) carried out a comparison of solid and ascitic tumor cells derived from inocula of the same cells. This kind of comparison seems uniquely valuable in view of the large number of studies of ascitic tumors as models of cancer. They found marked differences in the electrophoretic mobilities of the cells from the tumor grown in the two different forms. Incubation of ascites tumor cells with neuraminidase caused a marked reduction in their electrophoretic mobilities; in contrast, similar treatment of cells from the solid tumor caused no significant change in electrophoretic mobilities. In view of the facts that (1) it was necessary to disrupt the solid tumor mechanically and (2) there may have been different proportions of nonmalignant cells in the two tumors, this experiment seems somewhat inconclusive; however, the question addressed seems critical. With the electrophoresis equipment available in the early 1960s, it appears that the experiments performed were limited only by the state of the art, that is, the apparatus did not permit recovery of the electrophoresed cells for morphological and biological characterization in a fashion similar to that possible after free-flow electrophoresis.

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3. Lymphoma and Leukemia There have been many studies on the electrophoresis of cells from mouse and human lymphomas and leukemias. As in the several other organ systems described above, it appears that electrophoretic mobility is at least crudely related to the maturity of the cells under study. In both rats and humans, cells from bone marrow show a somewhat more rapid electrophoretic mobility than the more mature cells found in peripheral blood (Arnold, 1965; Ruhenstroth-Bauer, 1965). In human marrow (Ruhenstroth-Bauer, 1965), there is a small subpopulation of granulocytes with electrophoretic mobilities similar to those of granulocytes in the peripheral circulation; however, the majority of granulocytes from the marrow have greater electrophoretic mobility. In both virally produced leukemia in rats (Arnold, 1965) and naturally occurring leukemia in humans (RuhenstrothBauer, 1965), the transformation to acute leukemia is associated with increased electrophoretic mobility. Data obtained by Lichtman and Weed (1970) essentially confirmed Ruhenstroth-Bauer’s characterization of mature peripheral blood cells, marrow cells, and leukemic cells; in addition, they found that the electrophoretic mobilities of all tested cell types were decreased by treatment with neuraminidase. Leukemia cells, like normal blood cells and ascites tumor cells, have been used extensively to study the surface properties of cells as a function of pH (Mehrishi and Thomson, 1968; Cook and Jacobson, 1968) and as altered by neuraminidase, aldehyde fixation, and other chemical alterations (Cook and Jacobson, 1968). In contrast to the above-described findings, in a different buffer system, Schubert et al. (1973) noted that in a study of “bone marrow cells of patients with chronic myeloid leukemia, monocytic leukemia, myeloblast leukemia, chronic lymphatic leukemia, typical and atypical plasma cell myeloma and M. Waldenstrom. . . . Except for myeloma cells, the pathologic cells possess the same electrophoretic mobility as their normal counterparts. Changes in electrophoretic mobility have not been observed in chronic lymphocytic leukemia (Goldstone et a l . , 1974). Sabolovic et al. (1973) claims to have distinguished three types of chronic lymphocytic leukemia based on the electrophoretic mobilities of the nucleated cells from peripheral blood. There are several features of this report which lead us to interpret it with caution. For example, despite the fact that their initial preparation of cells on Ficoll-Isopaque was accomplished with an unorthodox modification of the technique, we are not told what proportion of the nucleated cells of the blood was recovered after this step. Knowledge of the proportion lost after cell separation on Ficoll-Isopaque is critical (Aiuti et al., 1974). After this separation, in contrast to the published experience with the conventional FicollHypaque technique (Zucker-Franklin, 1974), the purified cells from normal individuals contained only 510% monocytes. No mention is made of an effort to verify this unusual result with tests for phagocytosis or stains for peroxidase or ”

110

THOMAS G. PRETLOW, I1 AND THERESA P. PRETLOW

esterase. The purified lymphocytes were then electrophoresed. We are not told what proportion of the cells introduced into the electrophoresis apparatus was recovered at the end of the experiment. Based on the electrophoretic mobilities of an unspecified proportion of their peripheral blood lymphocytes, Sabolovic et al. (1973) concluded that “. . . the electrophoretic mobility allows distinction of three types of CLL.” One type was represented by a single patient. Smith et al. (1976, 1978) described differences between peripheral blood cells from normal and leukemic subjects using laser Doppler spectroscopy. They first purified mononuclear cells from peripheral blood “. . . by isopycnic centrifugation over Ficoll Hypaque [an internal contradiction] (specific gravity, l .078) by the method of Boyum . . . ” They then employed the laser Doppler spectroscopy technique discussed above for the characterizationof these cells. They found that “normal cells had an asymmetric, often bimodal, electrophoretic distribution; leukemic cells consistently had a single symmetric distribution. ” If one believes that this technique is useful, one can only be amazed by their finding that “analysis of both fresh and cryopreserved samples of each type showed that cryopreservation had no distinguishable effect on the electrophoretic distribution of either normal or leukemic cells. ” The effect of cryopreservation certainly merits reexamination with a technique which permits a more critical and thorough analysis of the electrophoretic patterns observed than is possible with laser Doppler spectroscopy. In particular, it would be important to calculate the recovery, examine the morphology, and test the viability of each subpopulation. Before leaving our discussion of cells from lymphoma and leukemia, we should mention the studies carried out by Ruhenstroth-Bauer and his associates with Hodgkin’s disease. While the reported studies of the electrophoretic mobilities of blood lymphocytes of patients with Hodgkin’s disease come exclusively from one laboratory and the samples are small (Ruhenstroth-Bauer et al., 1963; Ruhenstroth and Fuhrmann, 1962), patients with Hodgkin’s disease were consistently observed to have blood lymphocytes with the normal mobilities plus a considerable number of blood lymphocytes with electrophoretic mobilities which fell actually outside the normal range. A much larger unreported series of patients with Hodgkin’s disease were found to be consistent with those described in the reports cited above (Ruhenstroth-Bauer, verbal communication). Tognella and his associates (Tognella et al., 1975) observed that the sera of some patients with Hodgkin’s disease may alter the electrophoretic mobility of lymphocytes from peripheral blood. 4. Tissue Culture Observations

Heard et al. (1961) found that cultured fibroblasts from mouse embryos manifested greater electrophoretic mobility than cultured fibroblasts from adult animals. Fibroblasts from the 20-methylcholanthrene-treatedstrain-L fibroblasts

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111

exhibited an electrophoretic mobility intermediate between fibroblasts from embryos and adult mice. In the same vein, Forrester et a/. (1962) observed that polyoma-transformedhamster fibroblasts exhibited both more heterogeneous and higher electrophoretic mobilities than fibroblasts from the same population not transformed. Latner and Turner (1974) found smaller differences between virustransformed and normal BHK21 cells than have been characteristic of transformed and normal cells in other systems. Attempts have been made to correlate electrophoretic mobility with the aggressiveness of cells from different tumors. Purdom e?af. (1958) studied several sublines of the same transplantable tumor and concluded that “the selection for a biological characteristic, namely, the ease of producing the ascites form, which also leads to the appearance of early metastases, is correlated with the progressive increase in negative electrical charge. . . .” Bossmann et af. (1973) found that sublines of a melanoma developed by Fidler (1973) differed prior to confluency but became similar when the cultures reached confluency. The sublines differed with respect to their propensities to give rise to metastases in vivo; and in sparse cultures the line with the greatest propensity to metastasize exhibited the highest electrophoretic mobility. In one of the few studies of the electrophoretic mobilities of cells from human tumors, Vassar (1963) found no significant difference between cells from human carcinomas and cells from the corresponding normal tissues. He observed that “mesenchymal tumor cells have a higher surface negative charge than epithelial tumor cells (P € 0.01).” It should be noted that the apparatus available to Vassar in the early 1960s did not permit one to collect the cells following electrophoresis in order to perform a careful cytological examination. In our laboratory, cells obtained in suspension from human solid tumors are most often more than half stromal cells. The data reported in this excellent study by Vassar may have been influenced in some degree by the proportion of stromal cells in the suspensions of tumor cells. Schaeffer et af. (1973a) have suggested that electrophoresis might provide a valuable tool for the characterization of cells from different portions of developing embryos. They noted a correlation between “ . . . surface charge, morphogenetic movement, and adhesiveness, . . when adhesiveness was defined as the “degree of rounding of cells in situ. ” Further studies of embryo cells were reported by Schaeffer et al. (1973b). In summary, electrophoretic characterization of cells has, with few exceptions, shown that mature, normal cells have lower electrophoretic mobilities than cells from embryos, regenerating cells, and neoplastic cells. Most of the studies on cells other than those from the lymphoid and hemopoietic systems have been carried out with equipment which does not permit the separation and recovery of a sufficient number of cells to permit biochemical, morphological, and other ”

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THOMAS G. PRETLOW. I1 AND THERESA P. PRETLOW

kinds of characterization. With the development of free-flow electrophoresis, it should be possible to separate a sufficient number of cells to permit much more detailed characterization than was possible formerly. 5 . Synchronization of Cells by Electrophoresis There have been few reported evaluations of electrophoresis as a potential method for the synchronization of cells. In 1965, Mayhew and O’Grady (1965) studied the electrophoretic mobility of Roswell Park Memorial Institute no. 41 cells, a line obtained from a human osteogenic sarcoma, after the cells were partially synchronized by “double thymidine blocking. ” The mitotic peak after blocking contained a maximum of 15% cells in mitosis simultaneously. As compared with cells which had not been synchronized, the electrophoretic mobilities of the parasynchronous cells were increased during mitosis and during the postdivision phase and decreased during the DNA synthesis phase. The same trends in electrophoretic mobility as a function of the cell cycle were noted both in fixed cells and in cells prior to fixation with formaldehyde. Working with mitotic HeLa cells purified from other HeLa cells by a method dependent upon differential adherence to glass, Brent and Forrester (1967) found that populations containing 60-85% HeLa cells in mitosis exhibited a higher electrophoretic mobility than HeLa cells which were not purified with respect to mitotic cells. In contrast to HeLa cells and Roswell Park Memorial Institute no. 41 cells, cells from a lymphoid cell line (L5 178-Y) showed approximately constant electrophoretic mobility throughout the cell cycle; Shank and Burki (1971) hypothesized that the difference between L5178 and the other two cell lines may be related to the fact that L5178 has no tendency to adhere to glass. In summary, despite the relatively small number of cell lines studied, there is evidence that some cell lines may be able to be at least partially synchronized by electrophoresis. Some lines are not so affected, Electrophoresis deserves further study as a potential method for the synchronization of some cell lines.

VI. Viability and Function There are few published data to suggest that electrophoresis is harmful to cells. Except for a study performed with microorganisms (Hamilton and Sale, 1967), there has been little investigation of the mechanisms by which cells may be injured by exposure to electrical fields. There are considerable data which support the working hypothesis that electrophoresis performed properly does little harm to viabiIity or function. After electrophoresis, Boltz et al. (1973) found that cells exhibited 75% of the plating efficiency observed prior to electrophoresis. Zeiller et al. (1972a) observed little change in the number of mouse marrow cells with the capacity to form colonies in the spleens of irradiated mice after elec-

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113

trophoresis. Gamer et al. (1968) evaluated electrophoretically separated nucleated blood cells by observing their capacities to exclude dye and to exhibit phagocytosis; they estimated that 70-80% of the electrophoretically separated cells were viable. Zeiller and his associates, as well as others (Zeiller et al., 1970, 1972b,c, 1976; Schlegel et al., 1975a,b), have shown that antibodyforming cells still have the capacity to form plaques after electrophoretic separation. After electrophoretic separation, lymphocytes are able to function in the graft-versus-host reaction (Zeiller et al., 1971a, 1974; Andersson et al., 1973a), in cytotoxicity reactions (Hayry and Andersson, 1975; Hayry et al., 1973), in mixed lymphocyte cultures as stimulating cells (Hayry et al., 1975a; von Boehmer, 1974) and as responding cells (Andersson et al., 1973b; Hayry et al., 1973; von Boehmer, 1974), in cultures stimulated with various mitogens including PHA, bacterial lipopolysaccharide, concanavalin A, and pokeweed mitogen (Hayry et al., 1973, 1975b; Andersson et al., 1973b, 1975; Stein et al., 1973; Seiler et al., 1974; Stein, 1975b; Shortman et al., 1975), and as antibody progenitor cells detected by passive transfer into syngeneic recipients (Schlegel et al., 1975a,b).

VII. Effects of Lytic Enzymes on Electrophoretic Mobilities of Cells There have been many studies on the effects of various lytic enzymes on the electrophoretic mobilities of cells. While we review these studies briefly here, it is very difficult to evaluate their significance since most such studies were carried out with enzymes of unspecified purity. Most lytic enzymes can be obtained in a high degree of purity only with considerable difficulty, and most commercially available lytic enzymes are adulterated with other, unspecified lytic enzymes. Of the various commonly employed lytic enzymes, trypsin has been the subject of many reports which emphasize the facts that many commercially available trypsins are not pure and that the results obtained with pure trypsins are different from those obtained with crude trypsins (Rinaldini, 1958, 1959; Pine et al., 1969; Boeryd et al., 1968; Speicher and McCarl, 1974; Willson et al., 1976b). Purified bacterial collagenases have been noted to contain similar adulterating, unknown lytic enzyme activities (Hilfer and Brown, 1971; Kono, 1969). Many reports have demonstrated that commercial batches of trypsin or collagenase were valuable to the investigator for specific purposes not because of their specific trypsin or collagenolytic activities, but because of the adulterating lytic enzymes (Kono, 1969; Willson et al., 1976b). We emphasize the fact that alterations of electrophoretic mobility observed after cells are treated wiih lytic enzymes need not have resulted from the activity of the enzymes the investigator thought he or she employed. For example, if the electrophoretic mobilities of cells are decreased after treatment with commercially available neuraminidase of

TABLE I EFFECTOF ENZYMES A N D CHELATING AGENTSON ELECTROPHORETIC MOB~LITY

Enzyme

Cell

Trypsin Subtilisin EDTA

Chick retinal cells Chick retinal cells Chick retinal cells

DNase Trypsin Trypsin

Chick retinal cells WRE rat embryo cells XC virus-transformed rat embryo cells Human lymphocytes Human lymphocytes Human lymphocytes Human lymphocytes Human lymphocytes Human lymphocytes Human lymphocytes Mouse melanoma cell lines Mouse melanoma cell lines Mouse melanoma cell lines Human erythrocytes Horse erythrocytes

Sialidase Trypsin Maltose p-Glucosidase 8-Galactosidase Lecithinase Sulfatase Neuraminidase Trypsin Hyaluronidase Neuraminidase Neurarninidase

Papain EDTA Trypsin EDTA Trypsin

Chondroitinase ABC Neuraminidase Chondroitinase ABC

Lamb erythrocytes Human erythrocytes Calf erythrocytes Chicken erythrocytes Pig erythrocytes Human erythrocytes Slime mold cells HeLa cells HeLa cells Mouse embryo fibroblasts Adult mouse fibroblasts AH-1 30 rat ascites hepatoma AH-130 rat ascites hepatoma AH-130 FN and AH-130 FN rat ascites hepatoma

Change in electrophoretic mobility

Reference

Decreased Decreased Decreased slightly Decreased Increased Decreased

Barnard et al. (1969)

Reduced Increased Not affected Not affected Not affected Not affected Reduced Decreased

Bona (1975)

Boltz et al. (1976)

Bosmann er al. (1973)

Affected little Affected little Decreased Decreased slightly Decreased Decreased Decreased Decreased Decreased Decreased Not affected Decreased Not affected Not affected

Cook et al. (1961) Eylar et al. (1962)

Fike and van Oss (1973) Gingell and Garrod (1969) Hayry er al. (1965) Heard et al. (1961)

Not affected Reduced

Kojima and Yamagata (1971)

Not affected Reduced

(continued) 114

TABLE I (continued)

Enzyme Neuraminidase

Chondroitinase ABC Neuraminidase

Ribonuclease

Neuraminidase

Cell AH-130 FN and

Human erythrocytes Human erythrocytes Human erythrocyptes

Neuraminidase

Embryonic mouse fibroblasts Adult mouse fibroblasts L-strain mouse fibroblasts Ehrlich mouse ascites carcinoma Ehrlic h-Landsheetz mouse ascites strain HeLa cells Monkey kidney cells Human lymphocytes Cells from fourteen different human tumors Human lymphocytes Ehrlich ascites carcinoma Solid sarcoma Ascites sarcoma

Neuraminidase Sialidase

Neuraminidase RNase

Reference

Reduced

AH-130 FN rat ascites hepatoma Rat erythrocytes Line no. 41 from human osteogenic sarcoma Line no. 41 from human osteogenic sarcoma Ehrlich ascites Mouse liver cells

Trypsin Trypsin Papain

Neuraminidase Sialidase

Change in electrophoretic mobility

Not affected Decreased

Mayhew (1966, 1967)

Decreased

Mayhew (1967)

Decreased Decreased slighily Reduced Reduced Decreased

Mayhew and Nordling (1966)

Not affected

Ponder (1951) Seaman and Heard (1960) Seaman and Uhlenbmck ( 1963) Simon-Reuss er al. (1964)

Not affected Decreased Decreased Decreased

Decreased Decreased Decreased Decreased variably Decreased Decreased

Smith er al. (1978) Vassar (1963)

Vassar et af. (1973) Wallach and Esandi ( 1964)

Not affected Decreased

Mouse Ehrlich ascites carcinoma Three cell lines from human blood

Decreased

Wallach and Eylar (1961)

Decreased

Weiss and Horoszewicz (1971) (continued)

I15

116

THOMAS G. PRETLOW, I1 AND THERESA P. PRETLOW TABLE I (conrinued)

Enzyme RNase Neuraminidase Neuraminidase Neuraminidase

RNase

Trypsin Neuraminidase

Cell L1210 mouse leukemia Human erythrocytes Mouse lymphocytes Adult rat liver cells Fetal rat liver cells Rat hapatoma cells Addt rat liver cells Fetal rat liver cells Rat hepatoma cells BP8 mouse sscites tumor Mouse lymph no& cells Sensitized mouse lymph node cells Rat hepatoma cells Mouse lymphocytes

Change in electrophoretic mobility

Reference

Decreased

Weiss and Mayhew (1966)

Decreased Decreased Not affected Decreased Decreased Not affected Decreased Decreased Decreased

Weiss et al. (1972) Wiig (1974) Woo and Cater (1972)

Decreased slightly Decreased Decreased Decreased

Zeiller et al. (1975b)

unspecified purity and even when released neuraminic acid is quantitated in the supernatant, the decreased electrophoretic mobility is not proven to be the result of the action of neuraminidase. Alternatively, the altered electrophoretic mobility could result from the action of other lytic enzymes in the preparation or from the adsorption of any proteins in the preparation onto the surfaces of the electrophoresed cells. Because of the large proportion of the literature on the electrophoresis of cells which deals with the effects of lytic enzymes, we have listed selected features of representative reports in Table I. Before leaving our discussion of electrophoretic mobility of cells as affected by enzymic digestion, we should summarize the limited number of generalizations which are apparent from our review of the literature. First, malignant cells generally show decreased electrophoretic mobilities after exposure to neuraminidase. Cells from adult animals do not appear to follow any consistent rule in this regard. Trypsin has been observed to produce both increased and decreased electrophoretic mobility, and the effect observed probably depends upon a variety of undefined variables. Most other enzymes have not been tested sufficiently extensively to permit useful generalizations; however, it appears that treatment of cells with lytic enzymes produces decreased electrophoretic mobilities more often than increased electrophoretic mobilities.

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Whether or not treatment with lytic enzymes and consequent altered electrophoretic mobilities results in cell populations which are less separable into defined subpopulations remains a largely unanswered question. This question is important for investigators who want to purify cells from solid tissues which must be disaggregated into suspensions of single cells with lytic enzymes. It is interesting to note that Mayhew and Nordling (1966) found that treatment of parasynchronized subpopulations of RPMI no. 41 cells resulted in a reduction of ". . . their mobility by proportionately similar amounts"; that is, electrophoretic mobilities of rapidly and slowly migrating cells were reduced by an almost constant proportion of their original electrophoretic mobilities. From these data, one would predict that there was little loss in the susceptibility of subpopulations of these cells to separation by electrophoresis. Similarly, in our laboratory (Pretlow et al., in preparation), we have found that trypsin reduces the electrophoretic mobilities of all human tonsillar cells; however, morphologically recognizable subpopulations are separated from each other by approximately the same degree whether human tonsillar cells are obtained in suspension mechanically or with trypsin. Studies with laser Doppler spectroscopy (Smith et al., 1978) suggest that neuraminidase treatment increases the differences between the electrophoretic mobilities of T and B lymphocytes, but this needs verification by an electrophoretic method which allows direct analysis of the separated cells.

VIII. Adsorbable and Other Materials Since early in the history of electrophoresis, numerous investigations have been devoted to the study of alterations in the electrophoretic mobilities of cells as a function of solutes of one kind or another added to the medium for electrophoresis. In some cases, cells have been incubated with various substances prior to electrophoresis. When these substances have known biological effects (e.g., hormones, interferon), it is frequently difficult to determine whether changes in the electrophoretic mobilities of cells result from adsorption of the substance onto the cells, competitive elimination of a competitively adsorbed substance, or fundamental structural changes in the plasma membrane secondary to the biological effects of the added substance on the metabolism of the cells. Probably the simplest example of the effect of dissolved materials on the electrophoresis of cells is work with ions which alter the electrophoretic mobilities of cells. Earlier, we stated that we would refer the reader to other reviews for the theory of electrophoresis and that we would not discuss ions and counterions at length; however, it seems worthwhile to point out that the effects of different ionic compounds are different and not exclusively a function of ionic strength. This difference is well illustrated in a review by Brown and Broom (1935).

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THOMAS G. PRETMW, I1 AND THERESA P. PRETLOW

There have been several studies on the effects of antibodies or antisera on the electrophoretic mobility of cells. Abramson (1930) showed that the electrophoretic mobilities of sensitized red blood cells were different from those of normal red blood cells and that this difference was a function of the amount of immune serum employed. Forrester et al. (1965) showed that the electrophoretic mobilities of ascites tumor cells were altered by antibodies to these cells with and without complement. Bert et al. (1971) observed the differential alterations of the electrophoretic mobilities of mouse T and B cells with antilymphocyte antisera. In a very interesting report, Rhie and Sehon (1972) studied changes in the electrophoretic mobilities of immune lymph node cells in the presence and in the absence of strongly positively and strongly negatively charged antigens. The antigens which they employed included “. . . the strongly basic DNP,,Llysinenoo,molecular weight 66,000 (DNP-Lys); the strongly acidic DNP,-L-tyrosine,,-L-glutamic acid6,,-~-lysinelo3,molecular weight 98,000 (DNP-TGL); the strongly acidic N-(DNP-L-lysyl),.,-ethylene maleic acid,,,, molecular weight 31,000 (DNP-EMA); DNP,,-BSA, molecular weight 71,O00 (DNP-BSA).” They found that the . . . mobilities of the ‘naked’lymph node cells from rabbits immunized with the strongly negatively charged conjugates, DNP-TGL or DNPEMA, were somewhat lower than those of normal cells; by contrast the surface charge of the lymph node cells from rabbits immunized with the positively charged conjugate, DNP-Lys, was higher than that of normal cells. Moreover, exposure of the immunocytes to each of the antigens used for immunization resulted in a relative neutralization of their surface charges. Further, they found that immune lymphoid cells reacted with basic or acid polymers showed the expected alteration in electrophoretic mobility as a function of the pH of the medium. A variety of other substances have been shown to alter the electrophoretic mobilities of various kinds of cells. The systems studied include mixtures of viruses with erythrocytes (Hanig, 1948); adenosine diphosphate with human platelets (Nicholls and Hampton, 1972); Ehrlich ascites cells with polyadenylate, polyuridylate, and various RNAs (Mayhew, 1974); lymphoid cells with doublestranded polyadenylate-polyuridylate(Hanjan and Talwar, 1975; Donner et al., 1977); and human granulocytes with the cleavage product of the fifth component of complement, transfer factor, kallikrein, plasminogen activator, IgG, albumin, horseradish peroxidase, hydrocortisone, colchicine, and cytochalasin B (Gallin et al., 1975). “

”

IX. Miscellaneous Lymph node cells have been shown to be affected by incubation with antigen. Sundaram et al. (1967) observed that alterations in electrophoretic mobility by incubation with antigen could be reversed in some instances by washing the

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CELL ELECTROPHORESIS

antigen off of the cells. Hannig and Zeiller (1969) used electrophoresis as a method for detecting and characterizing new populations of lymph node cells during the course of immunization. Jenssen et al. (1974) described changes in the electrophoretic mobilities of lymph node cells during the course of induction of experimental allergic encephalomyelitis, Catsimpoolas et al. (1976) labeled human lymphocytes with radioactive chromium. After electrophoresis, they found that the radioactivities of subpopulations of lymphocytes varied, and they cautioned that, in view of the lack of uniform labeling, interpretation of cytotoxicity assays with such labeled target cells would be difficult. There have been attempts to correlate different electrophoretic mobilities with differences in partitioning coefficient as measured by countercurrent distribution. Based on their study of fresh and stored human red blood cells, Brooks et al. (1971) concluded that their experiments established “. . . that surface charge per se, or a charge-associated property or surface structure is directly involved in determining the partition behaviour of cells in two-polymer phase systems. It is important to point out that the only cells they studied were red blood cells. In related studies of L5178 mouse leukemia cells, Gersten and Bosmann (1974a,b) found a poor correlation between changes in partition coefficient and electrophoretic mobility. Unfortunately certain features of these reports by Gersten and Bosmann make their definitive interpretation difficult or impossible. They redefine the partition ratio as “the number of cellshnit volume in the total system before the phases separate divided into the number of cellshnit volume of the top phase after separation. ” They do not report the proportion of all cells placed in the system which were recovered at the end of the experiment and give no indication that the total recovery was ever examined. There is considerable surface tension at interfaces in two-polymer phase systems (reviewed by Pretlow et al., 1975); and, in fact, if the surface tensions of the solutions of the two polymers are not sufficiently different, no separation will occur. Numerous investigators have observed that cells accumulate at the interfaces and are lost in these systems, probably because of destructive forces at these interfaces. It seems likely that Gersten and Bosmann were measuring changes which were an undefined function of altered partition coefficient and alterations in the fragility of the cells. In any system for cell separation in which the actual separation is used to characterize cells, one must know what proportion of the total population was recovered in order to know what proportion of the total population was characterized. ”

X. Isoelectric Focusing

Most electrophoresis of cells has been dependent upon the different rates of migration of different cells in electrical fields. There have been a limited number

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THOMAS G. PRETLOW, I1 AND THERESA P. PRETMW

of reports of isoelectric focusing (IEF) of cells; one of the earliest reports is that of Sherbet et al. (1972). Five different cell types were analyzed including two types of tissue culture cells, ascites cells maintained in rats and mice, and normal rat liver cells. Two bands of cells were generally resolved by IEF: cells with PI values of 5.6-6.85 were primarily viable cells, whereas cells with PI values of approximately 5 were primarily dead cells. Approximately 90% of the cells were viable after IEF; HeLa cells have been grown in culture after IEF with plating efficiencies of 55-75%. A mixture of two types of tissue culture cells was separated by IEF, but the details of this experiment were not given. In 1974, Leise and LeSane reported several experiments in which they separated rabbit and human peripheral blood lymphocytes and rabbit thymocytes by IEF. They showed that the isoelectric distribution of peripheral blood lymphocytes from a normal rabbit was different from that of lymphocytes from a rabbit hyperimmunized with sheep erythrocytes. When lymphocytes obtained from a narrow region of a gradient were refocused on another gradient, the pH and density of their location in the second gradient had shifted somewhat from those observed in the first gradient; in addition (Leise and LeSane, 1974), “viability was considerably decreased as a result of this prolonged procedure. ” In some cases, a peak of lymphocytes in these experiments is separated from another peak by a single fraction; and one wonders how constant the peaks are from rabbit to rabbit and from experiment to experiment with the same rabbit. While we are told that most of the cells from the alkaline portions of the gradients were not viable as assessed by the exclusion of trypan blue, we are not told what proportion of the cells were viable from the fractions which contained the highest proportion of viable cells. It would be interesting to know what proportion of the cells introduced into the gradient were recovered after IEF. Cells from one fraction in an experiment with one rabbit are reported to have increased almost three-fold in number after incubation at 37°C in tissue culture medium for 3 days; cells from most fractions decreased in number during the same incubation. More recently, Hirsch et al. (1977) applied IEF to rqt peripheral blood lymphocytes. These lymphocytes were separated into two subpopulations;the major subpopulation was located at pH 4.6 in fraction 17, and the minor subpopulation was found at pH 3.8 in fraction 20. Evidence was presented that the major and minor subpopulations were enriched with respect to T and B lymphocytes. Eighty to 90% of the cells in these fractions excluded trypan blue after IEF, and an average of 88% of the cells applied to the column was recovered. Just et al. (1975) separated mixtures of human, rabbit, and mouse erythrocytes by IEF with a device somewhat similar to the device designed by Hannig for free-flow electrophoresis. The values of pH for zero electrophoretic mobility were consistent with the data of Heard and Seaman (1959) obtained from measurement of the electrophoretic mobility of erythrocytes at different pH values about 15 years earlier. In their study of mouse and rabbit erythrocytes, slightly

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different pH values for zero electrophoretic mobility were obtained with different ampholites. Interestingly, 55 years earlier, Coulter (1921) reported a slightly lower isoelectric point for sheep erythrocytes; the difference may reflect species differences or differences in buffer salts, ionic strength, and so on. In addition, it should be noted that Just et al. (1975) washed their erythrocytes with EDTA prior to electrophoresis; and it seems possible that this might have removed cations from their surfaces, thereby altering the net charges on cells and possibly even altering the configurations of some surface proteins.

XI. Concluding Remarks The electrophoretic mobilities of cells have been observed and used for over 75 years as an analytical probe to investigate a wide variety of biological phenomena. It has been only in the past decade that electrophoresis has become a preparative technique for the separation of cells in sufficient quantities to allow morphological, biochemical, or biological analysis. Free-flow electrophoresis has been the most widely used preparative electrophoretic method for the separation of cells. After free-flow electrophoresis, cells appear to be biologically functional as determined by a wide variety of tests. Most applications of this method have been limited to lymphoid, blood, or hemopoietic cells. The purification of proximal tubule cells by two laboratories suggests that this method may also be applicable to the purification of epithelial cells from solid tissues and warrants more extensive use. The analytical separation of many kinds of cells, one cell at a time, by electrophoresis in a cytopherometer, over the past several decades provides important background feasibility studies for the preparative purification of cells by free-flow electrophoresis.

ACKNOWLEDGMENTS This work was sup&rted by Grants CA-13148 and CA-23922 from the National Cancer Institute.

Dr.Thomas Pretlow is supported by NIH Research Career Development Award K4-CA-70584.

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Smith, B. A., Ware, B. R., and Weiner, R. S . (1976). Proc. Narl. Acad. Sci. U.S.A. 73, 23882391. Smith, B. A., Ware, B. R., andYankee, R. A. (1978). J. Immunol. 120, 921-926. Speicher, D. W., and McCarl, R. L. (1974). In Vim0 10, 30-41. Stein, G. (1975a). Biomedicine 23, 5-11. Stein, G. (1975b). Z . Immuniraersforsch. 150, 68-80. Stein, G., Had, H. D., Pabst, R., and Trepel, F. (1973). Biomedicine 19, 388-391. Straub, E. (1965). In “Cell Electrophoresis” (E. J. Ambrose, ed.), pp. 125-140. Little, Brown, Boston. Sundaram, K., Phondke, G. P., and Ambrose, E. J. (1967). Immunology 12, 21-26. Talwar, G. P., Hanjan, S. N. S., Mehra, V. L., andKidwai, Z. (1977). J. Immunol. 118,242-247. Tognella, S., Mantovani, G., Floris, C., Cengiarotti, L., Del Giacco, G. S., and Grifoni, V. (1974). Tumori 60, 203-210. Tognella, S., Mantovani, G., Cengiarotti, L., Del Giacco, G. S., Manconi, P. E., and Grifoni, V. (1975). Tumori 61, 45-52. Uzgiris, E. E., and Kaplan, J. H. (1974a). Anal. Biochem. 60, 455-461. Uzgiris, E. E., and Kaplan, J. H. (1974b). Rev. Sci. Insfrum. 45, 120-121. Uzgiris, E. E., and Kaplan, J. H. (1976a). J. Colloid Inrerfnce Sci. 55, 148-155. Uzgiris, E. E., and Kaplan, J. H. (1976b). J. Immunol. 117, 2165-2170. van Oss, C. J. (1975). Sep. Sci. 10, 47-54. van Oss, C. J., Fike, R. M., Good, R. J., and Reinig, J. M. (1974). Anal. Biochem. 60,242-251. Vassar, P. S. (1963). Lab. Invest. 12, 1072-1077. Vassar, P.S., Hards, J. M., and Seaman, G. V. F. (1973). Biochim. Biophys. A d a 291, 107-1 15. Vassar, P. S., Levy, E. M., and Brooks, D. E. (1976). Cell. Immunol. 21, 257-271. van Boehmer, H. (1974). J. Imrnunol. 112, 70-78. von Boehmer, H., Shortman, K.,and Nossal, G. J. V. (1974). J. Cell. Physiol. 83,231-242. Wallach, D. F. H., and Esandi, M. V. D. P. (1964). Biochim. Biophys. Acra 83, 363-366. Wallach, D. F. H., and Eylar, E. H. (1961). Biochirn. Biophys. Acra 52, 594-596. Ware, B. R., and Fiygare, W. H. (1972). 1.Colloid Interface Sci. 39, 670-675. Wass, M., Rawlins, G. A., Pentycross, C. R., and Bagshawe, K. D. (1977). Lancet 1, 171-172. Weiss, L. (1966). J . Narl. Cancer Insr. 36, 837-847. Weiss, L., and Horoszewicz, J. S. (1971). Inr. J. Cancer 7, 149-159. Weiss, L., and Mayhew, E. (1966). J . Cell. Physiol. 68, 345-360. Weiss, L., Zeigel, R., Jung, 0. S., and Bross, I. D. J. (1972). Exp. Cell Res. 70, 57-64. Weiss, L., Subjeck, J. R., and Glaves, D. (1976). Exp. Cell Res. 100, 172-176. Wiig, J. N. (1974). Scand. J. Immunol. 3, 357-363. Wiig, J. N. (1975). Clin. Exp. Immunol. 19, 159-165. Wiig, J. N., and Thunold, S . (1973). Clin. Exp. Immunol. 15, 497-506. Willson, J. K. V., and Luberoff, D. E., Pitts, A., and Pretlow, T. G. II (1975). Immunology 28, 161-170. Willson, J. K. V., Jr., Zaremba, J. L., Pitts, A. M., and Pretlow, T. G. 11 (1976a). Am. J. Pafhol. 83, 341-358. Willson, J. K. V., Pretlow, T. G. II, Zaremba, J. L., and Brattain, M. G. (1976b). Immunology 30, 157-160. Winchester, R. J., Fu, S. M., Hoffman, T., and Kunkel, H. G. (1975). J. Imrnunol. 114, 12101212. Woo, J., and Cater, D. B. (1972). Biochem. J . 128, 1273-1284. Zeiller, K., and Hannig, K. (1971). Hoppe-Seyler’s Z. Physiol. Chem. 352, 1162-1167. Zeiller, K., and Hansen, E. (1978). J. Hisrochern. Cyrochem. 26, 369-381. Zeiller, K., and Pascher, G. (1973). Eur. J. Immunol. 3, 614-618.

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LNTFJU’JATIONALREVIEW OF CYTOLOGY, VOL. 61

The Wall of the Growing Plant Cell: Its Three-Dimensional Organization JEAN-CLAUDE ROLANDAND BRIGITTE VIAN UniversitP de Paris, Paris, France I. Introduction . . . . . . . . . . . . . . . . . . . 11. Some Characteristics of Plant Cell Wall Growth . . . . . . A. Anisotropy of the Process . . . . . . . . . . . . B. Sites of Surface Expansion . . . . . . . . . . , m. Organization of the Expanding Cell Wall . . . . . . . . A. Interpretation in Terms of Disperse Texture . . . . . . B. Toward the Idea of an Ordered Texture . . . . . . . C. Interpretation in Terms of a Multi-ply Construction . . . D. Bow-Shaped Arrangements . . . . . . . . . . . . E. The Occurrence of a Disperse Texture and Its Relative Importance . . . . . . . . . . . . . . . . . . IV. Modalities of Cell Wall Expansion . . . . . . . . . . . A. A Classic Concept: The Multinet Growth Hypothesis . . . B. Difficulties with a Passive Behavior of the Cell Wall during Growth . . . . . . . . . . . . . . . . . . . C. An Alternative Roposal: The Ordered Subunit Hypothesis . V. Possible Factors in Cell Wall Morphogenesis . . . . . , . A. Pressure and Stresses . . . . . . . . . . . . . . B. Membrane and Microtubule Control . . . . . . . . . C. Self-Assembly Process . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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I. Introduction In higher plants, despite their well-known “indefinite” growth, the,possibility of expansion of a given cell is essentially a transient property. When cells leave the meristematic state, they engage in a program in which they increase more or less intensely their initial size. Many processes and, to some extent all cellular activity, can be considered to be involved in cell growth. This article deals with the ultimate step in expansion, which is characterized by an increase in cell wall surface. During the basically ephemeral period when growth is possible, the cell is surrounded by a thin wall called the primary wall (Bailey, 1938; Kerr and Bailey, 1934). The characteristic property of the primary wall is thus its plasticity. Knowledge about the relationship between the fine structure of the primary 129

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wall and its functional properties has been long dependent on technical approach and therefore has significantly changed during recent years. An earlier article in this series was devoted to cytoplasmic events occurring during the secretion of wall subunits and especially to the role of membranes (Roland, 1973). The present article focuses on the three-dimensional organization of the wall during growth and is centered on cells in tissues of higher plants.

11. Some Characteristics of Plant Cell Wall Growth A. ANISOTROPY OF THE PROCESS The embryogenesis of animals is marked by cell migration and the sliding of layers which are responsible for the ultimate shape of the organism. In plants, the cell wall cements the neighboring cells and prevents such displacements. Consequently the mature shape of the plant body is exclusively dependent on the orientation of cell divisions and the subsequent setting up of the cell growth axis (Erickson, 1976). The latter event is thus a key step in plant morphogenesis. The statement of Maclachlan (1977) that “plant cell expansion is not like blowing up a balloon” is illustrated by the only observation of an anatomical preparation of a plant organ. The variability of cell shapes indicates tissue specificity and cell specificity. This implies a highly controlled harmonization process at the cell wall level. Expansion requires both a driving force and a specific loosening of the wall. In the living cell turgor pressure has to be maintained above a critical threshold to keep the cell wall under stress. The regulation of internal pressure is related to the vacuolar system. The mechanical properties of the wall under stress have been extensively studied (see Masuda et al., 1974; Roland and Pilet, 1974; Cleland, 1971; Cleland e f al., 1972; Tepfer, 1977). For growth a complex balance exists between stress and cell wall loosening. It seems that both must occur simultaneously (Cleland, 1971; Rayle, 1973). Adjustment of the internal pressure could be a direct factor in growth rate variations (Cleland et al., 1977; Prat et al., 1977). Further detailing of this aspect is beyond our cytological purpose, but the importance of stress must be kept in mind in any attempt to explain cell growth. For example, it is noteworthy that turgor pressure is a nondirectional force, while the response is directional (Green, 1962). One of the problems is to find the sites and the mechanism of this polarized response. B. SITESOF SURFACE EXPANSION One explanation of the conflicting data is that the actual growth state probably is not always definitely controlled either at the organ level or at the cellular level. At the organ level growth of the intercalary type implies that various states of expansion are simultaneously present. When a growth gradient occurs (e.g., in a

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root or stem), only a small part of the organ is actually growing, and the major part is no longer expanding. This is an important point which is usually underestimated by authors, so that ambiguity remains in most of the published cytological data. Let us consider a hypocotyl of a legume, a common material for growth experiments. The size of the organ increases with age, and it is easy to check that this increment results from the expansion of only a narrow subapical zone (see Reis, 1975). Another underestimated question is whether in a growing organ all tissues drive expansion with the same intensity. For example, split-tests and peeling experiments from various stems and coleoptile indicate a regulating and limiting role for epidermis. Epidermis shows precisely one of the most ordered growing wall (see below). If specimens are regularly excised from the middle of the hypocotyl, for example, as is usually done, one is likely to observe cells no longer expanding. At the macroscopic level the growing or nongrowing state of the specimen can be roughly checked by simple means (the location of india ink marks followed at intervals of time, for example). At the present time more sophisticated measurements are possible with an apparatus using optical amplification (Nissl and Zenk, 1969; Evans and Ray, 1969; MacDowal and Sirois, 1977), the laser method (Metraux and Taiz, 1977), and mainly displacement transducers (Cleland et al., 1972; Penny et al., 1973; Rat, 1978 and bibliography therein). These methods emphasize that growth properties change significantly both in time and in space. Thus it becomes necessary to combine a refinement of ultrastructural approach with a concomitant improvement in knowledge of the physiological state of the studied specimens. At the cellular level, the question is whether, in a given cell from a zone actually growing, the wall studied increases overall. In bacteria (Frehel and Ryter, 1971) and in thallophytes (see for example Pickett-Heaps, 1975a; Green, 1958; Lacalli and Acton, 1974, for green algae; Bartnicki-Garcia and Lippman, 1969; Grove and Bracker, 1970, for fungi) the cellular surface is usually directly accessible to markers. The sites of expansion of the cell surface appear highly specific and often precisely localized both in unicellular and multicellular organisms. Waaland et al. (1972) and Waaland and Waaland (1975), showing the presence of “bipolar band growth” in red algae, provide a direct illustration of this accurate localization. For higher plants in which cells are associated in massive tissues the delimitation between growing and possibly nongrowing areas is more complex and difficult to analyze experimentally. Obviously cases exist where actual growth is localized in certain parts of the cell surface. For example, in elongating cells, longitudinal facets expand while the transverse ones retain their initial surface. Different types of cell growth have been distinguished (see Frey-Wyssling, 1950; Setterfield and Bayley, 1957; Wardrop, 1962). However, cells located at the surface of organs are the only ones directly accessible to markers. The cells whose growth sites have been the most clearly localized are those with free ends and apical growth. It is noteworthy that, even in these cases, delimitation of the

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growing and nongrowing parts remains unclear, as illustrated by observations on both cotton fibers (O'Kelley, 1953; Willison and Brown, 1977; Westafer and Brown, 1976; Ryser, 1977) and pollen tubes (Dashek and Rosen, 1966; Roggen and Stanley, 1971; Duhoux, 1977). For epidermal cells, the basic data are the measurements made by Castle (1955) of the spacing of metal particles deposited on the surface. An equal spacing of the particles was noted during expansion, suggesting growth of the entire surface and ruining the idea of apical localized growth for these cells. The observations of Castle (1955) were made at the light microscope level. When transposed to the electron microscope level, the technique shows minute zones where the spacing of the particles is in fact not uniform (unpublished results). Therefore the possibility of an arrhythmic surface increase at the ultrastructural level is not excluded. This is a field where further studies should be undertaken. When cells are located more deeply in organs, direct markers are not usable. So far, evidence for precise location of the growing part is restricted to observation of the gradual spacement of punctuations in the cell wall (Wilson, 1957, 1964), the so-called dilution of plasmodesmata (Juniper, 1977), or autoradiographic data. The last-mentioned have been mainly used at the light microscope level (Wardrop, 1956; Setterfield and Bayley, 1957; Gorham and Colvin, 1957). Though high-resolution autoradiographicstudies have been performed on the cell wall (Ray, 1967); Pickett-Heaps, 1967; Northcote and Pickett-Heaps, 1966; Wooding, 1968; Fowke and Pickett-Heaps, 1972; Paul1 and Jones, 1975; Rougier, 1976; Sandoz and Roland, 1976), it seems that both the turnover of certain polysaccharides (Franz, 1972; Gilkes and Hall, 1977; Labavitch and Ray, 1974) and the limit of resolution of the technique make the use of autoradiography difficult in the study of sites of expansion of the wall. The main result is evidence of the incorporation of matrix components throughout the cell wall (intussusception), whereas the deposition of cellulose is internal (apposition) (Ray, 1967). However, the delimitation of the zones of wall modification in the expanding cell remains an open question.

III. Organization of the Expanding Cell Wall A. INTERPRETATION IN TERMSOF DISPERSE TEXTURE'

Study of the higher-plant cell wall started at the electron microscope level 30 years ago with the observations of Frey-Wyssling et al. (1948). Later, in the 'Following the Cell Wall Meeting held in May 1978 at Nijmegen (Holland) an interesting discussion was published by Frey-Wyssling (1978) on the terminology used for primary cell walls. Based on physiological or structural d e f ~ t i o n smisinterpretations can emerge. The present article considers every kind of wall in higher plants engaged in the growth process.

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1950s and 1960s, numerous studies were devoted to this subject. Since the components of the growing cell wall-mostly polysaccharides-were weakly visualized with the heavy metals (lead, uranium, and osmium) used for staining other organelles, investigations were performed mostly by means of shadowcasting. This method, widely used, implies that the material undergoes particular conditions which have to be recalled because they can have consequences for the results. First, the specimens are macerated in order to separate the cells, remove the wall matrix, and clear the cellulosic skeleton. Because of the indestructibility of cellulose, strong treatments are used, mainly hot acid and alkali or concentrated hydrogen peroxide. The treatments unmask the fibrils, but at the same time break the chemical bonds and destroy the cohesion of the assembly. Second, the specimens are either spread directly on the specimen holder or embedded in a plastic medium for sectioning. In the latter case, since the plastic embedding is removed later on, a mixture of butyl and methyl methacrylate is generally used. This embedding medium has the disadvantage of changing volume during curing and thus creating local distortions within the specimen. Since the specimen is reduced to just a weak fibrillar framework, whatever the preparative procedure (direct spreading or embedding followed by disembedding) the risks of artifactual shifting are high. Consequently the shadowing method is convenient to reveal with sharp contrast the fibrillar subunits, but its limitations should not be overlooked when the three-dimensional texture of the wall is interpreted. The early observations of Frey-Wyssling et al. (1948) on many higher plants had revealed the presence of microfibrils and shown that both “primary and secondary walls display a beautiful fibrillar texture. ” Since then, the primary wall has been defined as being made of fibrils interwoven and associated in a loose network (Durchweben), whereas in the secondary walls the microfibrils are pressed close together and parallel. Soon after, Miihlethaler (1950a,b) confirmed, in young coleoptile tissues of maize and oats, that newly deposited cell wall microfibrils were interlaced in a very loose reticular network. During expansion this reticulum seems to be steadily strengthened by the addition of new cellulose fibrils which are interwoven into the original loose framework. When the cell reaches its final length, deposition of the secondary wall begins and, in opposition to those of the primary wall, the fibrils are parallel to one another. After these pioneer observations numerous studies were undertaken. The related papers have been reviewed several times (see Northcote, 1958; Wardrop, 1962; Setterfield and Bayley, 1961; Miihlethaler, 1967; Frey-Wyssling, 1976; O’Brien, 1972). The data are summarized in several books devoted to the cell wall (Roelofsen, 1959; Siege], 1962; Rogers and Perkins, 1968; Pilet, 1971; Preston, 1952, 1974; Frey-Wyssling, 1976). In this field, the book by Roelofsen (1959) remains an outstanding document of the data available from various species and tissues during this rich period. The fact that the primary wall is the part of the wall showing a disperse (or scattered) texture while the secondary wall is the part of the wall showing a parallel texture was apparently accepted. The different

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textures were related to the expansibility and growth properties of the wall, a disperse texture appearing more favorable to shifting and surface expansion than a parallel texture. The difference in texture could be explained by the difference in composition and especially in hydration of the primary and secondary walls. In the primary wall a loose arrangement of microfibrils may be due to their high hydration and their low percentage of cellulose. In the secondary wall, in which cellulose prevails, the microfibrils must be closely packed, and there is no space for dispersion (Frey-Wyssling and Miihlethaler, 1965). In the primary wall, despite the general disperse texture and wide angular dispersion of microfibrils, a preferred orientation was often statistically noted. On the basis of the terminology introduced with use of the polarizing microscope, the following textures were distinguished (see Frey-Wyssling, 1976): (1) a foliate texture in which the microfibrils were laid down with a completely random orientation, (2) a tubular texture in which microfibrils were oriented more or less transversely according to the growth axis, and (3) a fibroid texture with microfibrils more or less longitudinal. The foliate texture characterizes isodiametric cells growing in no preferred direction and whose shape is approximately a sphere. The tubular texture appears more usual in cylindrical elongating cells growing in a strongly oriented direction; in this case the wall expands roughly at a right angle to the main direction of the microfibrils. A longitudinal orientation of microfibrils characterizing the fibroid texture was illustrated by axial “ribs” at the cell comer of parenchyma, epidermis, and collenchyma (see Roelofsen, 1959; Setterfield and Bayley, 1958; Wardrop, 1962). When the cell wall was seen in face view, differences in microfibril orientation were noted whether the internal or the external face was observed. On the internal face, the microfibrils were said to be mostly transversely oriented, whereas on the external face they appeared to be more dispersed and even had a tendency to be longitudinal. This constitutes the basic argument of the multinet growth hypothesis (MGH). On this particular point, the following remarks should be made. (1) The identification of the observed face as internal or external is often difficult; (2) the diposition of the microfibrils within the median part of the wall is not usually visualized, and the hypothesis of a gradient of reorientation remains conjectural; (3) one should remember that any external shadowed view of the fibrillar network implies necessarily that it has been separated from the middle lamella; that is, it is dissociated. The fxst results obtained with the electron microscope were apparently in accordance with those obtained with the polarizing microscope (for details, see Veen, 1970). The two methods used for the study of the cell wall seemed to produce the same result, and this certainly might explain the general agreement about the disperse ‘texture. However, interpretation of the results obtained with the polarizing microscope requires prudence, because it gives a global view of the texture of the wall (Preston, 1974). According to Gertel and Green (1977) the

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polarizing microscope automatically yields a mean statistical measure of microfibril orientation within the thickness of the wall analyzed. The too-often neglected remark of Frey-Wyssling and Miihlethaler (1965) can be recalled in this connection: “If a sufficiently thick wall layer has the same texture throughout its width, the electron microscope confirms the textures derived from observations with the polarizing microscope. If however there are ultrathin lamellae with parallel textures criss-crossing each other, u disperse texture can be simulated in the polarizing microscope. This is actually the situation in multi-ply constructions encountered in the primary wall (Section 111, C). ”

THE IDEAOF A N ORDERED TEXTURE B. TOWARD

Although general agreement that growing cells were encapsulated by a tangle of microfibrils responsible for the expandability of the wall gradually emerged, early observations had shown that an ordered texture was not incompatible with active expansion. The striking example is undoubtedly the unicellular alga Valoniu. It has been known since Steward and Muhlethaler (1953) (see also Preston, 1974, and bibliography therein) that, as spores of Vuloniu grow, the new wall consists mainly of successive lamellae. In each lamella the microfibrils run in a single direction. The direction of the strands changes abruptly from lamella to lamella with an approximate angle of 120”. By repetition of this process, a multilayered wall is built up in such a manner that the principal direction tends to be repeated at every fourth lamella. According to the authors, during expansion the amorphous substance may serve as a lubricant between the successive lamellae which could thus move past each other. Among algae other examples of ordered textures have been described in growing walls. Oocystis has been well documented during recent years. At no time during the development of its layered wall were the microfibrils seen to have a random orientation (Robinson ef al., 1976) and, according to Montezinos (1977), slippage of microfibrils was possible without changes in the wall layer orientation. In higher plants the ribs of cellulose in which the microfibrils run parallel to the growth axis, observed at the cell comers of various young parenchyma and epidermis, were in the 1960s a source of dispute with respect to the idea that growth was supported by a disperse-textured wall (Bohmer, 1958; Wardrop and Cronshaw, 1958; Wardrop, 1962; Roelofsen, 1959; Setterfield and Bayley, 1958, 1961). Collenchyma provides an example of a cell wall supporting considerable expansion despite a conspicuous ordered organization. The texture of the early thickened wall has been repeatedly studied (Beer and Setterfield, 1958; Roland, 1966; Chafe, 1970; Chafe and Wardrop, 1970; Deshpande, 1976b). The layers can be counted in tens, and it has become evident that the cellulose is closely associated with the wall matrix in repeated multifibrillar complexes paral-

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lel during growth (Roland, 1966). The transverse lamellae are not restricted to a layer bordering the cell lumen but occur throughout the wall thickness, alternating with lamellae in which the orientation of cellulose microfibrils is longitudinal (Chafe and Wardrop, 1970; Chafe, 1970). The observations by Sterling and Spit (1957) on Asparagus fibers appear interesting in view of the ordered fibril hypothesis developed later. The fibers have been studied when increasing from 15-30 pm up to 700-900 pm in length. The microfibrils of the inner wall surface lie predominantly in an oblique, crossed arrangement. Despite the technique of preparation (maceration and shadowing) they appear to have a more-or-less alternating direction of orientation. According to the authors, the direction of growth should be independent of microfibrillar orientation. The general validity of these results was later questioned. Indeed, despite special care used in the dissection of tissues, identification of the observed surface of the wall was only presumptive, and an important dispersion actually existed in the fibrillar orientation. Furthermore, Veen (1971) has emphasized that Asparagus fibers are a small part of the stem; it is conceivable that the fibers are compelled to extend in a longitudinal direction by the growth of neighboring cells. The laticifers in Euphorbia provide another peculiar example in which the texture of the wall is seen to be laminated and crossed in actively growing cells (Moor, 1959). During intrusive growth the parallel textured wall supports areal extension, both in length and in diameter. Although the foregoing examples have been minimized or considered special cases, it is worth emphasizing that parallel textures can appear in growing walls, even after maceration. They show that the identification of growing and nongrowing parts of the wall is not possible with only morphological criteria. To avoid confusion created by identifying the random layer of microfibrils with the primary wall and the oriented layers of microfibrils with the secondary wall, Belford el a / . (1958) suggested that the terms “primary” and “secondary” be retained in their original, physiological sense (growing property) and that new terms be introduced for describing the two types of microfibrillar organization revealed by the electron microscope after shadowing. They proposed that a random layer of microfibrils be termed an a layer and that an oriented layer of microfibrils be termed a /3 layer, although this proposition was not retained later on. Perhaps it is regrettable, because such terms, easily used throughout the plant kingdom, probably would have avoided confusion between the texture and growth properties of the cell wall. Refinements of transmission electron microscopy techniques made it possible to reconsider cell wall architecture study, especially during growth, and to analyze the fine structure with different convergent techniques. The development of ultrastructural cytochemistry made it possible to visualize polysaccharides on ultrathin sections (for details see Roland, 1978), especially owing to the trans-

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position at the electron microscope level of the periodic acid-Schiff reaction (Seligman et al., 1970; PATAg reaction of Thiery, 1967). Along with a mild extraction (enzymic or chemical) the method allows sharp visualization of wall subunits with a minimum of disturbance (Reis and Roland, 1974). Moreover, a quantitative analysis with gas chromatography can be employed in the cytochemical study (Reis, 1976, 1978). Freeze-etching and ultracryotomy are two other ways to study the three-dhensional arrangement within the wall. The use of freeze-etching has long remained difficult in studying the plant cell wall (Northcote and Lewis, 1968; Chafe, 1970; Itoh, 1975a,b, 1976; Ameluxen et al., 1976). Results, especially those concerning the most recent part of the wall have greatly improved with the omission of cryoprotectants or faatives prior to freezing (Willison, 1975, 1976; Mueller et al., 1976; Willison and Brown, 1977; Mueller and Brown, 1977; Vian et al., 1978). Ultracryotomy, by avoiding the embedding of specimens, makes it possible to perform negative staining directly on ultrathin sections. Therefore the technique is highly suitable for visualizing the in situ fibrillar arrangement and the intefibrillar spacing within the wall (Fig.

FIG.1. In situ visualization of the texture of the growing cell wall by means of ultracryotomy. (A and B) Elongating zone of pea root. (A) ~ 7 2 . 0 0 0(B) . ~600,000.(C) Elongating zone of mung bean hypocotyl. Cortical parenchyma. x80,OOO. The texture appears more ordered than usually thought. ML, middle lamella; PM, plasmalemma.

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1). The results obtained from various materials and tissues led to the idea that the growing wall is probably more ordered than is generally claimed and that the randomization is, at least in part, due to the method of preparation. Using a combination of ultracryotomy and cytochemistry along with mild extractions, Roland et al. (1975) studied the architecture of walls from various types of actively growing cells (growing rate of about lO%/hour).They showed that in all examined examples the most characteristic feature was the highly ordered disposition of the subunits in successive layers (Fig. 2). Itoh (1975a,b), by means of freeze-etching and shadowing after freeze-drying, illustrated different types of crossed polylamellate structures of primary walls. He showed a great variety of microfibrillar crossing angles. Later Jtoh and Shimaji (1976) reported that these crossed arrangements existed in actively growing cells. Three main planes (transverse, longitudinal, and oblique) of microfibrillar orientation

FIG.2. Lamellation of the growing cell waU. Inner part of epidermis wall sectioned perpendicular to the direction of growth. Elongation zone of mung bean hypocotyl. PATAg staining. (A) Alternation of transverse (t) and longitudinal layers. (B) Twisted disposition (discontinuous line) of components recently incorporated into the wall. Arrows, Wall subunits before incorporation; PM, plasmalemma.

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were recorded throughout the developing wall. Desphande (1976a,b,c), using enzymic and chemical extractions followed by permanganate staining in Cucurbira stems also showed a layered, ordered organization of the wall of different tissues. The number and thickness of the layers vary according to the age and type of cell wall. The microfibrils are oriented within the plane of the lamella, but the orientation may vary in successive lamellae, and in many walls a crossed polylamellate condition has been detected. Sawhney and Srivastava (1975) and Srivastava and Sawhney (1975) have indicated that primary walls of epidermis and cortical cells of lettuce hypocotyl exhibit a thick polylamellate texture. Lamellae with a longitudinal fibrillar orientation alternate with lamellae having a transverse orientation. In each lamella the fibrils are not strictly in the plane of the lamella but at a slight angle to it. In epicotyl tissues of etiolated Alaska pea, Vanderwoude (1977) observed, with freezeetching, large areas of the wall’s inner surface. He showed that cellulose microfibrils were highly oriented. With the same technique, Mueller and Brown (1977), in Zea mays seedlings, showed layers of walls with contrasting orientation. From the observation of bamboo fibers, Parameswaran and Liese (1976) insisted on the similarity of the polylamellate texture of the primary and secondary walls. Roland et al. (1977), in the elongating zone of mung bean hypocotyl and pea roots, analyzed the threedimensional arrangement of walls, which appeared to be cell-specific. Using ultracryotomy, they showed that the arrangement was highly fragile, and that a mild extraction could introduce scattering in the ordered fibrillar arrangement. Liberman-Maxe (1978) found anordered texture in the wallof growing fern, specially in the protophloem. Interestingly, Itoh (1978, personal communication) reported that suspension-cultured cells of Rauwolfia and Nicoriana (i.e., cells without polarity of elongation) had regularly oriented microfibrils and that a crossed lamellate structure was often observed in their growing walls.

c. INTERPRETATION IN TERMSOF A MULTI-PLYCONSTRUCTION The foregoing data require modification of the current view that the texture of the primary wall is essentially scattered. Evidence has been progressively brought to light that, when mild techniques are used, the wall of growing cells is typically multilayered and ordered. Three-dimensional observation shows that the wall is built up with successive strata wherein the polysaccharide chains are parallel. The orientation of the chains changes in the successive layers. Thus the organization of the primary wall should not be considered different from the organization of the secondary wall, except that it is more fragile. The fragility is related to the lability of the chemical bonds between the wall subunits and to the great number of alkali-soluble polysaccharides. Both the disposition and the possible crossing of the subunits resemble the organization of plywood. Plywood is a manufactured material built up with

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superimposed thin layers of wood called veneers or plies (see Wood and Linn, 1946). It is a multi-ply construction in which typically an odd number of plies are glued together on both sides of a special layer called the core of the construction and arranged so that the grain of one layer runs usually more or less at right angles to that of any adjacent layer. The nature of the core can be different from that of the adjacent plies. The plies are disposed either symmetrically on both sides of the core (the so-called isotropic plywood of wood technicians) or assymetrically, each side being differently veneered (anisotropic plywood). An increasing number of plies (3- to 15-ply boards are usually manufactured) improves the mechanical properties of the material. When considering growing plant cells, the walls may be compared to a multiply construction, the number and the characters of the constitutive plies depending on the cellular type; that is, they are programmed by differentiation. One possibility is to consider each cell individually, its wall being comparable to a plyboard; as the organ grows as a whole, an apparently more appropriate conception is to consider the cells in relation to their neighbors. In the latter case, the, middle lamella can be considered the core of construction on both sides of which the successive plies are apposed. From this viewpoint the two following types of primary walls are distinguishable: (1) symmetric multi-ply constructions (Fig. 3), where the neighboring cells undergo the same differentiation, and (2) asymmetric multi-ply constructions (Fig. 4), where the neighboring cells are not of the same type or are in contact with the external medium. Symmetric constructions are seen in the majority of tissues of the growing plant. In the simplest case (meristematic cells, young parenchyma, and so on) a single multifibrillar layer is seen on both sides of the middle lamella, and the wall is like a three-ply construction (Fig. 3A). Such a construction could be called a primordial wall. The data obtained so far indicate that the first deposited multifibrillar layers are transversely oriented with respect to the growth axis (tubular texture), but further studies would be necessary to determine if such a disposition is a constant feature. Later, the cells either maintain a wall of this type (various piths), or new multifibrillar layers are added and the wall becomes a multi-ply construction (cortical parenchyma and collenchyma; Fig. 3B). Typical asymmetric constructions are found, for example, in epidermis; the internal part of the wall is layered and crossed, whereas on the external side of the pectin layer the wall is built up with cutinized layers (Martin and Juniper, 1970; Clowes and Juniper, 1968) and superficially veneered with cuticle and wax (Fig. 4A). Within the plant body, asymmetric constructions are found, for example, in protophloem; the nacreous wall of sieve cells is usually made of a single thick layer of transversely oriented microfibrils, whereas the neighboring cells display multilayered walls (Fig. 4B). The comparison between a growing wall and multi-ply construction presents the following advantages. (1) It provides a simple model usable for the various wall textures. The number, thickness, and chemical nature of the successive layers

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PM' .

/

A

B

FIG.3. Interpretation of primary wall layering in terms of a multi-ply construction. A case of symmetric construction. (A) Three-ply construction. Primordial wall. The middle lamella (pectin layer) represents the core (0).On both sides a single multifibrillar layer (ply 1) is apposed transversely. (B) Multi-ply construction. The successive layers (plies 1, 2, 3, and so on) display a crossed orientation. Double arrow indicates the direction of growth. PM, Plasmalemma.

can vary, but the general construction remains the same. Thus a collenchyma wall, instead of being a peculiar case needing a special explanation for growth, appears simply as an example of a wall with a great number of layers. (2) It draws attention to the mechanical properties of young plant organs. A veneer is often wrongly thought of as a sham or only a means of decoration. In fact, it is a material manufactured to combine lightness and strength. From the crossing of the grain results a remarkable increase in resistance in both directions. By manufacturing wood into plywood, the weaknesses of the original material (liability to split axially, failure to resist shear, and so on) are overcome. It is a balanced construction with outstanding flexibility properties. Flexibility is one of the striking characteristics of young and herbaceous plant organs.

D. BOW-SHAPED ARRANGEMENTS Interpretation of the crisscross texture of the wall is complicated by occurrence of the twisted or bow-shaped arrangements seen between the layers in several ultra-

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CI

0 1 2

3 4 5

PM A

n

FIG.4. Interpretation of primary wall layering in terms of a multi-ply construction. A case of asymmetric construction. (A) Outer cell wall of epidermis. Against the pectin layer (0)the internal face shows a typical crossed texture (plies 1 to 5); the other face is made up of cutinized layers (C1) and externally veneered with cuticle and wax (black). (B)Nacreous cell wall in protophloem. Against the pectin layer (0).one face displays the typical multi-ply construction (plies 1, 2 , and so on); the other face usually remains restricted to a single but thickened transverse layer (NA). Double arrow indicates the direction of growth. PM, Plasmalemma. Possible transitional strata are not drawn in Figs. 3 and 4.

thin sections (Figs. 2B, 6 and 7). Analysis of the twisted arrangementsunderlines the difficulty encountered by cytologists in establishing a three-dimensional organization from sections and fractures. Besides strictly morphological interest, an understanding of the twisted arrangements may help provide a better knowledge of wall morphogenesis. Bouligand (1965, 1972) and Bouligand et al. (1968) drew attention to the frequency of twisted arrangements in animal cells, especially in cuticles of arthropods and chromosomes of some protists. For example, sections of insect and crab cuticles show characteristic series of bow-shaped lines. Bouligand (1965, 1972) has shown that the arced appearance is an illusion. No fibril follows the curve and in fact spans the distance between two successive laminae of the curticle. From a detailed study of the geometry of the systems the author concluded that in each successive layer the fibril direction was rotated through a small angle and that the arced appearance was caused by overlapping planes of fibrils. The model in Fig. 5 shows how oblique and opposite sections related to an axis is perpendicular to the laminae give series of bow-shaped lines with inverted concavity. This type of assembly is characteristic of cholesteric

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mesophases (Bouligand, 1972; see also Neville and Luke, 1971; Neville et al., 1976; Livolant et al., 1978). In the literature of plant cell walls, the observations and interpretation of twisted arrangements was sporadic and confusing. The term “herringbone” was applied for the first time to the cell wall by Oort (1931), long before the use of the electron microscope. After a study of the spiral growth of Phycomyces with the aid of a cathetometer, the author assumed that theoretically “the direction of growth and structure need not coincide. It may for instance be imagined that the direction of growth coincides with the longitudinal axis, while the particles stand obliquely with respect to this axis. Compare for instance a herringbone clinkerpavement; this is a case in which the parts (clinkers) are oriented obliquely with respect to the main direction. . . .” According to this initial postulate the direction in which the pavement is laid out may be entirely independent of the direction in which the blocks are pointing. The subunits of the wall should be disposed along folding lines with regular “reversals,” giving the aspect of a chevron. When arced patterns were seen in the wall in the electron microscope, the idea that the subunits were arranged in a curved disposition was unquestioningly admitted. According to Mosse (1970) the resting spore wall of Endogone develops bands having “a curved fibrillar substructure, alternating with narrow

FIG.5 . Diagram showing how series of how-shaped lines appear in a laminated structure when obliquely sectioned. Rectangles, where pardel and equidistant straight lines have been drawn, are arranged in the form of a pyramid trunk. Oblique faces display a twisted arrangement. Note the inversion of the arcs between two opposite faces. (From Bouligand, 1965 and 1972.)

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bands of tangentially oriented fibrils. ” The twisted arrangements are usually described as “herringbone patterns,” following Oort (1931). Hoffman and Hofman (1975) call them “feathery patterns,” and Neville et al. (1976) “helicoidal systems. Evidence of a twisted wall structure in various cell types has been accumulating over the last decade. Probine and Barber (1966), Srivastava (1969), Mosse (1970), Chafe (1970, 1974), Chafe and Wardrop (1970, 1972), Chafe and Doohan (1972), Cox and Juniper (1973), Hoffman and Hofman (1975), Parameswaran and Liese (1975, 1976), Neville et al. (1976), Deshpande (1976b), Peng and Jaffe (1976), Sawhney and Srivastava (1975), Srivastava et al. (1977), Roland et al. (1975, 1977), Robinson and Herzog (1977), and Sargent (1978) have shown the presence of twisted arrangements in sections from various primary and secondary walls (twisted arrangements seem to be especially conspicuous in the wall of spores, possibly because of its thickness and spherical shape which increase the number of glancing sections). Bow-shaped patterns of cellulose fibrils were seen in ascidian tunica with a scanning microscope (Gubb, 1975). Recently bow-shaped patterns have been shown in the chitin-rich portions of cell walls of the alga Pithophora (Pearlmutter and Lembi, 1978, and personal communication). It is not rare to observe more-or-less conspicuous bow-shaped patterns in published micrographs of walls with no mention by the authors or misinterpretation. Appelbaum and Burg (1971) claim to have observed “ran”

FIG.6. Twisted arrangements seen with freeze-etching. Collenchyma of celery petiole. Moreor-less large, bow-shaped arcs on a ‘fracture traveling almost in the plane of the inner layers (il) of the wall (W).Note the imprints of microfibrils not yet associated in layers on the EF fracture face of the plasmalemma (PM). x23,800.

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domly distributed microfibrils" in the wall of Pisum sativum stem, while their micrographs show bow-shaped arrangements. Stereo observations (analyses of serial sections, sections made according to various orientations with respect to the cell axis, and observations of the same section from different angles by means of a goniometric stage) indicate that the bow-shaped appearance in the cell wall directly depends on the obliquity of the

FIG.7. Effect of tilting on arced patterns. Cancelation and revenal for three angles of the goniometric stage. Cortical parenchyma sectioned perpendicular to the growth axis. Mung bean hypocotyl. PATAg staining. Note three microtubules (mt) visible at +30° and +20"and disappearing at -40" (encircled area). PM, plasmalemma.

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observations (Figs. 6 and 7). Generally speaking it seems clear that the bowshaped patterns seen in the cell wall are relevant to the explanation of Bouligand indicated above and result from oblique sections through successive laminae in which the orientation of the subunits changes by a small angle. There is no real torsion on the subunits or microfibrils. The use of a goniometric stage directly emphasizes the importance of the angle of observation to the twisted appearance seen in the wall. Figure 7 shows, for example, that tilting produces a cancellation or even a reversal of the arcs; this demonstrates the illusory nature of the twisting and is characteristic of cholesteric and liquid crystal assemblies. At the present time the three-dimensional study of the bow-shaped patterns is not documented well enough in plant cells to establish the exact angle between the subunits of two neighboring laminae. An interesting calculation has been made recently by Peng and Jaffe (1976) during envelope formation in the egg of Pelvetia with the aid of a computer. In this case, the average orientation of microfibrils changed about 35” in each subsequent lamella of the wall. This slow change gives rise to typical bow-shaped arcs when the wall is obliquely cross-fractured. Concerning the growing cell properly said, Roland et al. (1977) and Vian (1978), analyzed the bow-shaped arrangements of primary walls in the elongating zone of mung bean hypocotyl and pea root and postulated that “transitional strata” might exist between the transverse and longitudinal multifibrillar layers previously described. Each stratum appears constituted of a thin sheet of rows of parallel fibrils. In the successive strata the fibril direction is rotated through an

FIG.8. Possible changes in the subunit orientation in a primary wall. (A) Typical crossed texture (orthogonal multi-ply construction). This indicates an abnrpt change in the rnorphogenetic activity of the cell. (B)Presence of transitional strata, which give rise to the bow-shaped appearance on oblique sections. This indicates a progressive change in the morphogenetic activity of the cell.

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FIG. 9. Whirlings and local disinclinations in the wall layering. (A and B) Same material as in Fig. 5. PM, Plasmalemma.

angle intermediate between orthogonal directions (Fig. 8). A certain asynchronism exists in the rhythm of layer deposition in cells of a given tissue. Figure 9 shows that defects of disinclination type can also occur, locally interrupting the ordering. This seems important in understanding the morphogenesis of the wall which could be considered a strengthened liquid crystal relevant to self-assembly processes (Section V).

E. THEOCCURRENCE OF A DISPERSE TEXTURE AND ITS RELATIVE IMF-ORTANCE

In the foregoing sections special attention was given to the ordering of the growing wall. Finally, the question arises whether a disperse texture and a random orientation of wall subunits actually exist and, if so, what their relative importance is compared to the ordered part of the growing wall. Visualization of a disperse texture surrounding the growing cell can be explained as follows:

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1 . A disperse texture is essentially an artifact introduced during the preparation of the specimen. If the wall is considered a highly three-dimensionallyorganized construction-with successive tiny, oriented layers-it is obvious that any local displacement will produce a random texture. 2. A disperse texture is an actual part of the growing wall. There are two possibilities: (a) it results from a formerly ordered texture dispersed during growth (a senescent wall); (b) it corresponds to part of the wall laid down randomly, before the establishment of adequate morphogenetic conditions (a juvenile wall). A direct illustration is given by naked plant cells regenerating a wall, such as eggs or spores of algae and fungi (Robinson, 1977; Sachs ef al., 1976; Brown and Montezinos, 1976; Montezinos, 1977; Pickett-Heaps, 1975a; Peng and Jaffe, 1976; Sassen er al., 1970; Kroh et al., 1976), and isolated protoplasts of higher plants (Willison and Cocking, 1975; Prat and Williamson, 1976; Herth and Meyer, 1977; Burgess and Fleming, 1974; Willison and Grout, 1978). In these cases the cells initially build a thin, nonordered envelope beneath which ordered secretions are subsequently deposited. The first envelope seems to act as a screen creating a defined space necessary for expression of the morphogenetic power of the cell (Fig. lo). For cells associated in tissues, the situation is apparently rather different. However, as in naked cells, the wall is built up de nova in the phragmoplast. Whatever the origin of an eventual disperse texture (senescent or juvenile), the question is to determine the relative importance of the random part compared to the ordered part in the growing wall. From the standpoint of growth it is known that the inner, recent part of the wall plays a major role in the expansion pattern of the cell (Richmond, 1977).

FIG.10. Regeneration of a juvenile disperse wall (naked eggs or spores, protoplasts, and possibly phragmoplasts). 1 , Naked state; 2, elaboration of an initial nonordered envelope (juvenile coat); 3, progressive ordering of new deposits beneath the juvenile coat; 4 and 5, further layering.

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IV. Modalities of Cell Wall Expansion A. A CLASSIC CONCEPT: THEMULTINET GROWTH HYFQTHESIS Most results supporting the idea of a disperse texture for the growing wall led to a general consensus about the MGH which can be considered the classic reference in the field. The MGH was proposed by Roelofsen and Houwink (1953) and progressively supplanted earlier concepts such as mosaic growth (Frey-Wysslingand Stecher, 1951), and bipolar tip growth (Miihlethaler, 1950b). This question has been discussed in numerous reviews (see, for example, Setterfield and Bayley, 1961; Wardrop, 1962; Roelofsen, 1959; Frey-Wyssling, 1950, 1976; Miihlethaler, 1959; Wilson, 1964), and just a summary of the basic elements of the concept is presented here. Initially proposed for cells extending freely into the medium (hyphae, cotton fibers, root hairs) and for cells more-orless loosely associated (arms of stellate cells of Juncus), the MGH was afterward extrapolated to all kinds of cells. According to the MGH microfibrils are deposited continuously with a transverse orientation against the internal face of the wall (i.e., according to the maximum stress in a tubular cell). During expansion the fibrils are passively shifted in an outward direction and, after a while, form a disperse net while additional fibrils are deposited underneath. Later they can be completely reoriented, becoming almost longitudinal. In this respect the wall “may be compared to a sheaf of fishing nets which gradually change in mesh and in direction of the twine” (Roelofsen and Houwink, 1953). Hence the growing wall resembles a more-or-less loose network, and the MGH is mainly supported by the supposed disperse texture of the cellulose skeleton seen in the wall after shadow-casting. The concept takes into accountat least for cylindrical cells-the stresses supported by the wall (Van Iterson, 1937; Castle, 1937, 1955; see Green, 1969, 1976). This is probably the reason why until recently analyses of plant cell growth were usually based on the MGH (see, for example, Appelbaum and Burg, 1971; Veen, 1970; Gertel and Green, 1977 and personal communication; Willison and Brown, 1977; Fujii el al., 1977).

B. DIFFICULTIES WITH

A

PASSIVE BEHAVIOR OF THE CELL WALL DURING GROWTH

According to the MGH the behavior of the wall should be essentially passive. Difficulties encountered with this concept are as follows. 1. A concept essentially mechanistic can hardly explain the multiplicity of cell shapes within a plant organ. A special difficulty is to explain how the cell is

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initially engaged in growth in a preferential direction. For example, in an elongating system, the texture is laid down before the cell assumes the cylindrical shape. If, therefore, as emphasized by Frey-Wyssling and Miihlethaler (1963, “we presuppose this shape for the explanation of the texture, this would amount to an inversion of cause and effect. ” Another problem is to understand, for example, the usual opposite directions of growth supported by primary meristematic derivatives (elongation) and cambial derivatives (enlargement) while they both present a tubular texture. 2. A concept essentially mechanistic can hardly explain the accuracy and the diversity of responses of a cell to various stimuli. The refinement of methods of growth measurement indicates that it is possible for a cell to change not only its growth rate but also to reverse its growth axis. The nature of compounds (nutrients, hormones, inhibitors, and so on) and concentrations produce very specific responses. The accuracy and the sequential characteristics of the response of the cells appear hardly reconcilable with a simple, passive tearing of the wall. When structural modifications have been noted following changes in cell growth direction, they can hardly be explained in terms of a reorientation of existing microfibrils (Veen, 1970; Ridge, 1973; Sargent et al., 1973, 1974; Osborne, 1976). Interpretations have been drawn from experiments with physically expanded cell walls (Cleland, 1971; Green, 1976; Gertel and Green, 1977). A change in structure can be noted, but it is difficult to extrapolate the results to what occurs for cells growing under physiological conditions. The situation had been clearly stated as early as 1935 by Bonner using the polarizing microscope. After a stretching of Avena coleoptiles he observed a change from negative to positive birefringence of the wall, that is, from a rhean transverse to a mean axial orientation of the subunits in the wall. In contrast, in a cell growing under physiological conditions, the wall behaves in a radically different way and remains negatively birefringent. 3. The MGH implies a wide angular dispersion of subunits within the wall. A large displacement and reorientation of a great number of subunits supposes a random rupture of bonds and subsequent dispersion of the chemical groups. These events result in spatial and thermodynamic problems. They seem incompatible with the highly organized molecular reticulum of the primary wall (see Albersheim, 1976; Keegstra et al., 1973; Monro et a!., 1976; Darvill et af., 1977). Sequences of interconnections between polysaccharides repeated many times in the molecular reticulum have to be accurately broken and after “creep” have to join adequate partners (Albersheim, 1975; Cleland et al., 1972; Davies, 1973). Such a process is probably dependent on a defined three-dimensional organization of the whole primary cell wall. 4. As detailed in Section 111, recent ultrastructural study indicates that the

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disperse texture is not as widely distributed as formerly thought, and conversely the wall is ordered in a manner rather inconsistent with the concept. Several authors consider that the difficulties are not sufficient to invalidate the MGH as a basic concept (see Veen, 1970; Preston, 1974; Sargent, 1978). However, the polylamellation throughout the wall thickness has led to the conclusion that “the orienting mechanism of the MGH cannot satisfactorily explain the microfibrillar orientations observed” (Chafe and Wardrop, 1972), and it has been suggested that the crossing of the microfibrils in the successive lamellae necessitates a modification of the MGH (Sawhney and Srivastava, 1975) or even an alternative explanation (Itoh, 1975b). According to Boyd and Foster (1975a, b), when each lamella of the wall is differentiated, randomly spaced bonds tend to develop between its adjacent microfibrils. Apparently the number of bonds so developed is related to the rate of expansion. Between unbroken bonds, tensions cause adjacent transverse microfibrils to bend to each side of their original orientation. Thus, within a lamella, they form lenticular or trellislike configurations. No general rotation of fibrils should occur. In this sense the trellislike concept is different from the MGH. Both concepts presuppose that expansion results of a behavior of the primary cell wall essentially passive.

C. AN ALTERNATIVE PROPOSAL: THEORDERED SUBUNIT HYPOTHESIS The foregoing structural data have led to comparisons of the primary cell wall to a more-or-less complex plywood panel (Section 111, C) but, compared to a plywood, it is an expandible material. When submitted to stresses, a plywood panel resists in both directions (Fig. 11). When submitted to stresses (turgor forces), the primary wall is able to increase its surface in preferential directions. This anisotropic expansion of the wall implies a specific loosening of the matrix and microfibrillar skeleton. It has been suggested that the wall is built up of successive, ordered multifibrillar layers and that its striking anisotropic growth is due to selective rupture of interfibrillar bonds within each layer (Fig. 12) (Roland et al., 1975, 1977). This ordered subunit hypothesis (OSH) is based on an in siru, three-dimensional analysis of an actual growing cell wall. The term “ordered subunit hypothesis” is preferred to “ordered fibril hypothesis” which was formerly used. The present concept is related to the whole architecture of the wall as it appears at the electron microscope level, including noncellulosic components. The terms “fibril’ ’ and “microfibril, ” which usually refer to cellulose, are ambiguous, since it appears that many noncellulosic polysaccharides of high molecular weight are microfibrillike (Franke et al., 1974; Reis, 1976, 1978; Herth and Meyer, 1977). In a

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FIG. 11. Toughness of a crisscross construction. The crossing of the grain of the neighboring layers gives the construction a striking flexibility and the greatest toughness in both directions. Made of successive multifibrillar layers associated with a crossed orientation, the primary wall is comparable to a plywood panel. Like manufactured plywood, the primary wall is mechanically strong. In addition, it expands differentially when submitted to stresses.

growth hypothesis it is important to take into account the noncellulosic components. The OSH can explain the multiplicity of cell morphogenesis and the specificity of responses of cells to regulators. The oriented growth of a plant cell appears as the terminal step in the following sequence. Selective loosener

Turgor pressure

-

1

ordered texture

r Morphogenesis

-

oriented growth

secretion Whether the loosening occurs within certain layers or not, the microfibrils resist or slide against each other. For example, during isotropic growth of spherical cells, the loosening should be rather equal throughout the wall thickness, whereas during strongly anisotropic growth of cylindrical cells the loosening should be different between the crossed layers. In the latter case the subunits running along the major internal stress (transverse) form annular bundles acting as rings opposed to cell enlargement. At the same time the subunits running along the longitudinal stress allow elongation by sliding against each other. According to the OSH,the texture of the wall appears to be more a cause of the direction of expansion than a consequence of such an expansion.

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FIG.12. Ordered subunit hypothesis. Differential loosening. Anisotropic surface expansion in response to stress can be explained by a selective loosening of bonds occurring in the successive ordered layers (the direction and intensity of stresses correspond to a cylindrical elongating cell). t, Transverse; 1 , longitudinal.

Further studies will be necessary to establish the validity of the unorthodox OSH proposed as a working hypothesis. Among the questions which remain unclear and which have to be thoroughly analyzed is the behavior of the twisted arrangement during expansion. Ultracryotomy shows that, for a long time, there is no evident change in the interfibrillar space and no gap within the layers. It seems likely that, owing to the organization of multifibrillar layers, local readjustments allow the fibrils to fill in the gap resulting from spacing (Roland et al., 1977). The consequence is a thinning of the multifibrillar layers toward the outer part of the wall without important modification of the microfibrillar orientation and ordering. The outlines of the OSH compared to the MGH are summarized in Fig. 13 for a cell doubling its length. Generally speaking, there is no evident reason for further considering the growing wall a purely mechanical and passive entity basically different from other organelles. The growing cell is a living level where a great variety of enzymes, including glucanases and lytic enzymes, exist (MacLachlan and Young, 1962; MacLachlan, 1977; Albersheim, 1975, 1976; Matile, 1975,

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FIG. 13. Comparison between the MGH (A) and the OSH (B). In (A) new fibrils laid transversely against the plasmalemma are reoriented and passively shifted in an outward direction. In (B) the subunits are laid down alternately in ordered layers (x and y). (The possible transitional strata are not represented.)

Chrispeels, 1976; Lamport, 1977) and where turnover of the critical linkages is very rapid (Darvill et ul., 1977). The latter can produce a series of minute breaks and repairs necessary for harmonized loosening of a three-dimensionally defined primary wall. Some cells in the plant body support evidently a passive expansion. A typical example is seen in the tracheids of protoxylem (annular and helical tracheids). The cell is dead, but the remaining primary walls expand between the discontinuous secondary thickenings. The protoplasm is removed, and the wall can be considered actually inert and passively shifted. The case of dead cells is undoubtedly peculiar, and the question arises as to whether all the cells grow according to the same mechanism. For some authors apical growth (cotton fibers) should definitely be related to the MGH (Ryser, 1977; Willison and Brown, 1977). Maybe the initial description of Roloefsen is suitable for these kinds of cells growing more or less freely in the medium and for walls expanding inertly, but it has been too widely generalized to all kinds of cells.

V. Possible Factors in Cell Wall Morphogenesis If the potentialities for growth of a given cell are dependent on the texture of its wall, the question is how can wall morphogenesis and the positioning of the wall submats be controlled?

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The aim of this article is not to detail the mechanisms of action of possible regulators of morphogenesis but only to indicate elements which can intervene in the positioning, in order to answer in advance objections that an ordered model of the primary walI should be rejected because of the difficulties in imagining how it is built up.

A. PRESSURE AND STRESSES

The possible orientation of the microfibrils according to the direction of greatest stress on the wall was mentioned in Section IV, A for the MGH, in which it is the exclusive factor. The process can hardly be considered for the secondary wall in which subunits show a specific, unquestionable orientation. In this case other processes have been conceived in which membranes and microtubules are generally implicated. In our opinion, it is artificial to consider a completely distinct mechanism of morphogenesis for primary walls and for secondary walls. It seems exaggerated to consider a solely mechanistic process for the positioning of wall subunits. However, it remains evident that a critical pressure is necessary on the one hand to cause expansion and on the other hand for adequate assembly of subunits. For example, plasmolyzed cells continue to release subunits which accumulate in the enlarged periplasm without further ordering (Roland and h a t , 1973). The pressure at least creates the necessary conditions for expression of the other subsequent morphogenetic factors.

B . MEMBRANE AND MICROTUBULE CONTROL This is a well-documented aspect of animal cells and is based on the fact the plasma membrane is a fluid structure in which the particles move (Singer and Nicolson, 1972). According to Edelman (1976), the “surface-modulating assembly” has a tripartite structure: (1) a subset of glycoprotein receptors that penetrate the membrane, (2) various actinlike microfilaments, and (3) dynamically assembling microtubules both to provide anchorage of the receptors and to allow propagation of movements. From the disperse, abundant literature available Heath (1974) has proposed a unified hypothesis to account for the various aspects of oriented fibril synthesis in plant cells. The hypothesis suggests that cellulose synthetase complexes located in the plasmalemma have an associated component which projects through the inner side of the membrane and interacts with the adjacent microtubules to generate a sliding force which moves the complex through the membrane along the tracklike microtubules. Figure 14 shows how a surface-modulating assembly could be imagined for plant cells during the secretion of fibrils. The direction of the synthetase move-

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FIG.14. Positioning of cell wall subunits via transmembrane control. (A) Mobility of synthetases (s) within the plane of the plasmalemma (PM). (B) Possible cooperation between microtubules(mt), contractile proteins (p), and synthetases (s); t, tubulin subunits; f, microfibrils. (The size of microtubule is underestimated.)

ment should determine the positioning of the growing ends of microfibrils (see Vian et al., 1976). The problem is to identify, at the ultrastructural level, a system able to orientate and function as a track. Data supporting this possibility are the following. 1. Observations on Plasmalemma

These observations are mainly documented with freeze-etching. The involvement of ordered particles in the biosynthesis and orientation of wall microfibrils was reported in Valonia and Chaetomorpha (Preston, 1974). Ordered particulate systems have been often found in algae and bacteria (Preston, 1974; Staehelin, 1968; Peng and Jaffe, 1976; Brown et al., 1976; Brown and Montezinos, 1976). The so-called granule bands, strings of particles, or rows of particles are supposed to serve as machinery for the assembly and orientation of wall subunits. Recently Montezinos and Brown (1976) have proposed for Oocystis a model showing how both a particulate complex at the end of each microfibril and rows of granule bands within the plasmalemma could be engaged in the building up of a highly defined, multilayered wall. For higher plants the existence of such arrangements is more debated. With the use of freezeetching without a cryoprotectant some short rows of particles have been described by Willison (1976) in root tips of soybean and by Mueller et al. (1976) in corn. Recently Robenek and Peveling (1977) reported that rows of plasmalemma-associatedparticles encountered on isolated protoplasts of Skimmia japonica may function as a linear complex for microfibril biosynthesis and orientation. However, the great majority of observations show a random disposition of

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particles within the plasmalemma (Chafe, 1970; Willison, 1976; Vian er al., 1978; Itoh, 1975), and the question remains open. 2 . Observations on Cortical Microtubules The literature on the microtubular skeleton underlying the plasmalemma of plant cells is abundant. It has been the subject of various studies, especially emphasizing the possible relation of microtubules to wall morphogenesis (see the reviews of Cronshaw and Bouck, 1965; Newcomb, 1969; Schnepf, 1974; Schnepf et al., 1975; Hepler and Palevitz, 1974; Hepler, 1976; Pickett-Heaps, 1975b). Different kinds of arguments are brought forward in which the microtubules are considered responsible for the orientation of wall subunits: (1) The disposition of microtubules mirroring the disposition of the subunits of the inner part of the wall suggests a causal relationship. The mutual orientation between cytoplasmic microtubules and wall microfibrils is evident in certain cells, for example, in tracheids, vessels, and developing stomata1 complexes. However, many exceptions have been reported (see Hepler, 1976). (2) Microtubuleinhibiting drugs induce a pronounced disturbance in wall texture. Srivastava et al. (1977), in lettuce hypocotyl, noted after colchicine treatment both random disposition and some degree of order of new wall fibrils. Recently Robinson (1977) criticized the generalization of a microtubule role for fibril Orientation. It should also be taken into account that drugs, especially colchicine, can interact with components other than microtubules, such as membrane-associated features (Hartand Sabnis, 1976). It is at the present time difficult to draw conclusions about a possible disturbance in the primary wall texture induced by drugs, since the texture of the primary wall itself is debatable. When observed in detail and three-dimensionally in growing cells, the microtubular skeleton follows a complicated pattern. In particular microtubules are not found only transversely as should imply a mechanistic concept (Fig. 15). Microtubules form bundles whose orientation changes from longitudinal to transverse. This is similar to the orientation of subunits in a multi-ply construction. A more detailed three-dimensional study is necessary to determine if the changes in orientation of microtubules can be exactly correlated with the changes in orientation in the primary wall. C. SELF-ASSEMBLY PROCESS The construction of macromolecules with complicated shapes, such as capsids of viruses and membrane subunits, is the result of self-assemblyprocesses (Kuschner, 1969; Bouck and Brown, 1976). For cell wall components, the possibility that self-assembly occurs has been little studied. Scarce data led to the idea that

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FIG.15. Diversity of cortical microtubule orientation in elongating cells. Mung bean hypocotyl. Underlying the plasmalemma (PM) parallel microtubules are associated in bundles. The orientation of the bundles varies between neighboring cells and within one cell. (D) The arrow points to a dense cross-bridge between a microtubule and the plasmalemma. W, Cell wall.

the spontaneous orientation of subunits was an important process in the ordering of the wall. Rees and Welsh (1977) have defined for polysaccharides, as for other macromolecules, the primary structure as a covalent sequence of monomer residues, the secondary structure as any geometric arrangement in space of this sequence, and the tertiary structure as the way in which these arrangements pack together in a compact whole. I n v i m studies of gels and cell wall fractions indicate that cross-linking of the covalent sequences, fibrillogenesis (parallel and regular as-

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sociation of sugar chains), and interfibrillar arrangements could result from spontaneous processes. Frey-Wyssling (1941, 1953), studying the properties of gels, emphasized that polysaccharide chains showed a strong tendency to self-orientate in parallel strands. Manley and Inoue (1965) have shown that “cellulose microfibrils can be regenerated in vitro by what is clearly a physicochemical process.” This was confirmed by Bittiger et at. (1969). Atalla and Nagel (1974) regenerated cellulose from solutions and recovered the native lattice spontaneously. Cross-linking of heterogeneous polysaccharides was obtained under acellular conditions (Rees and Welsh, 1977; Chanzy et al., 1978). Typical wall assembly has been obtained in bacteria (Beveridge and Murray, 1976; Sleytr, 1976) and Chlumydomonas (Hills, 1973; Hills et al., 1975; Catt et al., 1978). Quatrano and Stevens (1976) postulated that wall assembly in the Fucus zygote was initiated by the self-assembly of alginates into a gel on the surface of the cell, triggered upon fertilization by the release of alginate from cytoplasmic vesicles into the calcium-containing seawater. In growing cell walls of higher plants, ordered and twisted arrangements have been described in terms of cholesteric arrangements. Such arrangements result in self-assembly (see Bouligand, 1972, 1974; Neville et al., 1976; Livolant et al., 1978). The positioning of subunits in successive wall layers could result in spontaneous phenomena (Bouligand, 1972; Neville et ul., 1976; Roland and Vian, 1976; Roland et al., 1977). Reis (1976, 1978) and Reis et al. (1978) have shown that certain fractions of hemicelluloses are able to aggregate under acellular conditions in nodules whose texture is somewhat comparable to that of the wall arrangement. These authors conclude that in vivo the arrangement results basically from a self-assembly process occurring in the periplasm. The process is

FIG. 16. Assembly of cell wall subunits. The preexisting wall acts as a template for positioning. 1 , Synthesis of polymers; 2, sequential association of chains (s) in fibrillike components (f);3, stif-

fening and positioning of the chains. (From Reis, 1978.)

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modulated both by the wall, which acts as a template for the positioning of subunits, and the plasmalemma, which contains the synthetases (transmembrane control) (Fig. 16).

VI. Conclusions Figure 17 summarizes successive steps involved in the formation of a growing cell wall: (1) polymerization and release of wall subunits, (2) three-dimensional assembly of the subunits in the periplasm; positioning and cross-linking of subunits probably involving self-assembly and/or transmembrane control; and (3) anisotropic surface expansion of the wall implying selective loosening and sliding of the subunits when the wall is under stress. Figure 18 integrates the two cellular pathways necessary for growth (1) the exocytic route which contributes

FIG. 17. Diagram summarizing the successive main steps involved in the building up of a growing cell wall. ER, Endoplasmic reticulum; G, Golgi apparatus; sv, secretory vesicle; mt, microtubule; PM, plasmalemma; pe, periplasm; W, primary wall. 1, t, Longitudinal and transverse stresses.

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FIG.18. Membrane flow and plant cell growth regulation. Two apparently opposite cell levels are engaged in expansion. Internally the vacuolar system gives rise to the driving force, and externally the plastic primary wall is built up. In fact, both levels could be integrated into the same process of endoplasmic reticulum-Golgi apparatus membrane flow functioning in a dichotomic manner. ER, Endoplasmic reticulum; G, Golgi apparatus; sv, secretory vesicle; mt, mitochondria.

to plasmalemma enlargement and produces the subunits of the wall, and (2) the endocytic route which contributes to tonoplast enlargement and turgor pressure regulation. In this dichotomic flow, the endoplasmic reticulum and the Golgi body play a key role. Thus the main elements of growth regulation and wall morphogenesis, though acting at different levels in the cell, should result in a homogeneous sequential process (Prat et al., 1977). Knowledge about the organization and expansion of the plant cell wall has been renewed in recent years. In the future, closer cooperation among cytologists, biochemists, and physiologists seems highly desirable for a better understanding of the characteristic modalities of plant growth.

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Snvastava, L. M., and Sawhney, V. K. (1975). Int. Bot. Congr., I2th, p. 318. Srivastava, L. M., Sawhney, V. K., and Bonettemaker, M. (1977). Can. J . Bor. 55, 902-917. Staehelin, L.A., (1968). Proc. R. SOC. London 171, 249-259. Sterling, C., and Spit, B. J. (1957). Am. J . Bot. 44, 851-859. Steward, F. C., and Muhlethaler, K. (1953). Ann. Bot. 18, 301-310. Tepfer, M. (1977). Ph.D. Dissertation, Univ. of Washington, Seattle. Thiery, J. P. (1967). J . Microsc. 6, 987-1018. Vandenvoude, W. J. (1977). Plant Physiol. 59, 26. Veen, B. W. (1970). Proc. K . Neerl. Akad. Wet. 73, 113-117. Veen, B. W. (1971). Thesis, Groningen, Holland. Vian, B. (1978). Protoplasma 97, 379-385. Vian, B., Mosiniak, M., and Roland, J. C. (1976). Ann. Sci. Nat. Bot. 17, 105-118. Vian, B., Mueller, S., and Brown, R. M. (1978). Cytobios 22, 7-15. Waaland, S. D., and Waaland, J. R. (1975). Planta 126, 127-138. Waaland, S. D., Waaland, J. R., and Cleland, R. (1972). J . Cell Biol. 54, 184-190. Wardrop, A. B. (1956). Aust. J . Bot. 4, 193-198. Wardrop, A. B. (1962). Bot. Rev. 28, 241-285. Wardrop, A. B., and Cronshaw, J., (1958). Aust. J . Bot. 6, 89-95. Westafer, J. M., and Brown, R. M. (1976). Cytobios 15, 11-138. Willison, J. H. M. (1975). Planta 126, 93-96. Willison, J. H. M. (1976). Protoplasma 88, 187-200. Willison, J.H.M., and Brown, R. M. (1977). Protoplusma 92, 21-41. Willison, J. H. M., and Cocking, E. C. (1975). Protoplasma 84, 147-159. Willison, J. H. M., and Grout, B. W. U. (1978). Planta 140, 53-58. Wilson, K. (1957). Ann. Bot. 21, 1-11. Wilson, K.(1964). Int. Rev. Cyfol. 17, 1-49. Wood, A. D., and Linn, T. G. (1946). "Plywoods." Johnston, Edinburgh. Wooding, F. B. P. (1968). J. Cell Sci. 3, 71-80.

INTERNATIONAL REVIEW OF CYTOLOGY. VOL 61

Biochemistry and Metabolism of Basement Membranes NICHOLAS A. KEFALIDES, ROBERTALPER,AND CHARLES c . CLARK Departments of Medicine, and Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsy 1vania I. Introduction . . . . . . . . . . . . . . . . . . . . 11. Morphology and Distribution . . . . . . . . . . . . . . In. Functions of Basement Membranes . . . . . . . . . . . A. Role of Basement Membranes as Semipermeable Filters . . B . Role of Basement Membranes as Supporting andtor Boundary Structures . . . . . . . . . . . . . . . . . . . IV. Structural Chemistry of Basement Membranes . . . . . . . A. Isolation of Basement Membranes . . . . . . . . . . B. Morphological Properties of Isolated Basement Membranes . C. Solubility Properties . . . . . . . . . . . . . . . D. Amino Acid and Carbohydrate Composition . . . . . . . E. The Collagen Component . . . . . . . . . . . . . . F. The Noncollagen Components . . . . . . . . . . . . G. The Supramolecular Organization of Basement Membranes . H. The Subunit Composition of Basement Membranes . . . . V. Basement Membrane Metabolism . . . . . . . . . . . . A. Basement Membrane Biosynthesis . . . . . . . . . . B. Basement Membrane Turnover . . . . . . . . . . . . VI . Immunochemistry of Basement Membranes . . . . . . . . vm . Basement Membrane Changes in Disease . . . . . . . . . WI. Supramolecular Organization of Basement Membranes . . . . IX . Summary , . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . .

167 170 177 177 178 179 180 182 182 183 186 191 193 196 197 197 208 209 212 218 220 22 1 228

I. Introduction New knowledge concerning the nature of basement membranes has been accumulating through a series of biological and biochemical approaches which include morphological, physicochemical, pathological, immunological, functional, and biosynthetic studies. The literature up to 1973 on the chemistry, metabolism, and immunological behavior of basement membranes has been thoroughly reviewed by Kefalides (1971b, 1973). Since then, however, this literature has burgeoned, especially in the areas of basement membrane chemical structure and biosynthesis. This has prompted us to attempt to bring these latest observations into a unified, comprehensive, critical review. 167

Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form mservcd. ISBN 0-12-364461-5

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In this article, we propose to discuss briefly morphological features of basement membranes as they are seen by electron microscopy of the intact tissue as well as of preparations isolated by sonication and/or other means. We also discuss some functional aspects of basement membranes with particular emphasis on their permeability to large molecules and their role in maintaining tissue architecture in embryonic as well as mature tissues. The physicochemical, immunochemical, and biosynthetic properties of basement membranes are updated, and an attempt is made to construct from these data a basement membrane model. Finally, a review of the changes in basement membranes in human and experimentally induced disease is included. As indicated above, the more recent literature is emphasized in these areas of active research. Before proceeding, however, we feel that it would be appropriate to define our use of the term “basement membrane.” In 1857, Robert Todd and William Bowman, in a discussion of the histological appearance of the synovial and serous membranes, stated that their epithelium rested “immediately on a continuous basement membrane of excessive tenuity, apparently identical with that which supports the epithelium of mucous membranes. ” The same ahhors also stated that the digestive and respiratory epithelium rested “on a layer of membrane, hence basement membrane” and described it as distinctly homogeneous and transparent, and in some situations as finely fibrous. In discussing the development of teeth they further mentioned the presence of a homogeneous basement membrane upon which the columnar epithelium of the tooth papilla rested. Finally, in describing capillaries they reported that “in most mucous membranes, the basement membrane is placed between the capillaries and the epithelium. What Todd and Bowman observed at the light microscope level was an amorphous extracellular matrix contiguous with epithelial cells. With the advent of more advanced techniques, however, it became apparent that what in many cases was called a basement membrane in the light microscope actually was composed of a reticular layer as well as a basement membrane proper. There is now immunological evidence suggesting that the reticular fibers may represent a genetically distinct collagen type (Gay et a f . , 1975; Nowack et a f . , 1976) which differs from that found in basement membrane. In the electron microscope the basement membrane proper and the reticular fiber layer can be resolved and are now regarded as separate structures. Figure 1 shows a schematic diagram which depicts what we refer to as basement membrane in this article. There are several arguments in favor of the term “basement membrane” rather than either “basal lamina” or “basal membrane,” which have been suggested by others. It has been argued by some that the adjective “basement” is nondescriptive where the matrix coats the entire external surface of a cell, as is the case with muscle fibers and Schwann cells; and that the term “membrane” is confused with the lipoprotein limiting envelope of cells and intracellular organelles (Coggeshall and Fawcett, 1964; Pineda, 1965). Frankly, the adjective “basal” ”

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FIG. I . Diagram showing the relationship of an epithelial cell layer and its basement membrane to neighboring structures in the underlying connective tissue. The four layers depict: ( I ) the cell plasma membrane; the space between two cells is occupied by the ill-defined group of substances known as the cell coat; (2) the lamina lucida (LL), at times referred to as the lamina rara, most commonly seen at the epidermal-dermal junction, below the comeal epithelium and in the glomerular capillaries; the nature of the substances occupying the lamina lucida is not known, although it is speculated that the proteins which constitute the basement membrane proper also form this layer but are not cross-linked to the same extent; (3) basement membrane (BM), at times referred to as basal lamina; in the glomerulus this is referred to as the lamina densa to distinguish it from the lamina rara intema and the lamina rara extema lying on either side of it; (4) the subbasement membrane fibrous elements, at times referred to as the reticular layer; these include the anchoring fibrils (AF) and the microfibrils (MF‘) which are seen in the subepithelial layer of skin and of the mucous membrane of the mouth, vagina, and cervix; the chemical composition of these fibrils is unknown; collagen fibers (CF) with a typical periodic structure are seen in the region just below the basement membrane and are found in skin and in the loose connective tissue of mucous membranes, glandular epithelium, renal tubular epithelium, capillary-ependymal layer junction of the choroid plexus, and subendothelial layer of arteries and arterioles. It should be noted that the structures depicted here could only be seen as such with the electron microscope. Even immunofluorescent techniques are inadequate in distinguishing among the various structures.

does little to alleviate this problem; and, moreover, it seems too restrictive to argue that the term “membrane” be limited to denoting only the plasma membrane, and so on. Membranes, whether biological or synthetic, were known and the term used long before the cell plasma membrane assumed its prominence in the biological literature. Basement membranes are membranes in both the physical and physiological sense and belong, morphologically, chemically, and immunologically, to a class of substances which are clearly distinguishable from cell plasma membranes. For these reasons, and because of its prevalence in the literature, we prefer and continue to use the term “basement membrane” to refer to the dense, amorphous matrix observed in the electron microscope as lying between the lamina lucida

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and the fibrillar layer (Fig. 1) just below the basal layer of the epidermis. The term “basement membrane” is used to refer to the electron-dense matrix of the glomerular capillary and of the renal tubule and to the thick anterior and posterior capsule of the ocular lens, as well as to Descemet’s membrane of the corneal endothelium. The same term is applied to the amorphous matrix that lies below the respiratory and digestive epithelium, and the one that lies between the alveolar respiratory epithelium and the alveolar capillary endothelium, as well as to the electron-dense matrix that surrounds capillary endothelium in all tissues.

11. Morphology and Distribution

The morphological descriptions of basement membranes provided above by Todd and Bowman (Section I) were based on light microscope observations. Since the introduction of the electron microscope, basement membranes have been further described as continuous, homogeneous sheets of electron-dense ,Skin

Fat cells

- ipportlng Tissues

Cordiovasculor

FIG.2. Diagram of anatomic distribution of the basal lamina (depicted as a heavy line) in its typical location between parenchymal cells and the space occupied by connective and supportive tissues. Parenchymal cells include all epithelial cells of the epidermis and epidermal appendages, of the genitourinary, respiratory, and gastrointestinal tracts, and of all exocrine glands, as well as endothelial cells of the cardiovascular system, mesothelial cells of body cavities, cells comprising the central and peripheral nervous systems, endocrine cells, muscle fibers, and fat cells. The space occupied by connective and supportive tissue (shaded area) contains bone and associated cells, cartilage and associated cells, collagen, elastin, and fibroblasts. (Reprinted with permission from Vracko, 1974b.)

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material, which are distinct from the reticular layer (Fig. 1). Ultrastructurally, basement membranes appear to be composed of fine fibrils about 40 8, in diameter arranged randomly in a granular matrix (Farquhar, 1960; Vernier, 1964; Jakus, 1964). Some degree of fibrillar orientation is observed in the lens capsule (Jakus, 1964) and in Reichert’s membrane (Wislocki and Padykula, 1953; Jollie, 1968; Clark et al., 1975a), for instance, while a characteristic hexagonal pattern of nodes and filaments appears to be unique to the distal portion of Descemet’s membrane (Jakus, 1964). More recently, a fine meshwork structure has been demonstrated in negatively stained bovine and human glomerular basement membranes (Ota et al., 1977). The correlation of these morphological observations with the structure and function of basement membranes must await further investigation.

FIG. 3. Peripheral area of a glomerular capillary from a normal rat. The capillary wall is composed of three distinct layers: the endothelium (En) with its periodic interruptions or fenestrae (0;the basement membrane (B) which is a continuous layer, 100-150 pm in thickness; and the foot processes (fp) of the epithelium (Ep). In a number of places (arrows) a thin line or “slit membrane” can be seen bridging the narrow (250-A)gap between the foot processes. The epithelial filtration slits are defined as that portion of the space between foot processes extending from the level of the basement membrane to the level of the slit membrane. Cap, Capillary lumen; US, urinary spaces; RBC, red blood cell. Electron micrograph. X26,OOO. (Reprinted with permission from Farquhar, 1975.)

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As suggested by Todd and Bowman's initial discussion, basement membranes are ubiquitious extracellular matrices. The thickness of basement membranes varies from approximately 200 to 50,000 A. In mature tissues, basement membranes are found at the base of the epithelium lining the urinary, reproductive, respiratory, and digestive tracts; at &hebase of the endothelium lining the vascular tree; at the base of the luminal cells of endocrine and exocrine glands; and surrounding adipocytes, SChWaM cells, and skeletal and smooth muscle cells (Fig. 2). In most of these instances, basement membranes are interposed between an epithelial cell layer and the adjacent connective tissue matrix (Fig. 1). In addition, there are some instances-such as in the renal glomerulus, the pulmonary alveolus, and the rodent parietal yolk sac (PYS)-where portions of basement membranes are covered by cell layers on both surfaces. Specifically, the glomerular (Fig. 3) and alveolar basement membranes are covered by endothelial cells on one surface and epithelial cells on the other, while Reichert's membrane is covered by endodermal cells on one surface and trophoblasts on the other (Fig. 4). In embryonic tissue where electron-dense material is found between ectodermal and mesenchymal cell layers and where clear-cut delineation from the connective tissue elements is absent, an overlap between what appears to be basement membrane, collagen fibrils, reticulin, elastic fibers, and the connective tissue matrix takes place. There are also two well-documented instances in the mature animal where what is identified by light microscopy as basement membrane contains more than one type of structure when viewed by electron microscopy. One of these is the epidermal-dermaljunction (Fig. 5). Here, just below the epidermis, there is an electron-lucent zone, and below it an electron-dense layer which by any ultrastructural criteria is similar to the basement membrane described in other tissues. Adjacent and below this layer one finds filaments and fibrils of varying length and diameter. Some of these fibrous structures have been loosely identified as anchoring fibrils, dermal microfibril bundles, and collagen fibrils (Briggaman and Wheeler, 1975; Heaphy and Winkelmann, 1977). Since the histochemical stains used in light microscopy are not specific for proteins or large polysaccharides but rather for certain reactive groups on such molecules, the area stained should be referred to as a basement membrane zone rather than as basement membrane per se . The other instance is the corneal epithelium (Fig. 6). Here again one sees an electron-lucent zone just below the base of the cell, but not as clearly delineated as at the epidermal-dermal junction. Below this zone lies an electron-dense layer of granular, finely fibrillar material which has been identified as basement membrane. Adjacent to it there is a layer of striated fibrils which resemble collagen fibers. In addition to these cases, striated fibrils have been reported in the loose connective tissue juxtaposing such basement membranes as those of the renal glomer-

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FIG.4. Rat PYS. The basement membrane (Reichert’s membrane) lies between the endodermal cells and the giant trophoblast cells. Note the absence of a lamina lucida on either side of the basement membrane, as well as the absence of subbasement membrane fibrous elements. Light micrograph. X490. (Reprinted with permission from Clark et al., 1975a.)

ulus (Latta, 1961) and tubule (Mahieu and Winand, 1970), the PYS (Clark et al., 1975a), and the embryonic rat heart (Johnson et al., 1974). It should be noted, however, that when basement membranes are isolated from normal tissues and their cellular and reticular layers removed either by sonication (Krakower and Greenspon, 1951) or by strong detergents (Meezan et al., 1975), no striated

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FIG.5 . Epidermal-dermal junction. Basal lamina (BL) (basement membrane) and subbasal lamina fibrous components. Anchoring fibril (arrow) showing midzone assymetrical cross-banding. AF, Interlocking meshwork of anchoring fibrils; DMB, dermal microfibril bundle; C, collagen fibrils. Electron microphotograph. ~66,450.Marker, 0.5 pm. (Courtesy of Dr. R. A. Briggaman, University of North Carolina.)

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FIG. 6 . Corneal epithelial-stromal junction of the developing chick embryo eye. EC, Epithelial cell; LL, lamina lucida; BM, basement membrane; CF, collagen fibrils. Electron micrograph. ~ 9 0 , 0 0 0 .Marker, 0.2 p n , (Courtesy of Dr. R. Trelstad, Harvard University.)

J

COLLAGENASE

r

4

REDUCTION

FIG.7. The effects of reduction and alkylation and of collagenase digestion of a fraction obtained after hydroxylamine digestion (fraction A) of bovine ALC. Reduction and alkylation of either the intact fraction A or of its collagenase digest results in the loss of the collagen peptide, leaving a large polymer of noncollagen peptides. (Reprinted with permission from Alper and Kefalides, 1978b.)

TABLE I WHICHHAVEBEENISOLATED A N D ANALYZED REPRESENTATIVE BASEMENT MEMBRANES Organ

Tissue

Kidney

Glomerulus

Kidney

Tubule

Eye Eye Brain Brain

Lens Cornea Retina Cerebral cortex Choroid plexus

Lung

AIveolus

Testis Embryo

Seminiferous tubule PY s Intestine

Eye

Ascaris

Cell(s) Epithelium and endothelium Epithelium Epithelium Endothelium ? ?

Epithelium and endothelium Epithelium and endothelium Epithelium Endoderm Epithelium

Basement membrane

Reference

Glomerular

See Reviews: Kefalides (1971b, 1973)

Tubular Lens capsule Descemet's membrane Retinal vessels Vessels Choroid plexus

Ferwerda et al. (1974a,b); Meezan et al. (1975) See Reviews: Kefalides (1971b, 1973) Kefalides and Denduchis (1969) Meezan ef 01. (1975) Meezan et al. (1975) Kefalides and Denduchis (1969)

Alveolar

Kefalides and Denduchis (1%9)

Seminiferous tubule Reichert's membrane Intestinal

Denduchis er al. (1975) Clark et al. (1975a) Peczon et al. (1975, 1977)

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fibrils have been shown within the basement membrane proper. Thus unless morphological analyses have been performed on tissues to be used for studies of basement membrane, care must be exercised in interpreting chemical (Section IV) and biosynthetic (Section V) data. Table I lists representative basement membranes from a variety of tissues which have been isolated in a relatively pure state.

III. Functions of Basement Membranes A number of functions have been ascribed to basement membranes. These can be assembled into two broad, but not necessarily mutually exclusive, categories: (1) to act as a semipermeable filter and (2) to provide support and/or to serve as a boundary between different cell types (Figs. 3 and 4)or between cell layers and the underlying interstitial connective tissue (Figs. 5 and 6).

A. ROLEOF BASEMENT MEMBRANES AS SEMIPERMEABLE FILTERS This role of basement membrane is best exemplified by studies on the capillaries in the renal glomerulus (Latta, 1973; Farquhar, 1975; Ryan and Karnovsky, 1976) and to a lesser degree on the capillaries in other tissues (Majno and Palade, 1961; Karnovsky, 1968; Chinard et al., 1962). The transport of substances across the glomerular capillary wall depends on the collective contribution of three components-the endothelium, the basement membrane, and the epithelium. Based on studies with the tracer ferritin, Farquhar et al. (1961) have proposed that the basement membrane acts as the main filter, the endothelium as a valve which by the number and size of its fenestrae controls access to the filter, and the epithelium as a monitor which partially recovers proteins that leak through the filter. These conclusions were confirmed by Caulfield and Farquhar (1974) using dextrans as tracer particles. Another view proposed by Karnovsky and his associates is that the interpodocyte slit region of the epithelial layer also plays a role in barrier function (Graham and Karnovsky, 1966; Venkatachalam et al., 1970; Karnovsky and Ainsworth, 1973). Graham and Karnovsky (1966) and Karnovsky and Ainsworth (1973) have suggested that the basement membrane is a coarse filter for large molecules, while the interpodocyte slits restrict the passage of smaller proteins. At present, little is known about the factors which regulate the passage of molecules across the capillary wall. Ryan and Karnovsky (1976), using the immunoperoxidasetechnique, studied the distribution of endogenous albumin in the rat glomerulus under normal and abnormal hemodynamic conditions. During normal blood flow, reaction product specific for albumin was largely confined to the capillary lumen and endothelial fenestrae, with only small amounts in the

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lamina rara interna of the basement membrane. Latta and Johnston (1976) attributed these properties to the glycoprotein inner layer of the glomerular capillary basement membrane (GBM). When blood flow was interfered with by ligating the renal artery, reaction product was detected throughout the GBM and in the urinary space (Ryan and Karnovsky, 1976). Thus glomerular barrier function depends upon the maintenance of normal blood flow conditions. Studies by Rennke et al. (1975), using cationized ferritins as tracer particles, suggest the intrinsic negative charges are present on the endothelium, throughout the GBM, and on the epithelium, and that the barrier function of the glomerular capillary wall may be ascribed in part to its electrophysical properties. More recently, Caulfield and Farquhar (1976), using lysozyme (MW 14,000; PI = 1) to perfuse the kidney, concluded that anionic sites were present on the epithelial surface and throughout the basement membrane. Studies with muscle capillaries also indicate that the basement membrane functions as a filtration barrier (Majno and Palade, 1961). The permeability properties of the alveolar capillary wall were examined for water-soluble molecules, and it was shown that low-molecular-weight proteins (up to MW 40,000) traversed the endothelium and basement membrane and made their way into the epithelial cells, but did not enter the alveolar space (Chinard etal., 1962; Schneeberger-Keeley and Karnovsky , 1968). Finally, a speculative role for the basement membrane as a fdter to monitor the composition of the intracellular environment in differentiating cells has been proposed by Hay (1978). B. ROLEOF BASEMENT MEMBRANES AS SUPPORTING AND/OR BOUNDARY STRUCTURES The most obvious examples of basement membranes as supporting structures are the lens capsules of the eye and Bowman’s capsule of the renal glomerulus. In each case the basement membrane can be likened to a sack which holds and/or protects a more complex structure. There are also reports that basement membranes might contribute significantly to the structural rigidity of blood capillaries (Murphy and Johnson, 1975) and to the distensibility of renal tubules (Welling and Welling, 1978) in addition to their filtrative roles. Another major supportive role of basement membranes is that of an “extracellular scaffold positioned between parenchymal cells and connective tissue ” in a number of structures including skeletal muscle, lung, kidney, pancreas, nervous system, skin, and liver (see review by Vracko, 1974b). It is this role that Todd and Bowman originally observed (Section 1)-that of cells resting upon a basement membrane which separated them from the interstitial connective tissue (Fig. 1). Basement membranes also function as a required framework along which cells traverse during either normal tissue remodeling or injured tissue regeneration (Vracko and Benditt, 1972; Vracko, 1974a,b; Thorning and Vracko, 1977).

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This latter function also appears to be important in developing embryonic systems. It has been shown that basement membranes probably represent the earliest appearance of a connective tissue matrix (Low, 1967; Pierce, 1966, 1970; Trelstad et al., 1967), and diverse roles of basement membrane in development and differentiation have been suggested (Trelstad et al., 1967; Hay and Revel, 1969; Hay, 1968, 1973, 1978; Banerjee et al., 1977). These studies indicate that basement membranes may serve as substrata for various embryonic mesenchymal cells that migrate under and around the epithelial germ layers and epithelial organs, that they may be the medium through which epithelial cells influence the polymerization of underlying collagen fibrils, and that their component molecules may interact with the cell surface and control epithelial synthetic processes and lobular morphology. Finally, there is recent evidence to suggest that basement membranes play a role in the inductive effects of heterologous tissues (Kawakami et al., 1976; Tanaka et al., 1976). The critical role that basement membranes appear to play in many biological processes, including development and differentiation, the maintenance of tissue structure, function, and repair, makes these extracellular matrices worthy of the concern and continued curiosity which have led to a constantly increasing number of research studies in several laboratories.

IV. Structural Chemistry of Basement Membranes In attempting any discussion on the biochemical structure of a tissue component, criteria first must be established for the appropriate identification of that component. In the case of basement membranes, this should require isolation of the basement membrane being studied, its identification using morphological criteria and, finally, a definition of what will be considered a basement membrane component and what will not. This creates certain problems, some of which are simple to resolve and others of which are more difficult. For example, one of the functions of a basement membrane is that of a filter. Should the molecules trapped in transit at the time the basement membrane is isolated be considered basement membrane components? In this particular instance, we have taken the position that such molecules are not basement membrane components and that molecules adsorbed onto the surface of basement membrane (e.g., antibodies) should not be considered basement membrane components. A more difficult situation arises when, during the isolation of the basement membrane, substances such as lipids or glycoproteins are lost. Here it cannot be stated so definitively that these are not basement membrane components, for their presence or absence may depend solely upon the method of isolation. For the major part of this discussion we have chosen to consider as basement membrane components only those proteins which constitute the major components of a given preparation and which are not easily removed by mild, nondeg-

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radative procedures. There are a few exceptions to this and they are discussed separately. However, to better understand the problems associated with the chemical definition of a basement membrane, we include a short section on the methods of isolating basement membranes.

OF BASEMENT MEMBRANES A. ISOLATION

1. Glomerular Basement Membrane a. Isolation of Glomeruli. In 1951, Krakower and Greenspon described a procedure for the isolation of GBM which, with certain modifications, has been the method used most often in studies on the chemical composition of GBM. In this procedure, the renal cortex is forced through a sieve of defined mesh (depending upon the size of the glomeruli of the species used). This cleaves the pedicle and ruptures the capsule. The glomeruli are then separated by various means from other tissue elements. Spiro (1967a) modified this procedure by filtering the original mash through several steel meshes of different sizes, thus separating the glomeruli from larger and smaller tissue components including tubules and other tissue fragments. More recently, Daniels and Chu (1975) modified Spiro’s procedure by first grinding the cortex in a meat grinder and then homogenizing the mash using a mechanical homogenizer at a low speed prior to passage through the sieves. This modification apparently permits the processing of very large amounts of tissue in a short period of time. Freytag et al. (1976) used a mixture of protease inhibitors in their modification of the sieving procedure to minimize enzymic degradation during the processing of the tissues. Other methods for separating glomeruli from other tissue elements involve the prior perfusion of kidneys with a suspension of magnetic iron oxide (Misra and Berman, 1968; Meezan et al., 1975, 1978; Carlson et al., 1978a), followed by sieving. The glomeruli are then separated from the tubules and other tissue components by magnetic attraction. Under any of these conditions, it is important to take precautions against contamination with tissue components other than glomeruli. The best preparations are those in which this contamination is below 1%. b. Isolation of Glomerular Basement Membrane. After a good glomerular preparation has been obtained, the GBM can be isolated by one of several methods. Most workers have used sonic disruption (Krakower and Greenspon, 1951; Kefalides and Winzler, 1966; Spiro, 1967a; Westberg and Michael, 1970; Freytag et al., 1976) followed by isolation of the GBM by low-speed centrifugation and exhaustive washings. Meezan et al. (1975, 1978) and Carlson et al. (1978a,b) object to the use of sonication in the isolation of GBM. They feel that this procedure may alter the normal spatial arrangement of the component molecules of the GBM and that it may not completely remove contaminants such

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as cell membrane fragments and plasma proteins. Instead they have isolated GBM from glomerular lysates using digestion with nucleases followed by extraction with deoxycholate to remove any remaining cellular contaminants. They claim that their preparations maintain the ultrastructural integrity as seen in the electron microscope, while still retaining the chemical properties of preparations isolated after sonication. Furthermore, they suggest that this procedure is applicable to the isolation of basement membranes from other tissues such as retina and choroid plexus. A third procedure was used by von Bruchhausen and Merker ( 1965), Lidsky et al. (1967), and Kibel et al. (1976). These investigators isolated GBM from glomeruli by grinding in a Potter-Elvenhjem homogenizer in 0.25 M sucrose followed by isolation of the GBM by centrifugation in a discontinuous sucrose density gradient. The GBM was then treated with DNase and collected by centrifugation. These preparations were found to contain a significant amount of lipid material which has been claimed by a number of authors (Fung and Kalant, 1972; Kibel et al., 1976) to be a component of GBM. It is claimed that the use of sonication (and presumably detergents as well) results in a loss of the lipid material. 2. Renal Tubular Basement Membrane Proximal tubular basement membranes (TBMs) have been isolated from human (Mahieu and Winand, 1970), bovine (Ferwerda et a l . , 1974b), and rat (Krisko et a l . , 1977) kidneys using sieving and sonication procedures similar to those used for the isolation of GBM. It should be recognized that, in the isolation of GBM and TBM from different species, care should be taken to utilize sieves of appropriate sizes to accommodate the different dimensions of the structures undergoing isolation.

3 . Anterior Lens Capsule The anterior lens capsule (ALC) of the eye can be isolated in very large amounts in a very short period of time. The preparations are probably less contaminated with extraneous material than any other basement membrane preparations. Denduchis et al. (1970) and Fukushi and Spiro (1969) prepared bovine and ovine ALC by dissecting the ALC from the lens, carefully trimming away zonular fibers, and mildly sonicating to remove cellular components. Each bovine lens capsule can provide 1-2 mg of a highly purified product. 4. Parietal Yolk Sac Basement Membrane (Reichert’s Membrane) The PYS of the fetal rodent contains a thick basement membrane, Reichert’s membrane. Reichert ’s membrane has been isolated by Clark et al. (1975a) from rat fetuses and by Pierce et al. (1962) from mouse fetuses. In their procedure, Clark et al. (1975a) isolated PYSs from fetal rats by dissection under a micro-

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scope. The PYS consisted of Reichert’s membrane surrounded on one side by a layer of endodermal cells and on the other by a layer of trophoblastic giant cells. Reichert’s membrane was isolated by incubating the PYS in EDTA for a short period of time, followed by agitation on a vortex mixer. After three to four such treatments the Reichert’s membrane preparation was sonicated, and Reichert ’s membrane was isolated by centrifugation and washed. 5. Other The isolation and partial characterization of choroid plexus basement membrane was first reported by Kefalides and Denduchis (1969). Subsequently, Meezan et al. (1978) isolated retinal and brain vessel basement membranes using the detergent isolation procedure previously indicated for GBM. Descemet ’s membrane has been isolated by dissection by Dohlman and Balms (1955) and Kefalides and Denduchis (1969). Peczon et al. (1975) have isolated the intestinal basement membrane of the helminth Ascaris suum from a sonicate of the intestine. OF ISOLATED BASEMENT MEMBRANES B. MORPHOLOGICAL PROPERTIES

The major property of virtually every basement membrane preparation as seen under the electron microscope is that of an amorphous matrix, with the exception of Descemet’s membrane (Jakus, 1964). Fibrils are often observed within the matrix, but these apparently lack the characteristic striation patterns of collagen fibrils. When striated fibrils are observed, they are usually attributed to contamination of the preparation with interstitial collagen. Preparations of GBM isolated by sonication invariably lack the lamina densa and lamina rara seen in sections of tissue. When GBM is prepared using the detergent extraction procedures of Meezan ef al. (1975), the lamina densa and lamina rara can be observed. C. SOLUBILITY PROPERTIES

The solubility properties of basement membranes were established in the earlier literature and have been included in a number of comprehensive reviews (Kefalides, 1969a, 1971b, 1973; Spiro, 1972, 1973), and they need not be reviewed in depth here. Briefly, basement membranes are highly insoluble in buffers at neutral pH. They can be solubilized with alkali, but this is undoubtedly the result of degradation. A small amount of material can be brought into solution with acidic buffers. Treatment with chaotropes or strong denaturants can solubilize a small percentage of basement membrane material, but it is still not clear as to whether the extracted materials are components of the basement membrane or contaminants trapped in or adsorbed onto it. Reduction of disulfide

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bonds is only partially successful in bringing basement membranes into solution unless the reduction is performed in the presence of strong denaturants such as guanidine-hydrochloride urea, or sodium dodecyl sulfate (SDS). This has been the most successful approach to solubilizing basement membranes and, depending upon the source, brings from 60 to 100% of the material into solution. However, upon removal of the denaturant, the solution often becomes very unstable and reprecipitation is encountered. On the basis of these properties, it appears that the components of basement membranes are held together by hydrogen-bonding forces, disulfide cross-linkages, and perhaps other types of covalent cross-linkages.

D. AMINOACIDAND CARBOHYDRATE COMPOSITION Although the amino acid compositions of various basement membranes have been extensively reviewed, it is pertinent to this discussion to present a table (Table 11) showing the amino acid compositions of a number of basement membrane preparations. Newer procedures have facilitated the isolation of basement membranes from tissues previously considered too complex for the isolation of uncontaminated preparations. Examination of these amino acid compositions reveals a number of salient features common to all basement membranes. First is the presence in large amounts in every preparation (including that from a nonvertebrate species) of 4-hydroxyproline and hydroxylysine, indicative of the presence of collagenous components within the basement membrane. Second is the presence of considerable amounts of 3-hydroxyproline, resulting in an unusually high 3-hydroxyproline/4-hydroxyprolineratio. This appears to be a major characteristic of basement membranes and, based upon this observation, Man and Adams (1975) have proposed a method for estimation of the proportion of basement membrane in the collagenous components of tissues. A third feature is the presence of large amounts of half-cystine. Examination of the carbohydrate composition of various basement membranes reveals the presence of large amounts of sugars indicative of the glycoprotein nature of basement membranes. Present in particularly large amounts are glucose and galactose. All the glucose and most of the galactose have been shown to be covalently associated with hydroxylysine residues and thus are associated with the collagenous portion of the membrane (Kefalides and Winzler, 1966; Kefalides, 1969b; Spiro, 1967~).Subsequent studies have shown that about 80% of the hydroxylysine residues are glycosylated and that about 90% of the substituted hydroxylysine exists as glucosyl-a( 1+2)-galactosyl-~-O+hydroxylysine. This appears to be the single most characteristic chemical property of basement membrane collagen, since the hydroxylysine residues of other collagens are substituted to a far lesser degree (Butler and Cunningham, 1966; Kefalides, 1973).

TABLE II AMINOACIDAND CARBOHYDRATE COMFOSITIONS OF VARIOUS BASEMENT MEMBRANES~ ~

Component

Human GMBb

Canine ALP

Ovine Descemet’s membraned

Hydroxylysine Lysine Histidine Arginine 3-Hydroxyproline 4-Hydroxyproline Aspartic Threonine Serine Glutamic Roline Glycine Alanine Half-cystine Valine

24.5 26.0 18.7 48.3 7.0 66.0 65.0 40.0 60.0 103.0 62.0 227.0 58.0 23.0 36.0

32.0 11.0 12.0 38.3 18.0 80.0 57.8 31.0 48.0 95.0 66.0 288.0 46.1 18.0 35.0

20.0 24.0 11.6 39.5 6.8 77.0 58.0 36.4 42.1 94.0 95.0 230.0 53.0 11.0 45.0

Bovine renal TBM‘

28 18 16 38 20 83 60 40 57 96 55

218 54 11 39

Rat Reichext ’s membrane day 14.5’

Bovine retinal basement membrane’

18.4 33.0 19.9 45.0 6.0 46.2 84.0 48.0 67.0 107.0 61.5 179.0 62.0 18.8 44

27.4 18.3 9.0 55.6 n.d. 98.5 58.9 31.9 48.0

87.0 84.3 267.6 69.9 14.6 24.7

Ascaris s u m

Choroid plexusd

9.0 41 .O 16.0 61.0 Trace 48.3 68.0 39.0 52.8 94.0 69.0 235.6 104.5 10.0 37.0

intestinal basement membrane*

Mouse sarcomai

11.0 21.8 21.8 66.9

38

23.7 81.5 46.9 50.2 123.9 86.7 138.8 60.9 37.6 52.7

99 52 34 60 103 66 280 45 7 30

-

11 11

30 5

Methionhe Isolewine Leucine Tymsine Phenylalanine Glucose Galactose

Mmse Fucose Glucosamine Galactosamine Sialic acid

7.0 28.0 66.0 14.5 28.0 2.5 2.6 1.7 0.7 1.7 0.3 1.5

6.2 30.0 56.5

11.0 29.0 5.0 5.1 0.4

0.6 0.85 0.15 0.4

8.2 28.5 75.2 21 .o 25 .O 3.5 3.7 2.0 0.6 1.2 0.3 0.7

12 30 58 19 28 3.6 3.8 1.1 0.2 0.9 Trace 0.2

13.3 30 70 18.4 31 2.2 3.0 1.8 0.4

4.6 0.4 1.7

8.6 21.5 43.8 5.1

25.7 21.5’ 20.9 3.9 2.2’ 5.6*

-

9.4 22.5 50.0 12.0 21.5 1.4 1.9 0.8 0.2 1.1 0.5 1.5

10.9 35.0 61.2 25.8 20.1 1.5 1.8 0.4 0.4 0.5 0.3 -

“Amino acids expressed as residues per loo0 residues; carbohydrates expressed as grams of sugar per 100 gm unless otherwise noted. bKefaIides (1970). ‘Kefalides (1969b). dKefalides and Denduchis (1969). ‘Fenverda et al. (1974b). T l & ex al. (1975a). Tadson et al. (1978a). hPecmn et al. (1975). ‘Timpl et al. (1978). ’Expressed as residues per lOD0 residues. ”Total hexosamine as residues per 1000 residues.

12 25 54

9 31 n.d. n.d. n.d. n.d. n.d. n.d. n.d.

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In addition to glucose and galactose, glucosamine and, to a lesser extent, galactosamine, mannose, fucose, and sialic acid are present, indicating that in addition to the characteristic collagen-derivedcarbohydrates typical glycoprotein heteropolysaccharide units are also present. The most notable differences among the various basement membranes appear to reside in the proportion of the membrane taken up by the collagen component. This appears to be lowest in Reichert’s membrane and highest in the lens capsule and retinal basement membranes. Interestingly, the nonvertebrate basement membrane from A . suum apparently contains no 3-hydroxyproline although it, too, is collagenous (Hung et al., 1977). Based upon these gross observations, it appears that both collagenous and noncollagenous proteins are present in basement membranes, and this appears to be true for basement membranes from nonvertebrate animals as well as those from vertebrates. The question next arises as to whether the collagenous protein in basement membranes represents a distinct collagen type such as is found for types I, II, and I11 collagen.

E. THE COLLAGEN COMFONENT When native undenatured collagens are exposed to proteases such as pepsin, it is found that the triple-helical portions of the molecule are resistant to proteolytic attack, whereas the nonhelical portions are digested. This approach has been applied to the isolation and characterization of the basement membrane collagen component (Kefalides, 1968; Kefalides and Denduchis, 1969; Daniels and Chu, 1975; Dehm and Kefalides, 1978a,b,c). Upon treatment with pepsin under acidic conditions in the cold, the basement membranes are brought into solution. The collagen fraction is then isolated by salt precipitation and further purified. Studies of the physical properties of collagen preparations isolated from lens capsule and glomerular basement membrane revealed the high intrinsic viscosities and highly negative specific optical rotations characteristic of collagens (Denduchis et al., 1970; Kefalides, 1968). Circular dichroism spectroscopy revealed a melting temperature (T,) of about 40°C for triple-helical basement membrane collagen (Gelman et al., 1976). The amino acid compositions of basement membrane collagens are shown in Table III which compares the collagen fractions from a number of basement membrane sources. The analyses all show an amino acid composition typical of a collagen; that is, approximately one-third of all the residues are glycine and there is a significant content of 4-hydroxyproline and hydroxylysine. However, there are some distinctive features which are characteristic of basement membranes. First is the extremely high ratio of hydroxylysine to lysine, by far the highest for

187

BASEMENT MEMBRANES

TABLE III COMPOSITION OF COLLAGEN COMFQNENTS ISOLATED FROM AMINOACIDA N D CARBOHYDRATE VARIOUSBASEMENT MEMBRANES'

Hydroxylysine Lysine Histidine Arginine 3-Hydroxyproline 4-H ydroxyproline Aspartic Threonine Serine Glutamic Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Glucose Galactose Mannose Fucose Hexosamines Sialic acid

Human GBMb

Ovine ALC

Descemet 's membraneb

Mouse chondrosarcoma'

44.6 10.0 10.4 33.0 11.0 130.0 51.0 23.0 37.0 84.0 61 .O 310.0 33.0 8.0 29.0 10.0 30.0 54.0 6.0 27.0

57.0 10.0 8.0 27.0 12.0 120.0 50.0 20.0 38.0 92.0 67.0 330.00 32.0 8.0 26.0 10.0 20.0 43.0 2.0 30.0 6.0 6.3 Trace -

43.0 15.2 7.8 30.0 8.0 156.0 30.0 18.0 25.0 78.0 90.0 320.0 32.0 8.0 25.0 9.5 24.0 52.0 3.0 22.0 5.0 5.2 -

46 5 8 29 7 127 50 27.0 53 93 61 319 30 6 28 11 22 45

5.5

6.0 Trace

-

-

5

29 n.d. n.d. n.d. n.d. n.d. n.d.

"Amino acids expressed as residues per lo00 residues; carbohydrates expressed as grams of sugar per 100 gm. n.d., Not determined. bKefalides (1971a). 'Timpl et al. (1978).

any collagen previously isolated. Second is the unusually high content of 3-hydroxyproline and 4-hydroxyproline. The proportion of 3-hydroxyproline to total hydroxyproline (i.e., 3-hydroxyproline plus 4-hydroxyproline) can be as high as 12%. As will be seen in Section V, this has provided a valuable criterion for the demonstration of basement membrane collagen biosynthesis. Additional features include the presence of half-cystine, an unusually low content of alanine as compared with other collagens, and a low content of tyrosine.

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Carbohydrate analyses revealed a content of total neutral sugar of about 10% composed of nearly equimolar amounts of galactose and glucose with traces of mannose and hexosamine. This indicated that most of the sugars were attached to hydroxylysine, and in fact the proportion of glycosylated hydroxylysine observed for intact basement membranes was preserved in the collagen fraction. Upon fractionation of the collagen fraction on CM-cellulose, Kefalides (197la) found a single peak eluting in the region of type I collagen a chains. Gel filtration yielded a significant peak eluting with a molecular weight of about 108,000, suggesting f i s t that the basement membrane collagen was present to a significant extent as a triple helix composed of three identical a chains and, second, that the a chain was considerably larger than that from other collagen types. On the basis of these data it has been suggested that the collagen component of basement membranes is a true collagen, and it is now often referred to as type IV collagen. Daniels and Chu (1975) performed a similar set of studies on pepsin-digested bovine GBM. They utilized a unique homogenization procedure in the preparation of very large amounts of renal glomeruli and GBM, although they did not characterize their GBM preparation morphologically or chemically. After an exhaustive pepsinization of the GBM, followed by gel filtration on agarose gels, they isolated a series of collagen polypeptides ranging in molecular weight from over 300,000 to very small peptides. One of the polypeptide fractions, which accounted for about 10%of the pepsin digest, eluted in a position corresponding to a chains from type I collagen (MW 95,000), and this was chromatographically separated into two components one of which was retained on CM-cellulose and one of which was not. After reduction and alkylation of another of the highermolecular-weight fractions, a polypeptide fraction with a gel filtration elution position corresponding to about 140,000 daltons was obtained, and this too was resolved into two components on CM-cellulose. They hypothesized the presence of two different a-chain types in GBM on the basis of differences in amino acid composition and also suggested that each of the lower-molecularweight fractions were derivatives of a higher-molecular-weight fraction, arising presumably from more extensive proteolysis. Their data also suggested that these different a chains were linked by disulfide bonds. A closer examination of these studies reveals that the basis for inferring the I presence of two distinct collagen chains may not be a valid one. The differences observed in the amino acid compositions were based upon arginine. If the authors had chosen cysteine or aspartic acid for their comparison, they might have drawn an opposite conclusion. Their results may have been due to extensive overpepsinization of the GBM and to the fact that protease inhibitors were not utilized during its preparation and purification. Most workers using pepsinization procedures incubate no longer than 24 hours, whereas in this study the GBM was pepsinized for 8 days using a tremendous excess of pepsin. Thus it is difficult to

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accept the conclusion that two collagen chains exist in GBM. Nonetheless, the possibility that there is more than one collagen chain in basement membranes still remains. Dehm and Kefalides (1978b) modified the pepsin digestion procedure to isolate a collagenous peptide from bovine ALC. They utilized mild pepsinization followed by reduction and alkylation under nondenaturing conditions. The material was pepsinized a second time and then subjected to agarose gel chromatography. This three-step procedure resulted in the recovery of about 70% of the hydroxyproline in a single peak with a molecular weight of 95,000. This fraction was devoid of cysteine and had an amino acid composition typical of basement membrane collagen. When fresh lens capsules containing viable cells were incubated in the presence of labeled proline and then subjected to the three-step procedure, a labeled hydroxyproline-containing fraction was obtained which coeluted with the 95,000-molecular-weightpeptide. This has also been found for GBM (Dehm and Kefalides, 1978a). It was concluded from these studies that basement membranes contained a triple-helical collagen polypeptide composed of a chains, which was synthesized and secreted into the basement membrane matrix. Confirmatory evidence has recently been presented by Schwartz and Veis ( 1978), who prepared segment-long-spacing (SLS) aggregates of lens capsule collagen isolated by the three-step procedure. The 95,000-molecular-weight polypeptide obviously is a product of the proteolysis of a triple-helical collagen whose a chains must have a molecular weight greater than 95,000. The ends of this collagen molecule are composed of cysteine-containing sequences which apparently hold the molecules together through disulfide bridges and render the termini resistant to pepsin. It was only after mild reduction of these disulfide bridges that these terminal sequences were digested by the pepsin. It appears then that there is a collagen type which is characteristic for basement membranes and which is composed of d i k e chains. The question next arises as to whether this collagen contains only a single type of a chain. The 95,000-molecular-weight fraction elutes as a single peak on CM-cellulose chromatography and migrates as a single band on SDS gel electrophoresis (Dehm and Kefalides, 1978b). With the use of these criteria, basement membrane collagen appears to be composed of identical a chains. Final resolution of this question will require the preparation and characterization of cyanogen bromide peptides from the 95,000-molecular-weight fraction. Preliminary studies on cyanogen bromide peptides have been reported (Kefalides, 1972b). Cyanogen bromide peptides were prepared from pepsintreated (single-step) ALC. CM-cellulose chromatography revealed a peptide pattern different from those of the other collagen types. However, this starting material was inadequate for the purpose of comparing basement membrane collagens from different tissues or with different collagen types, and therefore these experiments should be repeated using the 95,000-molecular-weight (three-step)

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material. Once these data are obtained, firm conclusions should follow concerning the number of different collagen chains in basement membranes. Despite the shortcomings of the starting material, the data from cyanogen bromide studies demonstrated that of the 12 peptide fractions isolated, glycine accounted for one-third of amino acids, 4-hydroxyproline was present in all, hydroxylysine in 9, and 3-hydroxyproline in 7. These analyses revealed a relatively even distribution of 3- and 4-hydroxyproline and hydroxylysine. Only one peptide contained cysteine, and this peptide also contained glucosamine and mannose. Although no amino acid sequence data are yet available for basement membrane collagen, Gryder et al. (1975) sequenced a 3-hydroxyproline-containing tripeptide from a collagenase digest of swine renal cortex and detected 3-hydroxyproline in only one sequence, glycine-3-hydroxyproline-4hydroxyproline. Their data also suggested that the 4-hydroxyproline occurred predominantly, if not exclusively, in the Y position of the glycine-X-Y triplet sequence. were isolated from pepsin Recently, two collagen fractions termed d and CUB digests of human skin (Chung et al., 1976; Miller et al., 1977) and placenta (Burgeson et al., 1976). While the amino acid composition of these fractions had some similarity to that of basement membrane collagens, the chromatographic properties differed from those reported for collagen fractions of ALC and GBM. Glanville et al. (1979) isolated type IV (basement membrane) collagen and the d and aB fractions from placenta and demonstrated that they were unrelated, distinct collagens in that a4 and aB did not react with antisera against type IV collagen and have distinct cynogen bromide peptides. They suggested that the aA and aB fractions may be representative of yet another collagen type which they called type V. In addition to the d and CUB chains, Chung et al. (1976) isolated a 55,000molecular-weight fraction from human aortic intima also having an amino acid composition similar to that of a basement membrane collagen, although it lacked 3-hydroxyproline. It has been suggested that this fraction originates in endothelial basement membranes, whereas the CUA and aB chains are derived from * epithelial and smooth muscle basement membranes. However, Schwartz and Veis (1978) demonstrated that pepsin digest of ALC resulted in the formation of small amounts of a 55,000-molecular-weight peptide which appeared to be a cleavage product of the 95,000-molecular-weight peptide. Trelstad and Lawley (1977) found that they could remove interstitial collagens from pepsin digests of kidney, lung, and spleen by thermal gelation of the native collagens. Analysis of the supernate of the gels revealed that basement membrane-like collagen did not form gels and could be isolated in this manner from the supernate. Chromatographic and electrophoretic separation of the basement membrane-like collagen supernate revealed a group of molecules of similar composition but with different molecular sizes.

BASEMENT MEMBRANES

191

From these studies it might appear that basement membranes contain a heterogeneous class of collagens. However, this should be taken with caution. In each case, the collagen fractions were minor components isolated from a pepsin digest of whole tissue. The conclusion that these fractions are of basement membrane origin is based, for the main part, upon their apparent similarity in amino acid composition to collagens isolated from purified basement membranes. Unless it can be shown that the collagen chains can be isolated in reasonable yield from a purified basement membrane preparation, it cannot be assumed solely from their amino acid composition that they were derived from a basement membrane. It is just as probable that they were derived from different collagen types unrelated to basement membranes. It is possible, however, that basement membranes from different sources contain genetically distinct collagens, and this is presently an area of active interest. In summary, it appears that basement membranes contain unique collagenlike molecules, that these molecules are triple-helical in nature, that collagenous peptides equivalent in size to a chains can be isolated from digests of basement membranes, and that the triple helix appears to consist of identical a chains. It is not yet clear whether homologous basement membranes contain genetically identical collagen components. F. THENONCOLLAGEN COMPONENTS Upon examination of the amino acid composition of various basement membrane preparations (Table I) it becomes apparent that, in addition to the collagen component, basement membranes also contain noncollagen protein material. The proportions of this noncollagen material vary from basement membrane to basement membrane, the highest amounts being found in Reichert’s membrane of the fetal PYS and the lowest in the ALC and retinal basement membranes. It is conceivable that the major differences among basement membranes reside in the quantity and nature of the noncollagen components. It has been difficult to isolate a noncollagen protein from basement membranes without resorting to proteolysis of some sort. Kefalides (1966, 1972a) extracted GBM with 8 M urea and isolated a high-molecular-weight fraction which was poor in hydroxyproline and hydroxylysine and was rich in half-cystine, sialic acid, galactose, and mannose. Ohno et al. (1975) isolated a glycoprotein component from a guanidinehydrochloride extract of GBM. This component contained small amounts of 3- and 4-hydroxyproline and hydroxylysine and substantial amounts of aspartic and glutamic acids, half-cystine, and glycine. Carbohydrate constituted about 8.6% by weight, and there were four disaccharide and one heteropolysaccharide unit per 70,000-molecular-weight molecule. It was not clear if this fraction existed in polymerized form, since it was isolated after the reduction of disulfide bonds. The 3-hydroxyproline/4-hydroxyprolineratio was about 0.4 which is

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unusually high, even for a basement membrane. The behavior of this fraction in SDS gel electrophoresis was similar to that of collagen which migrates much more slowly than random coils of globular proteins. These studies suggested the presence of noncollagen subunits in basement membranes. However, the association of small but significant amounts of collagen-derivedamino acids in these fractions raises questions as to the origin of these proteins. It is now well known that collagen is secreted by cells as a precursor, procollagen (Section V). It is conceivable that the noncollagen glycoprotein fractions described above represent fragments of a procollagen-like molecule which may have been produced from proteolytic cleavages either in vivo or during isolation of the GBM (Spiro, 1976). Freytag et al. (1976) attempted to minimize degradation by isolating GBM in the presence of protease inhibitors. Their results suggested that some proteolysis could take place during the isolation of GBM, but they could not rule out the possibility of in vivo proteolysis. It is important to recognize that basement membranes are generally isolated as insoluble residues after extraction and sonication. If contaminating proteins remain adsorbed onto basement membranes, they will behave as noncollagen components. Thus Mohos and Skoza (1970) reported that epithelial foot processes, which remained associated with the GBM, were responsible for much of the sialic acid in the GBM and could have been the source of nephritogenic components. Of equal importance is the possibility that proteins are trapped in the GBM during renal filtration and that these can be extracted using chaotropes or denaturants, as has been reported by Marquardt ef al. (1973). In making comparisons of GBMs, such proteins tend to maximize differences and minimize similarities. Upon digestion of GBM with bacterial collagenase, Kefalides (1972a) reported the formation of an insoluble residue which could be solubilized by reduction and alkylation and was further purified by gel filtration. This glycoprotein fraction contained only traces of collagen-derived amino acids and had a carbohydrate composition consistent with that of a noncollagen glycoprotein containing heteropolysaccharide units. The collagenase-solubilizedmaterial gave rise to a number of glycopeptides of low molecular weight also containing only trace amounts of collagen-derived amino acids. When a rabbit antiserum against reduced and alkylated GBM was reacted with the immunogen by double diffusion, two precipitin lines formed (Kefalides, 1972a). It was found that the residue fraction formed a line of identity with one of these precipitin lines and that the collagenase-solubilized peptides formed lines of identity with the other precipitin lines. These data suggested the presence of at least two immunologically distinct noncollagen glycoprotein components in GBM, one of high molecular weight and one of low molecular weight.

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When Hudson and Spiro (1972a,b) attempted to fractionate reduced and alkylated GBM by gel filtration, they found a large number of polypeptide fractions ranging in size from 30,000 to greater than 700,000 molecular weight. The amino acid compositions of these polypeptides were very heterogeneous, some having large amounts of collagen-derived amino acids and others having much smaller amounts. In no instance was a fraction isolated which was devoid of collagen-derived amino acids. They also found that the heteropolysaccharideunits tended to be associated with the larger molecules. It is possible that the lowmolecular-weight collagenase-solubilized glycopeptides isolated by Kefalides (1972a) may have been derived from collagenase-resistant glycopeptide portions of larger molecules containing both collagenous and noncollagenous regions. There is evidence from biosynthetic studies that such peptides can be isolated from collagenase-resistant basement membrane procollagen (Clark and Kefalides, 1978b). A problem that should be emphasized here is the marked heterogeneity of basement membrane proteins solubilized by reduction (Hudson and Spiro, 1972a,b; Sat0 and Spiro, 1976; Freytag et al., 1976). In all these studies, sonication was used in one of the penultimate steps in isolation of the basement membrane. Sonic disruption could lead to molecular fragmentation, and the observed heterogeneity might be attributable, at least in part, to this effect. Although milder procedures have been developed which do not involve a sonication step, no evidence has been presented as yet to indicate that this reduces the degree of heterogeneity.

G. THE SUPRAMOLECULAR ORGANIZATION OF BASEMENT MEMBRANES From the preceding discussion, it should be obvious that basement membranes are composed of two types of proteins, collagen and noncollagen. The questions then arise: What are the origins of these proteins? What is the nature of their association with each other? Is there such an entity as a basement subunit? What models can be derived concerning the supramolecularorganization of a basement membrane that are consistent with available data? To answer these questions, it is necessary to draw on data obtained not only from chemical studies but also from morphological, immunological, and biosynthetic studies. In this section, we attempt to examine systematically the nature of the bonds holding basement membranes together and ultimately derive a model for their supramolecular organization. 1. Hydrogen Bonds The major forces which stabilize the collagen triple helix are those due to hydrogen bonding (Ramachandran and Ramakrishnan, 1976). It has been shown

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that the large amount of 4-hydroxyproline in collagens is of prime importance in this process (Jimenez et al., 1973; Berg and Prockop, 1973). If the hydroxylation of collagen is prevented, the triple helix will denature at a temperature of about 25°C as compared with the 37°C needed for the denaturation of fully hydroxylated collagen. In the case of basement membranes, it has been found that the melting temperature (T,) of its collagen component is even higher than that for type I collagen (Gelman et al., 1976). This is probably due to the significantly higher content of 4-hydrox yproline in basement membrane collagen. The resistance of basement membrane collagen (and other collagens as well) to the action of proteases is indirectly attributableto the hydrogen-bonding forces holding the triple helix together. Hydrogen bonding is probably involved in the association of noncollagen proteins as well, as is shown by the increased solubility of these compoents in the presence of urea or guanidine. There is one observed difference in the behavior of basement membrane collagen as compared to most other collagens with regard to the susceptibility to pepsin (Daniels and Chu, 1975; Chung et al., 1976; Dehm and Kefalides, 1978a,b,c; Orkin et al., 1977). Under certain conditions, basement membrane collagens can be degraded to molecules smaller than a chains. This may be particularly true for newly synthesized basement membranes (Orkin et al., 1977). It is not clear if this is due to a pepsin-susceptible amino acid sequence within the a chains, to a partial denaturation of the collagen during isolation, or to the removal of peptides which mask the site of action of the protease.

2 . DisuEfide Cross-Linkages It appears that cystinyl disulfide bonds are of major importance in maintaining the supramolecular organization of basement membranes. In the presence of strong denaturing agents, reduction and alkylation results in the nearly total solubilization of most basement membranes, whereas basement membranes are only sparingly soluble without prior reduction. Attempts to demonstrate the presence of free sulfhydryl groups in basement membrane preparations have not succeeded (Hudson and Spiro, 1972a), indicating that all the cysteine is tied up in disulfide bonds. Most of the cysteine in basement membranes is found in the noncollagen portions, and some cysteine may be located near terminal regions of the collagenous domain in the procollagen molecule (Alper and Kefalides, 1978a,b). It is probable that disulfide bonds are present within a single collagen (Y chain, between a chains, between a chains and noncollagen proteins, and between noncollagen proteins, and this serves to organize the basement membrane into an insoluble matrix. Dehm and Kefalides (1978a,b), isolated a 95,000-molecularweight collagen fraction from a pepsin digest of bovine ALC only after reduction

BASEMENT MEMBRANES

195

and alkylation was interposed between two pepsin digestions in their three-step procedure. The product contained no half-cystine (or at most a trace), indicating the removal by pepsin of a short peptide sequence which apparently contained interchain disulfide cross-linkages. This also indicated that the cysteine present in basement membrane a chains was located near the ends of the molecule. This is consistent with the observation of Olsen et al. (1973), who isolated a citratesoluble fraction from bovine lens capsule and showed that it consisted of a globular protein portion at the ends of a filamentous collagen portion. Upon reduction, some of the globular components were removed, suggesting that disulfide bonds linked collagen and noncollagen components of the lens capsule and, again, that the location of cysteine residues in the collagen portion may be near the ends of the a chain. Alper and Kefalides (1978a,b) isolated a very highmolecular-weight fraction from a hydroxylamine digest of bovine ALC. This fraction contained small but significant amounts of hydroxyproline, hydroxylysine, and half-cystine. Upon reduction and alkylation of this product, they found that most of the half-cystine and all of the collagen-derived amino acids were lost but that the fraction remained macromolecular. Upon treatment with bacterial collagenase, all the collagen-derived amino acids were removed, but the half-cystine remained with the macromolecular fraction. This suggested that there were noncollagenous peptide extensions at the ends of the collagen molecules and that they were linked through disulfide bonds to a highly crosslinked noncollagen protein fraction (Figs. 7 and 11). In addition, the collagenous peptides apparently were joined together by disulfide bonds. (This would explain the loss of cysteine after reduction but not after collagenase digestion.) It appears then that terminal portions of the collagen component of lens capsule bear some resemblance to those of procollagen molecules. It is interesting to note that the entire complement of amino acids of the lens capsule could be accounted for if the assumption were made that it consisted entirely of disulfide-crosslinked basement membrane procollagen. This can be calculated by assuming a content of 134 residues per lo00 4-hydroxyproline residues and molecular weights of 95,000 and 160,000 for the collagen and the procollagen molecule, respectively. 3. Other Covalent Cross-Linkages Tanzer and Kefalides (1973) demonstrated the presence of borohydridereducible covalent cross-linkages in a lens capsule collagen preparation similar to those found in interstitial collagens. This provides one explanation for the heterogeneity of reduced basement membrane fractions. The high-molecularweight fractions might represent cross-linked polymers of the collagen component.

196

NICHOLAS A. KEFALIDES ET AL.

Alper and Kefalides (1974) studied the effects of a selective cysteinyl cleavage procedure on ALC. This led to the isolation of a highly cross-linked noncollagen fraction. This fraction contained small amounts of hydroxylysine and little, if any, hydroxyproline. Radioactivity from sodium borotritide was incorporated into this fraction, but radioactive collagen cross-links could not be detected in hydrolysates of this fraction (Tanzer and Alper, unpublished observations). This study suggested the existence of covalent, noncollagen, nondisulfide crosslinkages within noncollagen portions of the lens capsule. This is consistent with the observation of a similar fraction from a hydroxylamine digest of ALC (Alper and Kefalides, (1978a,b). Thus another source of heterogeneity can be postulateddiffering degrees of covalent attachment of noncollagen proteins to either collagen or to other noncollagen components. This could also explain differences in the amino acid composition of the various peptides obtained after reduction and alkylation (Hudson and Spiro, 1972a, b; Ohno et al., 1975).

H. THESUBUNIT COMPOSITION OF BASEMENT MEMBRANES The observations that basement membranes contain collagenous and noncollagenous components and that they appear to be held together, at least in part, by disulfide cross-linkages have led to the concept that basement membranes are composed of a series of subunits associated with each other in a regular fashion through different kinds of cross-linkages. A number of models for the supramolecular organization of basement membranes have been presented (Kefalides, 1971, 1973; Spiro, 1976). A major problem encountered by investigators in attempting to unravel this subunit structure is the marked heterogeneity of the solubilized materials obtained after the reduction of disulfide bonds. Sat0 and Spiro (1976) have reported the presence of over 40 molecular species in a reduced and alkylated GBM fraction. Hudson and Spiro (1972a,b) and Freytag et al. (1976) have suggested that the sizes of the protein components vary and that there may be differences in the degree of association of collagen and noncollagen components. Ohno et al. (1975) have isolated a noncollagen glycoprotein from reduced and alkylated GBM after an exhaustive series of gel filtration experiments, and they have suggested that this, too, is a basement membrane subunit. These observations indicate that the supramolecular organization of basement membranes is extremely complex. When one examines biosynthetic systems, a much less complex situation is observed. Here it has been shown for a number of systems (Grant et al., i972a,b; Clark et at., 1976; Kefalides et af., 1976; Minor et a!., 1976b; Heathcote et al., 1978) that basement membrane collagen is secreted as a procollagen

BASEMENT MEMBRANES

197

molecule and that little or no cleavage of the procollagen extension peptides takes place. Thus basement membrane procollagen appears to be a major building block of basement membranes and may constitute the major subunit. Whether or not there is more than one type of basement membrane procollagen has yet to be demonstrated. It is feasible that at least some of the noncollagen components are derived from the procollagen extension peptides and that other proteins are synthesized independently and eventually are transported and organized into the matrix through some mechanism of cross-linking. It has been suggested that further modifications of the matrix occur, such as cleavage of peptide bonds within the collagen and noncollagen portions of the matrix (Spiro, 1976), although there is no evidence to distinguish this from turnover processes. Upon examination of the final product as isolated from tissues, the system appears to be much more complex than it really is. Some very important questions remain concerning the structure of basement membranes. Among these are: Do homologous basement membranes contain different collagen chains? What is the nature of the noncollagen components? Are they all derived from procollagen or are genetically distinct noncollagen proteins synthesized, secreted, and organized into the matrix of the basement membrane by the cells making the basement membranes? What controls the composition of basement membranes in different tissues?

V. Basement Membrane Metabolism In recent years, experiments dealing with basement membrane metabolism (i.e., synthesis and degradation) have been increasingly used as an approach to understanding the structure and function of these important connective tissue matrices. In this section, the emphasis is on recent literature, and an attempt is made to integrate the findings from a number of different laboratories into a coherent and credible account.

A. BASEMENT MEMBRANE BIOSYNTHESIS It is more or less accepted that the cells which rest upon a basement membrane are responsible €or its synthesis (Pierce, 1965, 1970; Pierce et al., 1964). In a number of laboratories, basement membrane biosynthesis has been studied using either in vivo or in vitro systems. A comprehensive, but certainly not exhaustive, list of such systems is presented in Table IV. Although the observations from all these biosynthetic systems have contributed to our knowledge and understanding

198

NICHOLAS A. KEFALIDES ET AL. TABLE IV SYSTEMS USED TO STUDY BASEMENT MEMBRANE BIOSYNTHESIS Tissue

Chick Embryonic lens capsule Embryonic heart Embryonic neuroepithelium Embryonic retinal pigmented epithelium Embryonic corneal epithelium Rat Embryonic PYS

Lens capsule Renal glomeruli

Schwann cells Mouse Embryonic PYS teratocarcinoma

Reference

Grant et a / . (1972a,b, 1973) Johnson et al. (1974) Cohen and Hay (1971) Newsome and Kenyon (1973) Dodson and Hay (1971); Trelstad ef al. (1974) Clark et al. (1 975b); Minor e t a / . (1 976a-d); Clark and Kefalides (1978b,c); Maragoudakis et al. (1975, 1976, 1978a,b) Heathcote et al. (1978) Cohen and Vogt (1972, 1975); Khalifa and Cohen (1975); Brown and Michael (1973); Killen et al. (1974); Wong et al. (1972); Bloodworth et al. (1977); Krisko and Walker (1974, 1976); Grant et al. (1975); Blau and Michael (1972); Romen e t a / . (1976); Williams et al. (1976); Striker et al. (1978); Cohen and Khalifa (1977); M. P. Cohen (1978); Foidart et al. (1978) Church et al. (1973); Nathaniel and Pease (1963); Thomas (1964) Martinez-Hernandez et al. (1974); Pierce and Johnson (1971); Johnson and Starcher (1972); Johnson and Warfel (1976); Pierce et al. (1962, 1963, 1964); Priest (1970); Johnson et al. (1978) (continued)

of basement membranes, a majority of the results have been obtained using isolated kidney glomeruli, corneal endothelium, embryonic lens capsule, and embryonic rodent PYS. Based primarily on structural and immunochemical studies (Sections IV and VI), it has been proposed that intact basement membrane is composed of a collagenous component and one or more noncollagenous glycoprotein components. Because of the relative ease and specificity in detecting collagen synthesis, the collagenous component of newly synthesized basement membranes is relatively well-characterized compared to the noncollagenous components(s). 1. Characterization of the Collagenous Component Most of our knowledge on the structure and biosynthesis of collagen (or procollagen) has come from sources other than basement membranes. The in-

199

BASEMENT MEMBRANES TABLE IV (continued)

Tissue

Reference

Embryonic submandibular epithelium

Bernfield and Banerjee (1972); Bernfield et al. (1972); Banerjee et at. (1977); Cohn et at. (1977) Pierce (1965) Laurent et al. (1978)

Epithelial tumors Lens capsule Rabbit Corneal endothelium Renal tubules Embryonic epithelial enamel organ Bovine Aortic endothelium Lens capsule Human Umbilical cord vein endothelium Skin Amniotic fluid cells Renal glomerular epithelium Pig Renal glomeruli Monkey Renal glomeruli

Perlman and Baum (1974a,b); Perlman et al. (1974); Kefalides et al. (1976) Kenney et af. (1978) Trelstad and Slavkin (1974) Howard et al. (1976); Macarak and Kefalides (1978); Chang et al. (1977) Hughes et al. (1975); Dehm and Kefalides (1978a-c) Jaffe et al. (1976) Briggaman er al. (1971); Briggaman and Wheeler (1975) Priest et al. (1977); Megaw et al. (1977); Johnson ef af. (1978) Killen et al. (1978) Krisko and Walker (1974) Striker et al. (1978)

terested reader is therefore directed to a number of recent reviews for details on these topics (Bornstein, 1974; Kivirikko and Risteli, 1976; Uitto and Lichtenstein, 1976; Prockop et al., 1976; Grant and Jackson, 1976). Since it is very likely that the processes involved in the synthesis of basement membrane procollagen are analogous to those elucidated for other types of procollagen, the emphasis in this section is on those features of biosynthesis which appear to be unique for basement membranes. Where possible, comparisons are made between basement membrane procollagen and other types of procollagen, primarily type I procollagen. a. Hydroxylation of Peptidylproline. Compositional studies have shown that basement membrane collagen differs from other collagen types in 3- and 4-hydroxyproline content (Table In).Results similar to these have been obtained in vitro by incubating tissues rich in basement membrane in the presence of labeled proline and measuring the production of labeled hydroxyproline. Table V

AMOUNTSOF 3COMPARIWN OF RADIOACTIVE

Tissue Chick embryo Lens cells Rat Glomerulus Lens capsule Rabbit Corneal endothelium Rat embryo

PYs Bovine Aortic endothelium

4-HYP x 100 total Pro

TABLE V 4-HYDROXYPROLINE AND GLYCOSYLATED HYDROXYLYSINE IN VARIOUS TISSUES SYNTHESIZING BASEMENT MEMBRANE AND

3-HYP total Hyp

~

100

Glycosylated Hyl x 100 total Hyl

Reference

5

11-15

92

8.5 3-6

14-16 5

91 n.d."

Grant et al. (1975) Heathcote et al. (1978)

11-14

14-16

94

Kefalides et al. (1976)

10-18 20 -26*

9-1 1

94-97

Clark er al. (1975b); Minor et al. ( 1976d)

91

Howard et al. (1976)

3

"n.d., Not determined. bTrophoblastwas removed prior to incubation.

10-13

Grant et al. (1972a)

20 1

BASEMENT MEMBRANES

summarizes the results for several basement membrane-synthesizing systems. Since basement membrane collagen is reported to have at least 60% of its total imino acid as 4-hydroxyproline (Table 111), it can be seen that from 5% (100 x 3/60) to 43% (100 X 26/60) of the total incorporated 14Cis in newly synthesized basement membrane procollagen in these systems. It is also possible to estimate from the above ratios the percentage of the newly synthesized protein represented by basement membrane collagen (Clark et al., 1975b). From such calculations, values range from a low of -1% in several tissues to a high of 6- 12% in the rat embryo PYS. Studies on other types of collagen have shown that only -45% of the total imino acid is 4-hydroxyproline (Table VI). In two systems widely used for studying the synthesis of type I (matrix-free chick embryo tendon cells) and type 11 (matrix-free chick embryo sternal cells) procollagens, the ratio of 4-hydroxy[14C]proline to total 14C averaged about 0.30 (Dehm and Prockop, 1971, 1973). Thus in these systems about two-thirds (100 x 30/45) of the total incorporated I4C is in newly synthesized procollagen. One characteristic of basement membrane collagen which distinguishes it from other types of collagen is its 3-hydroxyproline content (Table III). In most of the biosynthetic systems tested, the 3-hydro~y['~C]prolinecontent ranged between (Table V)-a value consistent with 10 and 15% of the total hydr~xy['~C]proline amino acid analysis (Table III). The corresponding value for other types of collagen is 1% (Table VI). b. Hydroxylation of Peptidyllysine and Glycosylation of Hydroxylysine. Another characteristic of basement membrane collagen is its content of hydroxylysine and glycosylated hydroxylysine (Table 111). In all the in vitro systems studied by labeling with [14C]lysine,greater than 80% of the hydroxy [14C]lysine was glycosylated and greater than 90% of the glycosylated hydr~xy['~C]lysine was glucosyl-galactosyl-hydroxy[14C]lysine(Grant et al., 1972a, 1975; Clark et al., 1975b; Minor et al., 1976d; Kefalides et a l . , 1976; Howard et al., 1976) (Table V). These values are consistent with quantitative hydroxylysine glycoside

-

TABLE VI SUMMARY OF

DIFFERENCES BETWEEN NEWLYSYNTHESIZED

INTERSTITIAL AND

BASEMENT

MEMBRANE PROCOLLAGEN

Collagen type

I I1 IV

3-Hyp total Hyp 1-3 1-3 10-15

loo

4-Hyp total Pro

44 44 60

S-S Bonding and helix formation

Secretion time

5-10 5-15 >45

20 35 60

total Hyl 21 67 95

202

NICHOLAS A. KEFALIDES ET AL.

analyses of intact basement membranes (Kefalides, 1970). The corresponding values for the hydro~y['~C]lysine glycosylation of type I is 21% and for type I1 is 67%. (Dehm and Prockop, 1973) (Table V). In studies using rat embryo PYS, it was briefly reported that either 8,9dihydroxy-7-methyl-benzo[b]quinolizinium(GPA 1734) (Maragoudakis et al., 1976) or L-dopa (Maragoudakis et al., 1975) specifically inhibited the formation of hydroxyproline and hydroxylysine in basement membrane procollagen biosynthesis without affecting protein biosynthesis in general. More recently, these authors have reported that, in contrast to the observations for interstitial procollagen (Prockop et al., 1976), unhydroxylated PYS basement membrane procollagen is secreted at a rate comparable to that of the hydroxylated molecule (Maragoudakis et al., 1978a,b). In addition, the newly synthesized unhydroxylated basement membrane procollagen does not appear to be deposited on Reichert 's membrane. c. Disulfide Bonding, Triple-Helix Formation, and Secretion. A common feature in the assembly of procollagen triple helices appears to be the initial formation of interchain disulfide bonds (Bornstein, 1974; Kivirikko and Risteli, 1976; Uitto and Lichtenstein, 1976; Prockop et al., 1976). The presence of disulfide bonds in basement membrane procollagen has been detected in chick embryo lens capsule (Grant et al., 1973), rat glomeruli (Williams et al., 1976), and rat embryo PYS (Clark and Kefalides, 1978b,c). Compared with the formation of disulfide bonds in systems synthesizing either type I or type II procollagen, the formation of disulfide bonds in basement membrane procollagen appears to occur relatively late in the intracellular processing (Grant et al., 1973; Williams et al., 1976). Since disulfide bond formation is closely related to triple-helix formation (Prockop et al., 1976), these observations are consistent with the finding that intracellular basement membrane procollagen is predominantly non-triple-helical as measured by susceptibility to limited protease digestion (Grant et al., 1973; Williams et al., 1976). In turn, these findings may help explain why basement membrane procollagen requires -60 minutes to be secreted (Grant et al., 1972a; Clark et al., 1975b; Kefalides etal., 1976), while type I1 and type I procollagens are secreted in 35 minutes (Dehm and Prockop, 1973) and 20 minutes (Dehm and Prockop, 197l), respectively. Table VI briefly summarizes some differences between newly synthesized interstitial and basement membrane procollagens. The intracellular processing of interstitial collagen has been shown to proceed from the rough endoplasmic reticulum, to the smooth endoplasmic reticulum, to the Golgi complex, and then to the extracellular space (Prockop et al., 1976). While relatively little work has been done in this area for basement membrane collagen, there is a report that the antigenic portion of murine Reichert's membrane does not pass through the Golgi complex (Martinez-Hernandez et al., 1974). This observation will eventually have to be substantiated by the isolation of subcellular organelles, followed by the biochemical characterization of their contents.

BASEMENT MEMBRANES

203

d. ExtracellularProcessing of Procollagen. Initial studies on the extracellular processing of basement membrane procollagen suggested that there was a time-dependent conversion of newly synthesized basement membrane procollagen to collagen (Grant et al., 1972b, 1975) analogous to that found for other types of procollagen. More recent studies, however, indicate that there is no such conversion (Minor et al., 1976b; Heathcote et al., 1978; Orkin et al., 1977) and that it is intact procollagen which is incorporated into the basement membrane matrix (Minor et al., 1976b). In the case of Reichert’s membrane, the morphological organization of the newly synthesized basement membrane matrix appeared to be similar to the preexisting basement membrane except for a lighter staining (Minor et al., 1976~). After incorporation of procollagen into the basement membrane matrix, it is likely that at least two types of covalent bonds are formeddisulfide and lysylderived cross-links. Pulse-chase experiments using rat embryo PYS in the presence or absence of P-aminopropionitrile (an inhibitor of lysyl-derived crosslinks), and extraction in the presence or absence of reducing agents, suggest that the integrity of the basement membrane is initially stabilized by disulfide bonds and is further reinforced by a time-dependent formation of lysyl-derived crosslinks (Minor et al., 1976a). These conclusions are in accord with structural studies (Section IV). e. Structure of Procollagen. The structure of the newly synthesized extracellular collagenous component of basement membrane has been studied primarily by SDS polyacrylamide gel electrophoresis, SDS agarose gel filtration, and sucrose density gradient rate zonal ultracentrifugation both before and after treatment with disulfide bond-reducing agents. As discussed previously, the procollagen molecule is disulfide-bonded, In the absence of reducing agents, this aggregate elutes in the void volume of an SDS agarose column (MW >300,000) (Grant et al., 1973; Williams et al., 1976; Clark and Kefalides, 1978b,c). Upon disulfide bond reduction, the molecular weight of the basement membrane pro-a chains which are liberated appears to be greater than that of the pro-a chains from the interstitial procollagens. Based on SDS gel filtration, the molecular-weight values for the former have ranged from 140,000 to 180,000 (Grant ef al., 1972b, 1975; Kefalides et al., 1976; Minor et al., 1976b; Heathcote et al., 1978; Clark and Kefalides, 1978b,c). Similar molecular weight values have been obtained by SDS polyacrylamide gel electrophoresis (Minor et al., 1976b; Clark and Kefalides, 1978~).These values are significantly higher than the values of 135,000-145,000 reported for types I or I1 procollagen, which were obtained by the same procedures (Harwood et a l . , 1977; Clark and Kefalides, 1978~).In addition, it has been shown by sucrose density gradient rate zonal ultracentrifugation that basement membrane pro-y and pro-a sediment significantly faster than the corresponding pro-y (I) and pro-a (I), respectively (Clark and Kefalides, 1978~). Specific proteolytic digestion of basement membrane procollagen and analysis

204

NICHOLAS A. KEFALIDES ET AL.

of the denatured products on SDS agarose in the absence of reducing agents show that a disulfide-bonded aggregate persists (Williams et al., 1976; Clark and Kefalides, 1978b,c). In this respect, basement membrane procollagen is similar to type 111 procollagen (Burke et al., 1977). Upon disulfide bond reduction and denaturation, the released basement membrane polypeptides have a molecular weight similar to that of intact interstitial pro-cu chains (i.e., -135,000) (Williams et al., 1976; Minor et al., 1976a; Clark and Kefalides, 1978b,c). It has recently been reported, however, that when bovine lens capsule procollagen is treated with pepsin as above and the disulfide bonds subsequently reduced under nondenaturing conditions, further pepsin digestion followed by denaturation generates polypeptides with molecular weights comparable to those of the cu chains of interstitial collagen ( D e b and Kefalides, 1978a-c). One implication of these results is that the collagenous domain (protease-resistant) of basement membrane procollagen is comparable to that found in interstitial procollagens, and thus the apparent larger molecular weight of the former may be due solely to the noncollagenous propeptide domain (s) (protease-susceptible). More evidence is needed to confirm this hypothesis. These results are in contrast to the findings for types I and I1 procollagen where specific proteolytic digestion followed by denaturation converts the disulfide-bonded molecule directly to a chains (MW -98,000). A summary of these findings is presented in Table VII. Digestion of procollagens with bacterial collagenase and characterization of the resultant propeptide domain has proven to be a useful tool in the elucidation of interstitial procollagen structure. Preliminary results of similar experiments using basement membrane procollagen are somewhat more complex, but one interpretation of the results suggests that, in contrast to types I and I1 procollagen, basement membrane procollagen contains a disulfide bond(s) in a hydroxyproline-containing,collagenase-resistant peptide (Clark and Kefalides, , 1978b,c) . From the results of experiments described in this section, a proposed structure of basement membrane procollagen is schematically represented in Fig. 8 along with that of type I procollagen. It must be emphasized that this model is only one of many which could be drawn and still be consistent with the data presently available. In spite of this shortcoming, the model is useful in highlighting the major differences between the structures of type I and basement membrane procollagen: (1) Basement membrane procollagen and pro-a chains behave as if they were larger than the corresponding interstitial procollagen components; this difference may be entirely due to the propeptide domain(s). (2) The basement membrane procollagen molecule has only a single type of pro-a chain. (3) After limited proteolytic digestion, basement membrane procollagen remains disulfide-bonded; only after disulfide bond reduction and redigestion with protease is basement membrane collagen generated. (4) After bacterial collagenase digestion, basement membrane procollagen yields a hydroxyproline-containing,

TABLE VII COMPARISON OF MOLECULAR PROPERTIES BETWEEN INTERSTITIAL A N D BASEMENT MEMBRANE PROCOLLAGEN A N D COLLAGEN COMRJNENTS Molecular properties Molecular weight after a single proteolytic Collagen type

Type 1 or I1 Basement memhrane

Molecular weight"

Component

135,000- 140,M)o ~400,000 170,OOO-180,000

Monomer Trimer Monomer Trimer ~~

>400,000

Reduced 98,000 n.a. 130,000-140,000 n.a.

Unreduced

mxu

Sedimentation coefficient, 520.w

n.a. n.a.

2.5 4.5 3.2

-4oo,oO0

5.6

~

aMolecular weights determined by SDS agarose gel filtration and/or SDS polyacrylamide digestion. bn.a., Not applicable.

206

NICHOLAS A. KEFALIDES ET AL.

+

+

Q!

BASEMENT MEMBRANE PROCOLLAGEN

A

BASEMENT MEMBRANE COLLAGEN

eon +B

1

BCase COOH

TYPE I PROCOLLAGEN

Ip 0

TYPE I COLLAGEN

FIG. 8. Schematic representation of the structures of basement membrane procollagen (A) and

type I procollagen (B) and the products arising after either bacterial collagenase (BCase) digestion or protease (P) digestion. The collagenous domains are represented by the coiled lines and the propeptide domains by either rectangular or oval shapes. Disulfide bonds are also indicated (SWS).Jn the case of basement membrane procollagen, P, and PI,refer to successive protease digestions with an intervening reduction step as described in the text.

disulfide-bonded peptide. In order to account for the observed differences in susceptibility to enzymic digestion, it is hypothesized that the propeptide region on one end of the basement membrane procollagen molecule may contain a collagenlike domain which protects an intermolecular disulfide bond-containing, noncollagenous domain from being digested. This collagenous domain, however, is susceptible to bacterial collagenase and thus yields a disulfide-bonded, hydroxyproline-containing peptide. Future experiments will determine the viability of this model.

BASEMENT MEMBRANES

207

f. Basement Membrane Collagen Heterogeneity. Compared to knowledge of the different genetic collagen types present in the interstitium, little is known about the possible types of collagenous components in basement membranes. The majority of the biosynthetic evidence accumulated thus far provides no basis for believing that there is collagen heterogeneity within a single basement membrane. However, recent data obtained from newly synthesized rat renal glomeruli show multiple hydr~xy['~C]lysine-containing polypeptides with apparent molecular weights ranging from <30,000 to >300,000 (Cohen and Klein, 1977). The origin of these components is still in question.

2. Characterization of the Noncollagen Component(s) As mentioned earlier, there is a paucity of information concerning the biosynthesis of putative noncollagen components of basement membranes. The major components which have been postulated are glycoproteins and glycosaminoglycans. a. Glycoprotein Components. A number of reports concern the glycoprotein secreted by cells which make an epithelial basement membrane (Johnson and Starcher, 1972; Hughes et al., 1975; Johnson and Warfel, 1976; Megaw et aE., 1977; Johnson et al., 1978). The glycoprotein component secreted by murine teratocarcinoma cells in vitro was antigenically identical to glycoprotein components either isolated from murine kidney homogenates (Johnson and Warfel, 1976) or secreted by human amniotic fluid cells (Megaw et a l . , 1977; Johnson et al., 1978). In addition, antibodies directed against epithelial basement membrane glycoprotein cross-reacted with the basement membranes of renal glomeruli and tubules (Johnson and Warfel, 1976). The epithelial glycoprotein isolated existed as an aggregate which could be disrupted by detergents and disulfide bondreducing agents, yielding a component with a molecular weight of 32,00034,000 and a carbohydrate content of 11-13% (Johnson and Warfel, 1976; Johnson et al., 1978). In contrast, Hughes et al. (1975) failed to find any low-molecular-weightglycoproteins related to bovine lens capsule but did find a major glycoprotein fraction with a molecular weight of 75,000-90,000. However, the relationship of this glycoprotein to the lens capsule was not established. There is very little information involving the use of radioactive tracers to study basement membrane glycoprotein biosynthesis. Krisko and Walker (1974, 1976) used labeled sugar precursors in incubations of rat glomeruli and found that it was incorporated into protein, but there was no evidence that the newly synthesized protein was indeed a component of basement membrane. However, Minor et al. (19764 showed by light microscope autoradiography that [3H]mannoseand [3H]glucosamine gave labeling patterns in Reichert 's membrane which were similar to those seen with C3H]proline. However, with the recent observations that the propeptide appendages of newly synthesized type I procollagen are glycoprotein in nature (Clark and Kefalides,

208

NICHOLAS A . KEFALIDES ET AL.

1976, 1978a; Duksin and Bornstein, 1977; Olsen et al., 1977), and that it is basement membrane procollagen which is incorporated into the basement membrane matrix (Minor et al., 1976b), it is apparent that the origin of any noncollagenous basement membrane glycoproteins must be carefully documented. b. Glycosaminoglycan Components. In several developing systems, most notably the embryonic chick and mouse, glycosaminoglycans (GAGs) have been postulated to be constituents of basement membranes. Specifically, GAGs have been detected in basement membranes of the chick embryo lens capsule, neural tube and notochord (Hay and Meier, 1974), and cornea (Trelstad et al., 19741, and in the basement membrane of the mouse submandibular gland (Bernfield and Banerjee, 1972; Bernfield et al., 1972; Banerjee et al., 1977; Cohn et al., 1977). However, GAGS were not detected in newly synthesized Reichert’s membrane of the embryonic rat PYS (Minor et al., 1976c) and are not found in basement membranes from mature animals. Thus the presence of such components in basement membranes is controversial. B. BASEMENT MEMBRANE TURNOVER Basement membrane turnover has been the subject of relatively few studies. Walker (1973) used an argyric technique to study the “natural history” of normal rat glomerular basement membrane. He concluded that a major component of the basement membrane was continuously removed in a slow process. The time for complete turnover of this matrix was on the order of 1 year. The mesangial cells were implicated in this process, but the possible mechanisms underlying basement membrane remodeling were not discussed. In an effort to learn about the relative contributions of synthesis and degradation to the turnover of basement membrane components, Minor et al. (1976d) used [14C]proline labeling of Reichert’s membrane in organ culture. In these cultures, synthesis and degradation of collagen and noncollagen components varied independently in response to environmental changes. The turnover of basement membrane procollagen as measured by loss of 4-hydro~y[~C]proline appeared to be much slower than that of the noncollagen proteins, and this difference had a major role in determining the composition of the newly synthesized matrix. A similar conclusion was reported by Price and Spiro (1977) using rat renal GBM. They found that the turnover of radioactivity in proline and hydroxyproline from GBM required more than 100 days, while the turnover of proline from other glomerular proteins took 9 days. In studies of the involuting murine breast by light and electron microscopy, Martinez-Hernandez et al. (1976) attributed the removal of basement membrane to enzymic hydrolysis. This activity was found in breast homogenates but not in liver or kidney. Moreover, the activity was abolished by heating at 100°C for 30

BASEMENT MEMBRANES

209

minutes, by removal of divalent cations, or the presence of diisopropylfluorophosphate; but the enzyme(s) was not characterized further. Slavkin and Bringas (1976) described basement membrane degradation in situ during embryonic and neonatal incisor and molar tooth organ development. Subsequently Sorgente el al. (1977) showed a mammalian-like collagenolytic activity in matrix vesicles lining the undersurface of the basement membrane. Although it was postulated that this activity might be responsible for basement membrane degradation, there was no direct evidence to support this contention. In this regard, it may be pertinent to note that neither rat PYS basement membrane procollagen (Clark et al., unpublished observations) nor murine tumor basement membrane collagen (Woolley et al., 1978)is susceptible to human skin collagenase. It is apparent therefore that much more work needs to be done in this very important area of basement membrane metabolism.

VI. Immunochemistry of Basement Membranes The antigenic nature of basement membranes and the immunological crossreaction between homologous and heterologous basement membranes has been adequately documented (for review see Kefalides, 197lb). The cross-reactions observed are due primarily to two general types of antigenic components-a collagenous glycoprotein and one or more noncollagenous glycoproteins (Denduchis and Kefalides, 1970; Kefalides, 1972a, 1975b). In this section we discuss recent studies on the immunochemistry of the collagenous and noncollagenous components isolated from basement membranes and the use of antibodies against the former component to study the immunochemistry of newly synthesized basement membrane procollagen. Recent studies by Bardos et al. (1976) with rat kidney confirm the antigenic complexity of GBM reported earlier by Kefalides (1972a) for canine GBM. Depending on the immunogen used, whether intact GBM, reduced and alkylated GBM, or fractions of GBM obtained after enzymic treatment, antigodies are produced against various antigenic regions of either the whole GBM or its component proteins. Although antibodies can be produced against either the collagenous or noncollagenous peptides of GBM or both, there is not always a direct correlation between the number and type of antibodies made and the chemical composition of the immunogen. A peptide fraction of GBM may contain both collagenous and noncollagenous regions, and the antibody or antibodies may be directed either against only the collagenous region, the noncollagenousregion, or both. Kefalides (1972a) prepared antibodies not only against whole GBM but also against noncollagenous fractions obtained after digestion of GBM with bacterial collagenase and showed the presence of at least two antigenic compo-

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nents, both noncollagenous glycoproteins in nature. Antibodies against the collagenous component of GBM and of ALC were obtained by Kefalides (1972a), Denduchis and Kefalides (1970), and Gunson and Kefalides (1976). Marquardt et al. (1973) solubilized GBM with chaotropes into fractions of varying amino acid composition and demonstrated the presence of at least four distinct antigens. Antibodies against a human GBM fraction were prepared by Mahieu et at. (1974). This peptide, with a molecular weight of 70,000, was obtained after solubilizing whole GBM by autoclaving at 110°C, followed by affinity chromatography and preparative gel electrophoresis of the solubilized material. This antigen had an amino acid and carbohydrate composition very similar to that of the whole GBM. Glycopeptides containing only either the hydroxylysinelinked glycosides or the heteropolysaccharide were prepared and were used as inhibitors in a radioimmunoassay employing lz5I-1abeled GBM fractions (GBM-Ag). Although both types of glycopeptides inhibited the binding of lz5Ilabeled GBM-Ag to anti-GBM antibodies eluted from Goodpasture syndrome kidneys, the hydroxylysine containing glycopeptide was more efficient. The presence of at least two noncollagen glycoprotein and one collagen antigenic components has been described for bovine tubular basement membrane by Ferwerda et al. (1974a,b). Similar observations were reported by Kefalides (1972a) for canine GBM. Johnson and Warfel (1976) isolated a noncollagen glycoprotein from mouse kidney cortex. Antibodies to this fraction cross-reacted with a glycoprotein isolated from the basement membrane of neoplastic epithelial cells (Johnson and Starcher, 1972) and localized on the basement membranes of renal glomeruli and tubules. The molecular weight of this glycoprotein was estimated at 32,000. Lectin-binding studies by Johnson and Faulk (1976) suggest the presence of a glycoprotein component or components in human placental trophoblastic basement membrane (TBM). Antiserum against TBM blocked binding to TBM. Collagenase digestion at a low concentration had no effect on TBM immunofluorescent staining, but at high concentrations staining was reduced. These data tell us little of the nature of the glycoprotein. Since we know that the nonhelical peptide extensions of procollagens contain mannose and glucosamine (Clark and Kefalides, 1976, 1978b; Oohira et al., 1975) the lectins could be binding to these regions of procollagen in addition to the noncollagen glycoproteins of TBM. The specificity of antibodies against the collagen component of bovine ALC, isolated after a single limited digestion with pepsin, was investigated by Gunson and Kefalides (1976). Using a radioimmunoassay, they demonstrated that the above antibodies were type-specific for basement membrane collagen (type IV) and did not cross-react with interstitial collagens type I, II, or III (Fig. 9). Using the same approach, Gunson et al. (1976) demonstrated that basement membrane procollagen synthesized by a variety of cell systems including rat PYS endoderm, rabbit Descemet's membrane endothelium, or vascular endothelium

BASEMENT MEMBRANES

21 1

Recipowlof ontiserum dilutbn

FIG.9. Radioimmunoassay of rabbit antiserum to native basement membrane (type IV) bovine collagen. Antigens are native type IV collagen (n), denatured type IV collagen (d), and native interstitial bovine collagens, types I, n, and III. (Reprinted with permission from Gunson and Kefalides, 1976.)

could be precipitated with the above antiserum. Antigenic cross-reaction between bovine ALC collagen and the procollagen synthesized by bovine aorta endothelial cells in culture was demonstrated by Howard er al. (1976). These studies demonstrate the tissue and species nonspecificity of the antibodies. Arbogast er al. (1976) showed, in addition, that the antigenicity of the basement membrane procollagen (type IV) depended on the hydroxylation of proline (Fig. lo), as well as on the presence of disulfide bonds. Since these studies were published, Dehm and Kefalides (1978a,b,c) have prepared basement membrane collagan from bovine lens capsule containing no half-cystine. Such a molecule will have to be used as an immunogen to test again the role of disulfide bonds in the antigenicity of basement membrane procollagen. It can be stated with certainty that there is immunological cross-reaction among homologous and heterologous basement membranes, that the antigenicity of basement membranes resides in glycoprotein components having the composition of collagen, procollagen, and noncollagen molecules, and that hydroxylation of proline as well as the integrity of disulfide bonds may influence the antigenicity of the collagen and procollagen components, possibly through their conformational effects on these molecules. In tissues where the presence of basement membrane has been demonstrated only by electron microscopy and its isolation has not been accomplished for one reason or another, the demonstration of one or more of its protein components must depend on the use of nonspecific antibodies prepared against basement

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NICHOLAS A. KEFALIDES ET AL.

Reciprocal of antiserum dilution

FIG.10. Radioimmunoassayof rabbit antiserum to native basement membrane (type IV)bovine collagen. Antigens are synthetic products of rat PYS endoderm and represent basement membrane [14C]procollagen,basement membrane [14C]protocollagen,and protocollagen proline hydroxylasehydroxylated basement membrane ['4C]protocoIlagen. (Reprinted with permission from Arbogast et al.. 1976.)

membrane components of other tissues. This situation is particularly true in tissues such as skin, aorta, and smooth and skeletal muscle fibers and in developing embryonic tissues.

VII. Basement Membrane Changes in Disease The ultrastructure and function of basement membranes are altered on several diseases (Kefalides, 1971b). These include poststreptococcal glomerulonephritis, disseminated lupus erythematosus, renal vein thrombosis, lipoid nephrosis, diabetes mellitus, Alport 's syndrome, and Goodpasture's syndrome. A number of mechanisms have been implicated including immunological injury, congenital and hereditary abnormalities, and mechanisms which remain largely unknown. In animal models of disease, selective use of immunological injury, such as anti-GBM antibody, and of a toxic agent, such as aminonucleoside, produced morphological and functional changes characteristic of nephritis and nephrosis. The administration of either alloxan or streptozotocin to rats caused diabetes mellitus but did not produce the exact counterpart of human disease. Any one of the structural components of the glomerular capillary wall, including the endothelium, epithelium, basement membrane, or mesangium may be affected. In muscle and skin capillaries the endothelium, basement membrane, and pericyte may be involved. The ultrastructural changes in GBM and in base-

BASEMENT MEMBRANES

213

ment membranes of other tissues may be characterized by diffuse thickening (Farquhar et al., 1959), splitting and localized rarefaction (Hinglais et al., 1973), deposition of electron-dense antigen-antibody complexes (Dixon et al., 1961), polyploid changes (Martin and Kissane, 1975), and wrinkling (Heptinstall, 1974). It has generally been assumed that the morphological and functional alterations of basement membranes in human and experimental disease must be accompanied by associated changes in their chemical composition and structure. Although this may be the case, compositional analyses of human GBM in a variety of glomerulopathies and of animal GBM in experimentally induced kidney disease produced incomplete and contradictory data. There are serious limitations encountered in the effort to carry out these studies which include the unavailability of adequate amounts of tissue in the early stages of human disease and the difficulty in obtaining pure basement membrane from patients with advanced disease. In chronic glomerulonephritis, the duration of the disease with its accompanying inflammatory and fibrotic changes makes the isolation of GBM free of interstitial and cellular contamination difficult (Misra and Berman, 1968; Mahieu, 1972; Blau and Haas, 1973), whereas in advanced diabetes mellitus it may result in a cleaner GBM preparation. In the latter instance, slightly higher levels of hydroxyproline and hydroxylysine are encountered in comparison with normal GBM (Beisswenger and Spiro, 1970; Westberg and Michael, 1973a; Kefalides, 1974a; Westberg, 1976). Initial studies by Misra and Berman (1968) on GBM from patients with advanced glomerulonephritis showed increases in hexosamine, reducing substances, cholesterol, lipid phosphorus, and the insoluble collagen fraction. The presence of cholesterol and lipid as an integral part of GBM has been questioned by most investigators. The presence of acid- and salt-soluble and acid- and salt-insoluble collagen fractions in GBM is also suspect, since repeated attempts to solubilize basement membranes with neutral salt or weak acid have failed (Kefalides and Winzler, 1966), although sodium citrate and acetic acid-soluble fractions have been obtained from ALC (Denduchis et al., 1970; Olsen et al., 1973). The two basement membranes, however, differ in their structural organization. Although Misra and Berman (1968) did not perform complete amino acid analyses, the soluble hydroxyproline-containingmaterial may have come from interstitial collagen. Mahieu (1972) reported that GBM in chronic glomerulonephritis contained increased levels of proline, hydroxyproline, and glycine, and decreased levels of lysine and hydroxylysine. This divergent change in the hydroxyproline and hydroxylysine content is difficult to reconcile, unless we assume a decrease in lysyl hydroxylase activity which, however, would have resulted in higher lysine levels. Studies by Westberg and Michael (1973b) on kidneys from four patients with chronic glomerulonephritis showed no significant changes in the composition of GBM, especially with respect to glycine, proline, hydroxy-

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NICHOLAS A. KEFALIDES ET AL.

proline, and hydroxylysine. In the GBM from kidneys of five patients with chronic membranoproliferative glomerulonephritis, and to a lesser extent in six patients with chronic pyelonephritis, the content of 3-hydroxyproline, 4-hydroxyproline, and hydroxylysine was reduced, whereas the lysine content was increased. A significant decrease in the concentration of glucose and galactose was noted in the group with chronic membranoproliferativeglomerulonephritisin accord with the decrease in hydroxylysine. No significant changes were noted in the content of the other sugars including sialic acid. In the same study, Westberg and Michael (1973b) analyzed hyalinized glomeruli from one kidney with chronic membranoproliferative glomerulonephritis and one showing chronic pyelonephritis. The 4hydroxyproline, hydroxylysine, and glycine content was higher than that of normal kidneys. The changes in the composition of GBM in advanced renal disease reported above are compatible with a relative decrease in the proportion of the collagen component. Whether this represents a decreased basement membrane collagen synthesis or increased synthesis of non-collagen glycoproteins is not certain at present. In some studies (Mahieu, 1972; Blau and Haas, 1973) the content of sialoprotein, a vague term for an unknown substance, has been reported to be decreased in GBM from patients with chronic glomerulonephritis. These reports refer to a decrease in histochemically detectable sialic acid using the colloidal iron reaction (Blau and Haas, 1973). This is a nonspecific reaction and cannot be relied upon to give quantitative measurements of specific reactants such as sialic acid or any other anionic substance in GBM (Nicholes et al., 1973). Mahieu and Galle (1975) examined the GBM from patients with “dense deposit disease” and found normal levels of hydroxyproline, glycine, and glucosyl-galactosyl-hydroxylysine.A decrease in cystine and an increase in sialic acid were noted. In Alport’s syndrome, McCoy et al. (1976) found that GBM from such patients was deficient in an antigen reactive with the anti-GBM antibody eluted from kidneys of patients with Goodpasture’s syndrome. Whether this is truly a lack of an antigen and therefore an inherited deficiency, or simply a loss or masking of antigen-reactive sites is not known (Scheinman et al., 1974). Analysis of GBM isolated from patients with congenital nephrotic syndrome, which is acquired by autosomal recessive inheritance, reveals an increase in hydroxyproline, hydroxylysine, and glucosyl-galactosyl-hydroxylysine content (Mahieu et al., 1976). Tryggvason (1977), however, analyzed the GBM from three patients with congenital nephrotic syndrome of the Finnish type and found a decrease in the relative amounts of 3-hydroxyproline, 4-hydroxyproline, hydroxylysine, and glycine. The lysine, arginine, and alanine contents were increased. The discrepancy between these two studies again is difficult to explain, but it may be due to differences in disease type. In this syndrome, there is also an

BASEMENT MEMBRANES

215

increase in the excretion of urinary GBM antigens (Huttunen et al., 1976), whose chemical characterization, however, remains unknown. Since the last review (Kefalides, 197lb), studies on the biochemistry of GBM in experimental glomerulonephritis are few. Lui and Kalant (1974) showed that in nephrotoxic serum nephritis there was a decrease in sialic acid content and an increase in the turnover of hydroxyproline. Blau and Michael (1972) studied the rat GBM in aminonucleoside nephrosis and found an increase in the incorporation of [3H]prolineand conversion to hydr~xy[~H]proline in GBM with the onset of proteinuria. However, the 4-hydroxyproline content of the collagen extracted with 5% trichloracetic acid from the GBM was lower and the proline content higher in the studies of Kefalides and Forsel-Knott (1970). Bartlett et al. (1975) demonstrated that in aminonucleoside nephrosis the content of dithioerythritolreducible disulfide bonds of GBM was significantly decreased. Subsequently, it was shown that in the kidney cortex of aminonucleoside-nephroticrats that the activity of glutathione reductase and levels of reduced glutathione were increased above normal (Bartlett and Joshi, 1976). Interference, therefore, with the posttranslational modifications of the protein components of GBM may be responsible for a series of structural changes which lead to proteinuria. Shibata et al. (1971) induced proliferative glomerulonephritis in rats by a single injection of a soluble glycoprotein isolated from homologous GBM. The glycoprotein was isolated after treatment of the GBM with trypsin. The composition of this fraction showed no hydroxyproline but contained more than one-third glycine. Its total hexose content was 11.06% with a glucose/galactose/mannose ratio of 1.0:0.38:0.03. In view of the high glycine and hexose content, as well as the low mannose content, one would suspect that this was a collagenous protein. Failure to detect hydroxyproline may have been due to technical problems occurring during the amino acid analysis. The pathogenesis of vascular lesions and particularly of the basement membrane thickening in diabetes mellitus is at present unknown. The relationship of the muscle capillary lesion to that seen in the glomerulus is not clear, although a common pathway may be operating to bring about the changes in both. The thickening of the peripheral capillary basement membrane and the increased amount of the mesangial matrix in the glomerulus of diabetic kidneys is thought to reflect the relative state of cell function. It is not known, for instance, whether the thickening of basement membrane is a result of increased synthesis by the epithelial and/or endothelial cells, whether it reflects failure of normal degradation and removal by mesangial cells, or whether it is due to a true increase in protein mass or simply edema. Similarly, the increase in the mesangial area from 8.4% of the total glomerular area in the normal adult to 12.7% in the diabetic patient with diffuse glomerulosclerosis (Wehner and Anders, 1969) may be the result of increased synthesis of mesangial matrix, decreased degradation and

TABLE VIII PARTIAL LISTOFTHE AMINO ACIDCOMPOSITION OFHUMAN GLOMERULAR BASEMENT MEMBRANE FROM NORMAL A N D DIABETIC PATIENTS Residues per lo00 residues Amino acid residue Hydroxylysine Lysine 3-Hydroxypmline 4-Hydroxypmline Proline Glycine Half-cystine

Residues per lo00 residues

Normal 24.9O 20.4 18.5 82.5 62.5 210.5 26.1

24.7b 25.4 8.9 80.6 74 214 20.3

awestberg and Michael (1973a). bBeisswenger and Spiro (1973). 'Kefalides (1974). dSato el af. (1975). 'Westberg (1976). 'Patients with early diabetes. gPatients with advanced diabetes.

24.5' 26 8.0 66 70 227 23

Diabetic 28.0' 23.9

84.2 70.9 191.0 20.1

25.4' 20.1 20.4 83.8 61.7 219.4 25.2

27' 22.2 16.8 88.6 59.5 220.1 19

29.5b 20

-

87.8 69.7 238.5 19.5

25.6' 30 8 71 63.2 242 18

29.0d 21.9

93.4 68.6 198.2 15.6

27.OCJ

-

18.6 88.6 58.4 206.5 18.9

27.3'7'

16.8 88.1 59.5 220.1 19.6

BASEMENT MEMBRANES

217

removal, or simply a result of trapping of serum proteins and their inefficient removal (Mauer et al., 1976). It is obvious that the changes in GBM may result from an abnormal metabolism of one, two, or all three types of cells found in the glomerulus. The biochemical basis for the thickening of GBM in diabetes still remains poorly understood. The initial claim of Beisswenger and Spiro (1970) that human diabetic GBM contained an abnormally high amount of hydroxylysine and hydroxylysine-linked glycosides without a concomitant increase in the other amino acids which characterize collagen, namely, 3- and 4-hydroxyproline and glycine, could not be substantiated subsequently by four independent studies (Westberg and Michael, 1973a; Kefalides, 1974; Sat0 et af., 1975; Westberg, 1976) (Table VIII). Later, Beisswenger and Spiro (1973) changed their view and published data in which not only hydroxylysine but hydroxyproline and glycine as well were increased in human diabetic GBM. An examination of Table VIII reveals that the increases in hydroxylysine, hydroxyproline, and glycine, whenever present, are small and represent a relative enrichment of the basement membrane by the collagen component (Kefalides, 1974). This view is compatible with the recent study by Klein et al. (1975) which showed that diabetic glomeruli from human kidneys were larger and heavier than nondiabetic glomeruli and that the former contained more collagen. It can be concluded that in diabetes the pathogenesis of the increased amount of GBM is still unknown and that this increase may be the result of either increased deposition or decreased turnover. The compositional and structural studies performed on GBM from diabetic kidneys provided little insight into the biochemical changes of microangiopathy in this disease. Another approach which has been used extensively to study the structural and metabolic aspects of connective tissue components has been that of biosynthesis in vitro. Biosynthetic studies have been used to study the pathway of synthesis and secretion of the various types of collagen by various cell systems, including those which synthesize basement membranes (Section V). Studies on the biosynthesis of basement membranes in diabetic kidneys are few and the results conflicting. The work of Khalifa and Cohen (1975) shows that the glomerular protocollagen lysyl hydroxylase activity is increased in streptozotocin-treated rats. Similarly, Cohen (1978) showed that GBM synthesis, as measured by the appearance of hydr~xy['~C]lysine in membranes obtained from sonicated glomeruli, is increased in preparations obtained from streptozotocin-treated rats. In the same study, Cohen (1978) found a significant increase in the activity of protocollagen lysyl hydroxylase, while no such increase was noted in the activity of prolyl hydroxylase. The latter finding is at variance with that of Grant et al. (1976), who reported increased activity for both lysyl and prolyl hydroxylases in streptozotocin diabetic rats. This increase correlated with a significant increase in the synthesis of hydr~xy['~C]lysineby

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NICHOLAS A. KEFALIDES ET AL.

glomeruli isolated from the diabetic animals. However, Beisswenger (1976) was unable to demonstrate an increased synthesis of hydr~xy['~C]lysine by glomeruli from streptozotocin-diabetic rats. Recently, Risteli et al. (1976) measured the activities of intracellular enzymes which catalyze the hydroxylation of proline and lysine and the glycosylation of hydroxylysine in the kidneys of rats with streptozotocin diabetes. When the activities of the four enzymes were expressed per milligram of protein in the 15,OOO X g supernatant of the kidney homogenates, there were no differences between control and diabetic kidneys. When the changes in activities were expressed as total enzyme activities per two kidneys, significant increases were noted at 12 weeks in all four enzyme activities, namely, prolyl hydroxylase, lysyl hydroxylase, collagen galactosyl transferase, and collagen glucosyl transferase. The data are consistent with an increased collagen synthesis in the diabetic kidneys and do not suggest that any specific increases take place in the level of hydroxylation of lysyl residues or glycosylation of hydroxylysyl residues in kidney collagen in diabetes. The data thus agree with those of Westberg and Michael (1973) and Kefalides (1974a) and do not support the claim of Beisswenger and Spiro (1970). This short review of the biochemical studies in human diabetic GBM illustrates the lack of definitive knowledge of the biochemical basis of microangiopathy in this disease and emphasizes the need for careful, controlled studies in appropriate animal models.

VIII. SupramolecuIar Organization of Basement Membranes A model for the supramolecular organization of basement membranes is presented in Fig. 11. *Wefeel that this model is consistent with most of the available biochemical, biosynthetic, and immunological evidence. The main building block is the triple-helical basement membrane procollagenlike molecule depicted in Section V (Figs. 8 and 11). The procollagen-like molecules polymerize through the introduction of at least two types of covalent cross-linkage: intermolecular disulfide bonds and lysine (and/or hydroxy1ysine)derived cross-linkages. The disulfide bonds are probably located within the noncollagenous peptide extensions of the procollagen molecule, whereas the lysinederived cross-linkages probably are found within the collagenous domain. A third type of covalent linkage may also be present, although the chemical nature of this cross-linkage has yet to be elucidated. This linkage is probably located within the noncollagen portion of the procollagen molecule and may be of importance in the polymerization of the basement membrane procollagen. In addition to the polymerized procollagen, genetically distinct, noncollagenous glycoproteins also may be present within the basement membrane matrix. These molecules would be linked to the procollagen molecule via disulfide bonds

219

BASEMENT MEMBRANES

Formation of microfibrils

07'I

Aggregates of microfibrils

-

:

-:.

~

Aggregates of microfibrils plus noncollagen glycoprotein

hw)

Example: Lens Capsule

FIG. 11. Diagram of a hypothetical model of the supramolecular organization of basement membranes. The newly synthesized and secreted procollagen molecule is composed of three identical (Y chains having noncollagen extensions at both the amino and carboxyl termini. There are interchain disulfide cross-links at the carboxyl terminus and intrachain disulfide cross-links at the amino terminus. Procollagen molecules a~ thought to form microfibrils which are stabilized by intermolecular disulfide cross-links (-S-S-) and covalent cross-links involving lysine and/or hydmxylysine (solid line). The microfibrils polymerize to form a larger matrix, as is the case in lens capsule. The.presence of a distinct noncollagenous glycoprotein within the basement membrane matrix has been postulated for certain tissues. This glycoprotein would be linked to the procollagen molecules via disulfide bonds and, possibly, through other types of covalent cross-linkages. It is suggested that the proportion of the noncollagenous glycoprotein varies among basement membranes including GMB, Descemet 's membrane (DM), and rat PYS.

and possibly through other types of covalent cross-linkages. It is not yet clear whether these glycoproteins are present in every basement membrane, for it is possible to account for the entire amino acid composition of at least one basement membrane, for example, the ALC, as a polymerized procollagen-like molecule without having to postulate the presence of distinct glycoprotein molecules. With the use of this model, it can be seen that proteolytic enzymes such as pepsin would digest away the noncollagen glycoproteins and procollagen extension peptides, leaving behind the pepsin-resistant, triple-helical collagenous portions of the procollagen chains associated with each other through the disulfide and lysine-derived cross-linkages which were not destroyed during the pro-

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NICHOLAS A. KEFALIDES ET AL.

teolysis. Similarly, treatment with bacterial collagenase would result in the formation of a series of glycopeptides. One of these would be derived from each of the noncollagen glycoproteins present within the basement membrane. Others would be derived from the noncollagerious extension peptides present at the ends of the procollagen-like molecules. If the structure of the individual chains of the procollagen-like molecule is analogous to that of type I procollagen, it would be expected that one peptide would represent the amino-terminal extension peptide and one peptide would represent the carboxyl-terminalextension (see Figs. 8 and 11). The latter peptides would probably be isolated as a triple-stranded peptide joined by disulfide bonds. Because of the possibility either or both of the extension peptide fractions could be cross-linked further by nondisulfide, covalent cross-linkages, it has not yet been established that a given glycopeptide fraction isolated after collagenase digestion of a basement membrane is derived from procollagen extension peptides or from a noncollagen glycoprotein. The crosslinking also complicates the interpretation of immunological data (Kefalides, 1972a) which suggested the presence of at least two immunologically distinct glycoproteins, one of high molecular weight and one of low molecular weight. It is probable that the multitude of heterogeneous fractions observed after reduction and akylation of basement membranes is attributable to a large extent to the random nature of the nondisulfide cross-linkages. Finally, it must be recognized that in vivo basement membranes undergo a degree of metabolic turnover. As pointed out in Section V, this process may proceed at different rates for the different molecular portions of the basement membrane, leading to still more heterogeneity.

IX. Summary Basement membranes or basal laminae are ubiquitious in the vertebrate and invertebrate world and constitute important structural and functional extracellular matrices. Basement membranes are composed of fine fibrils, 40-60 in diameter, arranged randomly in a granular matrix. Three general functions have been ascribed to basement membranes; they are thought to (1) act as a semipermeablefilter, (2) provide support, and (3) serve as a boundary between cell types or between cell layers and the underlying connective tissue. Physicochemical studies have shown that basement membranes are rather insoluble in buffers at neutral pH, although small amounts of material can be brought into solution with acid buffers. Complete solubilization can be accomplished by reduction and alkylation in the presence of denaturant or after limited proteolysis. Structural studies have demonstrated the presence of collagen molecules in basement membranes. These apparently exist as triple-helical procollagen molecules composed of identical pro-a chains. Collagenous peptides

BASEMENT MEMBRANES

22 1

equivalent in size to a chains can be isolated from digests of basement membranes. Noncollagenous glycoproteins are thought to be present in the matrix of most basement membranes; they are linked to the procollagen molecules via disulfide bonds and lysine- and/or hydroxylysine-derived cross-links. Metabolic studies indicate that basement membrane collagen is, like type I interstitial collagen, initially synthesized in a precursor form, procollagen. The molecular structure of basement membrane procollagen is consistent with a triple-helical collagen molecule having non-triple-helical precursor-specific appendages at the amino and carboxyl termini. Unlike interstitial procollagens, basement membrane procollagen does not undergo a time-dependent conversion to a smaller molecular form. Immunochemical studies have shown that there is immunological crossreaction between homologous and heterologous basement membranes. At least three major antigenic components have been identified: one noncollagenous, a second collagenous, both found on procollagen peptides, and a third, a noncollagenous glycoprotein independent of procollagen. The biochemical basis of basement membrane changes in diseases such as glomerulonephritis and diabetes mellitus is at present unknown. Compositional analyses of human GBM in a variety of glomerulopathies and in animal models of GBM disease produced incomplete and contradictory data. In diabetes mellitus the thickening of basement membrane may result from either increased deposition or decreased turnover. The collagenous component appears to be qualitatively unchanged. Future studies on basement membranes should be directed toward unraveling its structural organization, understanding its function in embryonic and adult tissues, and elucidating the steps which control their synthesis and secretion.

ACKNOWLEDGMENTS This work was supported by NIH Grants AM-20553, HL-15061, and HL-18827 from the U.S. Public Health Service.

REFERENCES

Alper, R.,and Kefalides, N. A. (1974). Eiochem. Eiophys. Res. Commun. 61, 1297-1303. Alper, R., and Kefalides, N. A. (1978a). In “Biology and Chemistry of Basement Membranes” (N. A. Kefalides, ed.), pp. 239-252. Academic Press, New York. Alper, R., and Kefalides, N. A. (1978b). In “Cellular and Biochemical Aspects in Diabetic Retinopathy” (F. Regnault and J. Duhault, Eds.), pp. 161-169. North-Holland, Amsterdam. Arbogast, B., Gunson, D. E., and Kefalides, N. A. (1976). J. Immunol. 117, 2181-2184. Banerjee, S . D., Cohn. R. H.,and Bemfield, M. R. (1977). J . Cell Biol. 73,445-463. Bardos, P.,Muh, J. P., Luthier, B., Devulder, B., and Tacquet, A. (1976). Comp. Eiochem. Physiol. 53B, 49-56.

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Pierce, G. B. (1970). In “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balms, ed.), pp. 471-506. Academic Press, New York. Pierce, G. B., and Johnson, L. D. (1971). In Virro 7, 140-145. Pierce, G. B., Jr., Midgley, A. R., Jr., Sri Ram, J . , and Feldman, J . D. (1962). Am. J . Parhol. 41, 549-566. Pierce, G. B., Jr., Midgley, A. R., Jr., and Sri Ram, J. (1963). J. Exp. Med. 117, 339-347. Pierce, G. B., Jr., Beals, T. F., Sri Ram, J., and Midgley, A. R., Jr. (1964). Am. J. Purhol. 45, 929-961. Pineda, A. (1965). Neurology 15, 536-547. Price, R. G., and Spiro, R. G. (1977). J . Biol. Chem. 252, 8597-8602. Priest, R. E. (1970). Nurure (London) 225, 745-746. Priest, R. E., Priest, J. H., Moinuddin, J. F., and Keyser, A. J . (1977). J . Med. Gener. 14, 157-162. Prockop, D. J., Berg, R. A., Kivirikko, K. I., and Uitto, J . (1946). In “Biochemistry of Collagen” (G. Ramachandran and A. H. Reddi, eds.), pp. 164-273. Plenum, New York. Ramachandran, G. N., and Ramakrishnan, C. (1976). In “Biochemistry of Collagen” (G. N. Ramachandran and A. H. Reddi, eds.), pp. 45-84. Plenum, New York. Rennke, H. G., Cotran, R. S., and Venkatachalam, M. A. (1975). J . Cell Biol. 67, 638-646. Risteli, J . , Koivisto, V. A., h e r b l o m , H. A,, and Kivirikko, K. I. (1976). Diabetes 25, 1066-1070. Romen, W., Schultze, B., and Hempel, K. (1976). Virchows Arch. B 20, 125-137. Ryan, G . B., and Kamovsky, M. J. (1976). Kidney Inr. 9, 36-45. Sato, T., and Spiro, R. G. (1976). J. Biol. Chem. 251, 4062-4070. Sato, T., Manakata, H., Hoshinaga, K., and Yosizawa, 2. (1975). Clin. Chim. Acru 61, 145-150. Scheinman, J. I., Fish, A. J., and Michael, A. F. (1974). J . Clin. Invest. 54, 1144-1154. Schneeberger-Keeley, E. E., and Kamovsky, M. J. (1968). J. Cell Biol. 37, 781-793. Schwartz, D., and Veis, A. (1978). FEES Len. 85, 326-332. Shibata, S., Nagasawa, T., Miyakawa, Y.,and N a m e , T. (1971). J . frnmunol. 106, 1284-1294. Slavkin, H. C., and Bringas, P., Jr. (1976). Dev. Biol. 50, 428-442. Sorgente, N., Brownell, A. G., and Slavkin, H. C. (1977). Biochem. Biophys. Res. Commun. 74, 448-454. Spiro, R. G. (1967a). J . Biol. Chem. 242, 1915-1922. Spiro, R. G. (196%). J . Biol. Chem. 242, 1923-1932. Spiro, R. G. (1967~).J. Biol. Chem. 242,4813-4823. Spiro, R. G. (1972). In “Glycoproteins” (A. Gottschalk, ed.), 2nd ed.,Part B, pp. 964-999. Elsevier, Amsterdam. Spiro, R. G . (1973). N . Engl. J . Med. 288, 1337-1342. Spiro, R. G. (1976). Diubetologiu 12, 1-14. Spiro, R. G., and Fukushi, S. (1969). J. Biol. Chem. 244, 2049-2058. Striker, G., Killen, P. D., Agodoa, L. C. Y.,Savin, V., and Quadracci, L. D. (1978). In “Biology and Chemistry of Basement Membranes” (N. A. Kefalides, ed.), pp. 319-334. Academic Press, New York. Tanaka. T., Noda, S., Kawakami, I., and Sato, A. (1976). Dev. Growrh Direrent. 18, 267-272. Tanzer, M. L., and Kefalides, N. A. (1973). Biochem. Biophys. Res. Commun. 51, 775-780. Thomas, P. K. (1964). 1.Cell B i d . 23, 375-382. Thorning, D., and Vracko, R. (1977). Lab. Invest. 37, 105-119. Timpl, R., Martin, G. R., Bruckner, P., Wick, G., and Wiedemann, H. (1978). Eur. J . Biochem. 84,43-52. Todd, R. B., and Bowman, W. (1857). “The Physiological Anatomy and Physiology of Man,” pp. 129, 523, and 660. Blanchard & Lea, Philadelphia. Trelstad, R. L., and Lawley, K. R. (1977). Biochem. Biophys. Res. Commun. 76, 376-384.

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Trelstad, R. L., and Slavkin, H.C. (1974). Biochem. Biophys. Res. Commun. 59, 443-449. Trelstad, R. L., Hay, E. D., and Revel, J. P. (1967). Dev. Biol. 16,78-106. Trelstad, R. L., Hayashi, K.,and Toole, B. P. (1974). J . Cell Biol. 62, 815-830. Tryggvason, K. (1977). Eur. J . Clin. Invest. 7, 177-180. Uitto, J., and Lichtenstein, J. R. (1976). J. Invest. Dermatol. 66, 59-79. Venkatachalam, M. A., Kamovsky, M. J., and Fahimi, H. D. (1970). J . Exp. Med. 132, 11531167. Vernier. R. L. (1964). In “Small Blood Vessel Involvement in Diabetes Mellitus” (M. D. Siperstein, A. R. Coiwell, and K. Meyer, eds.), p. 57, Am. Inst. Biol. Sci., Washington, D. C. von Bruchhausen, F., and Merker, H. J. (1965). Naunyn-Schrniedebergs Arch. Pharmakol. 251, 1-12. von Hempel, E., and Geyer, G. (1967). Anat. Anz. 120, 84-90. Vracko, R. (1974a). Diabetes 23, 94-104. Vracko, R. (1974b). Am. J . Puthol. 77, 314-346. Vracko, R., and Benditt, E. P. (1972). J. Cell Biol. 55,406-419. Walker, F. (1973). J. Pathol. 110, 233-244. Wehner, H.,and Anders, E. (1969). Verh. Dtsch. Ges. Path. 53, 380-383. Welling, L. W., and Welling, D. J. (1978). Am. J . Physiol. 234, F54-F58. Westberg, N. G. (1976). Diabetes (Suppl. 2) 25, 920-924. Westberg, N. G., and Michael, A. F. (1970). Biochemistry 9, 3837-3846. Westberg, N. G., and Michael, A. F. (1973a). Acru Med. Scand. 194, 39-47. Westberg, N. G., and Michael, A. F. (1973b). Acra Med. Scand. 194,49-57. Williams, I. F., Hanvood, R., and Grant, M. E. (1976). Biochem. Biophys. Res. Commun. 70, 200-206. Wislocki, G. B., and Padykula, H. A. (1953). Am. J. Anat. 92, 117-151. Wong, G., Killen, P., Quadracci, L. J., Striker, G. E., and Huang, T. (1972). J . Cell Biol. 55, 288a. Woolley, D. E., Glanville, R. W., Roberts, D. R., and Evanson, J. M. (1978). Biochem. J . 169, 265-276.

NOTEADDEDIN PROOF.Several papers with data relevant to the structure and metabolism of basement membranes have recently been published. Basement membrane procollagens from rat lens capsule [J. G. Heathcote, C. H. J. Sear, and M. E. Grant. (1978). Biochem. 1. 176,283-2941 and human amniotic fluid cells [E. Crouch and P. Bomstein. (1979). J . Biol. Chem. 254,4197-42041 have recently been characterized. In addition, characterization of basement membrane-related glycoproteins has been reported [A. E. Chung, R. Jaffe, I. L. Freeman, J.-P. Vergnes, J. E. Braginski, and B. Carlin. (1979). Cell 16, 277-287; R. Timpl, H. Rohde, P. G. Robey, S. I. Rennard, J.-M. Foidart, and G. R. Martin. (1979). J. B i d . Chem. (in press)], and an enzyme with a specificity for cleavage of basement membrane collagen has been documented [L. A. Liotta, S. Abe, P. G. Robey, and G. R. Martin. (1979). Proc. Natl. Acud. Sci. U.S.A. 76, 2268-2272).

INTERNATIONAL REVIEW OF CYTOLOGY,VOL. 61

The Effects of Chemicals and Radiations within the Cell: An Ultrastructural and Micrurgical Study Using Amoeba proteus as a Single-Cell Model M. J. ORD Department of Biology, The University of Southampton, Southampton, and Medical Research Council Toxicology Research Unit, Carshalton, Surrey, England

I. Introduction

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

11. Micrurgy . . . . . . . . . . . . . . . . . . . . . 111. The Site and Mode of Action of Toxic Agents within the Cell.

.

A. The Ameba Model: Using Chemicals in a Single-Cell System B. Cell Damage Characteristic of Particular Groups of Chemicals IV. Chemicals as Probes into Cellular Activities. . . . . . . . . A. General Nuclear and Cytoplasmic Damage . . . . . . . B. Specific Damage within the Nucleus or to Its Membrane . . C. Specific Damage within the Cytoplasm or to the Cell Membrane V. General Conclusions . . . . . . . . . . . . . . . . . VI. Evaluation of Amoeba proteus as a Model in Cell Biology and Toxicology . . . . . . . . . . . . . . . . . . . . A. Cell Biology . . . . . . . . . . . . . . . . . . B. Toxicology . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

229 23 1 233 233 231 248 249 250 255 273 214 214 276 211

I. Introduction The ability of Amoeba to withstand extensive micrurgy makes it invaluable as a model for studying the action of chemicals within the cell. With use of the nuclear transplantation technique not only is it possible to locate and assess damage to the nucleus and/or cytoplasm but, by using a series of transfers at different times after treatments, interactions between treated nuclei and cytoplasms can also be assessed. Examinations of this type are important both in understanding repair mechanisms and in predicting possible synergism between environmental chemicals and/or drugs in medical treatments. The ameba is not only amenable to nuclear transfers but also to microinjection. Injection systems in the past have been used principally to learn more about the ionic composition and pH of the cytoplasm (Kassel and Kopac, 1953; Chambers and Chambers, 1961; Lorch, 1973) and in inheritance studies (Hawkins and 229

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Cole, 1965; Hawkins, 1969, 1973a,b, 1976); Lorch and Jeon, 1969; Jeon and Lorch, 1973; Chatterjee and Rao, 1974). The need for a method of dosing cells with nonaqueous chemicals or chemicals having very short half-lives has more recently resulted in the use of injection for chemical treatments. Once technical difficulties-particularly in the accuracy of controlling the volume of a substance being injected-have been overcome, the future of microinjecting holds exciting possibilities. Not only can the results of direct application of injurious drugs and chemicals be studied, but treated cells can be subjected to injections of metabolic chemicals in attempts to reverse drug effects (cellular enzymes, substrates, or ions), or of control cell components in attempts to identify sites of critical damage (mitochondria, ribosomes, protein fractions, and so on). Future Amoeba experiments with chemicals could involve such combined operations as treatment of an ameba with a chemical, followed by stratification of its cell components by centrifugation, removal of some components by cutting, injection of a control cell fraction to replace the treated components removed by cutting, and exchange of the nucleus with that of a control cell. Operations of equal complexity in inheritance studies involving cell membrane, cytoplasm, and nuclear components from three different strains of Amoeba have already proved possible (Jeon et al., 1970). Although much can be learned using micrurgy with a subsequent study of the behavior and viability of the living cell, simultaneous study of living and fixed cells after chemical treatments is likely to prove far more illuminating than either micrurgy or ultrastructural studies on their own. At present, electron microscopy of amebas is in its infancy. The normal morphology of each cell component, with its cell cycle variations, must first be known in order to provide a reliable basis for comparative work on cell injury. As this basis gradually develops for each cell component, morphologically recognizable changes characteristic of particular cell lesions are emerging. Detailed studies of components such as the Golgi body (Flickinger, 1969, 1971a,b, 1972a; Wise and Flickinger, 1970a,b, 1971), mitochondria (Flickinger 1968a, 1973a,b; Smith, 1978a,b; Smith and Ord, 1979; Smith et al., 1979), and ribosomes (Ord, unpublished) are revealing links between physiological and morphological changes. The charting of an injured cell’s ultrastructural abnormalities should in time result in a means of identifying the site or cause of injury by a toxic agent. Amoeba can serve well as a research tool both in human toxicological studies, where the experimenter is generally interested in acute poisoning effects, and in ecotoxicology, where interest is more frequently directed toward continuous low-dose pollution possibly involving interactions between several chemicals. The ameba’s qualities-size, toughness, single cloning, handling ease for fixation, and nonspecialization-allow a unique opportunity to gain information about the site of action of chemicals within the cell. Where the main interest lies in the effects of toxic substances on humans, whether through medication or in the course of their daily work or life, information gained from ameba studies

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does not repeat, but rather complements, that gained from experiments using mammalian models; in particular, it helps in the interpretation difficulties encountered at the cellular level in multicellular systems. Where the main interest lies in the biosphere, it should be stressed that Amoeba is a free-living, singlecelled organism, and as such forms one of the many small links in the web of living organisms which create a balanced ecosystem; from it therefore predictions concerning other links in this web may become possible.

11. Micrurgy

The ameba nuclear transfer system is precise; two carefully selected cells are brought together using a microhook, and the nucleus of one (the donor cell) is pushed directly into the cytoplasm of the other (the host cell) with a microneedle (Comandon and deFonbrune, 1939; Jeon, 1970). A variety of nuclear and cytoplasmic combinations are possible using this technique, with the host cytoplasm either having its nucleus pushed out prior to the addition of the donor nucleus or having its nucleus retained along with the donated nucleus. The following nucleus and cytoplasm combinations illustrate the scope of biological and toxicological problems being tackled using Amoeba. 1. Homologous hybrids-the nucleus of one ameba plus the cytoplasm of a second ameba of the same strain. These hybrids are excellent for determining the interdependence of nuclear and cytoplasmic organelles (Ord, 1968a; Flickinger, 1969, 1973b; Jeon, 1975), the site of action of a chemical or radiations within the cell (Ord and Danielli, 1956a,b; Ord, 1968b, 1971a, 1974, 1976a; Hawkins and Willis, 1969a,b; Lorch and Jeon, 1969; Nikolaeva and Kalney, 1973; Chatterjee and Bhattachajee, 1975, 1976), the movement of chemicals such as toxic metals from one site to another within the cell (Ord and Al-Atia, 1979), and the interrelationship between the nucleus and cytoplasm in synthetic and repair activities (Ord, 1976a,b; Rao and Chatterjee, 1974). 2. Heterologous hybrids-the nucleus of one ameba plus the cytoplasm of a second ameba of a different strain. These hybrids are used in inheritance studies both for investigating nuclear and cytoplasmic inheritance patterns and for determining the nature of the lethal factor (Lorch and Danielli, 1950, 1953a,b; Danielli et a f . , 1955; Hawkins and Cole, 1965; Hawkins and Wolstenholme, 1967; Jeon and Lorch, 1973; Makhlin and Yudin, 1969, 1970; Ord, 1970, 1973a,b; Makhlin, 1971; Sopina, 1968, 1975; Yudin and Sopina, 1970; Yudin er al., 1971) and in studies of cytoplasmic organelles (Flickinger, 1970, 1973b, 1974b; Jeon and Jeon, 1975). 3. Homokaryons-the nucleus of one ameba transferred into a second whole ameba of the same strain. These are used to study the behavior of two nuclei in an identical cytoplasmic environment, for example, their ability to incorporate

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tritiated nucleic and amino acids when the homokaryon is exposed to these chemicals (Ord, 1970, 1971a) and the movement of such substances between the nucleus and cytoplasm or between nuclei-in studies where one of the nuclei contains a prelabeled component or where one nucleus has first been damaged by chemicals or radiations (Goldstein and Prescott, 1967, 1968; Prescott and Goldstein, 1968; Goldstein et al., 1973; Goldstein, 1974; Yudin and Neyfakh, 1973; Chatterjee, 1975; Ord, 1976a). 4. Heterokaryons-the nucleus of one ameba transplanted into a second whole ameba where host and donor cells may be of different strains, for example, composite cells with strain differences used to study nucleus-nucleus interactions in interspecific crosses, or where host and donor cells have other differences such as cell cycle phase, for example, composite cells with phase differences used to determine the mechanism of initiation, maintenance, and termination of DNA replication (Ord, 1969, 1970, 1971a, 1979a; Makhlin and Yudin, 1969; Makhlin, 1971; Yudin et al., 1971; Yudin, 1973). 5. The Amoeba system gives further scope for probing cell function by allowing separation of the nucleus and cytoplasm. Thus an isolated nucleus can be exposed to media of different compositions, stressed in different ways, or treated with chemicals before combining with anucleate cytoplasm (Ord and Bell, 1970; Ord, 1973a). Such experiments give a means (a) of determining the ionic strength and composition required to retain nuclear viability, (b) of exploring the normal functioning of the nucleus and its reaction to stress, and (c) of separating the direct and indirect effects of a chemical on the nucleus. Alternatively, anucleate cytoplasm can be used to obtain information on the maintenance of cell components in the absence of nuclear activity, their reaction to stress, and the direct effect of chemicals on them (Ord, 1968a; Flickinger, 1971a; Goldstein and KO, 1975; Jeon and Jeon, 1975; Chatterjee and Bell, 1976). 6. Binucleate cytoplasms as “test tubes. ” A Gz binucleate ameba has a volume of cytoplasm sufficient to allow the addition of up to 12 to 15 nuclei. Though these amebas would be nonviable in the long term, cell activities continue for hours or days. Such cytoplasm acts as a medium or test tube for studies of nuclear function (Ord, 1970, 1971). Alternatively Gz binucleate cytoplasm containing no more than 2 to 6 nuclei (a viable cell) can be used to determine the role of the nucleus in cell movement (Anderson, 1973) or its behavior at division (Ord, 1971a). 7. The cell components of living amebas can be stratified by centrifugation and the resultant amebas quickly cut to give living one-half, one-third, or onequarter cells. With careful cutting different cell components can be removed and their importance in cell functioning or cell repair determined (Daniels and Breyer, 1970; Hawkins, 1969, 1973b). 8. Amebas tolerate extraction of material by microsyringe. Studies using cytoplasmic transfers have been utilized in inheritance studies, particularly in

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determining the site of cell change which conveys drug sensitivity or resistance of an epigenetic nature (Hawkins and Cole, 1965; Hawkins, 1968; Hawkins and Hainton, 1972; Jeon et al., 1970). 9. Substances can be injected into amebas, for examples, cell metabolites, injurious chemicals, and cell fractions. Such studies are used both in understanding normal cell functioning and in studying the action of toxic substances (Goldacre and Lorch, 1950; Kassel and Kopac, 1953; Lorch, 1973; Jeon and Lorch, 1973; Flickinger, 1974a; Ford, 1976; Smith, 1978a).

III. The Site and Mode of Action of Toxic Agents within the Cell A. THEAMEBAMODEL:USINGCHEMICALS IN A SINGLE-CELL SYSTEM Chemicals are used in ameba studies for two distinct purposes: to investigate the site and mode of action of the particular chemical or of a group of chemicals, and to help to understand how different cell components function. It seems appropriate at this point therefore to divide this article into two sections: (1) a section dealing with the protocol being developed for use in investigating the activity of a chemical, and the information gradually emerging on particular groups of chemicals-carcinogens, antibiotics, uncouplers of oxidative phosphorylation, metals, and radiations; and (2) a section dealing with particular cell components whose normal and abnormal functioning is becoming better understood through the use of chemicals as probes. One of the very considerable advantages offered by a single-cell model is the repeatability of the dose; that is, a cell can be exposed to a certain concentration of a chemical for a defined time when in a known nutritional state and at a defined age or cell cycle phase. Breakdown of the chemical, and changes in the pH of the exposure medium can be checked during the course of the experiment. Electron microscope (EM) monitoring of cellular damage is possible both during treatment and at different times after treatment, giving a time course study of morphological changes taking place in each cell component. The importance of such a study when following changes in cell injury is shown in an EM study on ribosomes after treatment with N-methyl-N-nitrosourethane (MNU) (Fig. 1). Treatment regimes for use with the Amoeba model can be made far more precise than is possible with treatments of multicellular models. In a mammalian system, though the age, nutritional state, and sex of the animal can be controlled and a known dose of a chemical given, the distribution of the chemical to various tissues and its speed of detoxification cannot be controlled. There is little opportunity within a tissue to assess the actual dose received by a cell unless labeled chemicals are used in treatments. In EM studies it is frequently impossible to state the viability of individual cells when attempting to evaluate the importance

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HIGH

LOW

I

e n d of treatment

TIME Irnin)

FIG. 1 . A diagrammatic representation of the loss of attachment of the ribosomes from free and bound polysomes, and the loss of attachment of the nuclear riboprotein helices, after an 8-minute exposure of amebas to I mM MNU. The time course change was followed by fixing treated cells at 1-minute intervals (from treatment to 4 minutes), 2-minute intervals (from 4 to 20 minutes), and 5-minute intervals (from 20 to 60 minutes) after MNU exposure. Whereas at 2 minutes after treatment more than 90% of the ribosomes were attached as free or bound polysomes and nuclear helices were attached and located chiefly inside the nucleus, by 60 minutes after treatment more than 90% of the ribosomes were present as monosomes and almost all the nuclear helices were detached and had moved to the cytoplasm. 1, Free ribosomes; 2, riboprotein helices; 3, bound polysomes (RER), 4, free polysomes.

to be given to a cell component's loss or damage. With the single-cell model, however, gradations and variations in effects on cell components at a wide range of dose levels can easily be studied. Such a study is shown after treatment of Amoeba with MNU in Fig. 2. It is obvious from this figure that EM studies carried out at different dose levels suggest different ultrastructural damage by MNU and that multidose sampling is necessary when interpreting the results. The concept of dose as a combination of both concentration and exposure time (Ord, 1968b) may be difficult to grasp for workers using whole-animal systems. Though it is of considerable importance in these systems, since it may govern the distribution of a chemical within an animal's tissues, the degree to which different degradation pathways for the chemical are active (Gehring, 1977), or the availability of an efficient or an overworked cellular repair system, there is little opportunity to control it beyond the acute or chronic dosing of the animals. In single-cell systems both concentration and time are accurately controlled. Experiments with amebas show that the relationship between the two is not always a direct one. For example, the relationship between concentration and time is to 5 X lop4M direct with MNU treatment over a working range of 2 x but at concentrations above 2 X M cell components not normally affected can be severely damaged (e.g., the Golgi bodies), while at concentraM cell repair systems cope more efficiently with tions below 5 X

235

CHEMICALS AND RADIATIONS WITHIN THE CELL

-

DOSE

concentratmn

‘exposure

a

L E T HALITY/’

(mM1

ltme fmin)

5

2

I

6

12

0.5 0.25 0.1 0.05 0.03 0.01 0.0050.001 25

30

30

30

30

30

30

30

- phase

‘GZ-phase

1 b Ribosome

pattern

c Ri boprotein helices

.......

........

1

1

d Mitochondrial change

e Golgi bodies

FIG.2. A study combining ultrastructural and living cell changes observed after the treatment of amebas with MNU over a wide range of doses. The results are presented diagrammatically. By combining each concentration (Fist line) with its exposure period (second line) and following the vertical arrows, the comparative division and viability losses can be correlated with intracellular changes observed in the ribosomes, mitochondria, Golgi bodies, and ribonucleoprotein helices. (These in turn have been correlated in autoradiography studies with changes in DNA, RNA, and protein syntheses as indicated through loss of incorporation of tritiated nucleic or amino acids.) Living cell studies have shown repeatedly that (1) a 4 mM concentration of MNU for 5 minutes kills all cells, death seldom being delayed for more than 10-14 days; (2) a 2 mM concentration for 6 minutes, 1 mM concentration for 12 minutes, or a 0.5 mM concentration for 25 minutes produces long division delays, abnormal divisions, mutations, and approximately 50% mortality; and (3) concentrations of 0.25-0.001 mM each for 30-minute exposures cause decreasing division delays and decreasing mortality. The persistent patterns of low protein and RNA syntheses (as indicated by the loss of ribosome attachment and the absence of ribonucleoprotein helices in the nucleus), the sluggish behavior, and the slow return of normal division timings, may all relate to mitochondrial dysfunction, since the repair of damaged organelles and return of normal cell activities is dependent on energy being available in sufficient quantities to supply all cell functions and organelles. Horizontal bars indicate the following. (a) Lethality: loss of 50% or more of the cells; cells divide but do not form clones; note that S-phase cells are sensitive at lower dose levels than G,-phase cells. (b) Ribosome pattern: “OFF” ribosomes have detached from free polysomes and from ER; “ON-OFF” ribosomes have detached from some polysome formations,giving approximately 50%as free ribosomes and 50% attached to free or attached polysomes. (c) Riboprotein helices: helices have detached from their nuclear sites and moved into the cytoplasm; no nuclear replacement occurs for several days. (d) Mitochondrial change: form and density changes indicate persistent mitochondrial damage. (e) Golgi bodies: there is a breakup of the cisternae of the Golgi bodies; these are soon replaced. (f)Division delays: these occur between treatment and the F i t division after treatment and between the f i t and second divisions after treatment.

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some damage as it occurs (e.g., DNA damage). The relationship between concentration and exposure time with metal ions is more complex, and we have evidence to suggest that many factors are involved both in the amount of chemical taken in by the cell with time and with its secretion at safe sites within the cell (Ord and Al-Atia, 1979). An equally important factor which can be taken into account in single-cell work is the state of the individual cell; sufficient information is now available about the structure and the rapidity of changes in the cell membrane and the spectrum of internal changes occurring throughout the cell cycle to alert any cell biologist to the dangers of comparing the effects of a chemical or of radiations on two cells in different phases of the cell cycle. The feeding state of a single cell can also be accurately controlled, with both overfed, fragile-membranedamebas and starved amebas being avoided in treatments where repair of cellular damage would be expected to make immediate demands on metabolic precursor pools and in particular on membrane precursor pools. Single-cell models, in particular the Amoeba model, offer advantages over mass cell test systems only if care is taken to use them precisely. The following factors should be taken into account when carrying out chemical treatments with amebas. 1. Both concentration and exposure time should be accurately measured. 2. Whenever possible short time exposures should be carried out on cells of known age or cell cycle phase. 3. Heavily fed cells, or cells starved for periods of longer than 24 hours, wheat cultured cells with their more dubious viability and feeding, and cells contaminated with detergents or metal ions through glassware or tap water should all be avoided unless used deliberately for a particular purpose. 4. The pH of the solution and the integrity of the chemical should be monitored throughout the exposure period. 5 . Whenever possible, EM monitoring should accompany a study, since an examination of mitochondria, ribosomes, and Golgi bodies during the course of the treatment will give immediate indications of changes in cell activity, for example, through interference with energy supplies or new membrane components. Such changes in the activity of the cell during treatment could change both the effectiveness of the chemical and/or of any repair taking place during treatment. 6. Where treatment must be carried out in a buffer, or at a temperature or pH differing from that of normal culturing conditions (e .g., to minimize degradation of the treatment chemical or to give maximum efficiency by working at the pK of the chemical), EM monitoring is particularly important to evaluate any stress effects due to the abnormal conditions themselves. 7. EM examination of the strain of amebas in use should be made to assertain whether the cells contain endosymbionts which may interfere with treatments.

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This is particularly important with some of the American strains of amebas where large numbers of endosymbionts have been reported (Roth and Daniels, 1961; Jeon, 1972; Jeon and Jeon, 1976). Cultures invaded by foreign molds (as opposed to those molds with which the ameba coexists), or infected with yeasts, should also be avoided, as should cultures treated with antibodies or fungicides. 8. Cells should be retained until either death or clone formation. The policy of assuming that all cells which are not growing or dividing at the end of 1, 2, or 3 weeks are either dead cells or are viable cells can both give misleading results. Some treatments can cause division delays of weeks, even months, but the small almost immobile cells are still viable (Ord, 1974); some treatments which prevent DNA replication may not produce cell death for 1-2 months (e.g., dichlorodiammineplatinum(II), nitrogen mustard, and MNU),

B. CELL DAMAGE CHARACTERISTIC OF PARTICULAR GROUB OF CHEMICALS It requires information from studies using large numbers of different chemical and physical treatments to allow generalizations to be made on the production of different types of cell injury. Amoeba experiments thus far have included less than 50 such agents. Far more work is required before ameba studies can be used in a general way to predict the type of injury one might expect after treatment with a chemical of known structure but unknown activity. Since work up to the present has involved treatments which can be related either through their origins (e.g., antibiotics), common activity (e.g., carcinogens), or physical characteristics (e.g., radiations), some diagnoses of cellular injury are attempted here. 1. Antibiotics and Related Compounds This work includes results obtained and integrated from investigations using actinomycin D (ACT-D) (Hawkins, 1968; Flickinger, 1968c; Lorch and Jeon, 1969; Ord, unpublished results), puromycin (Daniels and Breyer, 1970; Sanders and Bell, 1970; Hawkins, 1973a; Ord, unpublished results), chloramphenicol (Ord, unpublished results), streptomycin (Cole and Danielli, 1963; Hawkins and Cole, 1965; Kalinina, 1969a), neomycin and kanamycin (Hawkins and Willis, 1969b), erythromycin and chloroquine (Hawkins and Hainton, 1972), cycloheximide (Hawkins and Hainton, 197l ) , rifamycin (Hawkins, 1973a), mitomycin C (Ord, unpublished results), ethidium bromide (Flickinger, 1973b), and dimidium bromide (Hawkins and Willis, 1969a). Though these substances have a wide range of activities, details of which are not considered here, the following generalizations can be made. 1. Exposure to these antibiotics generally inhibits growth and division either through inhibition of transcription or translation machinery-more rarely through interference with the energy machinery of the cell. One exception is mitomycin C

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which may inhibit division without affecting growth, so producing giant cells 10 to 30 times normal size. 2. The effects of these antibiotics are generally reversible over a wide concentration range; that is, continual exposure is required to retain the effect of the drug. 3. Some of these chemicals have the ability to induce cell resistance and/or sensitivity of an inheritable nature. Such characteristics once induced are relatively stable, are passed on by the cytoplasm and not by the nucleus, and have some similarity to the epigenetic inheritance observed by Kalinina (1969b) for certain chemicals. Resistance to streptomycin and neomycin (of an inheritable nature) has been isolated in the ribosome or protein fraction of the cell; it can be passed on by injection (Hawkins, 1976) of this fraction and is destroyed by RNase. Its action could be due to a direct effect on the transcription of DNA, or to an irreversible change in the RNA or protein of the ribosome; alternatively it could be concerned with the induction and synthesis of a scavenger protein, such as the metallothioneins in poisoning by metals (Shaikh and Smith, 1976; Webb, 1976). The cell fractionation experiments of Daniels and Breyers (1970) to locate the radioresistance factor of some strains of amebas implicate the involvement of a similar fraction. 4. When antibiotics are used at very high concentrations over long periods of time, a point may be reached where the effect is no longer reversible and cell division can no longer be resumed on their removal. This point of irreversibility has been examined in detail only for one antibiotic, ACT-D, and investigation showed that at the point of irreversibility the cytoplasm was lethally damaged. However, this point depends on a combination of concentration and exposure times and, if the experiment is taken to just beyond the point of irreversibility, the nucleus too is lethally damaged. 5 . Higher doses of many antibiotics are required to produce effects on amebas than are required by tissue cells in general. This difference possibly relates to the growth habitat of the free-living ameba: an aqueous environment shared with many types of molds. To secure its survival the ameba must have evolved a means of avoiding the action of at least some mold allelochemics. Whether this has resulted in multisite defenses, for example, exclusion from the cell by the mucopolysaccharidecoat, degradation within the cell by enzyme systems, and/or structural differences in the ameba ribosome or mitochondrion which would exclude insertion of the poison at a critical site, presents an interesting challenge to evolutionists, and a possible tool for the cell biologist or ecotoxicologist. For the toxicologist who wishes to compare his or her results with the mammalian system, this phenomenon is not only irritating but interposes a warning note on the extrapolation of results. This warning applies not only to antibiotics but to all toxic agents studied experimentally, and not only to amebas but to other cell models, in particular those with altered or defective coats. Altered coats or

CHEMICALS AND RADIATIONS WITHIN THE CELL

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membranes may exclude some chemicals but allow the entrance of abnormally high concentrations of others; in so doing they may change the site of activity of the chemical, for example, from a possible membiane interaction in the normal cell to an internal interaction in the defective cell. 2 . Metals Detailed information is only available from studies on methylmercury chloride and cadmium chloride and nitrate, though a small amount of work has also been carried out on mercury chloride and on zinc nitrate and chloride. These studies are being performed in our laboratory (T. Ford, I. Minassian, G. R. Al-Atia, and M. J. Ord) and have not yet been published. However, their importance is already evident, and since this is a field where Amoeba offers an excellent model system, they require some explanation. Several important points arise from metal studies using Amoeba. They are based chiefly on the work with methylmercury and cadmium. 1. The effects of both methylmercury and cadmium are evident over very wide concentration ranges with amebas sensitive to continual exposure of methylmercury at levels as low as lo-'* M . 2. The sensitivity of the ameba is related to the amount of change in its immediate microenvironment, that is, whether or not it is exposed to a continually moving flow of the toxic metal in solution. Thus amebas growing in a dish of M methylmercury chloride can attach and divide, possibly by reaching an equilibrium with their adjacent microenvironment; amebas growing under gently shaking conditions where the microenvironment of each cell is continually changing and where new cell membrane is constantly exposed eventually die off. This difference is of great importance to the ecotoxicologist when considering safety levels, since it suggests (a) that animals in the still layers of lakes and oceans may take up (from the same concentration levels) lower doses of metals than those in constantly agitating layers, (b) that animals which feed by creating currents (flagellates, ciliates, molluscs, tunicates) may take in greater quantities of metal ions than other animals in the same environment, (c) that animals which produce pseudopods may internalize more metal through a recycling of their membranes. This dose differential, if extrapolated to humans, suggests that certain body cells carry a greater potential risk than others, for example, a pseudopod-forming cell with a constant recycling of cell membrane (such as macrophages and white blood cells), a pinocytosing cell with an uptake of fluid and recycling of membrane (such as cells of the placenta or nerve cells with regional pinocytotic activity in the synaptic regions), a cell with a continually changing environment (such as cells in the kidney). 3. There is some evidence to suggest that a preliminary exposure of amebas to low doses of cadmium (e.g., loF8M) produces a limited resistance when the cell

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is exposed to subsequent doses of the metal (Al-Atia, 1978); this may be similar to the synthesis of metallothioneins by vertebrate cells (Shaikh and Smith, 1976; Webb, 1976). 4. Nuclear transfer studies suggest that the greater sensitivity to both cadmium and methylmercury is in the cytoplasm-cytoplasmic membrane complex, but that nuclear damage of a reversible nature occurs (Section IV). These studies also suggest that the relative nuclear and cytoplasmic sensitivities of cells exposed to a high concentration of metal over a short period differ when compared with those of cells exposed to a low concentration of metal over a long period.

3 . Uncouplers of Oxidative Phosphorylation Three uncouplers of oxidative phosphorylation have been studied using amebas, and the results from this work compared with data obtained with the more general respiratory poison potasium cyanide (Smith, 1978a; Smith and Ord, 1979; Smith et al., 1979). Most of this work has involved EM or cytochemical studies, though nuclear transfers were done on dinitrophenol (DNP)-treated cells to check for nuclear damage. These studies have led to the following conclusions. 1. Exposure to uncouplers is a reversible treatment, providing exposure does not continue until the cells round up and begin to cytolyze; once removed from the uncoupler the cells gradually attach to the substratum, resume movement, and rapidly return to normal. Only near the point of cytolysis is there a short period when irreversible damage without immediate cytolysis is evident. For these cells the mortality curve is of type 2 (Fig. 3a). 2. EM studies show that the morphology of the mitochondrion is rapidly altered by uncouplers (Smith and Ord, 1979), but in spite of striking changes in shape and cristae it is reversible (at least with some uncouplers) within hours (Section IV). 3. Rapid removal of nuclei from cells at the “near to” or “just lethal” dose level of the uncoupler DNP showed that nuclei had not been lethally damaged. The reverse operation was not practical, however, as these cells on being given a control nucleus were unable to close the gaps behind the outgoing treated nucleus andlor the incoming control nucleus to prevent leakage of cytoplasm to the medium. This suggests a lack of membrane and/or microfiber activity (Jeon and Jeon, 1975) probably due to the lesion in the energy-producing machinery.

4, Mutagenic, Carcinogenic, and Chemostatic Chemicals These studies include a range of substances from those with strong mutagenic but weak to no carcinogenic activity to those which though having some mutagenic or carcinogenic activity have proved useful in chemotherapy, and to those which are both mutagenic and carcinogenic. By far the most detailed study

, 10

20

30

40

Days

Ibl

Treatment I

Days

FIG.3. (a) Theoretical mortality curves. Curve 1: Typical mortality curve when death is due to damage to the cell membrane; that is, cells gradually cytolyze, generally with little to no recovery of normal attachment or movement. Curve 2: Typical mortality curve when death is due to the inhibition of nuclear and cytoplasmic interaction, that is, the viability of cytoplasm when no nuclear activity is present to contribute to the upkeep of essential membrane systems. Curve 3: Typical mortality curve when death is due to the inhibition of DNA replication but when other nuclear activities including transcription are near normal. In practice, death is generally due to a combination of more than one type of damage, that is, nuclear plus cytoplasmic, with curves falling between the theoretical curves. In spite of this, the shape of the mortality curve frequently indicates the main site of cellular damage. (b) Division delays. Theoretical curves to ilIustrate typical division delays after treatment with chemicals. Curve 1: Normal doublingpattern for A . proreus. strain T, or Da, growing at 20°C. Curve 2: Extended cell cycle of amebas growing under suboptimal conditions, for example, at 17°C or in low concentrations of antibiotic which only partially affect protein or RNA syntheses. Curve 3: Small delay to frst division after treatment, followed by a return to normal cell cycle times; typical of cells with reversible cytoplasmic damage and little to no nuclear damage. Curve 4: Division delay maximal between treatment and the first division after treatment; further divisions may return to normal immediately or remain slower for a further two to four cell cycles; typical of S-phase cells treated with a DNA-damagingchemical (cytoplasmic damage would be masked by the long delay before the first division). Curve 5: Division delay maximal between first and second divisions after treatment; further divisions may return to normal or remain slower for a further two to three cell cycles (Cytoplasmic damage would show as an initial delay between treatment and first division after treatment); typical of cells treated with LD,, to LD,, doses of DNA-damaging chemicals such as nitrosamides.

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has been that carried out on MNU (Ord, 1968c, 1971b, 1973a, 1974, 1976b) and, as this is a very potent carcinogen in mammalian systems, some of the following points, which have been derived from a study of this chemical only, may not always apply to all related chemicals. Early nuclear transfer studies led in 1976 to a general hypothesis concerning damage by carcinogens stating ‘‘that the requirements for a potent carcinogen are not only that it should cause nuclear DNA damage but that it should also damage cytoplasmic enzymes. The added damage should be such that it neither precipitates cell death nor causes a complete blocking of cell activity. Figure 4 presents a theoretical representation of different combinations of nuclear and cytoplasmic damage. Only the nucleus/cytoplasm sensitivity ratio in Fig. 4a should result after treatment with a porenr carcinogen (Ord, 1976a). So far this hypothesis has stood the test of experimentation well. In a study using five mutagenic or carcinogenic chemicals Chatburn (1977) found a gradual movement of sensitivity from a nearly equal nuclear and cytoplasmic lethality with the chemicals methyl methanesulfonate and dimethyl sulfate to a high nuclear but low cytoplasmic lethality with N-methyl-N-nitrosourea ( M r e a ) . The ICI compound 2( a-chloroisopropylamino)ethyl naphthylene hydrochloride (Tucker, 1968), which was activated by using a rat microsome preparation with the ameba treatment (Chatburn, 1977), fell between these two sensitivities. A fifth chemical, hydrazine sulfate, known to be carcinogenic in mammalian systems, failed to show lethal nuclear damage in transfer experiments. This chemical had a high cytoplasmic or membrane sensitivity, resulting in lysis of many of the cells. This may prevent the expression of nuclear damage, though the presence of nonlethal nuclear damage was shown by the division delays of cells with a hydrazinetreated nucleus in control cytoplasm. The situation illustrated by hydrazine presents a problem not uncommon with chemicals which cause significant cytoplasmic damage. In such cases, if the interpretation of results rested only on the nucleuslcytoplasm lethality ratio, the conclusion would be that no nuclear damage had occurred. Though in some cases this may be true, in others, in particular those where amebas with treated nuclei transferred to control cytoplasms showed a delayed first or second division after transfer, nuclear damage occurred but was masked by cytoplasmic damage. A number of explanations can be considered when interpreting results for chemicals producing this set of events. (1) The nuclear association of the chemical may be reversible and the chemical rapidly diluted out by the control cytoplasm in nuclear transfer experimentsdivision delay reflecting the interval required to reverse the effect. (2) The repair of nuclear damage from these chemicals may make use of error-free systems, or damage to the DNA strand may be sufficiently well spaced to avoid errors overlapping during repair (Cleaver, 1967; Witkin, 1969; Bresler, 1975; Bridges, 1977). (3) The excessive damage to cytoplasmic membrane may either prevent the uptake of the chemical, or the damage to the ”

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Increasing dose

FIG. 4. A diagrammatic representation of how different degrees of nuclear and cytoplasmic damage can combine to cause cell death. The curves represent ( I ) nuclear mortality, (dotted line), (2) cytoplasmic mortality (dashed line), and (3) whole-cell mortality (solid line), that is, death caused by a combination of both nuclear and cytoplasmic damage. This type of information can be built up from experiments on Amoeba, where nuclear transplantation can take place between treated and control cells. (a) Damage to both nucleus and cytoplasm but with the nuclear damage greater than the cytoplasmic damage; (b) damage to both nucleus and cytoplasm, as in (a), but with no clear separation of damage, so that at most dose levels both lethal nuclear and lethal cytoplasmic damage contribute to cell death; (c) damage to both nucleus and cytoplasm, but with cytoplasmic damage greater than nuclear damage; (d) lethal nuclear damage only, with little if any cytoplasmic damage. (Taken from Ord, 1976a.)

cytoplasm may inhibit repair so that the transferred treated nucleus receives no repair until it reaches control cytoplasm. In any of these situations cytoplasmic and membrane damage could prevent the expression of nuclear damage whether in treated cytoplasm or removed to control cytoplasm. From studies with this group of chemicals the following points have emerged, which when taken together give a picture of the type of damage expected after treatment of amebas with a carcinogen. 1. Nuclear damage is generally greater than cytoplasmic damage (Fig. 5 ) . 2. Attachment of ribosomes to mRNA decreases (Section IV). This loss is gradual and affects free polysomes more quickly than bound polysomes (Fig. 1). The increase in the free ribosome/polysomeratio suggests that damage to cytop lasmic enzymes may be the result of an upset in the protein synthetic machinery of the cell. This effect is reversible but requires time. 3. At least with the potent carcinogens MNU and MNUrea this loss of attachment aIso extends to the attachment of the nuclear helices (a riboprotein com-

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CELL S U R V I V A L

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CHEMICALS AND RADIATIONS WITHIN THE CELL

245

plex present in the Amoeba nucleus and believed to be the mRNA and/or rRNA packaged for export to the cytoplasm (Stevens, 1967; Georgiev and Samarina, 1971; Minassian and Bell, 1976a; Wise er al., 1972). Whereas in the normal cell only a few helices leave the nucleus at a time, in the treated cell all helices loose their nuclear attachments, move to the cytoplasm, and are not replaced by new helices in the nucleus for 1-4 days (Section IV). 4. Mortality curves for amebas can be of three general types (Fig. 3a) dependent on whether the main lethal damage is to the membrane, the cytoplasm, or the nucleus. Mortality curves falling between these theoretical curves suggest combinations of nuclear, cytoplasmic, and membrane damage. The mortality curves for most of the chemicals in this group are of type 3 (i.e., death due to nuclear damage) or fall between types 2 and 3 (i.e., death due to both cytoplasmic and nuclear damage). 5 . All the chemicals used which have been shown to have carcinogenic potential in mammalian systems produced a nucleus-dependent cell division delay. This delay proved longer for the more potent carcinogens. It could be positioned between treatment and the first division after treatment or between the first and second divisions after treatment (Fig. 3b), depending on the cell cycle phase when treated (Ord, 1971b, 1974; Chatburn, 1977). A delay also occurs in the division of cells with a control nucleus in treated cytoplasm, but this is seldom of more than 4-5 days’ duration, changes little with cell age, and is not closely related to the potency of the carcinogen in mammalian systems. So far, results from division delays have paralleled those from nucleus/cytoplasm lethality ratios; that is, the very potent carcinogens MNU and MNUrea produce the longest delays. Division delays have proved useful indicators of nuclear damage where no lethality has occurred. 6. Treatments with chemicals which cause more obvious nuclear than cytoplasmic damage generally result in abnormalities in division. Doses of chemicals ~~~

~

~~

FIG. 5 . The nucleus/cytoplasm sensitivity ratios for six chemicals. (a) Hydroxylamine hydrochloride (2 m M for x hours) (Chatburn, 1977); (b) methylmercury chloride (0.1 mM for x minutes) (Ord. 1976a); (c) methyl methanesulfonate (2 mM for x hours) (Chatburn, 1977); (d) dimethyl sulfate (1 mM for x minutes) (Chatburn, 1977); (e) MNU (1 mM for x minutes) (Ord, 1976a); (f) MNUrea (2 mM for x hours) (Chatburn, 1977). The treatment dose was increased by increasing the exposure time while keeping the concentration constant (similar curves are obtained by keeping the time constant while varying the concentration); sensitivity was measured using lethality as the criterion; separate nuclear and cytoplasmic sensitivities were obtained by means of nuclear transfers between treated and control cells. (e) and (f) tit the model in Fig. 4a, that is, nuclear damage is greater than cytoplasmic damage; there is a clear separation between nuclear and cytoplasmic damage, but some cytoplasmic damage is present. These chemicals are potent carcinogens in mammalian systems. (c) and (d) show treatments where nuclear and cytoplasmic damage are not clearly separable. These chemicals are recognized carcinogens in mammalian systems but are not single-dose carcinogens as are (e) and (0. (a) and (b) produce lethal cytoplasmic or cytoplasmic membrane damage; nuclear damage, if present, is masked (Section III,B,4). These are chemical which are not generally considered carcinogenic.

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M. I. ORD

producing nuclear damage that allows DNA transcription but not replication, and cytoplasmic damage that does not completely disrupt the energy or protein synthetic machinery, may result in giant cells. If nuclear division is not accompanied by cytokinesis, multinucleate cells result (Fig. 13c). 7. Treatments with the nitrosamides MNU and MNUrea showed a distinct early S-phase sensitivity. Since transfers show the difference to depend on increased nuclear sensitivity only, S-phase sensitivity results in an even greater separation between nuclear and cytoplasmic damage and could prove useful in distinguishing the more potent carcinogens.

5 . Radiations There are considerable similarities in the effects of radiations on amebas whether irradiation is by excitation or ionization, whether ionizing radiations are electromagnetic or particulate, or whether the action of particulate ionization is through the production of recoil electrons or through recoil protons (see review Ord, 1973b). Differences occur, for example, in the relative biological effectiveness of different radiations, but these are generally dependent on the intensity of the radiation pathway (Daniels and Vogel, 1958) or the relative nuclear and cytoplasmic sensitivities and apply in particular to ionizing radiations as opposed to ultraviolet radiation (the latter being absorbed at certain wavelengths by proteins in addition to the nucleic acids). The cell lesions resulting from different types of radiations have more similarities than differences, both in relation to each other and in relation to the lesions produced by carcinogens. The following generalizations can be made from studies on amebas. 1. Amebas are more resistant to radiations than tissue culture cells, for example, an LD50 dose for mouse fibroblasts of 0.24 kr (Whitmore et al., 1961) compared with an LD50 dose for Amoeba proteus, strain PTI, of 120 kr (Ord and Danielli, 1956b). This resistance is not unique to amebas, or to protozoa, but can be found in the effective doses used for other cells, for example, LD5o doses for Arbacia eggs of 30 kr compared with LD50 doses for Arbacia sperm of 250 kr (Henshaw et al., 1933). 2. Different species and strains of amebas show an equal disparity in their resistance to radiations with X and y radiations; for example, the two species Chaos carofinensis and C . iflinoisensis differ by a factor of 10 in their LD50 doses (Daniels, 1955), and the two strains of A . proteus, L and C, by a factor of 6 (Kalney et al., 1974). 3. Dose is directly dependent on intensity (Kalney, 1967). This intensity dependence is not unique to amebas but is found with other types of cells (Casarett, 1968). 4. With radiations given at low temperature, the dose required to produce a 50% killing is decreased. This temperature effect disappears when radiations are given at a high intensity (Paribok et a f . , 1968) and suggests that repair is

CHEMICALS AND RADIATIONS WITHIN THE CELL

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concurrent with treatment when irradiation exposure times are sufficiently long. However, a further factor cannot be entirely ruled out, and that is that the cellular activity taking place at the time of treatment and during the subsequent repair period influences the effects of the radiation damage; activity is minimal at a low temperature. 5. Amebas are insensitive to hydrogen peroxide; with doses as high as 50 mg/ml for 2 hours no deleterious effects were observed (Paribok et al., 1968). There is little indirect effect of radiations through the medium, whether treatment is with ionizing or ultraviolet radiations (Mazia and Hirshfield, 1951; Paribok et al., 1968). 6. Radiation causes division delays. In general the delay occurs between treatment and the first division after treatment, but it can take place between the first and second divisions after treatment, particularly with the use of radioresistant strains. Division delays increase with increasing dose to LD,, levels (Ord and Danielli, 1956b, Kalney, 1967). 7. Radiations cause cytoplasmic damage, which in itself is capable of damaging a control nucleus but which is rapidly reversible (1-4 days in duration depending on the dose and method of producing radiations) unless supralethal doses of radiations are reached (Ord and Danielli, 1956b). During this period of cytoplasmic damage the cell membrane is fragile and lacks normal repair capacity. Membrane fragility is particularly noticeable with micrurgy when the cell is frequently unable to close the resultant gap on the exit or entrance of a nucleus. This suggests that either the membrane itself is damaged or that it lacks energy or microfiber activity which aids in drawing the hole together (Jeon and Jeon, 1975). 8. Ultraviolet studies suggest disruption of both RNA and protein syntheses. This effect can be almost eliminated if cells are kept for 2 hours at 6°C after irradiation (Skreb and Eger, 1967). In the absence of a nucleus this damage may not be reversible. 9. The nuclei of amebas, though highly resistant to radiations when compared with the nuclei of other cells, are two or more times more sensitive to lethal damage than cytoplasm. At least some of the nuclear damage is indirect, that is, is caused by the irradiated cytoplasm; indirect damage may partly result from faulty nuclear repair in the irradiated cytoplasm, as suggested by improved survival of nuclei when removed from irradiated cytoplasm within minutes of treatment, and partly result from a more general injury effect as shown by the ability of irradiated cytoplasm to injure even the control nucleus (Ord and Danielli, 1956b). 10. Ultraviolet microbeam exposures of nuclei only (Jagger et al., 1969) suggest that, if nuclei are surrounded by unirradiated cytoplasm both during and after irradiation, that is, are exposed to an environment where nuclear repair is efficiently undertaken, irreversible nuclear damage is not produced until doses reach very high levels.

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11. Nuclear transfers between a radioresistant strain of A . proreus, strain C , and a radiosensitive strain, strain L, show that the hybrid cells behave according to the nucleus they contain; thus C nuclei in L cytoplasm behave similarly to C nuclei in C cytoplasm, and L nuclei in C cytoplasm behave similarly to L nuclei in L cytoplasm (Nikolaeva and Kalney, 1973). 12. Amebas are sensitive to visible and near-ultraviolet light. A dark-cultured strain has been found which is highly sensitive to these wavelengths. Nuclear transfers show that the sensitivity is present in both the nucleus and cytoplasm, and that only the G2 nuclei can be protected if transferred to the cytoplasm of a resistant strain. It is not known yet whether this is an isolated case of sensitivity dependent on the growth without light, whether it is a repair deficiency, or whether it is due to a change, for example, in endosymbionts (Chatterjee and Bhattacharjee, 1975, 1976). The high resistance of amebas to radiations may be dependent on a combination of three factors inherent in the ameba: (1) a highly developed repair system for radiation-type damage, (2) a presumed polyploidy, and/or (3) a very efficient scavenging of free radicals and/or hydrogen peroxide. The suggestion that polyploidy increases radioresistance (Astaurov, 1963) seems reasonable if one accepts that radiation death is due to gene damage, whether direct damage or damage during repair processes. However, the comparative DNA measurements made by Kalney et al. (1974) for the tetraploid nuclei of rat liver and the nuclei of A . proteus suggest that DNA content itself does not markedly differ between these very different cells. While the ameba nuclear volume is 200 times larger than that of the liver nucleus, the DNA content is only 2.9 times as great. However, these measurements give no indication of the more important figure, that is, the number of gene copies, though the finding that radioresistant and -sensitive strains of Amoeba show no differences in their DNA content is more difficult to explain. The possible helpful effects of a high cytoplasdnucleus volume ratio may be of considerable importance when considering radioresistance, particularly if cytoplasm contains thioneins similar to those used to chelate metal ions. As yet, all that can be concluded concerning the very different radiation sensitivities of amebas, or of protozoa in general, is that there is insufficient evidence available to draw any conclusions concerning the cause or site of radioresistance until further information is available on the repair enzymes involved in radiation repair, their situation in the cell, and their dependence (if any) on protein and/or RNA syntheses at a time of intensive repair activity.

IV. Chemicals as Probes into Cellular Activities One of the best ways of learning about cell function is to use chemicals as probes. This may entail a study of the activity, morphology, and cytochemistry

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of the cell after blocking particular functions with chemical inhibitors, increasing or decreasing rates of cellular activities with metabolic chemicals or stress situations, or taking the cell apart and studying its components in the test tube. Amoeba offers an excellent opportunity to take the cell apart in vivo-by combining control cell components with components which have been partially or wholly inactivated by chemicals. The following discussion attempts to collect some of the information which has been gained about cell components through the combined use of chemicals, micrurgy, and electron microscopy. A. GENERAL NUCLEAR AND CYTOPLASMIC DAMAGE'

In any study of cell components the fitst step in pinpointing damage is to separate cytoplasmic and nuclear damage by comparing the viability and behavior of CnTrcand TrnCc amebas with TrnTrc and control amebas. Lethal damage to cytoplasm is relatively easy to recognize by following CnTrc amebas. Thus (1) death is an indication of direct irreversible damage to the cytoplasm and/or cell membrane, (2) delays in division are an indication of reversible damage to the cytoplasm, and (3) the absence of both suggests that any cytoplasmic damage may be insignificant or easily repaired. However, situation 3 may not be as simple as nuclear transfers lead one to believe. Damage, insignificant in the presence of a control nucleus, may be debilitating in the presence of a damaged nucleus-a nucleus which itself requires repair. An ultrastructural study combined with nuclear transfers is necessary both to assess the relevance of cell death to direct cytoplasmic damage, and to recognize reversible cytoplasmic damage of a rapidly repairable nature. The situation for the nucleus is not as easily resolved. Nuclear transfers between treated and control cells performed between 4 and 24 hours after treatment give only a rough indication of nuclear damage as it relates to the TrnTrc situation. Ultrastructural studies resolve some of the complexities not obvious from these transfers, but not all damage is revealed. With many chemicals two types of chemical activity contribute to nuclear damage: that produced directly by the activity of the chemical within the nucleus, and that produced indirectly through exposure of the nucleus to cytoplasm which has been damaged by the chemical. The combination of these can be complex, but some separation is possible. For example, if (where treatments are of short duration) not one but two sets of Trn+Cc transfers are canied out-the first within 1 hour of treatment and the second 24 hours after treatment-then the early transfers with rapid removal of the damaged nucleus to control cytoplasm for repair assesses mainly direct damage to the nucleus, while the second set of late transfers which allows the treated IN, nucleus; C, cytoplasm; C,, control nucleus; C,, control cytoplasm; T r d e a t e d nucleus; Tr,, treated cytoplasm; 4, the movement of a nucleus from one cell to another; NC, a hybrid cell, with nucleus of one cell and cytoplasm of a second cell.

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nucleus to be repaired and/or resume its activity in the treated cytoplasm assesses direct plus indirect damage to the nucleus. This is the type of situation observed after treatment of amebas with MNU; it has been suggested that there is not only a direct but also an indirect effect of treatment with this chemical and that the indirect effect is due to faulty repair of DNA; where treatments were carried out during S phase, added lethality and mutations suggested both faulty synthesis and repair of DNA during the period of and following treatment (Ord, 1970, 1974, 1976b). In other treatments, where cytoplasmic damage predominates, no increase in nuclear damage may result on delaying the transfer of the nucleus to control cytoplasm, particularly if inactivation of enzyme systems inhibits the progress of nuclear activity and/or nuclear repair processes. The delay of nuclear repair until the nucleus reaches the control cytoplasm is a protection both against activation of an injured nucleus, so increasing its damage, and of repairing nuclear damage with cytoplasm which itself is in poor condition. This appears to be the situation which occurs after treatment with methylmercury, where transfers 0, 2, 4, 5 , 8, 11, and 14 days after treatment all gave equal division delays and cell survivals (Ord, 1976a).

B. SPECIFIC DAMAGE WITHIN

THE

NUCLEUS OR

TO

ITS MEMBRANE

1. The Nuclear Membrane Damage to the nuclear membrane may be recognized in the living cell by nuclear swelling and loss of rigidity felt on proding with a glass needle. This type of damage was recognized and assessed after treatment with nitrogen mustard and radiations through the use of double transfers [i.e., transfer of a control FIG.6 . [a-c) The change in nuclear membrane on isolation of nuclei into 35 mM potassium chloride for 2 minutes prior to fiation in osmium tetroxide (a), isolation directly into the fixative (b), and isolation into Chalkley's medium for 2 minutes prior to fixation in osmium tetroxide (c). While potassium chloride causes only occasional breaks along the length of the lipid bilayer and no disturbance in the honeycomb layer, Chalkley's solution causes rapid disruption of both the lipid bilayer and honeycomb layer even with only 1 to 2 minute contacts. Nuclei isolated straight into the fixative. solution show both layers intact and nuclear helices in the underlying nucleoplasm. (a) X8470. (b and c) ~ 2 0 , 8 0 0 (d) . Extensive RER produced by an ameba on exposure to an LD3,.,dose of hydroxylamine; cell fixed the day following the beginning of treatment; internal space contains an electron-dense material. ~ 4 7 7 5 (e) . Extensive lipid in a cell; one of many such offspring four-cell cycles after treatment with 0.25 mM MMNG. Cell was double-fixed in Kamovsky's fixative, followed by osmium tetroxide, but is unstained. The lipid in the cytoplasm stands out as large, dark patches. The central portion of figure contains the nucleus which-with its peripheral fringe of denser nucleoli, internal helix groups, and honeycomb lamina with a lipid bilayer membrane-appears . Vacuolation of the dense granular nucleolus of normal. N,nucleus: n, nucleolus; L, lipid. ~ 6 2 0(f) an ameba exposed to 1 mg/ml RNase for 1 hour at room temperature immediately before fixation. hcb, Honeycomb lamina; V, vacuole. X24,650.

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nucleus to treated cytoplasm and back again to control cytoplasm (Ord and Danielli, 1956a,b)]. Damage to the nuclear membrane should be more easily recognized and defined through ultrastructural studies, but EM examination of the nuclear membrane and of the underlying laminar or honeycomb layer of cells treated with about 20 or more chemicals rarely showed any morphologically visible change in this structure when treatment involved the whole cell. Treatment of isolated nuclei can produce marked changes in the nuclear membrane (Fig. 6). Such studies have been carried out on physically maltreated nuclei and on nuclei after isolation in different media. In the former Flickinger (1974b) showed the importance of the rough (RER) endoplasmic reticulum in the repair of nuclear membrane. In the latter Ord and Bell (1970) showed (on a return to cytoplasm) a greater loss of viability of nuclei isolated in sodium chloride or in Honda, Rusch, or Chalkley’s medium than in 35 mM potassium chloride. [Chalkley’s medium is a very weak salt solution used for growing amebas (See Ord, 1970).] Ord, Minassian and Mawson (unpublished), in an ultrastructure study of both the isolated nuclei and the isolated nuclei after a return to control cytoplasm, showed nuclear membrane, nucleolar and nucleohelical damage caused by contact of the “naked” nucleus with incompatible andor nonprotecting media even for periods as short as 1-3 minutes (Fig. 6a-c). 2. The Nucleolus

Amoeba proteus has a layer of many small nucleoli situated just inside the honeycomb layer of the nuclear envelope and visible in the living as well as the fixed cell. These nucleoli are compoased of a dense, granular central region surrounded by a granular plus fibrillar peripheral region (Flickinger 1968a). EM autoradiography studies have shown this peripheral region to contain nucleolar DNA (Minassian and Bell, 1976b). Damage to nucleoli by chemical treatments may result in (1) shrinkage of the nucleoli andor loss of the peripheral region, producing a sharp demarcation between nucleoli and nuclear sap; (2) vacuolation of the central core or more frequently the appearance of small vacuoles throughout the nucleolar body of the type present after the treatment of living amebas with RNase (Fig. 60; and (3) fusion of nucleoli. While types 1 and 2 are generally produced by high concentrations of chemicals, for example, MNU (Ord, 1976a)or ACT-D (Flickinger, 1968d), type 3 has occurred more often with low concentrations given over an extended exposure period, for example, ACT-D (Lorch and Jeon, 1969) and cadmium nitrate (Ord and Al-Atia, 1979). Though damage is generally examined using the EM, in some cases it is sufficiently extreme to be recognized even in the light microscope, for example, fusion of nucleoli after continuous exposure for 1 month to low doses of mitomycin C (Ord, unpublished results). Damage using low chronic exposures to chemicals, which allow the nucleoli to carry out their functions, require far more detailed study, particularly with the use of toxic metals.

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3 . The Riboprotein Helices of the Nucleus The mRNA andlor rRNA of the nucleus of Amoeba is packaged in a helical formation (Stevens, 1967; Wise et al., 1972; Georgiev and Samarina, 1971). Helices increase in frequency at certain times in the cell cycle (Stevens, 1967; Minassian and Bell, 1976a). Changes in the numbers and attachment of these riboprotein helices after treatment with chemicals could prove a sensitive indicator of changes in RNA synthesis in the nucleus. At present the changing frequency and distribution pattern of the helical groups is being studied in detail through the cell cycle of control cells to give a basis from which to assess damage by chemicals. Once such a basis is available, deviations from normal will be used to diagnose certain types of cell damage (Fig. 7a-d). Treatment of cells with MNU has helped to clarify some of the uncertainties in helical behavior which have been difficult to resolve with certainty from the static picture produced in EM studies of normal amebas. This chemical treatment results in a loss of attachment of the helices even when the chemical is used at very low concentrations (Fig. 2). The detachment of helices is followed by their mass exodus from the nucleus; unless the treated nucleus is moved to control cytoplasm, their resynthesis may be delayed for from 1 to 4 days (Ord, 1976a) (Fig. 7e-g). Furthermore, in the cytoplasm of the treated cell they retain the smooth helical form (Ord, 1976a) generally lost as the helix passes through the nuclear membrane (Minassian and Bell, 1976a). The mass movement of helices in treated cells has verified that (1) helices are attached in the control nucleus; (2) their movement is from nucleus to cytoplasm, with helices which move to the cytoplasm being replaced by new helices in the nucleus; and (3) some form of change of processing takes place as the helix passes through the nucleopore into the cytoplasm, which if absent allows the helix to retain its nuclear form in the cytoplasm. The presence in the treated cytoplasm of helices which appear to be partly processed supports the suggestion by Minassian and Bell (1976a) that the nuclear helices become the helical polysomes of the cytoplasm. Since the mass release of helices has only been found with two chemical treatments, namely, MNU and MNUrea (both carcinogenic nitrosamides in mammalian systems), it is possible that this release in combination with the detachment of the ribosomes (Section C,3) may prove a characteristic action of certain types of carcinogens. 4. The Chromosomes and Division

Many abnormalities have been observed in the division of living cells after treatment with chemicals [e.g., nitrogen mustard (Ord, 1956); MNU (Ord, 1968c, 197la); mitomycin C and dichlorodiammineplatinum(I1) (Ord, unpublished results)]. However, since it is possible to recognize different stages of division in the living cell (Ord, 1971b), amebas can also be used to study the direct action of a chemical on the dividing cell, exposing it to the chemical at

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FIG. 7. (a) A section through the nuclear membrane and honeycomb layer (left) into the nucleog lasm of a control ameba nucleus fixed at 23 hours of age. The nucleoplasmcontains a group of helices sectioned so that it appears to be composed of four clusters. At the upper left one helix is leaving the nucleus through a nucleopore. (b and c) Enlarged micrographs of helices leaving the nucleus. During

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known division stages and either treating from outside the cell or injecting the chemicals into the division sphere. This has been done with a number of chemicals including MNU, methylmercury, and ACT-D. A range of effects can be produced from failure of the cell to undergo cytokinesis to abnormalities during nuclear reconstruction or complete failure of the nucleus to reconstruct. This type of experiment has not yet been analyzed in terms of chromosome damage. The chromosomes of Amoeba are very small and numerous (L.G.E. Bell, see Ord, 1976a), and as yet methods for their study have not been developed. The patterns of DNA and RNA syntheses, worked out in the control ameba in some detail (Ord, 1968b, 1969, 1971a, 1973c), are used in assessing the effects of chemicals on DNA. Since both the configuration of the DNA at the time of exposure to chemicals, and the ability of the cell to continue synthesizing its DNA in the presence of a damaging chemical, may be of considerable importance in assessing DNA-damaging chemicals, much of the Amoeba experimental work includes a comparison between S-phase and G2-phase cells (Ord, 1974, 1976b; Chatburn, 1977) (Section III,B,4). C. SPECIFIC DAMAGE WITHIN

THE

CYTOPLASM OR TO

THE

CELLMEMBRANE

1. The Mitochondrion In spite of the early observations on mitochondria in the large free-living amebas (Pappas and Brandt, 1959; Pappas, 1959), little information has emerged the cell cycle there is a continual turnover of helices in the nucleus with the production of new helices and movement of helices from the nucleus to the cytoplasm at a rate of about one to three helices per nuclear perimeter per section of nucleus. Once the bound helix of the control nucleus detaches, it moves to the periphery of the nucleus, enters a cylinder of the honeycomb laminar layer (b), and exits through the electron-opaque plug of material at the nucleopore (c). Here it generally looses its smooth helical form. Close connections and a similiarity between the helix measurements (width, length, and pitch) led Minassian and Bell (1976a) to suggest that the helix of the nucleus may become the helical polysome of the cytoplasm (d). A study of polysomes and helices in the ameba after treatment with MNU would support this supposition. Thus treatment with MNU results in a detachment of all nuclear helices within 5-60 minutes. Their mass exodus frequently results in more than one helix entering the same nucleopore (e, right) and may be accompanied by mulberry bodies leaving the nucleus (e, left) (structure and function unknown). On passing through the nucleopore plug into the treated cytoplasm, helices are frequently able to retain, or to retain partially, their helical form. Thus, while the cytoplasm of the control ameba contains few if any smooth helices, the MNU-treated cytoplasm contains a gradation: smooth nuclear-type helices, smooth plus granular helices [as in (f)], and completely granular helices [as in (d), i.e., the helical fraction of the free polysomes]. Since most ribosomes are detached by the MNU,these smooth and granular helices stand out conspicuously in a sea of single ribosomes [see (g) and Fig. I le]. The ameba in (g) was fixed only 10 minutes after a 10-minute exposure to MNU and shows some helices still attached in the nucleus (extreme left). (a, d, e, and f) X26,950. (b and c) ~33,100.(g) x 13,100. H, Helix group (attached helices); h, single helix; M, mulberry body.

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FIG.8. A comparison between control mitochondria and mitochondria altered by different chemical treatments. (a) Control amebas showing Types I and I1 mitochondria; these occur in approximately a 5050 ratio. X 13,100. (b-g) Mitochondria of treated cells. All illustrations are at an equal magnification. X 19,250. (b) Mitochondria showing the two changes which can occur after treatment

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on their functioning. Structural observations, too, apart from species comparisons (Flickinger, 1973a, 1974c), have been minimal. This may result both from the type of fixation regimes used and from the use of unsynchronized masscultured cells where the irregularities in mitochondrial form relating to age and nutritional conditions give a poor framework from which to compare different activity states. The mitochondrial ultrastructure of cells in an active cell cycle is far more regular than that of starved, quiescent, or aging cells and is more reliable as a basis from which to compare mitochondria of chemically treated cells. In the past there has been a tendency for cell biologists or pathologists to dismiss differences in mitochondrial density (i.e., electron opacity differences occurring with glutaraldehyde fixation) as artifacts, doubt as to its natural existence being reinforced by the tendency in many animals for all the mitochondria of a particular tissue to assume an identical form at any one time, and by the failure of many fixation methods (in particular osmium tetroxide) to show density differences. Recently the appearance of density and constriction changes in isolated mitochondria, when fixed in different physiological states, has reemphasized the possibility that different mitochondrial functional states may be recognized as morphological shape and density differences in vivo. In Amoeba where several forms of mitochondria may exist side by side in glutaraldehyde or with nitrosamides. b, shows an electron-dense patch (see arrow) which seldom exceeds 800 A, and b2 a fiber bundle (see arrow) which with this treatment generally consists of 6 to 10fibers, can reach 0.8 p m in length and appears beaded when fixed in osmium tetroxide only. These two abnormalities can occur simultaneously and are not different orientations of a single abnormality. The structure is slow to appear, patches appear more quickly than fiber bundles, and mitochondria containing both abnormalities are seldom present until 10 or more days after treatment and require cell activity before the effect is precipitated. (c) Mitochondrion showing a typical internal cristae loss, that is, with peripherally located cristae and a clear central matrix. This change can occur 2-3 days after MNU treatment. It is also found in mitochondria 4 or more cell cycles after treatment with MNU, MNNG, or cadmium; frequently all cells of the clone contain these mitochondria and can be recognized by changes in their outward appearance: slower movement, oddly angular pseudopodia, and long cell cycles; such clones may die out or return to normal within 10 cell cycles. The mitochondrion shown is from an offspring of such a clone derived from an ameba treated with 0.5 m M MNNG. (d) Mitochondrion showing needlelike filaments. These develop within hours of treatment (e.g., with hydroxylamine), can reach the entire length of the mitochondrion, may contain from 2 to 10 or more fibers, and may be found in all mitochondria of a treated cell. (e) Mitochondria fixed immediately after a I-hour treatment with 8x 10-5M CCCP at pH 6. The form change shown takes place within 15 minutes and is reversible; though more extreme with this uncoupler of oxidative phosphorylation, form changes are found to a lesser extent with other uncouplers. (From Smith, 1978.) (r) Mitochondrion fixed immediately after a I-hour treatment with potasium cyanide at pH 6. The expanded cristae with internal vacuolated contents (see arrow) are a feature of this treatment but can be found after other treatments. They are less common than cristae changes which involve only loss or localization of cristae. (From Smith, 1978.) (g) Mitochondrion from a cell fixed immediately after a 15-minute treatment with lO-'M DNP at pH 4. Long mitochondria are an interesting abnormality seen after treatments of amebas with most uncouplers. (From Smith, 1978.)

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FIG.9. (a-d) The changes occurring with time in the mitochondria of cells treated with MNU. x 12,700. (a and b) Half-hour after a 12-minute exposure of amebas to 1 mM MNU (cells fixed at 23 hours of age). Mitochondria appear normal, except that there are far more of both intermediate Type I

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Karnovsky ’s fixed cells, their dismissal as artifacts has always been difficult. Even if considered a staining artifact a difference in stain penetration in itself would suggest a physiological difference between mitochondria. The suggestion of Flickinger (1968~)that two types of mitochondria may also exist in osmiumfixed material, but that osmium tetroxide is such a poor mitochondrial preservative that one form becomes swollen beyond recognition, is in agreement with our own findings. Two morphologically distinct forms of mitochondria coexisting in the same cell are clearly distinguished in Amoeba after fixation with Karnovsky’s fixative (Karnovsky, 1965). Type I is electron-opaque and has a thin, constricted form and wide cristae; Type I1 is less dense, is rounder in form, and has narrower cristae (Fig. 8a) (Smith and Ord, 1979). Between these dark and light mitochondrial forms are small numbers of intermediate mitochondria which may be nearer in form and density to either Type I or Type I1 (Fig. 9a and b) (Ord, 1976a). Under similar fixation and handling conditions the frequency of Type I and type II mitochondria and their ultrastructure (length, width, cristal form, and electron opacity) are repeatable (Smith, 1978a). A detailed study has been undertaken in our laboratory by Smith on the forms assumed by the mitochondria of A. proteus in different situations: in different cell cycle phases, under different metabolic stress conditions (e.g., anoxia, Smith et al., 1979), and during and after treatment with uncouplers of oxidative phosphorylation (Smith and Ord, 1979). All information gained so far from this study supports the theory that the different forms of mitochondria are due to differences in their activity state at the moment of fixation. If one accepts that this is so, then mitochondrial ultrastructure becomes an invaluable asset in studies of cell damage. A mitochondrial study based so far on treatments of cells with approximately 15 to 20 chemicals and 5 different stress situations (starvation, anucleation, temperature change, pH change, and anaerobiosis) allows a preliminary charting of mitochondrial change under different conditions. Three major changes have been observed.

(see arrow) (a) and intermediate Type II (see arrow) (b). All four types of mitochondria can be present in the same cell. (c) Seven hours after treatment for 12 minutes with 1 mM MNU (cells fixed at 23 hours of age). Ninety percent of the mitochondria of these cells were either light or light intermediate Type II. A single type I mitochondrion is shown in the micrograph. (d) Twenty-four hours after a 12-minute exposure of 1 mM MNU. All mitochondria have generally assumed a light Type I or Type I intermediate form by this time. (e and 0 Cytochemical staining of a control cell (e) and a MNU-treated cell (0.The photomicrograph shows the diaminobenzidine reaction for the demonstration of cytochrome oxidase activity. Incubation was for 2 hours on live material; cells were then washed and double-fixed in Karnovsky’s solution and osmium tetroxide. The reaction was carried out 5 days after MNU treatment. Though some cytochromeoxidase activitycan be seen in the MNU-treatedcells, it is extremely weak in comparison with that in the control cell and occurs in only small regions of the cristae. (From Smith, 1978.)

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1. A change in the ratio of the different forms of mitochondria: Type I, intermediate types, and Type 11. The change in the ratios of these different forms of mitochondria was seen to vary with different chemicals or stress situations. (a) Chemicals known to interfere with mitochondrial activity [potassium cyanide and antimycin A and the uncouplers of oxidative phosphorylation DNP, m-chlorocarbonyl cyanide phenylhydrazone (CCCP), and pentachlorophenol (PCP)] switched a large proportion of the mitochondria of treated amebas to an intermediate Type I or intermediateType 11density form (Smith and Ord, 1979). MNU treatment switched mitochondria to an intermediate Type I1 or a Type I1 form (Ord, 1976a) (Fig. 8e-g, uncouplers; Fig. 9a-d, MNU treatment), while cadmium nitrate switched 90% of the mitochondria to the Type I1 form with a considerable amount of form change (Ord and Al-Atia, 1979). (b) The stress situations-starvation , enucleation, and anaerobiosis-switched a large proportion of the mitochondria to the Type 11form. (c) The microinjection of ADP and of ADP plus succinate switched a large proportion of the mitochondria to the Type I intermediate form. (d) Other chemicals (e.g., caffeine, phenobarbitone, hydroxylamine), as well as low and high temperatures, and pH change, had less striking effects on the form ratios of the mitochondria but in some cases produced structural abnormalities. 2. A distortion of mitochondrial shape (Fig. 8g). This distortion was most extreme when cells were treated with CCCP (Fig. 8e) but was not limited to the uncouplers. In a time study with CCCP distortion was shown to take place within minutes and to be reversible within hours (Smith, 1978). A distortion of mitochondrial form as shown by extreme swelling with ultimate rupture of membrane is constantly observed after short exposures to cadmium at concentration levels M (Ord and Al-Atia, 1979). above 3. Alterations in the internal structure of the mitochondria, frequently with inclusions appearing within the matrix of cristae. (a) A single electron-opaque patch may appear in each mitochondria after treatment with MNU (Ord, 1976a) (Fig. 8b). This could be similar to the electron-opaque patch observed in interspecific hybrid amebas where RNA synthesis is inhibited (Jeon, 1975). (b) Long, needlelike fibers, intermediate-length fibers, or short, beaded fibers are commonly found. These could be present in all the mitochondria of a cell [e.g., after treatment with hydroxylamine (Fig. 8d)l or present in only a small proportion of the mitochondria [e.g., after treatment with DNP, MNU (b, in Fig. 8b), or cadmium nitrate or under anaerobic conditions]. Needlelike fibers can be produced in the mitochondria of cells fixed in Karnovsky 's solution by incubation in DNase, or they may be found in untreated but aging cells from mass cultures. It is probable that the needlelike fibers are of more than one composition and/or origin. (c) Prominent vacudes formed by dilation of the cristae. These are common after a number of treatments with chemicals including potassium cyanide (Fig. 8f), MNU, and uncouplers. (d) Cristae reduction generally with

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cristae only at the periphery of the mitochondrion and with a clear central matrix (Fig. 8c). These mitochondria are not only present in treated cells but also in whole clones of cells three to five cell cycles after treatment with MNU and N-methyl-N-nitro-nitrosoguanidine(MNNG). (e) Widening, straightening, or zigzagging of cristae. This is less common for mitochondria after chemical exposures and when present is confined to only a small number of mitochondria. Forms with similar cristae distortions have been reported for starved or just divided cells of Chaos chaos (Pappas and Brandt, 1959). (f) A centrally located electronlucent area traversed by a few fine filaments. This abnormality was observed by Flickinger after treatment of amebas with ethidium bromide (1973a). A number of different mitochondrial structural abnormalities are illustrated in Fig. 8. While it is clear that in many cases mitochondria with cristal abnormalities or distorted forms can gradually revert to normal, it is probable that in others damage reaches a point of irreversibility. At present there is little opportunity to verify this, though the presence of mitochondria in autophagocytotic vesicles after treatment with methylmercury suggests irreversible mitochondrial damage from this chemical. It is possible that a careful examination for autophagocytotic vesicles containing mitochondria would reveal other chemical treatments in which mitochondrial damage has contributed to cell death. No generalizations can yet be made concerning the cause of different mitochondrial changes after treatments with different toxic chemicals. Many sites could be vulnerable-membrane permeability and ion transport, the oxidative phosphorylation pathway, the mitochondrial ribosome, the transcription and translation pathways of mitochondrial DNA, or the phospholipids or proteins of the mitochondrial structure. As cytochemical staining techniques become available, they will be used to help in the identification of specific mitochondrial lesions [e.g., cytochrome oxidase activity (Smith, 1978b); Fig. 9e and fl. Variation in mitochondrial form is common in all types of cells on exposure to stress situations, chemical treatments, or disease (Munn, 1974). In spite of the prominence often given in the literature to ultrastructural abnormalities of mitochondria, however, little progress has been made in determining the importance of these changes in mitochondrial functioning or viability. It is hoped that, as more data become available on the treatment of mitochondria with toxic chemicals having known specific activities, it will be possible to correlate ultrastructural change with particular sites of damage within the mitochondrion. It may ultimately fall to the Amoeba model to prove the viability of these mitochondria, since at present it is the only model where the opportunity exists for removing the cell's damaged mitochondria and replacing them with control mitochondria. However, it should be stressed that, just as the relationship between nuclear and cytoplasmic activities makes it impossible to separate their damages, so the relationship between mitochondrial and other cellular activities makes it impos-

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sible to separate their lesions. This could mean that reversible damage to mitochondria is as likely to contribute to cell death, if it causes a temporary energy deficiency at a critical site or a critical time, as is actual lethal damage to the mitochondrion itself. 2. The Golgi Bodies The Golgi system of Amoeba is composed of many hundreds of Golgi bodies or dictysomes scattered throughout the large volume of cytoplasm (Fig. 10a). Each body consists of a stack of 5 to 10 cisternae. The cisternae are narrow in the central region but expanded at the distal or peripheral ends near which may be seen small, round or elongated vesicles. In cross section the stacks of cisternae can appear flat, semicircular, or completely circular (Fig. lob, and c). (The significance of the flat or circular forms is not at present understood, though the presence of one type far in excess of another after treatment of A . proteus with chemicals or with stress conditions, and the smaller differences which can be found at certain times in the cell cycle, rule out their dismissal as sectioning artifacts.) Studies on control ameba Golgi bodies have shown that, while the cisternae of the concave surface of the Golgi stack contain glycoprotein and acid mucopolysaccharideand have walls similar to those of coated vesicles and to cell surfaces, the cisternae of the convex surface of the stack contain acid phosphatase and thiamine pyrophosphatase activity possibly associated with such activities as packaging of hydrolases to form primary lysosomes (Flickinger, 1975; Wise and Flickinger, 1970 a,b, 1971; Revel and Ito, 1964; Stockem, 1969; Stockem and Korohoda, 1975). This division in form and cytochemistry suggests that the Golgi bodies of amebas are associated with both cell membrane turnover and with other enzymic processes such as those in digestion. The maintenance, abundance, and normal morphology of the Golgi bodies are dependent on nuclear activity. Lack of a nucleus or of nuclear activity results in a loss of all Golgi bodies within 3-4 (Flickinger, 1969) or 4-6 days (Ord, unpubFIG.10. Golgi bodies under normal and stress conditions. (a) A low-power micrograph of an area of cytoplasm from a 5-hour-old (mid4 phase) ameba. Though the numbers of Golgi bodies per area vary in different regions of the ameba, one could expect to cut through one or two Golgi bodies in a 15-pm2area of cytoplasm. ~6930. (b) A typical semicircular Golgi body of a G1control ameba with 6 to 7 cisternae and a smaller, flat Golgi body of a mid-S-phase ameba with 5 to 6 cisternae. (c) A circular Golgi body with 11 to 12 cisternae in a stressed ameba; the ameba received a 45-minute heat shock (35°C)1 hour before fixation; many Golgi bodies had 12 or more cisternae. (d) The shrinking Golgi body of an anucleate ameba 5 days after enucleation. Though this ameba contained a considerable number of Golgi bodies, in most 5-day anucleates the numbers of Golgi bodies were very low, numbers of cisternae per Golgi body were small, and all Golgi bodies had much narrower cisternae. (e) The Golgi bodies of an ameba 1 hour after treatment with 2 mM MNU were frequently distoned, with few cisternae and many scattered vesicles. (b-e) ~32,340.

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lished results) (Fig. 1Od). Renucleation of cells in which Golgi bodies have been allowed to disappear shows that their reappearance in the presence of a nucleus begins within 1/2 hour but, if RNA or protein syntheses are inhibited or if oxidative phosphorylation is interfered with, their numbers remain low (Flickinger, 1971a,b). The early disappearance on enucleation of the concave cisternae containing glycoprotein and associated with membrane turnover (Wise and Flickinger, 1971) suggests that damage to, or absence of, Golgi bodies may be expected to result in a fragility of the cell membrane. The increasing fragility of anucleate amebas with time substantiates this, as do ultrastructural studies of amebas exposed to chemicals. In the latter, treatments where rapid cytolysis is common are shown in EM studies to have reduced and/or greatly distorted Golgi bodies (e.g., hydroxylamine, methylmercury, and high concentrations of MNU). Treatment with MNU is of particular interest, since with doses of equal lethality, if the exposure involves high concentrations for very short exposure periods, the Golgi body is damaged and the cells fragile (Fig. 1Oe) and, if the treatment involves lower concentrations for longer exposure periods, the Golgi body remains undamaged and the cells are not fragile. In conclusion it may be assumed that any chemical which disrupts or destroys the Golgi bodies interferes with membrane turnover; in so doing it may not only produce cell fragility but may interfere with normal cellular activities requiring membrane, in particular cell movement and division. If new Golgi bodies are not resynthesized to replace those which have been damaged, cell death will result. In a less direct way, possibly by slowing growth and movement, substances which affect protein synthesis may also be expected to affect the cell through its Golgi bodies if the observations of Flickinger using ACT-D and emetine (1968b, 1972b), and of Sanders and Bell using puromycin (1970), apply to protein synthesis inhibitors in general. 3. The Ribosomes The ameba differs from many mammalian cells in having no functional specialization. Consequently it has a mixture of small and large free polysomes in addition to clear regions of RER where attached polysomes form a variety of spiral and circular patterns (Fig. 1l a and b). This gives it an advantage in general chemical studies over many of the cells from tissues where the ratios of free and bound polysomes may be greatly distorted, for example, at one extreme the liver cell with large quantities of RER and at the other the erythrocyte containing little to no RER and only limited amounts of free polysomes. While these latter cells are in an extended Go phase and synthesizing few if any growth or division proteins, the ameba is in an active cell cycle passing through all cycle phases and consequently making a large variety of proteins both for use within the cytoplasm and also for delivery to the nucleus and to the cell surface. When the ameba is fixed in situ (i.e., without removing it from the surface to which it is attached or

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stimulating violent movement with a microscope light) ultrastructural examination shows RER distributed in two main regions-a nuclear and a peripheral zone (Fig. 12a). The main internal region of the cytoplasm is occupied by free polysomes among which are scattered small patches of RER generally associated with the mitochondria and/or Golgi bodies. Though protein requirements change as the cell passes through the cell cycle biochemical differentiation imposing different demands on the synthesizing machinery of the cell (Mitchison, 1973)], the large variety of proteins being synthesized ensures that any fluctuations in their distribution on free polysomes or RER are minimal. Ninety percent or more of the ameba ribosomes under these conditions are attached. However, the attachment of ribosomes can change rapidly if the cell meets with adverse conditions. In a detailed ribosome study using chemical treatments to detach the ribosomes, and nuclear transfers to determine the part played by the nucleus in their detachment and reattachment (M. J. Ord, unpublished), the following changes have been noted. 1. The amount of peripherally located RER can be greatly increased, increased synthesis of bound polysomes possibly relating either to detoxification of the chemical or to the replacement of damaged membrane components (e.g., with hydroxylamine; Fig. 6d). 2. The ratio of RER to free polysomes can decrease, with most ribosomes attached to free helical polysomes and little RER remaining (e.g.. with long exposures to some metal ions). 3. The numbers of long polysomes can increase, increasing polysome length being associated with inhibition of termination of the polypeptide chain [e.g., with emetine (Flickinger, 1972b)l. 4. The amount of RER associated with or attached to the nucleus can increase, an effect which may denote increased nuclear activity and/or repair of nuclear damage (e.g., with methylmercury treatment; Fig. 12b and c). 5 . The attachment of ribosomes to mRNA can be lost, a gradual failure in the initiation of the polypeptide chain resulting in increased numbers of free ribosomes (e.g., after treatment with MNU; Fig. 1lc-e). Detachment of ribosomes is also a common characteristic of many stress situations and suggests that lack of initiation is acting as a negative feedback mechanism for protein synthesis in situations where cell growth would not be advantageous to the cell. With MNU treatment the rapid failure of the attachment of ribosomes to mRNA (i.e., 90-100% ribosomes free; Fig. 1) is more spectacular than that observed in stress situations, but it is not unlike that resulting on treatment of cells with chemicals which affect initiation of the polypeptide chain, chemicals which damage the endoplasmic reticulum (ER), or chemicals which alter the ribosome. However, although their “OFF” ribosome pattern appears similar in

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ultrastructural studies, separation is possible if accompanying ultrastructural changes are observed. Thus ER damage can be recognized by an “OFF” ribosome pattern accompanied by the breaking up of the ER (e.g., in cells given a heat shock) or inclusion of RER and smooth endoplasmic reticulum (SER) in autophagocytotic vesicles (e.g., in methylmercury-treated cells). Ribosome damage can be recognized by an “OFF” ribosome pattern accompanied by swelling of the ribosome and a decrease in its staining with uranyl acetate (e.g., in cells treated with RNase or in 2 to 3-day anucleate cells). Damage by a chemical such as caffeine, or stress conditions such as pH changes or suboptimal temperatures, produce an “ON-OFF’’ ribosome pattern, as do the very lowest effective doses of chemicals such as MNU (Fig. 1 lc); that is, there is an increase in free ribosomes with a corresponding decrease in the numbers of both free and bound polysomes. Damage by the two carcinogens MNU and MNUrea and by ultraviolet radiations differs from damage produced by all other treatments in that treatments not only cause detachment of ribosomes from both free and bound polysomes but are accompanied by detachment of the nuclear riboprotein helices. In most cases the detachment of ribosomes was reversible. Reattachment could be immediate on removal of the chemical or metabolic stress situation, or could require several days. Only where irreversible damage occurred to the ribosomes themselves, or to the ER, did the polysomes fail to reappear. Using (1) treatment of anucleate cytoplasms, (2) treatment of whole cells followed by enucleation, (3) transfer of control nuclei to treated cytoplasms, or (4) transfer of treated nuclei to control cytoplasms, a detailed study of the behavior of ribosomes after treatment with MNU established the following (M. J. Ord, unpublished). 1. Ribosome detachment was not nucleus-dependent but occurred as a direct result of chemical treatment. 2. Reattachment of ribosomes was able to take place in the absence of a nucleus.

FIG. 11. The ‘‘ON,’’ “ON-OFF,’’ and “OFF” patterns of bound and free polysomes shown in A . proreus after double-fixation in Kamovsky’s solution and osmium tetroxide followed by a 1-hour uranyl acetate staining before embedding. X20,400. (a and b) RER and free polysomes of a 23hour-old control ameba (early GZ),“ON” pattern. (c) “ON-OFF” pattern found in amebas exposed to 0.01 mM MNU for 30 minutes. These amebas had approximately 50% of the ribosomes detached in both regions of RER and of free polysomes (region of free polysomes on left, region of bound polysomes on right). (d) An “OFF” ribosome pattern in an RER region of an ameba exposed to 1 mM MNU for 12 minutes; fiation 24 hours after treatment. A lone coiled polysome can be seen (upper left). (e) An “OFF” ribosome pattern in a free polysome region of an ameba exposed to 1 mM MNU for 6 minutes and fixed 1 hour after treatment. Two nuclear-type helices can be seen in the lower region of the micrograph.

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3. Nuclear activity considerably increased the speed of reattachment of ribosomes. 4. In the long term the nucleus was necessary to maintain the integrity of the ribosome and the ER. 5 . Ribosomes did not detach simultaneously but gradually, and in the following order. Small free polysomes disappeared in 5-8 minutes; large free polysomes in 8-10 minutes; bound polysomes in from 20 to 90 minutes. This supports the idea that the ribosomes of the RER are attached more firmly than those of free polysomes (Sabatini and Blobel, 1970). However, the loss of 30-60% of both free and attached polysomes when very low doses of MNU were used suggests that, though the ribosome attachment to ER may protect it initially from detachment in the presence of the chemical, such protection is only temporary. Apart from their use as probes in learning more about ribosome attachment and initiation of protein synthesis, chemicals (particularly antibiotics) have been used to learn more about the ribosome itself. The structure of the ribosome can be important medically, since treatment of infections with many antibiotics depends on a difference in ribosome structure giving an added sensitivity to the bacterial ribosome without affecting the patient’s own ribosomes. However, ameba studies have as yet made only a limited contribution in this field. Reports on the size of the ameba ribosome and its relationship to the bacterial or mammalian ribosome are conflicting. Work on the ameba ribosome in explaining cytoplasmic inheritance is more promising but, as yet, these studies are in their infancy.

FIG. 12. (a) The RER of a 5-hour-old ameba growing and dividing in an environment of good food and fixed in siru without microscope light (which stimulates movement and breaks up the RER) or centrifugation. The micrograph shows a section through the nucleus (lower region) and the adjacent cytoplasm which contains large quantities of RER. Nuclear activity is indicated by both large numbers of nuclear helices in the nucleus and attachments between the nuclear membrane and RER seen on examination of the entire circumference of the nuclear section. Such attachments may join the internal space of the RER with the nucleus interior, or more commonly with the perinuclear space (i.e., the space between the inner and outer membranes of the nuclear envelope and possibly a site of much enzymatic activity). X5390. (b) One such attachment between the RER and the perinuclear space (confirmed by serial section). The attachment to the RER is between the plugs of (c) A similar type of RER-nuclear membrane electron-dense material of the nuclear pores. ~30,800. connection, but in an ameba with MNU-treated cytoplasm containing a control nucleus. The cell was fixed 24 hours after treatment and transfer, at a time when the detached ribosomes were beginning to reattach into polysome formations. The activity of the control nucleus speeds this reattachment. X57.750. N, Nucleus; n, nucleolus; H, groups of helices; g, Golgi body; L, lipid; F, bundle of microfibers; m, mitochondria; NM, nuclear membrane and underlying honeycomb laminar layer; pc, pennuclear cistema; dNp, dense material or plug in nuclear pore region; hcb, honeycomb lamina; RER, endoplasmic reticulum with attached polysomes.

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4. The Cell Membrane

Apart from information gained about lethal membrane damage from nuclear transfers and mortality curves, nonlethal effects of chemicals on the surface can be studied using both the living and fixed cell. Amoeba is a motile cell which attaches to a surface and moves by pseudopod formation-a movement not unlike that of macrophages, lymphocytes, and tissue cells in culture. Its nonrigid surface is not only of use in movement but also in division, in feeding, and in the intake of molecules or ions. This provides five membrane activities (adhesion, movement, phagocytosis, pinocytosis, and division) which can be used in living amebas to monitor the effects of chemicals. To this can be added other characteristics which are often most useful in recognizing membrane change: the state of the membrane felt on proding with a microneedle, its malleability, and its ability to both open before and close behind an outgoing or incoming nucleus in micrurgy. Such membrane properties depend both on the membrane integrity and on ,microfilament and Golgi activity within the cytoplasm. Membrane damage may be produced at several sites: (1) direct damage to either the mucopolysaccharide andor membrane-incorporated proteins or the lipid bilayer of the membrane itself; (2) indirect damage to structures, for example, microfilaments, which are involved both in movement and in repair of ruptured membrane; (3) damage to the machinery for membrane replacement, for example, the Golgi bodies; and (4) damage or inhibition of the ion-exchange channels (e.g., sodium-potassium or calcium channels) of the membrane. Few if any chemicals have proved so specific in their action that they affect any one of

FIG. 13. (a) Mutant strain of A. proteus produced by treatment of cells with MNU at 5-6 hours of age. The mutation is due to a lesion in the nuclear DNA (Ord, 1970, 1973a) but is recognized by a lack of triuret crystals in the cytoplasm, giving the ameba a ghostlike appearance. In the control ameba on the left the triuret crystals are in the form generally found in A . proteus, that is, as tiny, truncated, bipyramidal, isotropic crystals whose large numbers give an opaqueness to the ameba; in the SpG mutant on the right there is only a scattering of crystals (cubes, platelets, or bipyramidal crystals), and other cytoplasmic structures are clearly visible. N, Nucleus; fv, food vacuole. x60. (b) In the pale mutant (PM) produced by treatment of cells with MNU at approximately 30 hours of age, crystals are present as platelets (appearing as needles when viewed on edge). The control ameba is on the left and the PM mutant on the right. These amebas, though pale, can be distinguished from the more ghostlike SpG mutant, particularly if viewed through crossed polaroid filters where platelets are . Seven of the 10 nuclei present in this giant cell after treatment with seen to be birefhgent. ~ 8 5 (c) MNU. Such amebas arise when nuclear division takes place in the absence of cytokinesis. Frequently such cells round up to form a division sphere but then fail to separate their cytoplasm into two halves. Once a binucleate cell forms, future divisions result in a distribution of nuclei to give cells with one, two, three, or four nuclei (Ord, 1971a). In this way, as divisions continue in subsequent cell cycles, amoebas gradually appear with not 2 , 4 , 8 , or 16 nuclei but with many odd numbers of nuclei from 1 to 15. x85. (d) The crystals of the PM mutant viewed through crossed polaroid filters. Since the ameba was moving, the crystals tend to blur slightly with the long exposure time. ~ 8 5 .

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these alone. Thus methylmercury causes some cells to cytolyze almost immediately, others to cytolyze more slowly, and still others to become fragile but to survive providing no stresses are applied during the early post-treatment period; that is, the chemical acts directly on the outer cell membrane and indirectly on membrane replacement machinery, and on membranes of other cell components. Of the chemicals used to date, monocrotaline pyrrole (Mattocks and Ord, unpublished) has proved the most specific membrane poison. Given from outside the cell, it reacts rapidly with the outer mucopolysaccharide layer and polymerizes with it to form a stiff outer surface, at the same time inducing numerous deep pinocytotic channels into the ameba. As the cell pinches off vesicles from these channels, the surface plus the chemical enter the cell, but the very short half-life of the chemical precludes its entering the cell in an active form. With intensive pinocytosis, unless the exposure is of very short duration (less than 5 minutes), membrane and polymerized pyrrole soon accumulate to such an extent that the cell lyses, leaving behind a skeleton of stiff membrane. However, though possibly some tissues such as lung may be exposed to the toxic chemical from the outside and so show a membrane reaction, the chemical is normally produced inside the cell. Only injection of the chemical (an experiment not yet performed) will mimic the events found in, for example, the liver cell. Membrane activities of amebas have been studied by a large number of cell biologists over the past 30 years, and many of the ideas on ameboid movement, pinocytosis, and adhesion now being applied or rediscovered in tissue cells were originally explored and formulated using the ameba as a cell model. These lines of research are beyond the scope of this article, since, while yielding much information on cellular activities, they contribute only marginally to our understanding of the action of toxic chemicals. 5 . Other Cell Components The triuret crystals of the cytoplasm, identified by Granbaum et al., (1959) and Griffin (1961), are of interest in chemical studies because of the altered forms of crystallization which can be produced by nuclear DNA lesions (Ord, 1970, 1973a). A number of mutant lines of A. proteus have been produced by treatment with MNU, mutations which are recognized by a visible change in the cell’s phenotype due to a change in triuret crystals. These mutant lines are stable over a period of years and can be produced repeatedly at particular times in the cell cycle. The use or fate of the triuret crystals in the ameba has so far eluded biologists, and no attempt has been made to identify the biochemical lesion responsible for the A. proteus mutant change. However, the use of ammonia and the common carbamyl phosphate step as the starting point in both pyrimidine base and urea biosyntheses, as well as the replication and division lesions which always accompany the crystal change, suggest a possible link between the two. If it should prove possible to reverse the mutation with mutagens, the visible

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change in phenotype could make this mutation a most useful tool in mutagenic studies (Fig. 13a, b, and d). Another characteristic which may prove useful in assessing cell change is cell lipid. An increase in lipid suggests a change in the glycolysis and/or oxidative phosphorylation pathways of the cell. Lipid accumulation has been observed after treatment with metal ions and with nitrosamides (Fig. 6e). It can be seen as a change in both the treated cells and more particularly in the offspring of treated cells which contain accompanyingmitchondrial lesions. This characteristic has not yet been explored for use in assessing cell change with chemicals. Other structures such as food vacuoles and lysosomes (studied in detail by Chapman-Andresen, 1973) and the contractile vacuole (Pappas, 1959) are already proving useful measures of cellular activity, in particular of membrane availability, and are being used in the assessment of cell damage. However, as with the membrane studies, research has been aimed at understanding the cell rather than toxic chemicals and is not itself considered here.

V. General Conclusions Nuclear transplantation is not unique to the ameba. Methods of combining the nucleus and cytoplasm of two cells have been developed for a number of different animal cells: Ram (Briggs and King, 1952), Xenopus (Gurdon, 1974), Stentor (DeTerra, 1969). Each transplantation system serves well its own particular purpose, though each has limitations (Ord, 1979a). Of these models the one which is at present being used in studying the widest variety of problems is one which combines two or more cells, rather than transplanting the nucleus of one cell into another, that is, cell fusion (Harris, 1974). Its popularity is based on its versatility. Many types of cells can be fused: plant or animal, single-cell organisms, and cells of organized tissues. Many methods of fusion can be used: mechanical stirring, virus infection, and chemical treatment. Moreover, it is not only possible to fuse pairs of cells but also groups of cells or even parts of cells. Though cell fusion is proving invaluable in a variety of cell biology studies, it has two great drawbacks when applied to most toxicological studies. (1) It requires prior treatment with chemicals to synchronize the cells, subsequent treatment with viruses or chemicals to fuse the cells, and possibly further treatment and centrifugation of cells to separate them into parts. (2) It cannot be carried out on single cells; its “hit-or-miss” application requires a mixture of thousands of cells in order to obtain small numbers of the desired combinations. In spite of these drawbacks, however, it is extremely valuable as a means of examining the effects of chemicals on the nuclear DNA, that is, the chromosome at division, or (using the prechromosome condensation technique) (Johnson and Rao, 1970) the chromatin of the interphase nucleus (Hittleman and Rao, 1974 a,b; Johnson,

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1976; Collins et al., 1977). In this use it covers one cell component inadequately represented by the ameba and therefore complements this system rather than overlaps it in toxicological problems. The Amoeba model combines a versatility equal to that of fused cells and egg systems with a precision and ease of handling not found in any other nuclear transplant or fusion system. In spite of this it has attracted relatively little attention and is used by only a few groups of devoted cell biologists. This may be due in part to a lack of imagination among cell biologists in realizing the potentials offered by an unspecialized, free-living cell such as the Amoeba, and in part to a fear of difficulties inherent in the less known culturing and micrurgical techniques for Amoeba as opposed to the familiar techniques used for the culturing of tissue cells. The Amoeba model has limitations. Some of these, for example, the need for a vertebrate microsome system and the introduction of short-lived chemicals, are being overcome; others may be turned to advantage, for example, a probable dependence for the production of mutations in a polyploid cell on the functional configuration of the DNA (Ord, 1973a, 1974). From experimental work on the site and mode of action of chemicals so far undertaken, however, it is clear that, when the Amoeba model is chosen for problems which make use of its own unique qualities, few models can equal it. As more basic research on amebas accumulates, the opportunity to assess the physiological state of an organelle using changes in its ultrastructure as a criterion may become available. If so, alterations in cellular activity could be predicted from ultrastructural studies only and, with the accumulation through indepth ameba studies of a range of cell symptoms to characterize particular cell lesions, it may become possible to predict cell viability and activity changes without resort to micrurgy and autoradiography. An approach of this type is being made by some experimentalists in other fields, in particular in medicine where changes in the cellular membrane system of the cell (Golgi bodies, mitochondria, SER, RER, cell membrane, and lysosomes) are being used in assessing certain diseased states (Thrump, 1975). In view of the similarity in the pathology of many of the components of diseased and chemically treated cells, the two studies may in the future complement and aid one another.

VI. Evaluation of Amoeba proteus as a Model in Cell Biology and Toxicology A. CELLBIOLOGY

The value of Amoeba as a research tool in this field is unquestionable. A large, unspecialized, actively cycling cell, cultured in a simple saline medium without the tedium of sterile procedures or the addition of antibiotics or stress conditions,

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has limitless potential. The failure of researchers to make full use of it suggests a reluctance to tackle new techniques or a preconceived notion of their difficulty. In recent years the ameba techniques have been modified for ease and speed, and many of the hazards encountered during culturing identified. The tedium of cloning single cells has been reduced with the introduction of disposable plastic multiwell trays and the use of capillary cloning in a balanced ecosystem, and with the introduction of a syringe-pipette system for handling single cells or measuring their volumes (Ord, 1979b); the agar technique of Jeon and Lorch (1968) has simplified and speeded up the nuclear transplantation technique, microinjection now being possible using simple inexpensive manipulators; many activity changes can be followed in single cells with the availability of radioactive or fluorescent tagged chemicals, while the scaling down of chemical techniques such as electrophoresis is bringing chemical analyses down to the level of small numbers of, even single, amebas. One technical problem not yet tackled is that of large-scale mass culturing. Introduction of an axenic technique would standardize culturing techniques and could result in the use of amebas for investigating biochemical problems-work which would be of infinte use to the cell biologist. At present the varied conditions under which A. proteus will grow presents its chief danger in research. Shortcuts and carelessness in culturing, with contempt for a rigid regime (Section III,A), will stand in the way of Amoeba’s use as a precise model. It should be borne in mind that yeast, bacterial, mold, or algal infections, and detergent or metal contamination, are of equal danger in modifying ameba experimental results, as they are for tissue culture cells, though they may be slower to cause detectable changes. The bulk of ameba research has concentrated on two main problems, neither of which has as yet been satisfactorily resolved: (1) cytoplasmic inheritance, where initial results preceding knowledge of extranuclear DNA tended to set ameba aside as “different”; and (2) ameboid movement, where it is hoped that the many conflicting theories may now be resolved with the advent of tagged antibodies to contractile proteins. A smaller but more successful application has been in DNA research, where its use with other cell models-Xenopus eggs, fused tissue cells, Physarum, and Stentor-has helped in answering questions on cytoplasmic control of synthetic activities (see review, Ord, 1979a). But ameba offers much wider opportunities, and many fields are yet unexplored. Characteristicsof and techniques peculiar to amebas should be exploited. Many possibilities come to mind. Amoeba’s tolerance to radiation (a subject in itself of interest since it suggests highly efficient repair systems and/or welldeveloped scavenging mechanisms for hydrogen peroxide and free radicals) allows the use of radioactive tracers at far higher levels without interrupting normal cellular events; this gives a resolution in light and EM autoradiography for following synthetic events not possible with most cells. Direct injection of substances into the cytoplasm followed by EM fixation of individual cells allows

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the role of short half-life substances in chemical pathways, for example, oxidation-phosphyporylation, to be explored. The use of membrane-utilizing activities and controlled stress situations in conjunction with enucleation or removal of portions of cytoplasm could give invaluable information on membrane turnover. Living cell centrifugation with subsequent controlled removal of layered cell organelles offers many interesting opportunities for research. Lastly, not only is the ameba heterokaryon excellent for studying nuclear-nuclear and nuclear-cytoplasmic interactions (a field now receiving some attention in a number of laboratories in Great Britain, America, and Russia) (Section 11), but also the ameba presents a bag of cytoplasm into which foreign substances can be injecteddomplementing, possibly extending, some of the interesting RNA and DNA experiments using Xenopus egg cytoplasm (Gurdon, 1974).

B. TOXICOLOGY The use of A . proteus in toxicology research is more difficult to assess. Certainly its use in ecotoxicology cannot be disputed (Ord and Al-Atia, 1979). Free-living amebas represent one of the first links in both soil and freshwater food chains, their uptake and accumulation of toxic chemicals being passed on to herbivores, carnivores, and filter feeders alike. The value of Amoeba in investigating single chemicals in depth (where combined investigation through single-cell cloning, micrurgy , autoradiography, and EM microscopy can help to dissect the cell and evaluate separately the damage to and repair of the nucleus and cytoplasm) has also been shown (Ord, 1968c, 1970, 1974, 1976a,b). The extent to which A . proteus can be used in the screening of chemicals is less certain. The first two requirements for any screening test are met, namely, low cost and rapid results, but methods of interpreting and systematizing results require further work before Amoeba can be used on a wide scale with interchangeable data. As with all single-cell models the basic characteristics of the cell must be taken into account when considering amebas for general use in toxicology (Ord, 1979~).As a lower eukaryote, the organization of Amoeba falls between that of the bacteria and the tissue cell, that is, it has a clearly defined nucleus, cytoplasm, and cytoplasmic organelles, but its DNA organization is likely to present some differences. Amoeba proteus lacks specialization and therefore offers an overall picture of many cell activities. It does not undergo differentiation and therefore cannot be used to identify dedifferentiation (or transformation). It gives detailed information on division delays and viability loss, data which correlate well with available results from mammalian systems for some chemicals, but less well for others where mechanisms have evolved to safeguard the ameba against external damage in an environment less protective than that of tissue cells. The clarity of its cytoplasmic organelles when examined in the electron microscope

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(mitochondria, Golgi bodies, polysomes, microfibers, and membranes), and the ease with which their morphology is changed with damage, may yet prove its greatest asset (Ord, 1979~). Here at the Medical Research Council Toxicology Research Laboratory at Carshalton, A. proteus has been incorporated into a multiple cellular test system composed of the following.

1. Salmonella typhimurium, using the histidine back-mutation system of Ames (Ames et al., 1973,1975) for the detection of DNA damage and excision repair. 2. Amoeba proteus, using living cell studies to detect changes in cell activities, division, and viability, rapid EM sampling to find the extent of immediate damage by chemicals to particular organelles, and micrurgy to evaluate the relative sensitivity of the nucleus versus the cytoplasm to a chemical. 3. Rat liver cells, assessment of primary cultures being made to confirm the more extensive information on cell organelles which can be gained from amebas. 4. Human lymphocytes and tissue culture cells, for detecting chromosome aberrations and sister chromatid exchanges of DNA and also for obtaining information on the dose ranges to which mammalian cells show sensitivities. This combination is not yet fully comprehensive, since it lacks a good system for detecting both filament or microtubule disfunction and cell transformation, problems which are now being taken into consideration. The majority of single-cell toxic screening regimes aim at the detection of mutagenic or carcinogenic events, often with the cell engineered to exaggerate the extent of these types of damage. Though such tests are excellent for suggesting which chemicals may present a danger, they do not necessarily reflect what actually happens when the chemical meets most cells (Gehring, 1977). Which additional tests are most useful in imparting this information will depend both on recognition and understanding of the different repair pathways available in a normal cell, and the interactions which can take place between damaged organelles. For example, if a dose of a chemical interferes with energy flow by damaging the mitochondria, nuclear damage due to faulty repair may never take place.

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INTERNATTONAL REVIEW OF CYTOLOGY. VOL. 61

Growth, Reproduction, and Differentiation in Acanthamoeba THOMAS J. BYERS Department of Microbiology and Developmental Biology Program The Ohio State University, Columbus, Ohio I. Introduction . . . . . . . . . . . . . . . A. Classification . . . . . . . . . . . . . B. Pathogenicity . . . . . . . . . . . . . C. Research Areas . . . . . . . . . . . . 11. Growth and Reproduction . . . . . . . . . . A. Factors Influencing Culture Growth and Survival B . Membrane Structure and Transport . . . . . C. Respiratory Metabolism and Bioenergetics . . D. Nuclear Reproduction and Cytokinesis . . . . III. Molecular Biology and Differentiation . . . . . A. Classes and Metabolism of Macromolecules . . B. Regulation of Differentiation . . . . . . . IV. Beginningsof Acuntharnoeba Genetics . . . . . V. Conclusions . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .

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I. Introduction A. CLASSIHCATION

Amebas of the genus Acanthamoeba belong to a group of organisms referred to as soil amebas in the past but now more commonly called small free-living amebas. The name change reflects the realization that these amebas are widely dispersed in fresh water and salt water as well as in soil (Page, 1967). They are called “small” to distinguish them from large free-living amebas such as Amoeba proteus and Chaos chaos. The taxonomy of small free-living amebas has been in a state of confusion for a number of years, but some order seems to be emerging (Griffin, 1978). There are still important disagreements (Singh, 1975; Griffin, 1978), but most investigators are using the nomenclature recommended by Page (1967, 1976). Pussard and Pons (1977) distinguished 18 species of Acanthamoebu on the basis of cyst characters, and it would not be surprising if additional species were described from the many strains isolated by various laboratories around the world. Unfortunately, the confusion and disagreement over taxonomic relation283 Copyright @ 1979 by Academic Ress, Inc. All rights of repmduction in any form reserved. ISBN 0-12-364461-5

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ships has resulted in a literature filled with different names for the same organism. Page’s classification (1976) is accepted in this article, and currently accepted names consistent with this scheme are used. Table I lists equivalent designations for cases where the published name differs from the one used here.

B. PATHOGENICITY Early reports on the pathogenicity of small free-living amebas for humans were characterized by confusion over nomenclature (Griffin, 1978). It is now recognized that some species of Acanthamoeba, including the widely used A . castellanii, can be opportunistic human pathogens (Griffin, 1978; Willaert, 1977; Martinez, 1977). Infections that have been described include amebic meningoencephalitis, which is generally fatal, as well as nonfatal lung, ear, and eye infections (Martinez, 1977). There is a possibility that acanthamebas are involved in other infections as well; for example, Jadin (1975 and personal communication) suggested that Acanthamoeba may be a vector for mycobacteria, including Mycobacterium leprae, the causative agent of leprosy. Most knowledge about the human pathogenicity of Acanrhamaeba has been obtained within the last 15 years. At the present time, it is unclear whether infections by these amebas are rare or more common than realized. Furthermore, it is unclear whether potential pathogenicity is limited to certain species and strains. Although the general biology of Acanrhamoeba has been studied for years in many laboratories without a single report of infection in a laboratory TABLE I SPECIES NAMES USEDIN THISARTICLE A N D EQUIVALENT NAMESFOUND I N THE LITERATURE SINCE1960 Name used Acanrhamoeba castellanii Acanthamoeba culbertsoni Acaniharnoeba palesiinensis Acanthamoeba rhysodes

Previous or alternative names“ Harimannella casiellanii, Acanrhamoeba sp. Neff,b A . ierricola Harimannella-Acanthamoeba, strain A-1 , Harimannella culbertsonid Mayorella palestinensis, A . casiellanii, strain MP‘ Harimannella rhysodes. A . casiellanii, strain HRC

“The names used have been selected instead of the alternative names because of data and opinions in the references cited. bPage, 1967. eVisvesvara and Balamuth, 1975; Pussard and Pons, 1977. dSawyer and Griffin, 1975; Griffin, 1978. ‘The names most commonly found in current literature have been used. There is some evidence for differences among A . castellanii, A . palestinensis, and A . rhysodes (Pussard and Pons, 1977; Willaert ei al., 1978), but these organisms are very closely related and simply might represent different strains of the same species (Band and Mohrlok, 1973b; Criffiths et al.. 1978).

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worker (Griffin, 1978), it seems prudent for investigators to treat organisms in this genus with caution.

C. RESEARCH AREAS Acanthamoeba research has increased in recent years because of intensified interest in microbial models of cell differentiation and because of the relatively recent discovery of human pathogenicity. These organisms are attractive for study because they are readily grown in axenic culture and because differentiation is simple. Cells exist as ameboid trophozoites or cysts (Fig. l), and cyst formation (encystment) as well as cyst hatching (excystment) can be induced synchronously. These amebas are generally free of endosymbionts, although an exception may have been described (Proca-Ciobanu et al., 1975). Reproduction is thought to be asexual, thus creating problems for genetic analysis, but some progress has been made recently in the study of acanthameba genetics (Seilhamer and Byers, 1978). Research interest has focused mainly on the morphology and molecular biology of growth and encystment, the composition and function of the plasma membrane, contractile proteins, respiratory physiology, nutritional and physical requirements for growth, taxonomy, and pathogenicity. This article concentrates on the aspects of Acanthamoeba biology concerned with growth, reproduction, and differentiation. Several reviews of encystment are available (Neff and Neff, 1969; Griffiths, 1970; Krishna Murti, 1971; and Weisman, 1976) but, with the exception of a brief discussion of Acanthamoeba palestinensis (Lasman and Kahan, 1963), I am unaware of any reviews on the growth and reproduction of Acanthamoeba. A recent review on muscle proteins includes information about Acanthamoeba (Korn, 1978).

FIG. 1 . (a) Vegetative trophozoite of A . casrellanii Neff. Photographed with phase-contrast. (b) Cyst of A . castellanii Neff. Photographed with Nomarsky optics.

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11. Growth and Reproduction

A. FACTORS INFLUENCING CULTURE GROWTHAND SURVIVAL 1. Monolayer and Suspension Culture

Acanthamebas grow either as monolayer or suspension cultures. Most work is with asynchronous cultures, but methods for synchronization have been described (Lloyd et al., 1975; Chagla and Griffiths, 1978). Growth of A . castellanii in unagitated cultures continues as a monolayer until localized regions of confluent growth have been achieved (-1000 cells/mm2). Above this density, increasing proportions of the population tend to float or to form aggregates. In some, but not all clones, very extensive clumping occurs prior to the end of the log growth phase. Final cell densities may greatly exceed the density of a monolayer, reaching as high as 6000 to 7000 amebas/mm2in some cases. Individual amebas migrate extensively when A . castellanii is seeded in axenic medium on glass, plastic, or bacteria-free agar surfaces and, consequently, fail to form colonies. Colony formation could be observed when A . castellanii was grown in or on agar with yeast or bacteria (Jensen and Dubes, 1962; Dubes and Jensen, 1964; R. J. Neff, personal communication). This approach is useful for some studies, but a different approach using multiwell culture dishes will more likely be preferred for viability assays and clone selection (Seilhamer and Byers, 1978). Alternatively, clones are readily initiated with single cells picked off agar plates (Neff, 1958). Many laboratories utilize suspension cultures of Acanthamoeba because higher yields per culture flask can be achieved. Cells tend to have increased generation times when first suspended. In addition, greater variability in cell size is noted. Suspension cultures of Acanthamoeba rhysodes and A . castellanii may have relatively high levels of multinuclearity (Band and Machemer, 1963; James and Byers, 1967) and increased respiration rates (Rudick, 1970). Acanthamoeba castellanii, at least, eventually adapts to suspension growth, so that mononucleated cultures with low generation times can be obtained (Jensen, Barnes and Meyers, 1970; Stevens, personal communication). 2. Nutrient Requirements Acanthamebas readily grow on living or dead bacteria, and this is used to advantage in the isolation of organisms from the environment or from body fluids taken from patients suspected of having Acanthamoeba infections (Singh, 1975; Griffin, 1978; Willaert, 1977). For experimental work most, perhaps all, species can be grown axenically in complex media as used in a variety of laboratories (Reich, 1948, 1955; Adam, 1959; Band, 1959; Neff et al., 1964; Raizada and Krishna Murti, 1971). The Neff growth medium is routinely used in our labora-

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tory, and generation times of 6-10 hours are commonly achieved in monolayer cultures of A . castellanii and A . rhysodes grown at 30°C. Generation times close to 20 hours are observed for Acanthamoeba astronyxis. Others report low generation times for suspension cultures as well (Jensen et al., 1970; Stevens and Pachler, 1974). The first chemically defined medium for Acanthamoeba contained 18 amino acids, vitamin B,,, thiamine, acetate, citric acid, salts, and trace metals, and supported a mean generation time of 50-55 hours (Adam, 1959). Subsequently, it was discovered that 18 of 22 strains of Acanthamoeba could grow on a medium containing only 11 of the amino acids (DM 2) and that 12 strains could grow in a medium containing only 7 amino acids (DM 3) (Adam, 1964b). The mean generation time of A . castellanii Neff in DM 2 was 46 hours, whereas generation times greater than 100 hours were observed in DM 3. Pigon (1976) routinely used a slight modification of DM 2 for A. castellanii and observed generation times of 70-80 hours. Dolphin (1976) reduced the amino acid requirement of the Neff strain to 5 amino acids: arginine, methionine, leucine, isoleucine, and valine. A generation time of about 65 hours was achieved under the best conditions. Band (1962) grew A . rhysodes on the 5 amino acids used by Dolphin plus lysine and threonine. Growth rates were not determined because exponential growth was not seen. Amino acid requirements were dependent on other components of the medium. For example, glycine was required by the Neff strain when acetate was the carbon source, but could be eliminated when glucose replaced acetate (Dolphin, 1976). Likewise, glucose eliminated the phenylalanine requirement that was present when acetate was the major carbon source (Adam and Blewett, 1967). Growth on various carbon sources differed among nine strains of Acanthamoeba (Adam and Blewett, 1967). The Neff strain was able to utilize at least 15 carbon sources; glucose, maltose, trehalose, sucrose, raffinose, and starch gave the best growth. Fructose, mannose, cellobiose, lactose, melibiose, mannitol, glycerol, glycogen, and laminarin also supported some growth. In other studies, A . palestinensis utilized glucose, fructose, galactose, mannose, maltose, sucrose, glycogen, starch, dextrin, and, to a lesser extent, lactose (Eiger, cited in Lasman and Kahan, 1963). Six of the nine strains tested by Adam and Blewett were unable to use at least one of the carbon sources utilized by the Neff strain. Adam (1959) concluded that thiamine and B,, were the only vitamins required for growth by A . castellanii, A . rhysodes, and A . palestinensis. In contrast, Band (1961) found that biotin was required by A . castellanii, A . rhysodes, and A . palestinensis. Adam (1964a) later confirmed the biotin requirement and concluded that this vitamin must have been a contaminant in the amino acids used earlier. However, Pigon (1976) routinely grows A . castellanii in a modification of Adam's DM-2 medium to which no biotin is added.

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Further development of chemically defined media will depend on particular needs. For many, the currently popular undefined media will continue to be the best. For others, the ideal medium would be one that supports rapid multiplication, produces high yields, and is of minimum cost and minimum complexity. I have been trying to modify available chemically defined media to improve the generation times obtained and now have a medium that supports 15 to 20-hour doubling times for A. castellanii and A . rhysodes (Byers, in preparation). Unfortunately, the composition is relatively complex and the cost is relatively high. Nevertheless, substantial improvements should be possible.

3 . Termination of Exponential Multiplication Inocula of A. castellanii were able to grow equally well in fresh Neff’s medium or in medium recovered from post-log-phase cultures (Byers, Rudick and Rudick, 1969). Therefore it was concluded that the termination of log-phase growth was probably not due to nutrient deficiencies or to the accumulation of inhibitors. Rather, oxygen deficiency seemed a more likely cause. Unfortunately, the results are somewhat ambiguous and do not entirely rule out the possibility of either nutrient deficiency or accumulated inhibitors. Pigon (1970) used a slightly different nutrient medium and found that cells did not grow as well in recovered post-log-phase medium as in fresh medium. He found that growth of cells in post-log-phase medium could not be enhanced by prior removal of ammonia or by using high oxygen tensions during growth. The growth-supporting activity of post-log-phase medium was improved, however, when it was autoclaved or heated to 125°C for 30 minutes before inoculation. Thus Pigon concluded that exponential growth was terminated partly by a thermolabile growth-inhibiting factor (GIF) that accumulated in the medium during growth. Additional experiments were thought to rule out oxygen deficiency and to implicate high cell density (cells per milliliter) as causes for the termination of log growth, but the results were inconclusive. Pigon (1972) was unable to find evidence for GIF in post-log-phase chemically defined medium, but he did isolate from the medium a high-molecular-weightfraction that was transiently inhibitory to movement, attachment, and cytokinesis. Treatment of growing cultures with this fraction (Fraction 30) resulted in increased multinuclearity . A later study (Pigon, 1976) utilized polyacrylamide gel electrophoresis to identify five macromolecule bands, corresponding to molecular weights of 19,000 to greater than 200,000 daltons, in the post-log-phase medium. This material was thought to originate at the cell surface and might include Fraction 30, but no evidence correlating any of these bands with cytokinesis-inhibitingactivity was presented. Insufficient data are available to indicate what factors primarily are responsible for the termination of exponential multiplication. The problem is important, however, since it probably is related intimately to the initiation of encystment. It is possible that the primary factor differs in different growth media. Oxygen

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tension decreases in both unagitated monolayers and aerated suspension cultures and therefore remains a prime candidate (Byers et a l . , 1969; Edwards and Lloyd, 1977a). Cell crowding probably is important, but whether this is due to contact inhibition (Ambrose, 1958) or to some other reason is unclear. Pigon's observations indicate that the inhibition of growth by high-molecular-weightproducts of cell growth also may be an important factor. 4. Temperature Sensitivity The possibility that temperature sensitivity is an important factor limiting the pathogenic potential of various strains of Acanthamoeba has arisen in several studies. The relatively pathogenic Acanthamoeba culbertsoni grew on bacteria at 42°C whereas the less pathogenic A . castellanii and A . polyphaga only tolerated lower temperatures (Griffin, 1972, 1973). Acanthamoeba castellanii multiplied optimally from 29" to 32°C in axenic medium, but no growth was observed during 48 hours at 37°C (Neff et a l . , 1958). A study of 20 strains of Acanthamoeba isolated from soil or mammalian tissue culture found that most strains grew better at 30°C than at 25" or 35°C. However, only the three strains isolated from tissue culture showed significant growth at 35°C. Although the preceding results suggest that strains known to have a greater pathogenic tendency tolerate higher temperatures during growth, the distinction is not likely to be absolute, since it has been reported that A . castellanii can grow at 37°C (Dubes and Jensen, 1964). Encystment of A . castellanii in Neff's encystment medium was optimal between 30" and 32°C with an upper limit at about 37"-40°C (Neff et a l . , 1964; Griffiths and Hughes, 1969). Unfortunately, no data seem to be available on the influence of temperature on the encystment of more pathogenic species. Once formed, cysts survive for months at room temperature (Page, 1967; KneiflovaJirovcova, 1971), but it would be interesting to examine survival in the 35"-45"C range where distinctions among strains showing different levels of pathogenicity might be observed. Unfortunately, the only available data are for survival at higher temperatures. Cysts of A. castellanii survived at 70°C for 1 minute, 62.5"C for 30 minutes, and 55°C for 7 hours in a liquid suspension (KneiflovaJirovcova, 1971). In our laboratory, trophozoite survival of A. casteflanii in liquid suspension was less than after 1 hour at 53"C, but cyst survival was at least two orders of magnitude higher even after 2 hours at this temperature (Akins, unpublished observations). At 60"C, cyst survival was less than after 1 hour. Bodenheimer and Reich (1934) compared survival of A . palestinensis cysts in moist and dry soil. They reported that cysts in dry soil survived 6 hours at 75°C and 1 hour at 80°C but were killed after 2 hours at 80°C. Cysts in moist soil were more sensitive, being killed after 3 hours at 55°C or 1 hour at 65°C. No data on high-temperature survival of cysts are available for the more pathogenic isolates.

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5 . Radiation Sensitivity White light was found to inhibit cell multiplication in A . castellanii (Dolphin, 1970). Generation times increased by 7% of the control value for each increase of lo4ergs sec-I cmW2 of light intensity. The increase was linear up to at least 1.2 X lo5 ergs sec-l cmP2. Dolphin demonstrated that at least part of the growth inhibition was due to alteration of the growth medium by the irradiation. It was concluded that the additional inhibition was probably due to direct sensitivity of the amebas to light, but this was not demonstrated definitively. Few data are available on the x-ray or ultraviolet sensitivity of Acanthamoeba. Chatterjee (1968) reported an x-ray LDSoof about 142 krads for trophozoites and greater than 300 krads for cysts. The values for trophozoites fall within the range found for larger amebas (Ord, 1973). The LDSofor ultraviolet irradiation of trophozoites of A . castellanii measured in our laboratory was -1300 ergs/mm2, and the LDw,-2500 ergs/mm2at an irradiation intensity of 67.3 ergs sec-I mmU2 (Seilhamer, unpublished observations). These values are similar to those for larger amebas (Ord, 1973). Survival curves suggested the existence of a subpopulation with much higher than average ultraviolet resistance. A similar observation was made by R. J. Neff (personal communication). No reports are available on the effects of ultraviolet light on growth and encystment of Acanthamoeba, but ultraviolet light was shown to slow excystment of another small free-living ameba, Vahlkampfia (Schizopyrenus) russelli (Rastogi et al., 1972). There was some evidence in this study of an ability to reverse the ultraviolet effect. Excystment seemed to improve when cysts were incubated in the dark at 28°C in 0.5% sodium chloride for 1 hour after the irradiation and prior to the addition of nutrient medium. The improvement was not seen when the incubation was carried out at 0°C. B . MEMBRANE STRUCTURE AND TRANSPORT 1. Ultrastructure and Biochemistry a. Plasma Membrane Ultrastructure. The plasma membrane of A . castellanii is the usual trilamellar structure with a thickness of 70-100 A (Bowers and Korn, 1968; Thompson, 1977b). The technique of freeze-fracture replication revealed 6.5and 8.5-nm-diameter particles on the inner fracture face, whereas only the larger particles were prominent on the outer fracture face (Bowers and Olszewski, 1974). However, there also were numerous other particles and short filaments that projected only slightly above the fracture plane of the outer face. Elements 7 X 21 nm and 23 x 46 nm were observed on the cytoplasmic surface of the membrane. It was suggested that these structures might be sites of insertion for cytoplasmic elements. In fact, actin filaments sometimes are attached to the cytoplasmic side of isolated plasma membrane (Pollard and Korn, 1973).

DIFFERENTIATION IN ACANTHAMOEBA

29 1

The extracellular surface of the membrane has been characterized as “naked,” since usual methods of fixation and staining have failed to demonstrate an extracellular coat (Bowers and Korn, 1968). Numerous particles observed on the extracellular side of the membrane by freeze-etching (Bowers and Olszewski, 1974), plus staining of this surface by concanavalin A, ruthenium red, or phosphotungstic acid-chromic acid (Bowers and Kom, 1974), were thought to indicate the presence of carbohydrates. The latter were attributed to the presence of carbohydrate-containing lipophosphonoglycans in the membrane and were not thought to indicate the presence of a surface coat or glycocalyx (Bowers and Kom, 1974; Bowers and Olszewski, 1974). Recent studies by Dykstra and Aldrich (1978) combining Alcian blue and ruthenium red staining suggest that A. casrellanii has a patchy surface coat that was missed by earlier techniques. If so, the micrograph published by these workers suggests that the coat is not as thick as that observed for other amebas (Flickinger, 1973). b. Chemical Composition of the Plasma Membrane. Methods of acanthameba membrane isolation were reviewed recently (Thompson, 1977a). Plasma membrane fractions of relatively high purity have been isolated from A. castellanii (Schultz and Thompson, 1969; Ulsamer et al., 1971; Stevens et a l . , 1977). Membranes also have been isolated from A. palestinensis, but the purity has not been tested as extensively (Chlapowski and Band, 1971). Analyses of membrane composition have revealed a number of unusual features (Table 11). The plasma membrane composition of A. castellanii is relatively simple. One protein with a molecular weight of 15,OOO daltons is 40-60% of the total protein (Korn and Wright, 1973; Wilkins and Thompson, 1974). Polyacrylamide gel electrophoresis demonstrated at least 1 1 additional minor proteins (Wilkins and Thompson, 1974), and others are probably present. One unusual observation is the rarity or complete absence of glycoproteins. Actin is a major component in some preparations (Pollard and Korn, 1973), but actin-free membranes are readily prepared (Schultz and Thompson, 1969; Thompson, 1977a), and this protein is not considered a normal component of the plasma membrane. An association between actin and the cytoplasmic membrane surface could be a natural, physiologically important relationship (Pollard and Korn, 1973), however, proof of this is not yet available. Few enzyme activities have been identified in purified membranes. Alkaline phosphatase and 5‘-nucleotidase activities are found consistently in the plasma membrane fraction and increase in specific activity during membrane purification (Schultz and Thompson, 1969; Ulsamer et al., 1971). Cytochemical tests suggest, however, that alkaline phosphatase is localized in contractile vacuole membranes (Bowers and Korn, 1973), and therefore it has been suggested that the presence of this enzyme in the plasma membrane fraction probably is due to contamination by the closely associated vacuole membrane. Furthermore, it has been suggested that the observed 5’-nucleotidase

PLASMA

Major component" Protein (3596)

TABLE n MEMBRANE COMPoSWlON OF ACAMHAMOEBA CAsTELMh'll

Subcomponents 15,000-dalton polypeptide -15,000 to 70,000daIton polypeptides A l k a l i phosphatase 5'-Nucleotidase Mg'+-ATPase Phospholipases A, and A2 Acyl-CoAlysolecithin acyl transferast: Acyl-CoA hydrolase PalmitoylCoA transferase CDPcholiie: 1,2diiglyceride cholinephosphotransferase Lysophosphotipase

Subcomponent fraction*

40-60

Comments Major band obtained by SDS polyacrylamide gel electrophoresis 9 to 1 I minor bands obtained by SDS plyacrylamide gel electrophoresis Probably d w to contamination by contractile vacuole membranes May be a suboptimal activity of W i n e phosphatase

-

Referencef

2

3,4,5,11,12 3,4,5,12 3,4,5,12 6 6 6

6 6 6,13

Lipophosphonoglycan

Two species

-

(29%)

<

Sterols 13%)

Phospholipids (25%)

Ergosterol

60

Dehydropariferasterol Phosphatidylethanolamine Phosphatidylserine Phosphatidylcholine Acidic phosphatides Diphosphatidylglycerl Phosphoinostitide

40 47.2 26.9 18.6 5.0

2.6 0.2

Unusual macromolecule first discovered in Acanthamoeba membranes; contains neutral sugars, amino phosphonates, amino sugars,phytosphingosines, longchain fatty acids, inositol, and acid-hydrolyzablephosphate; species differ in neutral sugars Sterols occur in equimolar amounts with phospholipid

7,8,9,10,14

4

-

-

4

-

Percent of membrane weight is shown in parentheses. Values from Dearborn et ai. (1976). bProtein values are percentage by weight of membrane protein. Sterol and phospholipid values are mole fractions of the correspondjng major components. 1. Korn and Wright, 1973. 8. Kom et d.,1974. 2. Wilkins and Thompson, 1974. 9. Dearborn and Kom, 1974. 3. Schulfz and Thompson, 1969. 10. Dearborn er al.. 1976. 11. Bowers and Korn. 1973. 4. Ulsamer er a!., 1971. 12. Thompson, 1977a. 5 . Stevens et al., 1977. 6. Victoria and Korn, 1975a. 13. Victoria and Kom, 1975b. 14. Bowers and Kom, 1974. 7. Korn, Dearborn et al.. 1973.

294

THOMAS J. BYERS

activity is due to alkaline phosphatase operating under suboptimal conditions (Ulsamer et al., 1971). Thus neither alkaline phosphatase nor 5'-nucleotidase is considered a likely component of the plasma membrane. A Mg2+ activated ATPase is found in the membrane fraction and may be a normal component, but this is difficult to determine since it also occurs in other membrane fractions and only shows a small increase in specific activity during plasma membrane purification (Schultz and Thompson, 1969; Ulsamer et al., 1971). Several enzyme activities such as adenyl cyclase and Na+; K+-ATPase, which are found in mammalian plasda membranes, were not found in acanthameba membranes (Ulsamer et al., 1971). In addition, acanthameba plasma membranes may be deficient in transport proteins found in other cell types, since pinocytosis is thought to be the major mechanism for uptake of dissolved nutrients and there is no evidence for other forms of active transport (Bowers and Olszewski, 1972). At present, the enzymes considered most likely normal components of the membrane are several enzymes of phospholipid metabolism: phospholipase A, lysophospholipase, acyl-CoA hydrolase, and palmitoyl-CoA synthetase (Victoria and Korn, 1975a). These enzymes could play a role in the endocytotic activity that is so prominent, however, there is no evidence for this role (Victoria and Korn, 1975a,b). Approximately 29% of the plasma membrane mass is due to two lipophosphonoglycans (Kom et al., 1974b; Dearbom and Korn, 1974). These phosphaterich lipocarbohydrates were first described for membranes from A . castellanii and have not been found in membranes of higher animals. The complete composition of these lipophosphonoglycans is unknown, but so far the carbohydrate moieties are the only differences between the two variants present (Dearbom et al., 1976). Cytochemical tests reveal carbohydrates on both the cytoplasmic and extracellular sides of the membrane (Bowers and Korn, 1974). Presumably this is due to the presence of lipophophonoglycanson both sides, possibly spanning the membrane. Lipids comprise about 38% of the membrane mass (Table 11). Phospholipids, including predominantly phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine, are about 55% of the lipid mass (Ulsamer et a l . , 1971). Glycerides plus sterols, including ergosterol and dehydroporiferasterol,represent another 37% of the mass. The sterols and phospholipids occur in equimolar amounts. The absence of the phospholipid sphingomyelin seems to be a significant difference between acanthameba and mammalian plasma membranes. c. Plasma Membrane Antigens. Antisera against purified plasma membrane fractions isolated from A . castellanii and A . culbertosni have been used as diagnostic tools for examining taxonomic relationships among species of Acanthamoeba (Stevens et a l . , 1977). Results of these tests indicate that Acanthamoeba castellanii, A . rhysodes, and A . polyphaga share surface antigens. Acanthamoeba culbertsoni A- 1 appears to share surface antigens preferentially with two other isolates from human tissue cultures, Acanthamoeba sp. HA and

295

DIFFERENTIATION IN ACANTHAMOEBA

Acanthamoeba sp. KA. A newly described species, Acanthamoeba royreba, showed some cross-reactivity with both A . culbertsoni and A. castellanii. d. Variations in Composition among Membrane Systems. Comparative studies of various membrane systems in Acanthamoeba have been limited. U1samer et al. (1971) reported the only extensive data comparing the composition of several membrane fractions. Their data, partially compiled in Table 111, suggest that there are significant differences among the several fractions. Nevertheless, electron microscope cytochemical studies demonstrate that interpretation of the composition data is not simple. For example, as indicated in Section II,B, 1,b, the preponderance of alkaline phosphatase activity in the plasma membrane fraction compared to that in mitochondria and microsomes is misleading, since the plasma membrane never stains for this enzyme (Bowers and Kom, 1973). In fact, the activity present probably is due to contaminating contractile vacuoles. Likewise, the apparent high acid phosphatase activity in the mitochondrial fraction is misleading, since mitochondria do not stain for acid phsophatase (Ryter and Bowers, 1976). This enzyme is localized primarily in cytoplasmic vacuoles, some vesicles of the Golgi apparatus, mitochondria-like bodies (peroxisomes?), and phagosomes. Thus the acid phosphatase activity in the mitochondria1

TABLE 111 COMPARISON OF PLASMA, MITOCHONDRIAL, MICROSOMAL, A N D PHAGOSOME MEMBRANE FRACTIONS IN ACANTHAMOEBA CAS7ELLANII"

Component RNA Protein-bound sugarb Phospholipid Sterolb Lipid-bound sugarb Succinic dehydrogenaseP NADH-cytochrome c reductase' NADPH-cytochrome c reductase' Acid phosphatase' Glucose-6-phosphatas' Alkaline phosphatase' Alkaline phosphatased Acid phosphatase" 5'-Nucleotidased

Plasma membrane

Mitochondria

0.005 0.052 0.43 0.212 0.024 0 0

0 1.32 0.22 197 365 6 33

'Data from Ulsamer et al., 1971. *Expressed as milligrams per milligram of protein. =Expressed as enzyme units per milligram of protein. dExpressed as enzyme units per micromole of sterol.

0.043 0.01 1 0.25 0.015 0.0012 2.32 0.44 0.53 13.2

Microsomes

Phagosome

0.26

0.22 0.026

0.35 1.06 0.50 6.6 1.01

-

18

-

-

334 171

-

48

296

THOMAS J. BYERS

fraction is probably due to contaminants. Combined cytochemical and biochemical analyses of membrane fractions might help resolve this question. Nevertheless, quantitative comparisons will always be complicated by the problem of selecting an appropriate basis for determining relative abundance: Should it be expressed per milligram of protein or per milligram of sterol, or is there any satisfactory common base? e. Plasma Membrane Changes during Growth and Differentiation. Formation of a cyst wall must involve extensive secretion through the plasma membrane. In fact, when cells that have ingested latex beads are placed in encystment medium, phagocytosis is discontinued and the beads are ejected by exocytosis (Stewart and Weisman, 1972), suggesting the possibility of an alteration in membrane function. As cells pass from log growth into stationary phase, there is a decrease in the rate of water expulsion by the contractile vacuole (Pal, 1972)-possibly another indication of changed membrane function. This observation plus the probability that alkaline phosphatase primarily is associated with the contractile vacuole, explain why the alkaline phosphatase activity of plasma membrane fractions in stationary-phase cells is greatly reduced and the lost activity appears in internal membrane fractions (Thompson, 1977b). Efforts to find substantive modifications in the plasma membrane that might be associated with changed function have revealed only that the major membrane protein is more readily extracted from stationary-phase than from log-phase membranes (Thompson, 1977b). 2. Membrane Transport a. Pinocytosis and the Transport of Water and Water-Soluble Substances. Rates of transport across the plasma membrane have been determined in A. castellanii for water expulsion (Pal, 1972); fatty acid uptake (Weisman and Korn, 1966); and glucose, leucine, inulin, and human serum albumin uptake (Bowers and Olszewski, 1972). Albumin uptake was proportional to the concentration up to 4 mg/ml. Electron microscope studies indicated that horseradish peroxidase, and therefore probably albumin, was taken up by pinocytosis. Leucine uptake was proportional to the concentration up to 10 mM, the increase in rate lessening above this concentration but probably not reaching saturation by 100 mM. This lack of saturation suggested a transport mechanism involving either passive diffusion or bulk uptake. When the uptake rates of leucine (MW 131), glucose (MW 180), inulin (MW 5000-5500), and serum albumin (MW 65,000) were compared by assuming that all were transported by pinocytosis, the calculated rates of medium uptake were 0.65, 0.5, 0.59, and 0.39 pl/106 amebas per 15 minutes, respectively. The similarity of rates for molecules of such different size and character, plus the evidence that protein was taken up by pinocytosis, suggested that all these molecules were transported by this mechanism (Bowers and Olszewski, 1972).

DIFFERENTIATION IN ACANTHAMOEEA

297

The possibility that pinocytosis is the major mechanism for the uptake of dissolved substances, or at least some classes of dissolved substances, is interesting and unusual. Bowers and Olszewski (1972) point out that the rate of this process in Acanthamoeba is much higher than rates observed for several mammalian systems, but data available for comparison were limited. The rates of pinocytosis determined by Bowers and Olszewski (1972) would result in an uptake of water of 1.6-2.6 pl/106 amebas per hour at 30°C. Pal (1972) measured the rate of water expulsion by the contractile vacuole in A. castellanii at 7.6 p1/106 cells per hour at 23°C. Both laboratories used the Neff strain, but slightly different culture conditions. Thus the differences in rates of water uptake and expulsion might be related to differences in culture methods, but there is a strong probability that pinocytosis is the minor mechanism of water uptake and that most water transport is by osmosis as assumed by Pal. The influence of nonelectrolyte concentration on the rate of contractile vacuole output was also examined (Pal, 1972). Equimolar concentrations of methyl alcohol, ethylene glycol, urea, and glycerol differentially affected the output rate. It is unlikely that this would have been observed if all these compounds were primarily entering by pinocytosis. Furthermore, relative rates of penetration were inferred from the assumption that slowly penetrating substances would affect the osmotic balance more than rapidly penetrating substances. The sequence in order of decreasing rate of penetration was methyl alcohol, ethylene glycol, urea, and glycerol-a sequence common to biological membranes. While it is probable that all dissolved substances can and do enter by pinocytosis, this is not necessarily the quantitatively important mechanism for all compounds. An interesting question is how the contents of pinosomes enter the cytosol. The answer may be in the observations of Pal (1972), in which he describes two phenomena: the fusing of small vesicles with larger ones, and the collapse of vesicles. In the former case, pinosome contents may be released into lysosome vesicles. In the latter case, vesicles may release their contents by bursting, a mechanism that obviates any problems of transport across the membranes. Pinocytosis in A. castellanii is an energy-dependent process that is inhibited by 2,4-dinitrophenol (DNP), sodium cyanide or potassium cyanide, sodium azide, and low temperatures, but not sodium fluoride or iodoacetate (Chambers and Thompson, 1976; Bowers, 1977). b. Transport of Lipid-Soluble Substances. When A. castellanii was incubated in the labeled fatty acid [14C]palmitate complexed with human serum albumin, the fatty acid was taken up while the albumin was not (Weisman and Korn, 1966). The palmitate apparently was transferred from the albumin to the cell and seemed to enter two cellular compartments: one easily removed by washing in 1% albumin, and the other resistant to washing. The mechanism of fatty acid uptake is unclear, but direct incorporation into membranes as seen for the phospholipid palmitoyl lysolecithin (Hax et al., 1974) seems likely. Elegant

298

THOMAS J . BYERS

experiments by Batzri and Korn ( 1975) demonstrated two different mechanisms for the uptake of phospholipid vesicles. Dipalmitoyl lecithin vesicles fused with the plasma membrane, releasing their contents both to the cell interior and exterior. In contrast, egg lecithin vesicles were ingested by endocytosis and carried their contents entirely inside the cell. The reasons for the differential response of the cells to the different phospholipids is unknown, but the authors point out the possible chemotherapeutic benefits from being able selectively to transfer vesicle membrane components to the plasma membrane or vesicle contents to the cytosol. c. Transport of Solids by Phagocytosis. Acanthamebas readily phagocytose particulate matter. The rate of uptake for six sizes of latex beads (0.126-2.68 pm in diameter) was proportional to bead concentration (Weisman and Korn, 1967). As with pinocytosis, uptake was inhibited by anaerobiosis, low temperatures, DNP, sodium azide, and cyanide, but not by sodium fluoride or iodacetate. Uptake showed saturation kinetics, and the bead concentration (in micrograms per milliliter) giving half-maximum rate of uptake was the same for all bead sizes. The number of beads per phagosome seemed inversely related to bead size (Korn and Weisman, 1967). It was proposed that the rate of bead uptake was controlled by a mechanism that packaged a constant volume of beads per phagosome regardless of bead size (Weisman and Korn, 1967). Chemically modified sheep red blood cells were phagocytosed more rapidly than unmodified cells (Rabinovitch and DeStefano, 197 l), thus indicating the important role of surface properties. In the same study, the rate of uptake of red blood cells from different species decreased in the following order: horse, guinea pig, rat, mouse, sheep, and human. The importance of surface properties is indicated by the fact that the relative sizes of these red blood cells, based on volume (Mitruka and Rawnsley, 1977) or diameter (Altman and Dittmer, 1964), decrease in the following order: human, guinea pig, rat, mouse, horse, and sheep. The maximum variation in volume was 3-fold. The rate of uptake varied over 100-fold, but the largest (human) and the smallest (sheep) cells were taken up at nearly the same rate. There is some evidence that the preferential uptake of horse red blood cells is due to binding of the cells to carbohydrate-sensitivesites on the ameba surface (Brown et al., 1975). d. Interactions between Pinocytosis and Phagocytosis. Pinocytosis is a continuous process in A. castellanii in nutrient medium, however, the rate is reduced whenever phagocytosis occurs (Bowers, 1977; Weisman and Kom, 1967). Pinocytosis of [3H]inulin was reduced 50% when the uptake of latex beads was maximized (Bowers, 1977). Chambers and Thompson (1976) proposed that pinocytosis and phagocytosis were separate processes under different controls. Their main evidence was the observation discussed below that phagocytosis was discontinued during stationary phase, whereas pinocytosis was only reduced. Bowers (1977), however, has emphasized the similarity and interdependence of

DIFFERENTIATION IN ACANTHAMOEBA

299

the two processes. She found that the rates of pinocytosis ([3H]inulin uptake) and phagocytosis (polyvinyltoluenelatex bead uptake) were inversely related, showed the same temperature dependence, were inhibited in a similar way by the lectin concanavalin A, and were inhibited to the same extent by a series of metabolic inhibitors. Bowers calculated that the combined volume of pinosomes plus phagosomes taken up per 15 minutes remained constant under conditions that resulted in a sevenfold increase in the volume of beads phagocytosed and a 46% decrease in the volume of inulin pinocytosed. Neither pinosome surface, phagosome surface, nor total surface of endocytic vesicles remained constant under these conditions. Bowers found that some very small beads (0.2 pm in diameter) were taken up in single endocytic vesicles and therefore questioned the validity of the hypothesis that constant volumes of beads were packaged in each phagosome (Weisman and Korn, 1966; Korn and Weisman, 1967). Instead, she suggested that the total endocytic vesicle volume, including phagosomes and pinsomes, was the critical variable regulated. A mechanism for regulation has not been proposed. Pinocytosis is counterbalanced by water expulsion through the contractile vacuole, whereas phagocytosed materials are not so readily removed (Bowers, 1977). Thus it is not surprising that cells phagocytosing beads become saturated. It appears that the saturation level is a volume of beads about 15%of the total cell volume. Endocytosis causes a large turnover of membrane. The total plasma membrane surface turns over 3 to 40 times an hour (Bowers and Olszewski, 1972). It is unknown how much of the turnover is due to redistribution of membrane components between the surface and the internal compartments and how much is due to new membrane synthesis. Efforts to detect enhanced membrane synthesis by comparing phagocytosing with nonphagocytosing cells have shown no enhancement during phagocytosis (Ulsamer et a l . , 1969; Thompson, 1977a). This result is not surprising, however, since it is now recognized that the continuous high background rate of pinocytosis probably obscures any phagosome membrane synthesis (Bowers, 1977; Victoria and Korn, 1975a). Nevertheless, it has been calculated that the rate of nutrient uptake is insufficient for de novo synthesis of plasma membrane for all the endocytic vesicles that a cell can form (Korn et a l . , 1974a). Therefore it is probable that recycling of membrane between the surface and internal compartments is extensive. e. Changes in Transport during Culture Aging and Diflerentiation. Phagocytosis of latex beads came to a virtual halt, whereas pinocytosis of inulin was only reduced about 50%, as A. castellanii progressed from log growth phase to stationary phase (Chambers and Thompson, 1976). The reason for the change in activity is unknown. Phagocytosis of latex beads also stopped when A . castellanii was transferred to encystment medium (Weisman and Moore, 1969). Cells that ingested beads prior to encystment expelled them before the completion of cyst walls (Weisman and Moore, 1969; Stewart and Weisman, 1972). Pinocytosis apparently has not been examined during encystment, but it would be interesting

300

THOMAS J. BYERS

to know the relationships among pinocytosis, water expulsion, and bead expulsion in view of Bowers’ hypothesis that there is a maximum endocytic vesicle volume. Continued pinocytosis in the absence of phagocytosis might shift the balance toward bead expulsion. Alternatively, bead expulsion might simply be indicative of an increase in exocytosis required for cyst wall formation. C. RESPIRATORY METABOLISM AND BIOENERGETICS 1. Respiration Rates, Mitochondria1 Structure, and Differentiation Acanthamebas are obligate aerobes for both growth (Neff er al., 1958; Band, 1959; Byers et al., 1969) and encystment (Band, 1963; Neff et al.. 1964). The oxygen tension in growth media begins to decrease at mid-log phase in both agitated (Edwards and Lloyd, 1977a) and unagitated (Byers et al., 1969) cultures. Byers et al. have suggested that this decrease is one signal that initiates encystment, however, there is no definitive evidence for this proposal. Changes in overall respiration rates may occur during vegetative multiplication (Edwards and Lloyd, 1977a), but the most significant changes occur during encystment when rates first increase slightly and then drop to minimal values (Band and Mohrlok, 1969; Chagla and Griffiths, 1974). Trophozoite oxygen uptake rates reported for three species of acanthamebas fall within the relatively narrow range of 5 to 18 nmoles/106cells per minute for log-phase trophozoites (Table IV). The agreement is especially good, since three different methods of measurement were used and both agitated and unagitated cultures were studied. Cyst rates were approximately 20% of the vegetative cell values (Table IV). Studies of synchronous cultures of A . castellanii indicated that respiration rates of trophozoites oscillated continuously throughout a single cell cycle (Edwards and Lloyd, 1978b). Pool levels of ATP, ADP, and AMP also oscillated, although not necessarily in phase with respiration. The total adenylate charge varied between 0.63 and 0.88. It was concluded that the oscillations reflected endogenous regulation of respiration. Changes in overall respiration rates during encystment are accompanied by ultrastnrctural modifications of the mitochondria. Vegetative cells have mitochondria with tubular cristae typical of protozoans (Vickerman, 1962; Bowers and Korn, 1968; Idzikowska and Michejda, 1969). During encystment, the number of mitochondria in A. castellanii remained relatively constant (Bowers and Kom, 1968), although the organelles became smaller, more spherical, and often elaborated intracristal granules (Vickerman, 1962; Bowers and Korn, 1968). Mitochondria isolated from cysts of A. rhysodes had lower respiration rates on several substrates when compared to mitochondria from log-phase cells (Table IV; Band and Mohrlok, 1969). It is likely that the decreased respiration and the ultrastructural changes are related, but the basis for the respiratory deficiency is unknown.

301

DIFFERENTIATION IN ACANTHAMOEBA TABLE IV RESPIRATION RATESO F CELLS IN PEITONE-BASED CULTURE MEDIA Oxygen uptake (nmoledminute per lo6 cells) Species

Log phase

Acanthamoeba castellanii (Neff)

6.8 17.0 5-6 7-8 18.1 8-13 12.8 8.1 12.3

Acanthamoeba palestinensis Acanthamoeba rhysodes

cysts

References

-

Neff et al., 1958n Byers et al., 1969* Chagla and Griffiths, 1974c Chagla and Griffiths, 1974d Hamburger, 1975' Edwards and Lloyd, 1977a' Bowers, 1977' Reich, 1948h Band and Mohrlok, 1969

2 -

1.5

2.5

"Measured in buffer but said to be the same as in growth medium; 120 pVhour per milligram of nitrogen; 28°C. *25.4 pLYhour per lo6 cells; 30°C. rDetermined from Fig. 2; 30°C. dValues are for Mg'+-supplemented growth medium; 30OC. '2.7 X l O P pLYhour per cell; 30°-31"C. 'Determined from Figs. 4 and 6; 30°C. '19.2 pVhour per lo6 cells; 29°C. 117.3 pVhour per IO'cells for trophozoites of unspecified growth phase; 22 pVhour per IO'cysts; 27°C.

Band and Mohrlok )1969) found that respiration increased during early encystment by an amount approximately equivalent to the residual respiration of cysts. They proposed that the initial increase might represent activation of a new respiratory pathway used for encystment. Little evidence is available on this point; Hryniewiecka (1967) found a 30-fold decrease in the ability of cyst mitochondria to use NADH, whereas the ability of the mitochondria to use succinate decreased only 2-fold. There is, however, evidence for alternative electron transport pathways in vegetative cells, and the possibility that changes in the respiratory chain occur during encystment needs further exploration. 2. Electron Transport Pathways and Oxidative Phosphorylation Reich (1955) observed that respiration in vegetative A . palestinensis was relatively insensitive to potassium cyanide and sodium azide; concentrations of 1-2 mM inhibited oxygen uptake less than 50% in a phosphate buffer, whereas higher concentrations were lethal. Respiration was inhibited 15% by 100 pM potassium cyanide or 100 pM azide in the buffer, but the addition of glucose

302

THOMAS J . BYERS

raised the rates slightly above that for uninhibited controls. In contrast, peptone stimulated aide-inhibited respiration but failed to enhance cyanide-inhibited respiration. Reich concluded that the respiratory enzymes included some that were inhibited by cyanide or azide and others that were stimulated by these agents. Neff et al. (1958) reported a single experiment in which 46 pM potassium cyanide inhibited respiration in A , castellanii, a result that seemed to agree with that of Reich. Band and Mohrlok (1969), however, found that the respiration of vegetative amebas, starved amebas, encysting amebas, and cysts of A. rhysodes was stimulated at least transiently by 2 mM potassium cyanide when respiration was examined in culture medium. Greater than 40% stimulation was observed with all cell stages except vegetative amebas, where the increase was less than 10%. The apparent inconsistency of these results with those of Reich and Neff et al. is not surprising since, as discussed below, it is now known that the effect of cyanide depends on cell age and is altered by several environmental factors. Low-temperature spectroscopy indicated that mitochondria of A. castellanii contained cytochromes b555,bSe2,b565,c549,and somewhat unusual a-typ cytochromes (Edwards and Lloyd, 1978a; Edwards, et al., 1977;Lloyd and Griffiths, 1968). Two cyanide-reacting and several carbon monoxide-reacting components seemed to be present (Edwards et al., 1977). It appears that acanthamebasutilize a branched electron transport system (Fig. 2) in which one branch probably follows a relatively conventional cytochrome b-c-a sequence and the other branch leads to an alternative terminal oxidase (Edwards and Lloyd, 1977b; 1978a; Hryniewiecka and Michejda, 1977). The conventional pathway may be inhibited by cyanide at the site of the a-type cytochromes, but the alternative EXOGENOUS

MALATE GLUTAMATE a -KETOGLUTARATE PYRUVATE

-NADH

SUCCINATE

1

Ill

-UQ--CYT

\ BYPASS/

B-CYT

SHAM

C-CYT

A-02

A

CN-

t 02 FIG.2. Hypothetical electron transport pathway for mitochondria of Acunrhurnoebu. The scheme is a slightly simplified version of the model proposed by Edwards and Lloyd (1978a). These authors point out that there is at least one additional site where oxygen may be reduced and that the data do not rule out the possibility that exogenous NADH can be oxidized by a cytochrome involved in a pathway that bypasses the scheme shown. SHAM, Salicylhydroxamic acid; UQ, ubiquinone; cyt, cytochrome; 1 4 1 , coupling sites for ATP synthesis; NADH, the intramitochondrial fraction of this compound.

DIFFERENTIATION IN ACANTHAMOEBA

303

pathway is insensitive to cyanide. Thus Reich’s observation (1955) of relatively low cyanide sensitivity in A . palestinensis can now be explained by the presence of the alternative pathway. This pathway can be inhibited by salicylhydroxamic acid (SHAM), and 100% inhibition of respiration can occur when potassium cyanide and SHAM are both present (Edwards and Lloyd, 1977b; 1978a). The existence of similar branched pathways is known for other microorganisms (Knowles, 1976) and some higher plants (Solomos, 1977). At present, there is agreement on the existence of a branched pathway in A . castellanii, but there is some controversy over whether the relative dependence on each branch changes during population growth and aging. Edwards and Lloyd (1977b) observed that respiration in young log-phase cells was stimulated by potassium cyanide, an observation in agreement with Band and Mohrlok (1969), but that cyanide inhibited respiration in older cultures. They suggested that conventional and alternative terminal oxidases both could be utilized during early log phase, but that a shift to dependence on the conventional pathway occurred after mid-log phase. The shift would explain the inhibition of respiration by cyanide. This conclusion was consistent with other evidence indicating that respiratory metabolism changed during log phase. For example, respiration of mitochondria utilizing malate or glutamate (endogenous NADH-linked substrates) produced 1.9 moles of ATP from ADP for each gram-atom of oxygen consumed (i.e., exhibited an ADP/oxygen ratio of 1.9) when isolated from log-phase cells, and 2.6-2.7 moles when isolated from stationary-phase cells (Table V; Evans, 1973). Rotenone failed to inhibit respiration in early log-phase cells but was inhibitory after mid-log phase (Edwards and Lloyd, 1977b; Evans, 1973). Early log-phase cells produced high levels of AMP, whereas much lower quantities were produced after mid-log phase (Edwards and Lloyd, 1977a). The high AMP production resulted in an unusually low energy charge ratio of about 0.1 during early log phase, but subsequent decreases in AMP synthesis raised the energy charge ratio up to about 0.8, a more nearly normal value for growing cells (Chapman and Atkinson, 1977). In addition to these changes, it was reported that the rates of respiration and glucose utilization increased significantly at mid-log phase (Edwards and Lloyd, 1977a,b). The data presented in these cases, however, were not critical rests of whether changes in these parameters really occurred. Nevertheless, it is clear that significant differences in inhibitor sensitivity, ADP/oxygen ratios, and AMP production occur between early and late log phase in cells grown under the conditions used in the preceding experiments. The main question is whether these changes are necessary correlatives of population growth and aging. Edwards and Lloyd (1977b) proposed that the rotenone insensitivity of young cells could be explained by assuming that the electron flow in the mitochondria of these cells bypassed the first site of ATP synthesis (Fig. 2 ) and therefore that a maximum ADP/oxygen ratio of 2 could be expected. The appearance of rotenone sensitivity in older cultures was thought to mean that the electron flow was pass-

TABLE V RESPIRATION OF ISOLATED MITOCHONDRIA” ~

Log phase Species Acanthamoeba castellanii

Acanthamoeba

Substrate

Stationary phase

cysts

RO,

RCR

ADPloxygen

RO.

RCR

ADPloxygen

Succinate Succinate Succinate Succinate

53.5 0.3 28.0 -

2.5 1.6 -

1.7

79.7 -

2.4 -

1.7 1.8

Succinate Succinate

63.2 60.8

1.4 1.4

1.5

66.7 68

1.9 1.5

1.4 1.6

NADH-linked

9-32

1.9

9-35

NADH-Iied NADH-linked

46.6 51

3.23.4 3.0 1.81.9

2.4 2.52.7

62.6 4568

1.64.0 4.2 2.23.1

2.62.7 2.4 2.63.6

1.7 1.21.5 1.4

RO,

RCR

-

1

6.9

ADPIoxygen

Reference

5 6

rhysodes Acanhmoeba castellanii Acanthamoeba rhysodes

1

8.18.7

-

5

1

6

“Respiration rate (RO,) in the presence of ADP expressed as nanomoles of oxygen per minute per milligram of protein. Respiratory control ratio (RCR) expressed as respiratory rate with ADP relative to rate after ADP is exhausted. ADP/oxygen is the ratio of ADP phosphorylated to oxygen utilized. 1. Evans, 1973. NADH-linked substrates used were malate, glutamate, and a-ketoglutarate. 2. Fouquet, 1973a. 3. Lloyd and Griffiths, 1968. 4. Budzinska and Michejda, personal communication. Twenty percent higher values could be obtained for ADP/oxygen using a hexokinase trap to prevent recycling of ADP. 5. Edwards and Lloyd, 1978a. 6. Band and Mohriok, 1969. Values listed for stationary phase m for starved cells; NADH-linked substrates were pymvate plus malate, or a-ketoglutarate.

DIFFERENTIATION IN ACANTHAMOEBA

305

ing through site 1 and therefore explained why ADP/oxygen ratios greater than 2 were observed (Table V). Rotenone inhibited respiration in young cells following the addition of potassium cyanide. Therefore it was suggested that cyanide, in addition to inhibiting electron flow at cytochrome a, might also shift the electron flow to site 1 from the hypothesized site-1 bypass. Recent experiments suggest alternative explanations for some of the observed changes in respiratory metabolism in A . castellanii. It was shown that depletion of iron could occur during culture aging and that an iron deficiency inactivated the electron transport leading to the cyanide-insensitiveterminal oxidase (Hryniewiecka et al., 1978; Jenek and Hryniewiecka, 1979). This iron deficiency caused an increase in cyanide sensitivity in older cultures, since respiration depended on the conventional terminal oxidase. Supplementation of the growth medium with at least 8 pM ferrous sulfate prevented loss of the alternative pathway. Functioning of the alternative pathway also was sensitive to AMP concentration (Edwards and Lloyd, 1978a). In contrast to whole cells, where potassium cyanide always stimulated respiration if sufficient iron was present, cyanide inhibited mitochondrial respiration unless AMP was added (Hryniewiecka and Michejda, personal communication). The increased respiration following the addition of AMP to potassium cyanide-inhibited mitochondria was completely inhibited by SHAM and therefore was attributed to the alternative pathway. Edwards and Lloyd (1978a) found that both AMP and ADP stimulated this pathway. It has been suggested that the loss of alternative oxidase activity observed by Edwards and Lloyd (1977b) during culture aging might be related either to a developing iron deficiency in the culture medium or to the greatly different rates of AMP production in early and late log-phase cells (Hryniewiecka et al., 1978). In any case, loss of the alternative oxidase activity is not a necessary correlate of culture aging. Budzinska and Michejda (1978 and personal communication) found that mitochondria isolated from either log phase or stationary phase were sensitive to 8 p M rotenone when respiring on endogenous reserves or on substrates such as malate and glutamate, but were relatively insensitive when succinate or exogenous NADH was the substrate. Rotenone sensitivity of log-phase mitochondria respiring on a-ketoglutarate was observed by Edwards and Lloyd (1978a). These results for A. castellanii, along with the fact that ADP/oxygen ratios greater than 2 could be obtained for log-phase mitochondria of A. rhysodes and A. castellanii respiring on endogenous NADH-linked substrates (Table V), suggest that a functional site 1 is present and can be used in log-phase mitochondria. Site 1 would be bypassed by succinate and exogenous NADH as observed in other systems (Palmer, 1976). The results conflict with reports that log-phase mitochondria of A . castellanii are insensitive to 10 pA4 rotenone and have ADP/oxygen ratios less than 2 (Evans, 1973; Lloyd and Griffiths, 1968). The reason for the discrepancy is unclear. It is possible that a site-1 bypass is present, but that the pathway of electron flow is readily modified by such factors as culture conditions, mitochon-

306

THOMAS J. BYERS

drial isolation methods, and conditions for measuring respiration. Fouquet (1973a) found evidence for great flexibility in the pathways of electron flow in mitochondria of A . castellanii when they were studied with artificial electron acceptors and various inhibitors. In addition, he found that inhibitors could act at unexpected sites. For example, antimycin A, which normally inhibits electron flow at cytochrome b, was found to inhibit NADH oxidase (Fouquet, 1973b). This kind of observation simply accentuates the potential problems in the interpretation of any inhibitor studies.

D. NUCLEAR REPRODUCTION A N D CYTOKINESIS 1. Mitosis The mode of nuclear division is one useful criterion for the classification of small free-living amebas (Pussard, 1973; Singh, 1975), although there is disagreement on the relative importance of this criterion (Singh, 1975; Griffin, 1978). Techniques for the photography of mitosis have been described (Molet and Kremer, 1976), and excellent photographs have been published for’Acanthumoeba lenticularu (Molet and Ermolieff-Braun, 1976), A . cornmondoni, and A . astronyxis (Pussard, 1972, 1964). In general, nuclear reproduction in Acanthurnoeba is by metamitosis, a relatively typical form of mitosis characterized by formation of a bipolar spindle and disappearance of the nucleolus and nuclear envelope (Pussard, 1973). Although typical centrioles have not been described by electron microscopy, centriole-equivqent bodies can be visualized by phasecontrast microscopy (Pussard, 1972; Molet and Ermolieff-Braun, 1976). Dense material said to have similarities to centriolar satellites described in other organisms has been found in association with Golgi bodies in A . castellanii (Bowers and Korn, 1968) and A . royreba (Willaert er al., 1978). In contrast to the situation in Acanthumoeba, the nucleolus and/or the nuclear envelope may persist during mitosis in other small free-living amebas (Pussard, 1973). Chromosomes are visible during mitosis but are small and often clustered too tightly to permit accurate counts (Pussard, 1964, 1972). However, Volkonsky (1931) estimated about 80 chromosomes for A . castellanii. Cytokinesis occurs during mitosis, but nuclei reform prior to complete cell separation. A final stage in division results in daughter cells connected by a fine filament. According to Pussard (1964), remnants of the spindle pass through this filament in A. commondoni. This observation is interesting when compared with observations on amitosis (see Section II,D,2). 2. Amitosis Several different types of atypical nuclear division or uncoupling between nuclear division and cytokinesis have been described. Tripolar mitotic figures, as

DIFFERENTIATION IN ACANTHAMOEBA

307

well as the production of anucleate cell fragments, were found in A. commondoni (Pussard, 1964). The latter phenomenon seemed to occur in dense cultures. A phenomenon called amitosis can occur with relatively high frequency in suspension cultures of A . rhysodes under some conditions (Band etal., 1970; Band and Mohrlok, 1973a,b). In amitosis, the nucleus locates in the region of the cytoplasmic division furrow and pulls apart into two halves as cytokinesis progresses. The half-cells formed typically are held together by a very long connecting filament until final separation occurs. Each division product receives part of the nucleus, but the majority of the visible nucleolus usually ends up in one of the daughters. Both products eventually die. Frequently, one product undergoes a second amitotic division before the first is complete, and additional divisions are possible before death occurs. Band and Mohrlok (1973a) described one case in which amitosis resulted in an average of three anucleolate products and one nucleolate product. Division products often are very different in size. Electron micrographs have shown that the nucleus is continuous through the long amitotic connecting filaments (Band et a l . , 1970). Nuclear events in amitosis have not been described in detail, but the liklihood that the nuclear envelope remains intact is interesting, since this is a normal aspect of mitosis in some other genera of free-living amebas (Pussard, 1973). Band and colleagues induced amitosis by agitating cells for 24 hours in glucose-deficient growth medium and then transferring them with continued agitation to normal growth medium supplemented with dilute agar. The agar was thought to provide suspended amebas with a surface for attachment and subsequent amitotic division; only mitosis was observed in the absence of agar. Amitosis also could be induced in a low-glucose chemically defined medium and in a nonnutrient salt medium. The phenomenon could be observed in unagitated cultures following glucose starvation, but better synchrony was obtained in suspension cultures. Band et al. (1970) were unable to induce amitosis in the Neff or Jensen strains of A. castellanii or in A . palestinensis, but I have observed it commonly in the Neff strain grown on chemically defined media. In contrast to the observations of Band and colleagues, I found amitosis in the presence of high glucose during amino acid starvation. Possibly the aberrant nuclear behavior can be induced by a variety of selective nutrient deficiencies. Amitosis following dilution of late log-phase cells in fresh growth medium has also been observed for A. castellanii in our laboratory. Band and Mohrlok (1973b) observed that induced amitosis never involved an entire population and reasoned that the potential for this phenomenon might be related to the cell cycle. Experiments on cells released from colchicine blocks indicated that amitosis occurred most readily in mid-Gz phase. Amitosis is interesting as a subject for studies on the control of nuclear replication, but also is a practical problem, since it produces a subpopulation of incomplete and dying cells. Band and Mohrlok (1973a) demonstrated that this subpopu-

308

THOMAS J. BYERS

lation could be relatively large under some conditions. It is not clear how great a variety of environmental conditions can induce amitosis, and therefore investigators should be alert to its possible presence under any new set of conditions. 3. Uncoupling of Nuclear Division and Cytokinesis Actively growing acanthamebas in unagitated cultures typically have one nucleus with a prominent nucleolus (Page, 1967). A few binucleates sometimes are present during log phase in A. castellanii (Byers et al., 1969), but cells with several nuclei more often are found in post-log-phase cultures (Neff, 1957; Byers et al., 1969, Pigon, 1972). A suspension culture consisting mainly of mononucleates has been reported for A. castellanii (Jensen et al., 1970), but suspension of previously unagitated cultures of A. rhysodes and A . castellanii led to relatively high levels of multinuclearity (Band and Machemer, 1963; James and Byers, 1967; Kjellstrand, 1968). James and Byers observed up to 62% of cells with 2 or more nuclei and occasional cells with 20 or more (unpublished data). It is generally assumed that the multinuclearity resulted from continued nuclear division in the absence of cytokinesis. In fact, there was no definitive evidence to rule out the possibility that some multinucleates arose from cell fusion. This possibility seems unlikely, however, since our deliberate efforts to fuse cells of the Neff strain experimentally have been unsuccessful and seem to indicate that cell fusion can occur less readily in this organism than in numerous other cell types. This fact may be related to the unusual composition of Acanthamoeba plasma membranes (Section II,B, 1,b). Pigon (1972) isolated a fraction from conditioned cell culture medium that was able to induce multinuclearity in growing populations of A. castellanii. The process was observed by time-lapse photography and appeared to result from a failure of cytokinesis. In a few cases there was a possibility that two postdivisional daughter cells fused, but it was uncertain whether cytokinesis actually had been completed. In other experiments, Pigon induced multinuclearity in a mixture containing unlabeled cells and cells that had been labeled for 8 to 10 generations with [14C]oroticacid. He then used autoradiography to examine labeled cells for the presence of unlabeled nuclei. In a total of seven experiments, 4.3 ? 4.2% of the labeled multinucleated cells contained unlabeled nuclei. He concluded that most multinucleates arose by failure of cytokinesis, but that some fusion was also possible. When multinucleated cells from agitated cultures were permitted to settle out and attach to a surface, cytokinesis occurred repeatedly until the mononucleated state was restored (James and Byers, 1967). The division events that occurred resembled plasmotomy as described for other protozoans (Kudo, 1966). The variety of possible relationships between nuclear division and cytokinesis can be a practical problem for the researcher, but it also presents an opportunity for studies of factors that normally coordinate nuclear and cytoplasmic division.

DIFFERENTIATION IN ACANTHAMOEBA

309

4. Cell Cycle Phases Information about cell cycle phases has been obtained by several methods. The primary objective in most of the studies has been to discover whether there is any correlation between specific phases of the cell cycle and the ability to differentiate. Neff (1971) observed that short pulses of [3H]thymidine([3H]TdR) labeled 3-5% of the cells in an exponentially growing population of A. castellanii. Using this information and the methods of Sisken (1964), he estimated the percentages of time in each portion of the cell cycle: M, 2%; GI, 10%; S, 3%; and Gz, 85%. A second estimate based on analyses of encystment in response to 2-deoxy-5-fluorouridine (FUdR) gave M, 2%; G1, 20%; S, 3%; and G2,74% (Neff and Neff, 1972). Our experience indicates that cell division lasts approximately 10 minutes out of a total generation time of 6-8 hours. Thus the 2% estimate for M is relatively consistent with our experience. We tend to obtain higher percentages of nuclei labeled during [3H]TdR pulse-labeling. When pulse-labeling was combined with microspectrophotometric analyses of DNA distributions in Feulgen-stained nuclei, no evidence was obtained for a G1 population (King and Byers, in preparation). Instead, our data suggest that the portion of time attributed to G1in Neff’s 1971 estimate should be added to the estimate for S phase. Our data do not entirely rule out a G, phase, but it certainly cannot be as high as 10%. Data from work on A. rhysudes also indicate the lack of a G, phase (Band and Mohrlok, 1973b). Using [3H]TdRlabeling plus inhibition of the cell cycle with colchicine, these authors concluded that nuclear mitosis occupied 2% (24 minutes) and S phase 2% (20 minutes) out of a total generation time of 18 hours. Evidence that DNA synthesis began immediately following mitosis was obtained from two observations. First, cells blocked with colchicine, presumably in metaphase, did not incorporate [3H]TdR into nuclei in the presence of drug, even though the label was incorporated into cytoplasmic DNA. When the drug was removed, nuclear mitosis occurred immediately and [3H]TdR incorporation was detected in newly forming daughter nuclei before the nucleoli reformed. These data suggest that DNA synthesis does not occur in metaphase, but sometime soon after. The second observation was on mitosis that occurred when nuclear division was uncoupled from cytokinesis by agitation. In this case, metaphase nuclei did not label when exposed to r3H]TdR, but again, label appeared in the daughter nuclei before nucleoli reappeared. Recently developed methods for obtaining synchronous populations of acanthamebas should prove useful in the analyses of cell cycle phases (Chagla and Griffiths, 1978). Early studies indicate that cells undergo a relatively long lag before DNA synthesis and that cell division occurs soon after the doubling of DNA. These results can be interpreted as indicating that there is a relatively long G1phase in the cell cycle. This conclusion seems inconsistent with the best information from other sources. Thus it is appropriate to seek other explanations. One possibility is that the initial lag before visible DNA doubling is due to disturbances caused by the method

310

THOMAS J . BYERS

used. This seems rather unlikely as discussed by the authors. Another possibility is that the synchronous DNA increase observed is in nonnuclear DNA (Section III,A, 1). A third possibility is that suspension cultures are heterogeneous and that the selection method isolates an atypical subpopulation of cells. Until these possibilities have been explored, it is reasonable to accept the evidence that GIis relatively short or absent in acanthamebas. It has been proposed that encystment is initiated from late S phase in A . castellanii (Neff and Neff, 1969; 1972; Neff, 1971). This conclusion was based on inhibitor studies and consequently depended on the sometimes difficult problem of correctly predicting the molecular consequences of inhibitor treatments. Our studies, based on decreases in cellular DNA, suggested that encystment might occur from G,or early S phase (Byers et al., 1969; Rudick, 1971). More recently, microspectrophotometric analyses of nuclear DNA distributions during starvation-induced encystment demonstrated that cyst and log-phase nuclei had similar DNA content and distributions, both cell types probably being primarily in G, (King and Byers, in preparation). In our laboratory, independent assays of DNA obtained from cell lysates after centrifugation in cesium chloride gradients favor some decrease in nuclear DNA during encystment. The loss appears to be due to degradation of the DNA. This is consistent with the microspectrophotometric data which do not favor a cell cycle shift. It is possible that inhibitor-induced encystment differs from starvation-induced encystment, but in the latter case it appears that differentiation may occur primarily from G,phase. Since the data of Band and Mohrlok (1973b) indicate that DNA synthesis begins immediately following or during late mitosis, it is possible that starvation or drug treatments permit cells to enter early stages of S without allowing completion of cell division. In this case, cysts might be blocked in S but would contain G, amounts of DNA plus whatever DNA had been replicated during the second round of synthesis.

111. Molecular Biology and Differentiation

A. CLASSES AND METABOLISM OF MACROMOLECULES 1. DNA Autoradiographic studies revealed the surprising fact that [3H]TdR was more actively incorporated into the cytoplasm than into the nucleus of Acanthamoeba (Ito eral., 1969; McIntosh and Chang, 1971). Only 40-50% of the cytoplasmic label seemed to be associated with mitochondria (It0 et al., 1969). It was suggested therefore that acanthamebas might possess cytoplasmic DNA in the

31 1

DIFFERENTIATION IN ACANTHAMOEEA

form of an episome or a defective virus in addition to the nuclear and mitochondrial DNA. There have been several efforts to characterize DNA isolated from Acanthamoeba, the most extensive studies being on DNA from A . castellanii Neff isolated from soil. The strains studied by Chang and colleagues were isolated from humans, and the difference in origin could be importit. Possible endosymbionts were identified in A. castellanii Snagov isolated from humans (ProcaCiobanu et a f . , 1975), but to date no endosymbionts have been reported in the Neff strain. Page (1967) noted a possible cytoplasmic infection in A . polyphaga isolated from fresh water, consequently, the presence of nonmitochondrial cytoplasmic DNA is a possibility that should be considered for all strains regardless of origin. Whole-cell DNA is resolved into two bands by the use of cesium chloride density gradient centrifugation. Nuclear DNA is found in the main band at a bouyant density ( p ) of about 1.72 gm/cm3 (57-62% G C) for A. castellanii, A . palestinensis, and A . polyphaga, and about 1.71 (50% G C) for A. astronyxis (Table VI). Estimates of the G + C content based on melting temperature (T,) were several percent different from those based on p, therefore suggesting the presence of modified bases in the DNA (Bohnert and Henmann, 1974). Mitochondria1 DNAs (mtDNAs) of A. castellanii and A . ustronyxis were found in the minor band at a density of about 1.69 gm/cm3 (30-35% G C). The minor bands of two other species were slightly heavier and had correspondingly higher estimates of G + C (Table VII). The minor band accounted for 10-30% of purified whole-cell DNA in measurements using an analytical centrifuge. In contrast, when cells were exposed to long-term [3H]TdR labeling, lysed directly in cesium chloride, and then centrifuged in a preparative ultracentrifuge, 60-70% of the total label appeared in the minor band (Kuhns, 1976). This distribution could be explained by differences in the TdR content of the DNA if the TdR were labeled to the same specific activity in each band and the bands contained approximately equal amounts of DNA. More recent studies of [3H]TdRlabeling patterns and specific activity measurements of DNA in our laboratory favor the lower minor band estimates in the range of 10-30% of the whole cell DNA. Alternatively, large natural variations in the amounts of minor band DNA also are possible. There are some differences in estimates of kinetic complexity in both major and minor bands. Marzzoco and Colli (1975) concluded that nuclear DNA was almost entirely composed of unique sequences with a kinetic complexity of 1.46 x 10" daltons. Other workers found 20-30% repetitious nuclear DNA se' daltons for the unique sequences quences with a complexity of about 2 X 1OO and 0.2-4 x los daltons for the repetitious sequences (Table VII). Kinetic complexity was determined in all these studies by using the relatively insensitive optical methods to study reassociation kinetics of denatured DNA. A more accu-

+

+

+

TABLE VI CHARACTERISIICSOF NUCLEAR DNA

Species Acanthatnoeba castellanii

Acanthamoeba palestinensis Acanhzmoeba P o W w Acanthamoeba astronyxis Acanrhamoeba culbertsoni

Percent G + C based on

Bouyant density (gm/cm3)

T, ("C)

p

1.720

806

61

63

1.717 1.720 1.716

-

75.5*

58 61 57

-

-

-

61

22

1.720'

-

2.1 x loio (U), 0.17-4.1 X l P ( R )

61

-

-

-

-

69'

-

-

94.2d

T,

53=

Kinetic complexity (daltons)"

1.46 x 1011

1.9 x 1010 (u), -1.5 x 108 (R)g

Repetitive sequences

None detected

30

References Adam et al., 1969 Mamma and Colli, 1975 Hettiarachchy and Jones, 1974 Bohnert and Hemnann, 1974

Kuhns, 1976 Jantzen, 1973

-

-78.56

-

1.721

-

62

-

-

-

-

-78.5'

57

-6a -

-

-

Band and MohrIok, 1973b Adam et al.. 1969 Band and Mohrlok, 1973b Adam er al.. 1969

50

-

-

-

Band and Mohrlok, 1973b

-

47

-

-

Band and Mohrlok, 1973b

1.716

-

1.709

-

-

-73b

-60

'U, Unique DNA sequences; R, repetitious sequences. bMeasured in 10% standard saline citrate (SSC). 'Calculated with equations from Mandel, Schildkraut and Marmur (1968). dMeasured in SSC. 'Measured in a preparative ultracentrifuge. 'Measured in 4 x SSC; 50% formamide. 'Calculated value: kineticcomplexity = C,r% (=0.33) X molecular weightof basepair(=618) x K (=7.14 x lW), where K wasdetermined from data for the unique fraction.

TABLE VII CHARACTEMSTICS OF MITOCHONDRIAL DNA %G+C based on

Bouyant Species Acanrhamoeba casreliunii

density (gmlcmg) 1.693

p

T,

66" a00

34

-

30b 64b

6 3 " ~ ~ 23

-

Kinetic complexity (daltons) -1

X

lo*

4.3 x 10' ( l a w 1.2 x 1010 (a21)d 3.4 x 107

Total DNA in Length of minor band (8) genome ( p m ) 30

References Adam et a!., 1969

-

16

1.697

-

37

-

Marzmco and Colli, 1975 Hettiarachchy and Jones, 1974 Bohnert and Heman, 1974 Adams et al., 1969

1.702

-

43

-

Adams era!.. 1969

1.689

-

30

-

Adams et al., 1969

1.692

Acanthamoebu palestinensis Acunthamoeba PO!YPhOgo Acanthamoeba astronyxis

T, ("C)

1.694

83'

35

34

1.690

82'

31

31

2.57 x lo7

-

16.7

10-18

12.7

"Measured in 10% SSC. bCalculated from equations in Man&l er al.. 196%.Two species of DNA were present. CValue is average for entire minor band; three subfractions with T, of 62", 65", and 75°C were identified. d T ~ species o were in the mtDNA; a third species (complexity of 2.5 X lo6 daltons) was reported in the minor band but absent from the mitochondria. 'Measured in SSC.

3 14

THOMAS J . BYERS

rate measure of unique and repetitious fractions would require other techniques (Britten et al., 1974). The methods of Marzzoco and Colli (1975) could not rule out the presence of small amounts of repetitious DNA, however, and it is unlikely that nuclear DNA is composed only of unique sequences. A single kinetic class of DNA was isolated from mitochondria when organelles were treated with DNase prior to extraction of the DNA (Table VII; Bohnert and Herrmann, 1974; Hettiarachchy and Jones, 1974). The kinetic complexity measured for this DNA ranged from 2.6 to 3.4 X lo7daltons. Up to 80% of the DNA could be isolated as circular molecules (Bohnert and Herrmann, 1974). Isolated molecules averaged 12.7 or 16.7 nm in length (Table VII). Recently, Bohnert and von Gabain (personal communication) have confirmed the smaller size by additional measurements and extensive fragment mapping following digestion of the mtDNA with restriction enzymes. Two kinetic classes were found when mtDNA was isolated from organelles that had not been pretreated with DNase (Adam et al., 1969; Marzzoco and Colli, 1975). The presence of subfractions with a high T , and relatively high kinetic complexity (Table VII) suggests that nuclear DNA was contaminating the mitochondria1preparations. A third fraction with a kinetic complexity of 2.5 X lo6 daltons was found by Marzzoco and Colli (197s) when minor band DNA was isolated from whole cells without prior isolation of mitochondria. These authors suggested that this fraction might be a nonmitochondrial cytoplasmic DNA, as postulated by Chang and colleagues to explain their autoradiographic results. It would be worthwhile to study this fraction further. One approach would be to utilize more sensitive methods of studying reassociation kinetics (Britten et a l . , 1974). Another approach would be to fractionate the cytoplasm and then to try to extract DNA from nonmitochondrial fractions. The existence of cytoplasmic DNA probably should not be taken too seriously until it has been verified by other methods. Nevertheless, until the question is finally resolved, it is appropriate to avoid equating mtDNA and minor band DNA unless the DNA is extracted from mitochondria. 2. DNA Synthesis and Degradation The DNA content of Acanthumoeba varies with culture age and growth conditions (Byers et al., 1969; Chagla and Griffiths, 1974). Variability may be caused by changes in nuclear number, duration of cell cycle phases, or DNA synthesis and turnover, but the correct explanations have not always been clear. Most studies have reported values between 1.2 and 2.3 pg/ameba for log-phase cultures of A . castellanii (Table WI). DNA values of 4.5 pg/cell have been reported for A. rhysodes and of 4.4 pglcell for A . polyphaga (CouIson and Tyndall, 1978). Careful studies in our laboratory indicate that variable measurements are common (King and Byers, in preparation). The reasons for this are not entirely clear. Byers et al. (1969) reported that DNA per cell decreased during

315

DIFFERENTIATION IN ACANTHAMOEBA

late log phase in unagitated cultures. Recently, we have confirmed these results and demonstrated a further loss of DNA when post-log-phase cells encysted. Decreases also have been reported (Table VIII) for suspension cultures in PYG medium (Chagla and Griffiths, 1974) and for encystment in nutrient-free medium (Neff and Neff, 1969). In contrast, no changes were observed during the growth of suspension cultures in Neff’s growth medium (Roti Roti and Stevens, 1975), and increases were observed during the induction of encystment by Mg2+in the nutrient-rich PYGS medium (Chagla and Griffiths, 1974). It remains to be seen whether the different patterns of change are developmentally significant or only trivial differences unimportant for development. Changes in DNA content during the transition from log phase to post-log phase and during encystment are interesting because of their possible significance in differentiation. Neff and Neff (1969) proposed that a 25-30% decrease in DNA seen during starvation-induced encystment in A. casfellunii might be due to degradation of mtDNA. This suggestion is consistent with observations that mitochondrial structure changes and that some mitochondria are found in digesTABLE VIII DURING GROWTH A N D DIFFERENTIATION OF WHOLE-CELL DNA CONTENT ACANTHAMOEBA CASTELLANII

DNA content (pg/cell)c Culture conditions“ M M M M S S S S S S

Mediumb

LP

PL

PC

c

Reference

GMd GM EM GM GM GM‘ Mg-GM‘ EM GM’ GM

1.6 1.4

0.84 0.89

-

-

-

-

Byers et al., 1969 King and Byers, in preparation King and Byers, in preparation Coulson and Tyndall, 1978 Tomlinson, 1962 Chagla and Griffiths, 1974 Chagla and Griffiths, 1974 Roti Roti and Stevens, 1975 Marzzoco and Colli, 1974 Agrell et al., 1969

3.6g 1.0 1.7 1.2 2.3 10.6 0.66

1.4

-

0.65

2.3 2.3

-

0.62 -

1.9

-

“M, Monolayer culture; S, suspension culture. bGM, Growth medium; EM, nutrient-free encystment medium; Mg-GM, growth medium supplemented with Mg2+ to induce encystment. “LP, Log-phase; PL, post-log phase under conditions that do not favor encystment; PC, pre-cyst phase defined as post-log phase under conditions that favor encystment; C, cyst phase. dThe published values have been lowered to be consistent with the more recent and accurate standard curves used by King and Byers. ‘Values corrected for multinuclearity. ’values for isolated nuclei. ’Fluorescent assay of intact cells.

316

THOMAS J. BYERS

tive vacuoles during encystment (Section II,C, 1). Since available estimates indicate that a maximum of 10-5096 of whole-cell DNA could be mitochondrial (Table VII and Section J.II,A,l), a large fraction of mtDNA would have to be degraded. Byers et al. (1969) proposed an alternative explanation suggesting that the decrease could be due to the synchronizationof cells at an early stage in the cell cycle. Blockage of the cycle at either GIor early S would result in a net decrease in DNA per cell, because most cells in a log-phase population are in G2 and thus have the replicated amount of DNA. For reasons discussed in Section II,D,4, it appears that encysting cells are not likely to be blocked in GIor early S phase, and consequently this cannot explain the decrease in whole-cell DNA observed during population aging and encystment. If this conclusion is correct, then part of the loss may be in cytoplasmic (mitochondrial?) DNA. Recently, evidence for the differential degradation of minor band DNA during encystment has been obtained in our laboratory (King, unpublished results). It is possible also that differential production of cytoplasmic DNA might explain the one case in which whole-cell DNA increased during encystment (Table VIII;Chagla and Griffiths, 1974). DNA replication in A. castellanii has been examined with the use of [3H]TdR (Kuhns, 1976; Kuhns ef al., 1976). Kuhns (1976) found that [3H]TdRincorporation was greatly reduced in both main band DNA and minor band DNA during the log phase-post-log phase transition in unagitated cultures of A. castellanii. She concluded that synthesis of both nuclear DNA and mtDNA was inhibited. Roti Roti and Stevens (1974) also observed decreases in whole-cell incorporation of TdR during culture aging and during encystmentbut noted that some incorporation continued late into the encystment period. Since the DNA content of the cells did not change, they proposed that synthesis was balanced by degradation. They also concluded that inhibition of synthesis was not required for the initiation of encystment. Subsequently, it was discovered during studies of mitomycin C(MC)-induced encystment that [3H]TdR incorporation into minor band DNA continued long after incorporation into nuclear DNA was inhibited (Kuhns, 1976). Thus it is possible that the continued incorporation observed by Roti Roti and Stevens was into mbDNA, probably mtDNA. 3. RNA and Ribosomes Ribosomes and rRNA from A. castellanii have unusual properties. Ribosomes were isolated from trophozoites and cysts in 300 mM potassium chloride as 66s monomers, a large 53s subunit, a small 40s subunit, and a smaller component X (Parish and Hall, 1972). When ribosomes were isolated in 25 m M potassium chloride, the 53 and 40s subunits were relatively unchanged, but the monomer sedimented at 97s. It was speculated that the latter was simply a more condensed form of the 66s particle. Extensive degradation of RNA by endogenous RNase was observed in all the particles except component X.This component contained 26 and 18s rRNA, but its relationship to the other particles was unknown.

DIFFERENTIATION IN ACANTHAMOEBA

317

RNA isolated from the small ribosomal subunit sedimented at 18s and had an anomalously large molecular weight of 0.88 X lo6 daltons (Loening, 1968; Stevens and Pachler, 1972). RNA from the large subunit contained 26 and 4-5s components (Stevens and Pachler, 1972). The 26s component (1.52 x lo6 daltons) could be dissociated into three smaller components, 18s (0.88 x lo6 daltons), 16s (0.6 X lo6daltons), and 6 s (4.3 X lo4daltons), by treatments that disrupted base pairings (Stevens and Pachler, 1972). The fragments may have originated from single-strand scissions during maturation of the 26s molecule, but no evidence is available on this aspect. In the mature subunit, the three fragments apparently unite by noncovalent bonding to produce the 26s rRNA. RNAs from log-phase trophozoites and starvation-induced precysts of A . castellanii Vc were studied by reverse phase chromatography (McMillen et al., 1974). The number of isoaccepting species of tRNAs for 12 amino acids was examined. Trophozoites had four species of tRNA for arginine and leucine; three species for lysine, serine, phenylalanine, isoleucine, and glycine; two species for valine, methionine, alanine, and asparagine; and one species for tryptophan. The number of isoaccepting species for 10 of the amino acids was the same for trophozoites and precysts. Differences were found in the seryl-tRNAs and, possibly, the isoleucyl-tRNAs. Whereas three seryl-tRNAs were present in trophozoites, only two were found in precysts. There was some evidence for a difference in isoleucyl-tRNA species, but the possibility of a trivial explanation for the differences was not entirely ruled out. In these studies the trophozoites were from late log phase. The use of such high-density populations is a convenience because large numbers of cells can be harvested; however, late log-phase cells already have undergone extensive metabolic changes, and it is likely that differences between trophozoites and precysts or cysts would be greater if younger trophozoites were used. It is interesting that the most significant difference was in seryl-tRNA, since serine seems to be one of the best amino acids for the induction of excystment (Section III,B,4). Poly-A-containing RNA has been isolated from whole-cell RNA and from mtRNA (Jantzen, 1974b; Kuhns, 1976). The poly-A sequences from whole cells were about 80 nucleotides long (Jantzen, 1974a). No changes in the total amount of poly-A RNA occurred during encystment (Jantzen, 1974a). It is presumed that this fraction represents mRNA, and in fact it can be used in in v i m translation (Jantzen, personal communication). No information is available on the precursors, processing, or turnover of mRNA in acanthamebas. 4. RNA Synthesis and Degradation The three classes of eukaryotic DNA-dependent RNA polymerases have been isolated and purified from A. casfellanii (Spindler, 1978a,b; Detke and Paule, 1975). This organism is an excellent source of eukaryotic polymerase, because large quantities of each class can be isolated easily and quickly. Each polymerase contains a large number of putative subunits: 10 in polymerase I (Detke and

318

THOMAS J. BYERS

Paule, 1978a; Spindler et al., 1978b); 13 in polymerase I n (Spindler et al., 1978a); and 11 in polymerase I1 (Paule, personal communication). There may be two forms of polymerase 11, and there is a possibility that certain lowermolecular-weight subunits are shared by polymerases I, 11, and 111. When RNA polymerase activity was studied in isolated nuclei during encystment, it was observed that the activity rose during the first 10 hours of starvation and then decreased (Rudick and Weisman, 1973; Detke and Paule, 1975). However, polymerase activity was not limited to the nucleus and, when the activity of whole cells was examined, there was no change in the activity of polymerase I1 or I11 during the first 10 hours of encystment (Detke and Paule, 1978a,b). Polymerase I, which was associated with the nucleolus and presumed to be involved in rRNA synthesis, was examined in detail to determine whether any changes occurred during encystment. The possibility of changes was considered because cessation of rRNA synthesis was observed within the first 7 hours of starvation (Stevens and Pachler, 1974). In spite of the dramatic change in synthesis of rRNA, no changes in the number or structure of polymerase I molecules were detected (Detke and Paule, 1975; 1978a). Thus the regulation of rRNA synthesis would have to depend on more subtle changes in polymerase I structure or on some other mechanism. Total cellular RNA increased during the transition from log-phase to postlog-phase growth in monolayer and suspension cultures of A. custellunii (Byers et al., 1969; Stevens and Pachler, 1974; Jantzen, 1973). Most of the increase in the monolayer cultures was in rRNA, although low-molecular-weight RNA (-4s) exhibited a greater relative increase (Rudick, 1971). Synthesis of total RNA decreased and then continued at a reduced rate during the log phase-postlog phase transition (Rudick, 1971; Stevens and Pachler, 1974). Consequently, Stevens and Pachler suggested that the accumulation of RNA could be explained if the rate of RNA synthesis decreased less than the rate of cell multiplication. In suspension cultures, where encystment occurred spontaneously during post-log phase, the period of RNA accumulation was followed by a period of degradation, the RNA decreasing to 50% of the log-phase amount (Stevens and Pachler, 1974). A similar decrease was observed when A. castellanii (Neff and Neff, 1969; Jantzen, 1973; Stevens and Pachler, 1974) or A. culbertsoni (Raizada and Krishna Murti, 1972a) was induced to encyst by starvation. Radioactive precursors continued to be incorporated into RNA throughout the first 24 hours of starvation-induced encystment, the period during which total RNA decreased 50% (Park and Loggins, cited in Neff and Neff, 1969). The RNA loss was not inhibited by actinomycin D (AmD) and therefore probably did not depend on the synthesis of new RNA (Jantzen, 1973). The RNA made after the first 7 hours seemed to be low in molecular weight, whereas rRNA synthesis terminated early during starvation- or ethidium bromide (EB)-induced encystment (Stevens and Pachler, 1974; Jantzen, 1974a).

DIFFERENTIATION IN ACANTHAMOEBA

319

It is uncertain whether RNA synthesis is required for cyst formation. Rudick (1971) observed greater than 70% encystment when early to mid-log-phase cultures of A. castellunii were transferred to encystment medium, but cyst formation was reduced to as low as 10% when AmD was present. The cells used had not completed the RNA accumulation characteristic of late log phase before they were placed in encystment medium. In contrast, cells that had completed the accumulation phase before transfer gave maximum encystment in the presence of the drug (Rudick, 1971). Apparently these cells had completed AmD-sensitive processes prior to drug treatment. Increases in the activity of P-glucan synthetase and in cellulose synthesis were depressed during the inhibition of encystment by AmD according to Potter and Weisman (1972). These authors did not indicate the age of the cultures used but observed that AmD sensitivity was lost 14 hours after the transfer of cells to encystment medium. It was demonstrated that AmD inhibited RNA synthesis in these experiments, and therefore it is possible that enzymes for cyst wall synthesis depend on transcription. It is unlikely, however, that encystment requires a large increase in the fraction of active genes (see below). It was demonstrated by RNA-DNA hybridization that 12-14% of the nuclear genome (6-7% of the nuclear DNA) of A . custeflunii was active in RNA synthesis during log phase, whereas the active fraction rose to about 48% in stationary-phase suspension cultures (Jantzen, 1973, 1974a). When the latter were placed in encystment medium plus [3H]uridine, the RNA remaining after 7.5 hours of labeling hybridized with 42% of the genome; the RNA remaining after 17 hours of labeling hybridized with only 34% of the genome (Jantzen, 1974a). Thus some of the RNA sequences synthesized early in encystment were lost subsequently. When stationary-phasecells were concentrated to 10' amebas/ ml, additional RNA species were synthesized until 92% of the genome was represented by complementary RNA copies. It appears therefore that crowding somehow regulates stationary-phase RNA synthesis. New RNA species were also produced, however, when log-phase cells were transferred to encystment medium; therefore nutrient depletion may also be a factor regulating RNA synthesis. The new RNA species were produced at the same time that total RNA synthesis was decreasing (Jantzen, 1974a). The possibility that the new synthesis was essential for encystment was diminished by the discovery that there was no significant increase in the variety of RNA species produced during encystment induced by EB (Jantzen, 1974a). It appears that any new RNA required for encystment by log-phase cells would represent no more than 1% of the nuclear genome. Nevertheless, the possibility that some RNA synthesis is required follows from the observation that EB-induced encystment was inhibited by AmD (Byers and Kuhns, 1973). The new RNA species synthesized during stationary phase or when log-phase cells were placed in nutrient-free medium did not possess poly-A tails. This

TABLE IX INDUCTlON OF ENCYSTMENT IN MONOLAYER CULTURES OF ACANTHAMOEBA CA.TEILAh'II BY INHIBITORS OF NUCLE~C ACID AND PROTEIN SYNTHESIS Level of encystment" Neff Compound

LP

Inhibitors of DNA synthesis MC

5

Trenirnm MethotreKate

5

2

Berenil

-

EB

-

Hydroxyurea FudR

1

DPL

Byers LP

DPL

Mode of inhibitor action in eukaryotes

Cross-Iinks DNA, affecting nuclear and mtDNA in Acunfhurnoebu; little effect on RNA or protein synthesis Probably similar to that of MC Inhibits nucleotide (especially thymidylate) synthesis by inhibiting dihydrofolate reductase Binds preferentially to organelle DNA; site of inhibition unclear Binds to DNA; inhibits organelle DNA and RNA synthesis; can affect nuclear rRNA processing at high concentration Inhibits reduction of ribonucleotides to deoxyribonucleotides; inhibits RNA synthesis in Acunthnmwba Converted to 5-fluorodeoxy-UMP which inhibits thymidylate synthetase; incorporated into RNA; may inhibit maturation of rRNA; inhibits RNA synthesis in Acunthumoebu

5-fluorouridine Iododeoxyuridine

0 1

Similar to FudR, incorporation into RNA may & extensive but is not necessarily inhibitory; data on effects in Acmtharnoebu unavailable Similar to that of 5-fluorouracil Replaces thymidine in DNA

Reference"

Cytosine arabinoside

-

4

1

Chloroquine

l

Inhibitor of RNA synthesis AmD

0

4

0

2

Binds to DNA; primarily inhibits RNA synthesis; can inhibit DNA and protein synthesis secondarily

Inhibitors of protein synthesis Cycloheximide

0

0

0

0

Inhibits translation on 80s (cytoplasmic) ribosomes; inhibits DNA synthesis and in some organisms, rRNA synthesis Same as for cycloheximide Releases nascent polypeptides from 80 and 70s ribosomes Inhibits translation on 70s (organelle) ribosomes Inhibits translation on 70s ribosomes; taken up into lysosomes; stimulates protein degradation in

Emetine Puromycin Chloramphenicol Erythromycin

o

Inhibits conversion of cytidylic to deoxycytidylic acid; inhibits DNA polymerase Binds to DNA; inhibits DNA polymerase and, to a lesser extent, RNA polymerase; may inhibit protein synthesis (can dissemble ribosomes); taken up into lysosomes and induces autophagy in macrophages

-

_ 2

-

-

-

-

0

1

b

_

0

-

-

1

3 2

3 3

1

11

6 1,12 1,6,13,14

Acanthamoeba

"Data are for similar test systems used in two laboratories (seetext). LP, Drugs added to unagitated cultures at mid-log phase; DPL, drugs added to unagitated post-log phase cells diluted to mid-log concentrations. 0, (1%; 1, 1-20%; 2, 21-408; 3, 41-6096, 4, 61-8076; 5 , 81-10045. Levels reported are for highest values 3 days (Neff) to 4 days (Byers et al.) after addition of drug to c u h r e . Data from: Neff and Neff, 1972; Byen and Kuhns, 1973; Kuhns, 1976; Akins ef al., unpublished observations. bobserved at 5 days. 8. Heidelberger, 1965. e l . Gale et al., 1972. 9. Hoffman and Post, 1973. 2. Kuhns eta!., 1976; Kuhns, 1976. 10. Hahn, 1975. 3. Schlaeger and Hilz, 1969. 11. Grollman and Jardovsky, 1975. 4. Newton, 1975. 12. Pestka, 1975. 5. Waring, 1975. 13. Oleinick, 1975. 6. Alman and Katz, 1976. 14. Nies, 1976 (unpublished observations). 7. Roti Roti and Stevens, 1975.

322

THOMAS J. BYERS

could mean that the new RNA (1) was not mRNA, (2) was incomplete mRNA, or (3) was mRNA that normally did not contain ply-A tails (Jantzen, 1974b). The first possibility could account for some, but probably not all, of the new RNA sequences. The fact that a simple concentration of cells can increase the RNAsynthesizing fraction of the genome from the post-log-phase value of -50% to a new level at 92% is an interesting problem in gene regulation that may be pertinent to studies of contact inhibition in other cell systems and should be investigated further. The causes of RNA degradation and its significance in encystment are unknown. Acid RNase activity decreased during post-log-phase growth in monolayer cultures of A. castellanii to about 50% of the log-phase value (Martin and Byers, 1976). The remaining activity decreased to less than 10%of the log-phase value during the first 20 hours after cells were placed in encystment medium. It is curious that the highest RNase activity was found in log-phase cells where total RNA turnover was minimal (Martin and Byers, 1976; Stevens and Pachler, 1974). It probably is inappropriate, however, to look for correlations between total RNA turnover and total RNase activity, since it can be anticipated that turnover characteristics differ among RNA classes and since RNase occurs in at least two compartments: particle-associated (lysosomal?) and soluble forms (Martin and Byers, 1976). Nevertheless, RNA degradation is found during encystment in both nutrient-rich and nutrient-free media (Neff and Neff, 1969; Jantkn, 1973; Stevens and Pachler, 1974), and further study of the process would be worthwhile. Erythromycin and chloroquine, two agents that induce encystment, affect or at least are taken up by lysosomes in other systems (Table IX) and the possibility that lysosomal enzymes are important for the initiation of encystment should be considered.

5. Protein Synthesis and Degradation Two-dimensional gel electrophoresis of log-phase and differentiating A. casfellanii revealed at least 800 different proteins (Jantzen, personal communication). The contractile protein actin is the most abundant species, accounting for 15-20% by weight of the total protein of trophozoites (Gordon et al., 1976). Total cellular protein increased 10-50% during the log phase-post-log phase growth transition of monolayer cultures (Byers et al., 1969; Martin and Byers, 1976), whereas the protein content of either monolayer or suspension cultures dropped about 30% below log-phase values during starvation-induced encystment (Neff and Neff, 1969; Martin and Byers, 1976). Very little is known about the protein degradation. Proteolytic activity was studied in A. palestinensis but was not correlated with differentiation (Edelstein et al., 1968). There is relatively little published information on the regulation of protein synthesis during encystment. Cycloheximide and AmD inhibited the usual 30fold increase in the activity of P-glucan synthetase during encystment of A.

DIFFERENTIATION IN ACAMHAMOEBA

323

castellanii (Potter and Weisman, 1972) and A . culbertsoni (Raizada and Krishna Murti, cited in Krishna Murti, 1971). These results suggest the possibility that both RNA and protein synthesis are required for the increase in activity. Practically nothing is known about the drug sensitivity of changes in activity of other enzymes in Acanthamoeba. Extensive studies on in vivo and in vitro protein synthesis in A . castellanii indicate, however, that regulation of translation may be more important quantitatively than regulation of transcription (Jantzen, personal communication).

6. Cyst Wall Composition and Cellulose Synthesis The mature cyst walls of A . castellanii, A . rhysodes, and A . palestinensis contain 36-45% protein and 20-34% carbohydrate (Barrett and Alexander, 1977). The bulk of the carbohydrate, in A . castellanii at least, appears to be authentic cellulose (Neff and Neff, 1969; Tomlinson and Jones, 1962; Blanton and Villimez, 1978). Efforts to degrade the walls of several species of Acanthamoeba enzymically were only partially successful (Barrett and Alexander, 1977). Proteases and cellulase caused some degradation, but chitinase and /3-1,3-glucanase had no effect. Complete destruction of cyst walls of A . culbertsoni was accomplished with enzymes secreted by a mold, Alternaria sp. (Verma et al., 1974b). The effective mixture contained proteases, cellulase, and chitinase. It was subsequently reported that A . culbertsoni secreted these three enzymes during excystment (Kaushal and Shukla, 1976), a fact probably responsible for the observed degradation of empty cyst walls. Cellulose synthesis is the only aspect of cyst wall formation that has been examined at the molecular level. The glucose monomers of cellulose are derived from glycogen reserves in A . culbertsoni (Verma and Raizada, 1975) and A . castellanii (reviewed by Weisman, 1976). B . REGULATION OF DIFFERENTIATION 1 . Natural Environmental Factors That Enhance Encystment Although low levels of encystment occur in actively growing populations (Byers et al., 1969), significant enhancement of cyst formation is associated with adverse environmental conditions. Natural conditions shown or thought to induce encystment include starvation (Band, 1963; Neff et al., 1964; Raizada and Krishna Murti, 1971), desiccation or high salt concentrations (Band, 1963), decreased oxygen tension (Byers et al., 1969), and high pH (Byers, unpublished results; Neff et al., 1964). More than one of these conditions may exist and be required in any particular situation. It is possible that some or all of these factors directly affect common response systems that can initiate encystment. Alternatively, each factor may affect a different response mechanism. Since there has

324

THOMAS J . BYERS

been very little effort to compare the effects of different environmental factors on encystment, it is difficult to elaborate on these possibilities. Discussions of the requirements for encystment can be found for the following species: A . castellanii (Neff et al., 1964; Griffiths and Hughes, 1968, 1969), A . culbertsoni (Raizada and Krishna Murti, 1971), A . rhysodes (Band, 1963), and A. palestinensis (Lasman and Shafran, 1978). 2. Cyclic AMP and Encystment A transient two- to threefold increase in cyclic AMP (CAMP) levels was observed in suspension cultures of A. castellani when growth was terminated during the normal log phase-post-log phase transition, following the addition of EB to log-phase cells, or after transfer of cells to nutrient-free media (Gessat and Jantzen, 1974;Achar and Weisman, personal communication). The timing of the increase was dependent on prior culture history but seemed most closely associated with termination of cell multiplication. In each of the preceding situations, encystment followed growth termination. The cAMP levels dropped back to log-phase values either before or during the appearance of mature cysts. Phosphodiesterase activities exhibited transient changes roughly in parallel with the cAMP levels. The possibility that changes in cAMP were associated with encystment followed from the observation that encystment could be induced by theophylline, an inhibitor of phosphodiesteraseand therefore an agent that should maintain high CAMPlevels (Gessat and Jantzen, 1974). Theophylline also was found to inhibit phagocytosis of latex beads, a phenomenon associated with growth termination and with encystment (Section II,B ,2,e). The bioamines epinephrine, 5-hydroxytryptamine, dopamine, and tyramine have been used to induce encystment in A. culbertsoni, and each of these agents was found to elevate adenyl cyclase activity and cAMP levels (Verma et al., 1974a). Epinephrine also may have depressed cyclic nucleotide phosphodiesterase activity (Kaushal et al., 1976). Either cAMP or dibutyryl cAMP could be used directly to induce encystment (Raizada and Krishna Murti, 1972b). These data were interpreted to mean that cAMP played a role in the initiation of encystment (Krishna Murti, 1971; Raizada and Krishna Murti, 1972b). The nature of the role is unknown, although a possible involvement in regulation of glycogen levels has been considered (Weisman, 1976; Gessat and Jantzen, 1974). 3. Effects of Inhibitors of Macromolecule Synthesis on Direrentiation a. Inhibitors of DNA Synthesis. Several inhibitors of DNA synthesis are known to enhance encystment in A. casfellanii (Table I X ) . Neff and Neff (1972) studied the effects of over 40 compounds on differentiation and concluded that inhibitors of (nuclear) DNA synthesis were the most effective inducers of encystment and that cessation of DNA replication might be a key event in the initiation of cyst formation. The Neffs used monolayer cultures and tested the

DIFFERENTIATION IN ACANTHAMOEBA

325

effects of drugs on (1) mid-log-phase cells in nutrient medium and (2) diluted post-log-phase (DPL) cells in nutrient medium at mid-log-phase cell concentrations. Relatively little encystment can be induced in undiluted monolayer post-log-phase populations, possibly because of the low oxygen tension (Section II,C, 1). Upon dilution, these cells resume exponential multiplication unless encystment-enhancing drugs are present. Since little spontaneous encystment occurs in the absence of drugs in either mid-log-phase or DPL test populations, it is possible to determine unambiguously whether a compound induces encystment in this system. A slightly modified Neff system has been used in our laboratory with results that tend to confirm and extend the Neffs’ original observations (Table IX). Variability in cell responses to drugs has been a problem in our studies. Spontaneous changes in drug sensitivity occurred during routine subculturing over periods as short as 1 month. In the most common trend, the tendency to encyst in a drug decreased with time. In some cases, both multiplication and encystment became insensitive to the drug. In other cases, the encystment response was lost, but multiplication sensitivity was retained. Although this variability can be a problem, we have assumed it at least partly represents genetic variation potentially useful for analyzing molecular mechanisms of encystment. When proper measures were taken, it was possible to isolate clones with relatively stable phenotypic characteristics (Seilhamer and Byers, 1978). It should not be surprising, however, that laboratories working with different isolates of the same species, or different subclones of the same strain, do not always obtain the same result for a particular drug. The data in Table IX summarize results from two laboratories that have used similar procedures as well as descendants of the same subclone of the Neff strain of A. castellanii. The data are for subclones that were responsive to the drugs. All inhibitors of DNA synthesis tested induced significant levels of encystment under some conditions (Table IX). Mitomycin C, trenimon, methotrexate, berenil, and EB induced more than 20% encystment when mid-log-phase cells were tested. The same drugs, with the exception of trenimon which was not tested, also induced at least 20% encystment in DPL cells. Untreated populations typically had less than 1% cysts in the experiments performed in our laboratory. Studies with EB and berenil indicated that less than 20% encystment was obtained routinely if early log-phase rather than mid-log-phase cells were used (Akins and Byers, in preparation). This appears to be a problem of cell concentration or medium conditioning rather than physiological differences in the cells. Hydroxyurea, FUdR, and iododeoxyuridine reproducibly gave greater encystment with DPL cells. In this case, since testing was in essentially the same medium at the same cell concentration, the difference between mid-log-phase and DPL cells probably was due to physiological differences. These drugs all gave more than 20% encystment when DPL cells were treated.

326

THOMAS J. BYERS

Relatively little is known about the molecular effects of inhibitors of DNA synthesis in Acanthamoeba. MC and EB inhibited [3H]TdR incorporation into nuclear DNA (Kuhns et al., 1976; Kuhns, 1976). The effect of EB is interesting because mtDNA is the major target of this drug (Table IX); consequently, the nuclear effect may be secondary. Relatively high levels of [3H]TdRincorporation by mtDNA continued following treatment with MC, even though incorporation by nuclear DNA was inhibited (Kuhns, 1976). This fact probably explains why Roti Roti and Stevens (1974) found continuing [3H]TdR incorporation during MC-induced encystment in suspension cultures and is reason to question their conclusion that inhibition of (nuclear) DNA synthesis cannot be a prerequisite for the initiation of encystment. Hydroxyurea and FUdR both stimulated incorporation of [3H]TdR into the DNA of A . castellanii (Rudick, 1969; Kuhns, 1976; King, unpublished results). In the case of FUdR, the stimulation was shown in both nuclear and mtDNA (Kuhns, 1976). Similar data are not available for hydroxyurea, but the stimulation of overall synthesis in each drug probably represented increased dependence on exogenous Tdr as the drugs repressed the de nova synthesis of thymidylate. Experiments using [32P]phosphoricacid rather than [3H]TdR as a label clearly indicated that hydroxyurea and FUdR blocked both DNA and FWA synthesis (Roti Roti and Stevens, 1975). No information is available on the molecular consequences of treatment with other inhibitors of DNA synthesis. EB-induced encystment also has been reported for suspension cultures of A . castellanii, where 100% encystment was observed (Jantzen, 1974b). There have been reports, however, that some inhibitors of DNA synthesis can block encystment. Low concentrations of MC inhibited starvation-induced encystment of A . culbertsoni (Raizada and Krishna Murti, 1971; 1972a). Roti Roti and Stevens (1975) found inhibition of starvation-induced encystment by FUdR. They observed induction of encystment when the drug was added to log-phase suspension cultures but felt that it was inhibiting differentiation because the rate of encystment following termination of cell multiplication was less than that observed after log phase terminated in the absence of the drug. Actually, FUdR can have both stimulatory and inhibitory effects on differentiation. The effect that prevails might depend on differential effects of the drug on DNA and RNA synthesis. It may be stimulatory in situations in which DNA synthesis is the main function affected and inhibitory in situations in which RNA synthesis is relatively strongly inhibited. Stevens and O’Dell (1974) found that 5-fluorocytosine inhibited encystment that occurred during stationary phase in suspension cultures of A . castellanii but stimulated encystment under the same conditions in A. culbertsoni. Since fluorocytosine is probably converted in vivo to FUdR (Heidelberger, 1965), it also may inhibit both DNA and RNA synthesis in Acanthamoeba. It would be interesting to determine whether the two species respond differently to the drug because of differences in sensitivity of the DNA and RNA synthetic pathways.

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Although it is possible that inhibition of nuclear DNA synthesis is a key event in the initiation of encystment, as suggested by Neff and Neff (1969, 1972), it is unclear what role this inhibition plays. It was suggested that MC, the DNA cross-linking agent, might induce encystment by activating genes necessary for differentiation by causing conformational changes in the chromatin. The assumption that nuclear DNA is physically altered by MC was confirmed by Kuhns (1976). Furthermore, she found that the drug stimulated [3H]uracilincorporation while inhibiting nuclear DNA synthesis; thus gene activation could have occurred. The Neffs’ explanation for MC seems inadequate for inhibitors such as FUdR, hydroxyurea, and AmD, since they inhibit RNA synthesis as well as DNA synthesis (Table IX) and consequently should block gene activation. In fact, I have pointed out that these three inhibitors are only really effective inducers of encystment in older monolayer cultures in which RNA synthesis needed for encystment has already been completed. It cannot be disputed that inhibitors of DNA synthesis can induce differentiation under some conditions, but alternatives or modifications of the Neffs ’ explanation are required. One possible explanation is a modification in which it is assumed that acanthamebas lack a GI phase of the cell cycle, that DNA synthesis begins before mitosis is completed, and that completion of cell division depends on a segment of DNA synthesis during early S phase that is not completed in the presence of inhibitors. In this modification inhibitors of DNA synthesis would block cell division and favor differentiation, provided that transcription or translation essential for cyst components had been completed or could proceed. Encysting cells would have G2amounts of DNA plus an additional amount due to whatever part of S phase had been completed. A second alternative to the Neff hypothesis suggests that inhibitors of DNA synthesis exert their influence on differentiation as a result of interactions with nonnuclear, presumably mitochondrial, DNA. This possibility is suggested by observations that “mitochondrion-specific’’inhibitors such as EB and berenil are among the best encystment inducers (Table IX); preferential degradation of mtDNA occurs during starvation-inducedencystment (King and Byers, in preparation); and substances released from isolated mitochondria under the influence of berenil greatly enhance encystment by early log-phase cells (Akins and Byers, in preparation). One suggestion is that nucleotides released by degradation of mtDNA are modified to become regulatory signals. The possibility that nucleotides are involved in the control of encystment already has been suggested by the work on CAMP(Section III,B,2). Other studies indicate that encystment can be induced by deoxyadenosine (Neff, 1971, 1978). We have not yet characterized the encystment-enhancing activity released from mitochondria, but similar activity has been extracted from the media of cells encysting in response to berenil. In this case, the activity seems to be a relatively small molecule, possibly a nucleotide because of its sensitivity to snake venom phosphodiesterase and its resistance to a variety of other enzymes and treatments. In the medium, however,

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the activity is associated with a larger molecule that is sensitive to a-amylase. The latter observation is interesting, because Sin (1975) reported that a nondialyzable substance released from mitochondria of heat-shocked Drosophila cells could activate specific nuclear genes. A third possible explanation for the enhancement of encystment by agents that affect DNA metabolism is that they exert their influence through effects on processes other than DNA synthesis: for example, stimulation of transcription or translation and initiation of cell degradation processes. The possibility that MC could enhance transcription, as suggested by Neff and Neff (1969), has been discussed. Work on other systems indicates that AmD may, in some circumstances, stimulate translation (Leinwald and Ruddle, 1977). The stimulation of protein degradation by erythromycin has also been discussed. If this general explanation is correct, then the fact that these inhibitors affect DNA metabolism may be relatively unimportant for encystment. Very little work has been done on the effects of various inhibitors on excystment. Hydroxyurea failed to block excystment in A . castetlanii, but control excystment levels were low and the possibility of a requirement for DNA synthesis during this step may not have been adequately tested (Mattar and Byers, 1971). Bromodeoxyuridine, which replaces TdR during DNA synthesis (Gale et al., 1972), had little effect on the growth and encystment of A. castellanii but inhibited excystment of cysts that had been exposed to the inhibitor for seven generations prior to the induction of encystment (Roti Roti and Stevens, 1974). The significance of this observation is unknown, albeit interesting. b. Inhibitors of RNA and Protein Synthesis. AmD induced encystment in A . castellanii when the drug was added to DPL cells (Table IX). No encystment was observed when the drug was added to mid-log-phase or younger cells. Furthermore, AmD blocked encystment by mid-log-phase cells that had been induced by EB (Byers and Kuhns, 1973) or starvation (Rudick, 1971; Potter and Weisman, 1972). Likewise, AmD inhibited encystment in A . culbertsoni induced by dibutyryl CAMP or starvation (Raizada and Krishna Murti, 1971; 1972a; 1972b). The fact that AmD inhibits DNA synthesis in Acanthamoeba has been discussed above, but this agent is primarily an inhibitor of RNA synthesis, and the inhibitory effect that it can have on encystment by log-phase cells suggests that they require some RNA synthesis for differentiation (see also Section II,A,4). Cycloheximide and emetine, both inhibitors of cytoplasmic protein synthesis on 80s ribosomes, induced no encystment with either mid-log-phase or DPL cells (Table IX). Furthermore, cycloheximide inhibited encystment induced by other agents in A . castellanii (Potter and Weisman, 1972; Byers and Kuhns, 1973) and A. culbertsoni (Raizada and Krishna Mwti, 1971, 1972a, 1972b). Using cesium chloride gradients, Nies observed that [3H]TdR incorporation was reduced 64% in both nuclear and mtDNA after 5 hours of treatment at 24 pg cycloheximide/ml. Thus, even though cycloheximide is another inhibitor of

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DNA synthesis, its primary effect is inhibition of encystment. Presumably this is due to inhibition of protein synthesis required for encystment. Tetracyclines, which block translation on both 80 and 70s ribosomes (Gale et al., 1972), inhibited starvation-induced encystment in suspension cultures of A. castellanii (Griffiths and Hughes, 1969). In contrast, puromycin, another probable inhibitor of both 80 and 70s translation, induced low levels of encystment in monolayer DPL cultures (Table IX). The 70s-specific translation inhibitor chloramphenicol was reported to inhibit starvation-induced encystment in A. castellanii (Griffiths and Hughes, 1969) and A. rhysodes (Band, 1963) but, more recently, this agent and erythromycin, another 70s inhibitor, were observed to induce high levels of encystment in A . castellanii (Table I X ) . It appears, then, that the response of cells to inhibitors of 70s translation is less predictable than the response to specific inhibitors of 80s translation. This may be partly due to the fact that A . castellanii, at least, readily loses sensitivity to the former inhibitors and rarely to the latter (Seilhamer and Byers, 1978; and unpublished data). Furthermore, there is evidence that the 70s inhibitors affect multiple cell processes; consequently, the cell response promoted may reflect the processes most sensitive in a particular cell clone. Some of the observed consequences of drug treatment include alteration of mitochondrial structure by chloramphenicol (Hryniewiecka, 1979), stimulation of whole-cell protein degradation by chloramphenicol and erythromycin (Nies, unpublished data), and stimulation of r3H]TdR incorporation into mtDNA while incorporation into nuclear DNA decreases (Nies, unpublished data). In addition, it is likely that these inhibitors affect cell multiplication and encystment by different mechanisms. Evidence for this comes from the observation that resistance to inhibition of multiplication by inhibitors of 70s translation is drug-specific, whereas changes in the ability to encyst affect several inhibitors of mitochondrial translation and transcription simultaneously (Seilhamer, Akins and Byers, unpublished data). The problem ahead is to determine which, if any, of these observations will help reveal mechanisms by which inhibitors such as chloramphenicol and erythromycin can induce encystment. It is a reasonable guess, nevertheless, that agents affecting 70s translation may induce cyst formation either by blocking the production of encystment-regulatingproteins or by stimulating the production of encystment-enhancing factors. A model for the former effect has been suggested elsewhere (Byers et al., 1977). Excystment of A . castellanii was inhibited by both AmD and cycloheximide (Mattar and Byers, 1971). These results may indicate the need for both RNA and protein synthesis, but the matter requires much more study. 4. Excystment Factors

Media taken from encysting cultures of several strains of Acanthumoeba inhibited colony production by cysts from the same strain (Dubes and Jensen, 1964). The active factor was insensitive to proteases, RNase, and DNase. The bulk of

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the activity was nondialyzable, but a small portion could pass through a dialysis membrane. The inhibitory activity was labile in acid and base and after boiling for 10 minutes. To my knowledge, the substance has not been further characterized. The inhibitor was formed either slightly before or during encystment in yeast-supplemented nutrient medium. This raises the interesting question of whether the agent is primarily an inhibitor of excystment, as suggested by Dubes and Jensen, or an inducer of encystment. These authors did not indicate whether the active factor could induce cyst formation. In our laboratory, factors with similar properties were produced during berenil-induced encystment and were excellent enhancers of encystment by early log-phase cultures of A . castellanii (Akins and Byers, in preparation). Further research is needed to clarify relationships between encystment enhancers and excystment inhibitors. Excystment of A . culbertsoni was stimulated by heat-stable factors from a variety of bacteria and several fungi, as well as by peptone, proteose peptone, tryptone, or amino acids (Kaushal and Shukla, 1977a). The active factors in bacterial extracts appeared to be amino acids-glutamic acid being the most active, followed by serine, threonine, and alanine (Kaushal and Shukla, 1977b). Investigations of the structural requirements of compounds able to induce excystment in A . culbertsoni have suggested that the best inducers are molecules closely related to a-aminobutyric acid (Kaushal and Shukla, 1977c), however, additional work is required before the requirements can be identified more precisely. It may be significant that all four active amino acids have been identified as nonessential for the growth of several species of Acanthamoeba (Band, 1962; Adam, 1964b), including A . culbertsoni (Acanthamoeba sp. Lilly).

IV. Beginnings of Acanthamoeba Genetics Species of the genus Acanthumoeba generally are presumed to be asexual. This conclusion is based on the absence of structural evidence for meiosis or genetic exchange. Unfortunately, the very small chromosomes that can be seen (Molet and Ermolieff-Braun, 1976; Volkonsky, 1931) are difficult to count. Volkonsky estimated approximately 80 chromosomes for A. castellanii. The small size and large number of chromosomes make it difficult to determine whether any changes characteristic of meiosis occur. There have been no reports of naturally occurring cell fusion, but neither have there been any systematic searches for mating types. The absence of appropriate mutants has prevented the use of genetic techniques for testing whether any exchange of genetic markers occurs. Preliminary studies in our laboratory with drug sensitivity and resistance markers failed to provide evidence for marker exchange between mutant lines (Seilhamer, unpublished observations), but the markers used may have been mitochondrial genes (see below). Therefore, while it is possible that

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Acanthamoeba does not have a sexual means of reproduction, the possibility that it does have some natural genetic exchange has not entirely been ruled out. An estimate of the ploidy level in Acanthamoeba can be obtained from knowledge about the nuclear DNA content, the fraction of the DNA that contains unique sequences, and the genomic complexity of the unique sequences. Unfortunately, some rather large differences have been reported for these parameters (Section III,A,2). Nevertheless, a reasonable calculated ploidy level for cells in the G2portion of the cell cycle is approximately 12n. [The estimate has been calculated for cysts with 0.62 X gm DNA per cell with the assumptions that 90% of the cyst DNA is nuclear, 74% of the nuclear DNA contains unique sequences, and 2 X 1O'O daltons is the kinetic complexity of the unique sequences. Ploidy ( n ) = (0.74 X 0.90 X 0.62 X 10-l2) gm unique DNA per nucleus + (2 X 1O1O/6.O2 X loz3) gm unique DNA per genome.] If the higher value for kinetic complexity obtained by Marzzoco and Colli ( I 974) is used, the ploidy level drops to 212. Gzvalues are used because of data discussed in Section II,D,4. In fact, uncertainty in estimates for the various parameters probably makes it impossible to be certain whether these organisms are diploid or polyploid. If the indications that G2is the predominant portion of the cell cycle are correct (Section II,D,4), then most cells should have at least two copies of the basic genome. Jensen and Dubes (1962) described the first mutants of Acanthamoeba, several lines that differed in colony morphology when grown in agar with bacteria or yeast. It is probable that numerous mutant lines of Acanthumoeba are available but not recognized as such. It has been the experience in our laboratory that strains can be quite unstable in phenotypic character, and others have observed variability among subclones of cells (e.g., Thompson, quoted in Pigon, 1976). We were bothered for a number of years by instability of drug sensitivity. For example, Byers and Kuhns (1973) reported that EB inhibited multiplication and induced encystment in A . castellanii. Today, descendants of the strain used in those studies still fail to multiply in the drug but also fail to encyst. Selection techniques allowed us recently to isolate from this strain a new clone, 0 s - 4 , that encysts very actively in the drug again. The phenotype of 0 s - 4 has been very stable, but it also has been possible following ultraviolet irradiation to isolate stable subclones of this line that will not encyst in the drug. A number of cell lines that differ in ability to multiply or encyst in several different drugs have now been isolated (Akins, unpublished observations). Some of these mutants represent a first step toward obtaining a collection of mutants with developmental defects. Thus there is reason to hope that mutants useful for the study of encystment will eventually be available. The origin of Acanthumoeba mutants is of considerable interest. All of our drug-resistantmutants have been obtained in the absence of mutagenesis. Several lines that have lost the ability to encyst in response to specific drugs have been

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obtained following ultraviolet irradiation, but we have been unable to obtain any evidence that the irradiation caused the mutations. Likewise, efforts to increase the frequency of drug-resistant mutants by treatment of cells with the chemical mutagens nitrosoguanidine and methyl methanesulfonate have not been successful (Seilhamer, in preparation). Of many drugs tried, resistance developed most readily to agents known to affect mitochondrial processes. Only one putative nuclear mutant, a cycloheximide-resistantcell line, has been reported (Chisholm and Vaughn, 1979). This fact may be due to polyploidy, but drug resistance was chosen as a marker because of the possibility that it would arise from a dominant mutation. Efforts to determine the nuclear or mitochondrial origin of available drug-resistant mutants by the use of cell fusion, microinjection of mitochondria, or phagocytosis of mitochondria have been unsuccessful to date but should resolve the question following technical improvements. Development of a method for artificial exchange of genetic material is one of the major problems at the present time. Effective mutagenic methods are also needed before genetic approaches will be very useful in studies of encystment. One promising genetic approach is the use of gene cloning. While this has not been applied to the problem of encystment, Paule (personal communication) has been successful in cloning nuclear genes of A. castellanii.

V. Conclusions Acanthamebas are attractive models for the study of several major problems in eukaryotic cell and developmental biology. The ease with which a variety of strains can be grown in axenic or chemically defined media and the simplicity of the single-cell differentiation are very useful attributes. The ability to interpret experimental results has been hampered by insufficient background data, but the situation is improving considerably as more laboratories become interested in the potential of these organisms. It is becoming evident that acanthamebas have many characteristics that are unusual, although not necessarily unique, in the world of animal cells. The occurrence of lipophosphonoglycan instead of glycoprotein in the plasma membrane, the prominent role of pinocytosis in soluble nutrient uptake, the branched mitochondrial electron transport chain, the phenomenon of amitosis, and the induction of differentiation by inhibitors of mitochondrial macromolecule synthesis are all unusual features. The extensive work on contractile proteins (Korn, 1978) and RNA polymerases (Section III,A,4) indicates the great potential of these amebas for biochemical studies of purified proteins having widespread significance. Other areas of study that offer interesting potential include observations that acanthamebas have chemotactic responses (Kane, unpublished results), the effect of visible light on cell multiplication (Section II,A,5), and the influence of cell concentrationon nuclear gene

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expression (Section III,A,4). A major drawback at present is the current inability to apply standard genetic analyses. This problem can be circumvented partially by utilizing modem approaches such as gene cloning, but it can be assumed that interest in acanthameba research and consequently progress in understanding the biology of this organism, would be greatly enhanced by the discovery of methods for achieving genetic crosses.

ACKNOWLEDGMENTS The preparation of this article was assisted greatly by discussions that occurred during the first international Acanthamoeba Conference held in Columbus, Ohio, June 1978. I thank all those individuals who helped educate me by sharing both their published and unpublished work. Any errors in the interpretation of their ideas or results are solely my own.

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Lasman, M., and Shafran, A. (1978). J . Protozool. 25,495. Leinwand, L.,and Ruddle, F. H. (1977). Science 197, 381. Lloyd, D., and Griffiths, A. J. (1968). Exp. Cell Res. 51, 291. Lloyd, D., John, L.,Edwards, C., and Chagla, A. H. (1975). J. Gen. Microbiol. 88, 153. Loening, U. E. (1968). J. Molec. Biol. 38, 355. Mandel, M., Schildkraut, C. L., and Marmur, J. (1968). In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 12B, p. 108. Academic Press, New York. Martin, S. M., and Byers, T. J. (1976). J . Prorozool. 23, 608. Martinez, A. J. (1977). In “Proceedings of the International Conference on Amebiasis,” p. 65. Inst. Mex. Seguro SOC.,Mexico City. Marzzoco, A., and Colli, W. (1974). Biochim. Biophys. Acra 374, 292. Marzzoco, A., and Colli, W. (1975). Biochim. Biophys. Acra 393, 525. Mattar, F. E., and Byers, T. J. (1971). J . Cell Biol. 49, 507. McIntosh, A. H., and Chang, R. S. (1971). J. Prorozool. 18, 632. McMillen, J . , Nazario, M., and Jensen, T. (1974). J. Bacreriol. 117, 242. Mitruka, B . M., and Rawnsley, H. M. (1977). “Clinical Biochemical and Hematological Reference Values in Normal Experimental Animals,” Masson, New York. Molet, B., and Ermolieff-Braun, G. (1976). Protistotogica 12, 571. Molet, B . , and Kremer, M. (1976). Bull. SOC. Sci. V i r . Mid. Comp. (Lyon) 78, 215. Neff, R. J. (1957). J . Prorozool. 4, 176. Neff, R. J . (1958). J. Prorozool. 5, 226. Neff, R. J . (1971). In Virro 6, 300. Neff, R. J. (1978). J . Cell Biol. 79, 38a. Neff, R. J., and Neff, R. H. (1969). Symp. SOC. Exp. Biol. 23, 51. Neff, R. J., and Neff, R. H. (1972). C. R . Trav. Lab. Carlsberg 39, 1 1 1 . Neff, R. J . , Neff, R. H., and Taylor, R. E. (1958). Physiol. Zool. 31, 73. Neff, R. J., Ray, S. A., Benton, W. F., and Wilbom, M. (1964). In “Methods in Cell Biology” (D. M. Prescott, ed.), p. 55. Academic Press, New York. Newton, B. A. (1975). In “Antibiotics HI. Mechanism of Action of Antimicrobial and Antitumor Agents” (J. W. Corcoran and F. E. Hahn, eds.), p. 34. Springer-Verlag, New York. Oleinick, N. L. (1975). In “Antibiotics III. Mechanism of Action of AntiMicrobial and Antitumor Agents” (J. W. Corcoran and F. E. Hahn, eds.), p. 396. Springer-Verlag, New York. Ord, M. 1. (1973). In “The Biology of Amoeba” (K. W. Jeon, ed.), p. 401. Academic Press, New York. Page, F. C. (1967). J . Protozool. 14, 709. Page, F. C. (1976). Freshwafer Biol. Assoc. Sci. Publ. #34. Ambleside, Cambria. Pal, R. A. (1972). J. Exp. Biol. 57, 5 5 . Palmer, J. M. (1976). Annu. Rev. Plant Physiol. 27, 133. Parish, J. H., and Hall, L. (1972). J . Gen. Microbiol. 73, 365. Pestka, S. (1975). In “Antibiotics III. Mechanism of Action of Antimicrobial and Antitumor Agents” (J. W. Corcoran and F. E. Hahn, eds.), p. 370. Springer-Verlag, New York. Pigon, A. (1970). Protoplusma 70, 405. Pigon, A. (1972). Exp. Cell Res. 73, 170. Pigon, A. (1976). Cyrobiologie 13, 107. Pollard, T. D., and Kom, E. D. (1973). J . Biol. Chem. 248, 448. Potter, J. L.,and Weisman, R. A. (1972). Dev. Biol. 28, 472. Roca-Ciobanu, M., Lupascu, G., Petrovici, A., Ionescu, M. D. (1975). Int. J . Parasitol. 5 , 49. Pussard, M. (1964). Rev. Ecol. Biol. Sol. 4, 587. Pussard, M. (1972). J. Prorozool. 19, 557.

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Subject Index A

collagen component, 186-191 isolation of membranes, 180-182 morphological properties of isolated membranes, 182 noncollagen components, 191 -193 solubility properties, 182-183 subunit composition, 196-197 supramolecular organization, 193-1 96, 218-220 Bioenergetics, of Acanrharnoeba. 300-306 Blood Celts, separation by electrophoresis, 99-103

Acanthamoeba growth and reproduction factors influencing culture growth and survival, 286-290 membrane structure and transport, 290-300 nuclear reproduction and cytokinesis, 306-3 10 respiratory metabolism and bioenergetics, 300-306 molecular biology and differentiation classes and metabolism of macromolecules, 3 10-323 C regulation of differentiation, 323-330 beginnings of genetics, 330-332 Carbohydrates, basement membrane composiclassification of, 283-284 tion, 183-186 Cell biology, evaluation of ameba as model, pathogenicity of, 284-285 research areas, 285 274-276 Amino acids, basement membrane composition, Cell electrophoresis 183-186 adsorbable and other materials, 117-1 18 Amoeba proteus applications to specific cell separations micrurgy, 231-233 lymphoid, blood and hemopoietic cells, site and mode of action of toxic agents, 23396-107 neoplastic, dividing and immature cells, 237 107-112 effects of chemicals and radiations, general apparatus conclusions, 273-274 cytopherometer, 89 evaluation as model electrophoresis in columns, 92-93 in cell biology, 274-276 in toxicology, 276-277 endless-belt electrophoresis, 93-94 free-flow electrophoresis, 94-96 B laser Doppler spectroscopy, 89-91 effects of lytic enzymes on mobilities, 113117 Basement membranes isoelectric focusing, 119-121 in disease, 212-218 functions medium for, 88 miscellaneous, 118-1 19 as semipermeable filters, 177-178 as supporting and/or boundary structures, theory, 86-88 viability and function, 112-1 13 178-179 immunochemistry of, 209-212 Cellular activities, chemicals as probes into, metabolism 248-249 biosynthesis, 197-208 general nuclear and cytoplasmic damage, turnover, 208-209 249-250 morphology and distribution, 170-177 specific damage 10 cytoplasm or cell memstructural chemistry, 179-180 brane, 255-273 amino acid and carbohydrate composition, specific damage to nucleus or its membrane, 183-186 250-255

340

SUBJECT INDEX

Collagen, basement membrane and, 186-191 Culture growth, of Acanthumoebu, 286-290 Cytokinesis, in Acanrhamoeba, 306-3 10 Cytopherometer, 89

Isoelectric focusing, cell electrophoresis and, 119-121 Immunochemistry, of basement membranes, 209-212

D

L

Deoxyribonucleic acid, membrane-associated eukaryotes, 8-17 prokaryotes, 2-8 transformation and transfection, 17-2 1 Differentiation, regulation in Acanthamoeba , 323-330 Disease, basement membrane in, 212-218 Dividing cells, separation by electrophoresis, 110-1 12

Lymphoid cells, separation by electrophoresis, 96-99

E Electron cytochemical stains immunocytochemical methods, 74-76 incipient instability, 64-65 possible developments, 76-80 postchelation nonosmiophilic methods, 65 osmiophilic methods, 65-71 robust complex formation, 71-74 Electrophoresis in columns, 92-93 endless-belt, 93-94 free-flow , 94-96 Enzymes, lytic, effect on cell electrophoretic mobilities, 113-1 17 Eukaryotes membrane-associated DNA of, 8-17 membrane-associated RNA of. 25-34

G Genetics, of Acanthamoeba, 330-332

H Hemopoietic cells, separation trophoresis, 96-105

by

elec-

I Immature cells, separation by electrophoresis, 110-1 12

M Macromolecules, classes and metabolism in Acanrhamoeba, 3 10-323 Membrane Acanthamoeba, structure and transport, 290-300 DNA associated with eukaryotes, 8-17 prokaryotes, 2-8 transformation and transfection, 17-21 RNA associated with, 21-22 eukaryotes, 25-34 prokaryotes, 22-25

N Noncollagen components, of basement membranes, 191-193 Neoplastic cells, separation by electrophoresis, 107-1 10 Nucleic acids, membrane-associated, additional theoretical considerations, 34-39

P Pathogenicity, of Acanthamoeba, 284-285 Plant cell wall expansion modalities of difficulties with passive behavior during growth, 149-151 multinet growth hypothesis, 149 ordered subunit hypothesis, 151-154 organization of bow-shaped arrangements, 141-147 interpretation in terms of disperse texture, 132-135 interpretation in terms of a multi-ply construction, 139-141

341

SUBJECT INDEX occurrence of disperse texture, 147148 ordered texture and, 135-139 Plant cell wall growth, characteristics of anisotropy of process, 130 sites of surface expansion, 130-132 Plant cell wall morphogenesis, possible factors for, 154-155 membrane and microtubule control, 155-157 pressure and stresses, 155 self-assembly process, 157-160 Postchelation; of electron cytochemical stains nonosmiophilic methods, 65 osmiophilic methods, 65-7 1 F’rokaryotes membrane-associated DNA of, 2-8 membrane-associated RNA of, 22-25

R Reproduction, in Acanthamoeba, 306-3 10 Respiration, in Acunrhamoeba, 300-306

Ribonucleic acid, membrane-associated, 2 1-22 eukaryotes, 25-34 prokaryotes, 22-25 5

Spectroscopy, laser Doppler, 89-91

T Toxic agents, site and mode of action within the cell ameba model, 233-237 damage characteristic of certain chemicals, 237 -248 Toxicology, evaluation of ameba as model, 276-277 Transfection, membrane-associated DNA and, 17-21 Transformation, membrane-associated DNA and, 17-21

This Page Intentionally Left Blank

Contents of Previous Volumes Volume 1 Some Historical Features in Cell BiologyARTHURHUGHES LEONARD HUSKINS Nuclear Reproduction<. Enzymic Capacities and Their Relation to Cell Nutrition in Animak-ciEoRGE W. KIDDER The Application of Freezing and Drying Techniques in Cytology-L. G . E. BELL Enzymatic Processes in Cell Membrane PenetraAND w. WILBRANDT tion-TH. ROSENBERG Bacterial Cytology-K. A. BISSET Protoplast Surface Enzymes and Absorption of Sugar-R. BROWN Reproduction of Bacteriophage-A. D. HERSHEY

The Folding and Unfolding of Protein Molecules as a Basis of Osmotic Work-R. J . GOLDACRE

Nucleo-Cytoplasmic Relations in Amphibian Deve1opment-G. FRANK-HAUSER Structural Agents in Mitosis-M. M. SWANN Factors Which Control the Staining of Tissue Sections with Acid and Basic Dyes-MARCUS

Quantitative Histochemistry of PhosphatasesWILLIAML. DOYLE Alkaline Phosphatase of the Nucleus-M. CH~VREMONT A N D H. FIRKET Gustatory and Olfactory Epithelia-A. F. BARADIAND G . H. BOURNE Growth and Differentiation of Explanted Tissues-P. J . GAILLAQD Electron Microscopy of Tissue Sections-A. J . DALTON A Redox Pump for the Biological Performance of Osmotic Work, and Its Relation to the Kinetics of Free Ion Diffusion across Membranes-E. J. CONWAV A Critical Survey of Current Approaches in Quantitative Histo- and CytochemistryDAVIDGLICK Nucleo-cytoplasmic Relationships in the Development of Acerabulan'a-J. HAMMERLING Report of Conference of Tissue Culture Workers Held at Cooperstown, New York-D. J. HETHERINGTON AUTHOR INDEX-SUBJECT

INDEX

SINGER The Behavior of Spermatozoa in the Neighborhood of Eggs-LORD ROTHSCHILD The Cytology of Mammalian Epidermis and Sebaceous Glands-WILLIAM MONTAGNA The Electron-Microscopic Investigation of Tissue Sections-L. H. BRETSCHNEIDER The Histochemistry of Esterases-G. GOMORI

Volume 3

The Nutrition of Animal Cells-CHARITY WAYMOUTH Caryometric Studies of Tissue Cultures--OTTo BUCHER The Properties of Urethan Considered in Relation to Its Action on Mitosis-IvoR CORNMAN AUTHOR INDEX-SUBJECT INDEX Composition and Structure of Giant Chromosomes-MAX ALFERT How Many Chromosomes in Mammalian SoVolume 2 matic Cells?-R. A. BEATTY Quantitative Aspects of Nuclear NucleoproThe Significance of Enzyme Studies on Isolated teins-HEWSON SWIFT Cell NUCki-ALEXANDER L. DOUNCE Ascorbic Acid and Its Intracellular Localization, The Use of Differential Centrifugation in the Study of Tissue Enzymes-CHR. DE DUVE with Special Reference to Plants-J. CHAYEN Aspects of Bacteria as Cells and as OrganA N D J . BERTHET iSmS-STUART MUDDAND EDWARDD. DE Enzymatic Aspects of Embryonic DifferentiaLAMATER tion-TRYGGvE GUSTAFSON Ion Secretion in Plants-J. F. SUTCLIFFE Azo Dye Methods in Enzyme HistochemistryA. G. EVERSONPEARSE Multienzyme Sequences in Soluble ExtractsMicroscopic Studies in Living Mammals with HENRYR. MAHLER The Nature and Specificity of the Feulgen Transparent Chamber Methods-Roy G. WILLIAMS Nuclear Reaction-M. A. LESSLER

344

CONTENTS OF PREVIOUS VOLUMES

on Cytokinesis and Amoeboid MovernentThe Mast C e l l - G . ASBOE-HANSEN DOUGLAS MARSLAND Elastic Tissue-EDWARDS w. DEMPSEYA N D Intracellular pH-PETER C. CALDWELL ALBERTI. LANSING The Composition of the Nerve Cell Studied with The Activity of Enzymes in Metabolism and Transport in the Red Cell-T. A. J. PRANKERD AND New Methods-SVEN-OLOE BRATTGARD Uptake and Transfer of Macromolecules by Cells HOLGERHYDEN with Special Reference to Growth and DeAUTHOR INDEX-SUBJECT INDEX velopment-A. M. SCHECHTMAN Cell Secretion: A Study of Pancreas and Salivary VoIume 4 c. J. JUNQUEtRA A N D G. c. Glands-L. HIRSCH Cytochemical Micrurgy-M. I. KOPAC The Acrosome ReaCtiOn-JEAN C. DAN Amoebocytes-L. E. WAGGE Problems of Fixation in Cytology, Histology, and Cytology of Spermatogenesis-VIsHwA NATH The Ultrastructure of Cells, as Revealed by the Histochemistry-M. WOLMAN Electron Microscope-FRITIoF S. SJOSTRAND Bacterial CytOlOgy-ALFRED MARSHAK AUTHOR INDEX-SUBJECT INDEX Histochemistry of Bacteria-R. VENDRELY Recent Studies on Plant Mitochondria-DAVID P. HACKETT The Structure of Chloroplasts-K. MOHLE- Volume 6 THALER

Histochemistry of Nucleic Acids-N.

B. KUR-

NICK

Structure and Chemistry of Nucleoli-W. S. VINCENT On Goblet Cells, Especially of the Intestine of Some Mammaiian Species-HARALD MOE Localization of Cholinesterases at Neuromuscular Junctions-R. COUTEAUX Evidence for a Redox Pump in the Active Transport of Cations-E. J. CONWAY AUTHOR INDEX-SUBJECT

INDEX

Volume 5 Histochemistry with Labeled Antibody-ALBERT H.COONS The Chemical Composition of the Bacterial Cell W a l l - C . S. CUMMINS Theories of Enzyme Adaptation in Microorganisms-J. MANDELSTAM The Cytochondria of Cardiac and Skeletal Musck-JoHN w . HARMON The Mitochondria of the NeUTOn-WARREN ANDREW The Results of Cytophotornetry in the Study of the Deoxyribonucleic Acid (DNA) Content of the Nucleus-R. VENDRELY A N D C. VENDRELY

Protoplasmic Contractility in Relation to Gel Structure: Temperature-Pressure Experiments

The Antigen System ofParamecium aure1ia-G. H. BEALE The Chromosome Cytology of the Ascites Tumors of Rats, with Special Reference to the Concept of the Stemline Cell-SAJIRo MAKIN0

The Structure of the Golgi Apparatus-ARTHUR A N D PRISCHIA F. POLLISTER W. POLLISTER An Analysis of the Process of Fertilization and Activation of the Egg-A. MONROY The Role of the Electron Microscope in Virus Research-ROBLEY C. WILLIAMS The Histochemistry of Polysaccharides-ARTHUR J. HALE The Dynamic Cytology of the Thyroid Gland-J. GROSS Recent Histochemical Results of Studies on Embryos of Some Birds and Mammals-ELio BORGHESE Carbohydrate Metabolism and Embryonic Determination-R. J . O’CONNOR Enzymatic and Metabolic Studies on Isolated A N D R. M. S. SMELLIE Nuclei-G. SIEBERT Recent Approaches of the Cytochemical Study of Mammalian Tissues-GEORGE H. HOGEBOOM,EDWARD L. KUFF, A N D WALTERC. SCHNEIDER The Kinetics of the Penetration of Nonelectrolytes into the Mammalian ErythrocyteFREDABOWYER

345

CONTENTS OF PREVIOUS VOLUMES AUTHOR INDEX-SUBJECT

INDEX

CUMULATIVE SUBJECT INDEX (VOLUMES

1-5)

Volume 7 Some Biological Aspects of Experimental Radiology: A Historical Review-F. G. SPEAR The Effect of Carcinogens, Hormones, and Vitamins on Organ Cultures-ILsE LASNITZKI Recent Advances in the Study of the Kinetochore-A. LIMA-DE-FARIA Autoradiographic Studies with S35-Sulfate-D. D. DZIEWIATKOWSKI The Structure of the Mammalian Spermatozoon-DON w . FAWCETT The Lymphocyte-4. A. TROWELL The Structure and Innervation of Lamellibranch Muscle-J. BOWDEN Hypothalamo-neurohypophysid NeummretionJ . C. SLOPER Cell Contact-PAUL WEISS The Ergastoplasm: Its History, Ultrastructure, and Biochemistry-FRANGolSE HAGUENAU Anatomy of Kidney Tubules-JOHANNES RHODIN

Structure and Innervation of the Inner Ear Sensory Epithelia-HANS ENGSTROMA N D JAN WERSALL The Isolation of Living Cells from Animal Tissues-L. M. RINALDINI AUTHOR INDEX-SUBJECT

INDEX

Volume 8 The StWCtUre

A Survey of Metabolic Studies on Isolated Mammalian Nuclei-D. B. ROODYN Trace Elements in Cellular Function-BERT L. VALLEE A N D FREDERIC L. HOCH Osmotic Properties of Living Cells-D. A. T. DICK Sodium and Potassium Movements in Nerve, Muscle, and Red Cells-I. M. GLYNN Pinocytosis-H. HOLTER AUTHOR INDEX-SUBJECT

Volume 9 The Influence of Cultural Conditions on Bacterial Cytology-J. F. WILKINSON A N D J . P. DUGUID

Organizational Patterns within ChromosomesBERWIND P. KAUFMANN, HELENGAY,A N D MARGARET R. MCDONALD Enzymic Processes in Celk-JAY BOYDBEST The Adhesion of Cek-LEONARD WElSS Physiological and Pathological Changes in Mitochondrial MorphologypCH. ROUILLER The Study of Drug Effects at the Cytological Leve1-G. B. WILSON Histochemistry of Lipids in Oogenesis-VIsHwA NATH Cyto-Embryology of Echinoderms and Amphibia-KUTSUMA DAN The Cytochemistry of Nonenzyme ProteinsRONALD R. COWDEN AUTHOR I N DE X-SU

Of

INDEX

BJ ECT INDEX

CytOplaSm~HARLESOBER-

LING

Wall Organization in Plant Cells-R.

D. PRES-

TON

Submicroscopic Morphology of the SynapseEDUARWDE ROBERTIS The Cell Surface of Pararneciurn-C. F. EHRET A N D E. L. POWERS The Mammalian Reticulocyte-LEAH MIRIAM LOWENSTEIN The Physiology of Chromatophores-MILTON FINGERMAN The Fibrous Components of Connective Tissue with Special Reference to the Elastic FiberDAVIDA. HALL Experimental Heterotopic Ossification-J. B. BRIDGES

Volume 10 The Chemistry of Shiff's Reagent-FREDERICK H. KASTEN Spontaneous and Chemically Induced Chromosome Breaks-ARUN KUMARSHARMA AND ARCHANA SHARMA The Ultrastructure of the Nucleus and Nucleocytoplasmic Relations-SAUL WISCHNITZER The Mechanics and Mechanism of CleavageLEWISWOLPERT The Growth of the Liver with Special Reference to Mammals-F. DOLJANSKI Cytology Studies on the Affinity of the Carcinogenic Azo Dyes for Cytoplasmic Components-YosHIMA NAGATANI

346

CONTENTS OF PREVlOUS VOLUMES

Epidermal Cells in Culture-A.

GEDEONMA-

TOLTSY

INDEX

AUTHOR INDEX-SUBJECT

CUMULATIVE SUBJECT

INDEX

(VOLUMES 1-9)

Volume 11 Electron Microscopic Analysis of the Secretion Mechanism-K. KUROSUMI The Fine Structure of Insect Sense OrgansELEANOR H. SLIFER Cytology of the Developing Eye-ALFRED J. COULOMBRE The Photoreceptor Structures-J. J . WOLKEN Use of Inhibiting Agents in Studies on Fertilization Mechanisms-CHARLES B. METZ The Growth-Duplication Cycle of the Cell-D. M. PREXOTT Histochemistry of Ossification-RoMuLo L. CABRINI Cinematography, Indispensable Tool for Cytology-€. M. POMERAT

Sequential Gene Action, Protein Synthesis, and Cellular Differentiation-REED A. FLICKINGER

The Composition of the Mitochondria1 Membrane in Relation to Its Structure and Function- ERICG. BALLA N D CLIFFED. JOEL Pathways of Metabolism in Nucleate and Anucleate Erythrocytes-H. A. SCHWEIGER Some Recent Developments in the Field of Alkali Cation Transport-W. WILBRANDT Chromosome Aberrations Induced by Ionizing Radiations-H. J. EVANS Cytochemistry of Protozoa, with Particular Reference to the Golgi Apparatus and the Mitochondria-VlsHwA NATHA N D G . P. DUTTA Cell Renewal-FELIX BERTALANFFY AND CHOSEN LAU AUTHOR INDEX-SUBJECT

INDEX

Volume 14

Inhibition of Cell Division: A Critical and Experi. mental Analysis-SEYMOUR GELFANT Electron Microscopy of Plant Protoplasm-R. BUVAT Volume 12 Cytophysiology and Cytochemistry of the Organ Sex Chromatin and Human Chromosomes--of Corti: A Cytochemical Theory of HearJOHN L. HAMERTON A N D L. K. TITOVA ing-J. A. VINNIKOV Chromosomal Evolution in Cell Populations-T. Connective Tissue and Serum Proteins-R. E. C. Hsu MANCINI Chromosome Structure with Special Reference to The Biology and Chemistry of the Cell Walls of the Role of Metal IOnS-DALE M. STEFFENHigher Plants, Algae, and Fungi-D. H. SEN NORTHCOTE Electron Microscopy of Human White Blood Development of Drug Resistance by StaphyloCells and Their Stem Ceh-MARCEL BESSIS cocci in Vitro and in ViVO-MARY BARBER AND JEAN-PAUL THIERY Cytological and Cytochemical Effects of Agents In Vivo Implantation as a Technique in Skeletal Implicated in Various Pathological CondiBiology-WILLIAM J. L. FELTS tions: The Effect of Viruses and of Cigarette The Nature and Stability of Nerve Myelin-J. B. Smoke on the Cell and Its Nucleic AcidFINEAN CECILIE LEUCHTENBERGER A N D RUDOLF Fertilization of Mammalian Eggsin Vitro-C. R. LEUCHTENBERGER AUSTIN The Tissue Mast W~I-DOUGLASE. SMITH Physiology of Fertilization in Fish Eggs-ToKI-o AUTHOR INDEX-SUBJECT INDEX YAMAMOTO AUTHOR INDEX-SUBJECT

INDEX

AUTHOR INDEX-SUBJECT

INDEX

Volume 13 The Coding Hypothesis-MARTYNAS YEAS Chromosome Reproduction-J. HERBERT TAYLOR

Volume 15 The Nature of Lampbrush Chromosomes-H. G. CALLAN The Intracellular Transfer of Genetic Information-J. L. SIRLIN Mechanisms of Gametic Approach in Plants-

347

CONTENTS OF PREVIOUS VOLUMES LEONARD MACHLIS A N D ERIKARAWITSCHERKUNKEL The Cellular Basis of Morphogenesis and Sea AND L. Urchin Development-T. GUSTAFSON WOLPERT Plant Tissue Culture in Relation to Development CytOlOgy~ARLR. PARTANEN Regeneration of Mammalian Liver-NANCY L. R. BUCHER Collagen Formation and Fibrogenesis with Special Reference to the Role of Ascorbic AcidBERNARDs. GOULD The Behavior of Mast Cells in AnaphylaxisIVANMOTA Lipid Absorption-ROBERT M. WOTTON

The Cells of the Adenohypophysis and Their Functional Significance-MARC HERLANT AUTHOR INDEX-SUBJECT

INDEX

Volume 18

The Cell of Langerhans-A. S. BREATHNACH The Structure of the Mammalian Egg-ROBERT HADEK Cytoplasmic Inclusions in Oogenesis-M. D. L. SRIVASTAVA The Classification and Partial Tabulation of Enzyme Studies on Subcellular Fractions Isolated by Differential Centrifuging-D. B. ROODYN AUTHOR INDEX-SUBJECT INDEX Histochemical Localization of Enzyme Activities by Substrate Film Methods: Ribonucleases, Deoxyribonucleases, Proteases, Amylase, and Volume 16 Hyaluronidase-R. DAOUST Ribosomal Functions Related to Protein SyntheCytoplasmic Deoxyribonucleic Acid-P. B. GAsis-ToRE HULTIN HAN AND J. CHAYEN Physiology and Cytology of Chloroplast FormaMalignant Transformation of Cells in Virrotion and “Loss” in Euglena-M. GRENSON KATHERINE K. SANFORD Cell Structures and Their Significance for AmeDeuterium Isotope Effects in Cytology-E. boid Movement-K. E. WOHLFARTH-BOTTFLAUMENHAFT, S. BOSE,H. I. CRESPI,AND J . ERMAN J. KATZ Microbeam and Partial Cell 1rradiation-C. L. The Use of Heavy Metal Salts as Electron SMITH Stains<. RICHARDZOBEL AND MICHAEL Nuclear-Cytoplasmic Interaction with Ionizing BEER Radiation-M. A. LESSLER AUTHOR INDEX-SUBJECT INDEX In Vivo Studies of Myelinated Nerve FibersCARLCASKEYSPEIDEL Respiratory Tissue: Structure, Histophysiology, Volume 19 Cytodynamics. Part I: Review and Basic Cyto“Metabolic” DNA: A Cytochemical Study-H. morphology-FELIX D. BERTALANFFY AUTHOR INDEX-SUBJECT INDEX ROELS The Significance of the Sex Chromatin-MuRRAY L. BARR Volume 17 Some Functions of the Nucleus-I. M. MITCHIThe Growth of Plant Cell Walls-K. WILSON SON Reproduction and Heredity in Trypanosomes: A Synaptic Morphology on the Normal and Degenerating Nervous System-E. G . GRAY A N D R. Critical Review Dealing Mainly with the AfW. GUILLERY rican Species in the Mammalian Host-P. J. WALKER Neurosecretion-W. BARGMANN The Blood Platelet: Electron Microscopic Stud- Some Aspects of Muscle Regeneration-E. H. BETZ, H. FIRKET,A N D M. REZNIK ies-]. F. DAVID-FERREIRA The Gibberellins as Hormones-P. W. BRIAN The Histochemistry of MucopolysaccharidesPhototaxis in PlaIItS-wOLFGANG HAUFT ROBERTC. CURRAN Phosphorus Metabolism in Plants-K. S. Respiratory Tissue Structure, Histophysiology, Cytodynamics. Part 11. New Approaches and ROWAN AUTHOR INDEX-SUBJECT INDEX Interpretations-FELIX D. BERTALANFFY

348

CONTENTS OF PREVIOUS VOLUMES

The Dynamism of Cell Division during Early Cleavage Stages of the Egg-N. FAUTREZThe Chemical Organization of the Plasma MemFIRLEFYN AND J. FAUTREZ brane of Animal Cells-A. H. MADDY Lymphopoiesis in the Thymus and Other Tissues: Subunits of Chloroplast Structure and Quantum Functional Implications-N. B. EVERETT AND Conversion in Photosynthesis-RODERIC B. RUTHW. TYLER (CAFFREY) PARK Structure and Organization of the Myoneural Control of Chloroplast Structure by Light-LEsJunction-€. CoERs TER PACKER AND P A U L - A N D SIEGEN~ The Ecdysial Glands of Arthropods-WILLIAM THALER S. HERMAN The Role of Potassium and Sodium Ions as Cytokinins in Plants-B. I. SAHAISRIVASTAVA Studied in Mammalian Brain-H. HILLMAN AUTHOR INDEX-SUBJECT INDEX Triggering of Ovulation by Coitus in the RatCUMULATIVE SUBJECT INDEX (VOLUMES 1-21) CLAUDE ARON,GITTAASCH,AND JAQUELINE Roos Cytology and Cytophysiology of Non-MelanoVolume 23 phore Pigment Cells-JosEPH T. BAGNARA Transformationlike Phenomena in Somatic The Fine Structure and Histochemistry of ProstaCells-J. M. OLENOV tic Glands in Relation to Sex HormonesRecent Developments in the Theory of Control DAVIDBRANDES and Regulation of Cellular Processes-ROBCerebellar Enzymology-LuclE ARVY ERT ROSEN AUTHOR INDEX-SUBJECT INDEX Contractile Properties of Protein Threads from Sea Urchin Eggs in Relation to Cell DivisionVolume 21 HIKOICHISAKAI Electron Microscopic Morphology of OogeneHistochemistry of Lysosomes-P. B. GAHAN sis-ARNE NqRREVANG Physiological Clocks-R. L. BRAHMACHARY Dynamic Aspects of Phospholipids during ProCiliary Movement and Coordination in Ciliatestein Secretion-LOWELL E. HOKIN BELAPARDUCA The Golgi Apparatus: Structure and FunctionElectromyography: Its Structural and Neural H. W. BEAMSAND R. G. KESSEL Basis-JOHN V. BASMAJIAN The Chromosomal Basis of Sex DeteminationCytochemical Studies with Acridine Orange and KENNETHR. LEWISAND BERNARD JOHN the Influence of Dye Contaminants in the AUTHOR INDEX-SUBJECT INDEX H. Staining of Nucleic Acids-FREDERICK KASTEN Experimental Cytology of the Shoot Apical Cells Volume 24 during Vegetative Growth and Flowering-A. Synchronous Cell Differentiation-GEORGE M. NOUGAREDE A N D IVANL. CAMERON PADILLA Nature and Origin of Perisynaptic Cells of the Mast Cells in the Nervous SySteffl-yNGVE Motor End Plate-T. R. SHANTHAVEERAPPA OLSON AND G. H. BOURNE Development Pha$es in Intermitosis and the AUTHOR INDEX-SUBJECT INDEX Preparation for Mitosis of Mammalian Cells in Vitfo-BLAGOJE A. NESKOVIC Volume 22 Antimitotic Substances-GuY DEYSSON The Form and Function of the Sieve Tube: A Current Techniques in Biomedical Electron MiProblem in Reconciliation-P. E. WEATHERcroscopy-SauL WISCHNITZER LEY AND R. P. C. JOHNSON The Cellular Morphology of Tissue Repair-R. Analysis of Antibody Staining Patterns Obtained M. H. MCMINN with Striated Myofibrils in Fluorescence MiStructural Organization and Embryonic Differencroscopy and Electron Microscopy-FRANK tiatiOdAlANAN v. SHERBET AND M. s. A. PEPE LAKSHMI

Volume 20

349

CONTENTS OF PREVIOUS VOLUMES Cytology of Intestinal Epithelial Ceh-PETER G. TONER Liquid Junction Potentials and Their Effects on Potential Measurements in Biology Systems-P. C. CALDWELL AUTHOR INDEX-SUBJECT

INDEX

Volume 25 Cytoplasmic Control over the Nuclear Events of Cell Reproduction-NOEL DE TERRA Coordination of the Rhythm of Bedt in Some Ciliary Systems-M. A. SLEIGH The Significance of the Structural and Functional Similarities of Bacteria and MitochondriaSYLVAN NASS The Effects of Steroid Hormones on Macrophage Activity-B. VERNON-ROBERTS The Fine Structure of Malaria Parasites-MARIA A. RUDZINSKA The Growth of Liver Parenchymal Nuclei and Its Endocrine Regulation-RITA CARRIERE Strandedness of ChrOmOSomeS-SHELDON WOLFF Isozymes: Classification, Frequency, and SignifiCanCe-CHARLES R. SHAW The Enzymes of the Embryonic NephronLUCIEARVY Protein Metabolism in Nerve Cells-B. DROZ Freeze-Etching-HANS MOOR AUTHOR INDEX-SUBJECT

INDEX

Volume 26 A New Model for the Living Cell: A Summary of the Theory and Recent Experimental Evidence in Its S U P P O ~ ~ ~ I L N. B ELING RT The Cell Periphery-LEONARD WEISS Mitochondria1 DNA: Physicochemical Properties, Replication, and Genetic Function-P. BORSTAND A. M. KROON Metabolism and Enucleated CdlS-KONRAD KECK Stereological Principles for Morphometry in Electron Microscopic Cytology-EwALD R. WEIBEL Some Possible Roles for Isozymic Substitutions during Cold Hardening in Plants-D. W. A. ROBERTS AUTHOR INDEX-SUBJECT

INDEX

Volume 27 Wound-Healing in Higher Plants-JACQUES LIPETZ Chloroplasts as Symbiotic Organeks-DENNIS L. TAYLOR The Annulate Lamellae-SAUL WISCHNITZER Gametogenesis and Egg Fertilization in Planaria n s 4 . BENAZZILENTATI Ultrastructure of the Mammalian Adrenal CorteX-SlMON IDELMAN The Fine Structure of the Mammalian Lymphoreticular SyStem-IAN CARR lmmunoenzyme Technique: Enzymes as Markers for the Localization of Antigens and Antibodies-STRATIS AVRAMEAS AUTHOR INDEX-SUBJECT

INDEX

Volume 28 The Cortical and Subcortical Cytoplasm of Lymnaea EggXHRISTIAAN P. RAVEN The Environment and Function of Invertebrate A N D R. B. Nerve Cells-J. E. TREHERNE MORETON Virus Uptake, Cell Wall Regeneration, and Virus Multiplication in Isolated Plant ProtoplastsE. C. COCKING The Meiotic Behavior of the Drosophila 00CYte-ROBERT c . KING The Nucleus: Action of Chemical and Physical Agents-RENB SlMARD The Origin Of Bone Cek-kfAUREEN OWEN Regeneration and Differentiation of Sieve Tube Ekments-WILLIAM P. JACOBS Cells, Solutes, and Growth: Salt Accumulation in Plants Reexamined-F. C. STEWARD AND R. L. MOTT AUTHOR INDEX-SUBJECT

INDEX

Volume 29 Gram Staining and Its Molecular MechanismB. B. BISWAS,P. s. BASU,A N D M. K. PAL The Surface Coats of Animal Cells-A. MARTfNEZ-PALOMO Carbohydrates in Cell SUrfaCeS-RICHARD J. WINZLER Differential Gene Activation in Isolated Chromosomes-MARKUS LEZZI Intraribosomal Environment of the Nascent Peptide Chain-HlDEKo KAJI

350

CONTENTS OF PREVIOUS VOLUMES

Location and Measurement of Enzymes in Single Cells by Isotopic Methods Part I-E. A. BARNARD

Location and Measurement of Enzymes in Single Cells by Isotopic Methods Part 1 1 4 . C. BUDD Neuronal and Glial Perikarya Preparations: An Appraisal of Present Methods-PATRICIA v . JOHNSTON AND BETTY1. ROOTS Functional Electron Microscopy of the Hypothalamic Median Eminence-HlDEsHI KOBAYASHI, TOKUZOMATSUI.A N D SUSUMl ISHI1 Early Development in Callus Cultures- MICHAEL M. YEOMAN AUTHOR INDEX-SUBJECT

INDEX

Highly Repetitive Sequences of DNA in Chromosomes-W. G. FLAMM The Origin of the Wide Species Variation in Nuclear DNA Content-H. REESA N D R. N. JONES Polarized Intracellular Particle Transport: Saltatory Movements and Cytoplasmic Streaming- LIONELI. REBHUN The Kinetoplast of the Hemoflagellates-LARRY SIMPSON Transport across the Intestinal Mucosal Cell: Hierarchies of Function-D. S. PARSONS AND C. A. R. BOYD Wound Healing and Regeneration in the Crab Paratelphusa hydrodromous-RITA G. ADIYODI

Volume 30 High-pressure Studies in Cell Biology-ARTHUR M. ZIMMERMAN Micrurgical Studies with Large Free-Living Amebas-K. W. JEONA N D J. F. DANIELLI The Practice and Application of Electron Microscope Autoradiography-J. JACOB Applications of Scanning Electron Microscopy in Biology-K. E. CARR Acid Mucopolysaccharides in Calcified TisSUeS-SHINJIRO KOBAYASHI AUTHOR INDEX-SUBJECT

Volume 32

The Use of Ferritin-Conjugated Antibodies in Electron MicrOSCOpy
Metabolic DNA in Ciliated Protozoa, Salivary Gland Chromosomes, and Mammalian Cells-S. R. PELC AUTHOR INDEX-SUBJECT

INDEX

INDEX

CUMULATIVE SUBJECT INDEX (VOLUMES

1-29)

Volume 33

Visualization of RNA Synthesis on ChromoSOmeS-0. L. MILLER,JR. A N D BARBARA A. Volume 31 HAMKALO Studies on Freeze-Etching of Cell MembranesCell Disjunction (“Mitosis”) in Somatic Cell KURT M ~ H L E T H A L E R G . DIACUMAKOS, Reproduction-ELAINE AND PAULINE PECORA SCOTTHOLLAND, Recent Developments in Light and Electron Microscope Radioautography-G. C. BUDD Neuronal Microtubules, Neurofilaments, and Morphological and Histochemical Aspects of Microfilarnents-RAYMOND B. WUERKER Glycoproteins at the Surface of Animal A N D JOELB. KIRKPATRICK Cells- A. RAMBOURG Lymphocyte Interactions in Antibody ReDNA Biosynthesis-H. S . JANSZ, D. V A N DER sponses-J. F. A. P. MILLER MEI, A N D G. M. ZANDVLIET Laser Microbeams for Partial Cell lrradiationCytokinesis in Animal Cells-R. RAPPAPORT MICHAEL W. BERNSAND CHRISTIAN SALET The Control of Cell Division in Ocular Lens-€. Mechanisms of Virus-Induced Cell FusionN. J . UNAKAR, GEORGE v . HARDINC, J . R. REDDAN, POSTE A N D M. BAGCHI Freeze-Etching of BaCtWki-cHARLES C. REMThe Cytokinins-HANS KENDE SEN A N D STANLEY w . WATSON Cytophysiology of the Teleost Pituitary- MAR- The Cytophysiology of Mammalian Adipose TIN SAGE A N D HOWARD A. BERN Cek-BERNARD G . SLAVIN AUTHOR INDEX-SUBJECT

INDEX

AUTHOR INDEX-SUBJECT

INDEX

35 1

CONTENTS OF PREVIOUS VOLUMES

tigophora and Opalinata (Protozoa)--G. P. DUTTA The Submicroscopic Morphology of the InterChloroplasts and Algae as Symbionts in Molphase Nucleus-SAUL WISCHNITZER AND RICHARD IUSCS-LEONARDMUSCATINE The Energy State and Structure of Isolated W. GREENE Chloroplasts: The Oxidative Reactions InvolvThe Macrophage-SAIMoN GORDONAND ZANing the Water-Splitting Step of PhotosyntheV I L A. COHN sis- ROBERTL. HEATH Degeneration and Regeneration of NeurosecreTransport in Neurospora-GENE A. SCARtory Systems-HORST-DIETER DELLMANN

Volume 34

BOROUGH

AUTHOR INDEX-SUBJECT

INDEX

Mechanisms of Ion Transport through Plant Cell Membranes-EMANUEL ERSTEIN Cell Motility: Mechanisms in Protoplasmic Volume 37 Streaming and Ameboid Movement-H. KOMNICK, W. STOCKEM, A N D K. E. WOHLE- Units of DNA Replication in Chromosomes of FARTH-BOTTERMANN Eukaroytes-J. HERBERT TAYLOR The Gliointerstitial System of MoIIuscs- GHIS- Viruses and Evolution-D. C. REANNEY LAIN NICAISE Electron Microscope Studies on Spermiogenesis Colchicine-Sensitive Microtubules-LYNN MARin Various Animal SpeCieS-GONPACHIRO GULIS YASUZUMI AUTHOR INDEX-SUBJECT INDEX Morphology, Histochemistry, and Biochemistry of Human Oogenesis and Ovulation-SARDuL S. GURAYA Volume 35 Functional Morphology of the Distal LungKAYEH. KILBURN The Structure of Mammalian ChromosomesComparative Studies of the Juxtaglomerular ApELTONSTUBBLEFIELD SOKABE AND MIZUHO paratus-HIRoFuMI Synthetic Activity of Polytene ChromosomesOGAWA HANSD. BERENDES The Ultrastructure of the Local Cellular Reaction Mechanisms of Chromosome Synapsis at Meiotic to Neoplasia-IAN CARR A N D J. c. E. UNDERProphase-PETER B. MOENS WOOD Structural Aspects of Ribosomes-N. NANScanning Electron Microscopy in the UltrastrucNINGA tural Analysis of the Mammalian Cerebral Comparative Ultrastructure of the Cerebrospinal Ventricular System-D. E. SCOTT, G. P. Fluid-Contacting Neurons-B. VICH A N D 1. KOZLOWSKI, AND M. N. SHERIDAN VIGH-TEICHMANN AUTHOR INDEX-SUBJECT INDEX Maturation-Inducing Substance in StarfishesHARUOKANATANI The Lirnoniurn Salt Gland: A Biophysical and Structural Study-A. E. HILLA N D B. S. HILL Volume 38 Toxic Oxygen Effects-HAROLD M. SWARTZ Genetic Engineering and Life Synthesis: An AUTHOR INDEX-SUBJECT

INDEX

Volume 36 Molecular Hybridization of DNA and RNA in Situ-WOLFGANG HENNIG The Relationship between the Plasmalemma and Plant Cell Wall-JEAN-CLAUDE ROLAND Recent Advances in the Cytochemistry and Ultrastructure of Cytoplasmic Inclusions in Mas-

Introduction to the Review by R. Widdus and C. AUlt-JAMES F. DANIELLI Progress in Research Related to Genetic Engineering and Life Synthesis-Roy WIDDUS A N D CHARLES R. AULT The Genetics of C-Type RNA Tumor VirusesJ. A. WYKE Three-Dimensional Reconstruction from Projections: A Review of Algorithms-RICHARD GORDONAND GABORT. HERMAN

352

CONTENTS OF PREVIOUS VOLUMES

The Cytophysiology of Thyroid Celk-vLADIMIR R. PANTIC The Mechanisms of Neural Tube FormationPERRYKAWUNKEL The Behavior of the XY Pair in MammalsJ. SOLARI ALBERTO Finestructural Aspects of Morphogenesis in Acetabularia-G. WERZ Cell Separation by Gradient Centrifugation-R. HARWOOD SUBJECT INDEX

Volume 39 Androgen Receptors in the Nonhistone Protein Fractions of Prostatic Chromatin-TuNc YUE WANGA N D LEROYM. NYBERG Nucleocytoplasmic Interactions in Development of Amphibian Hybrids-STEPHEN SUBTELNY The Interactions of Lectins with Animal Cell Surfaces-GARTH L. NICOLSON Structure and Function of Intercellular Junctions-L. ANDREW STAEHELIN Recent Advances in Cytochemistry and Ultrastructure of Cytoplasmic Inclusions in Ciliophora (Protozoa)-G. P. DUTTA Structure and Development of the Renal Glomerulus as Revealed by Scanning Electron MicrOSCOpy-fkANCO SPINELL1 Recent Progress with Laser Microbeams- MICHAEL w . BERNS The Problem of Germ Cell Determinants-H. W. BEAMSA N D R. G. KESSEL SUBJECT INDEX

Volume 40 B-Chromosome Systems in Flowering Plants and Animal Species-R. N. JONES The lntracellular Neutral SH-Dependent Protease Associated with Inflammatory ReactionsHIDEOHAYASHI The Specificity of Pituitary Cells and Regulation of Their Activities-VLADIMIR R. PANTIC Fine Structure of the Thyroid Gland-HIsAo FUJITA Postnatal Gliogenesis in the Mammalian BrainA. PRIVAT Three-Dimensional Reconstruction from Serial Sections-RANDLE w. WARE AND V I N C E N T LOPRESTI SUBJECT INDEX

Volume 41 The Attachment of the Bacterial Chromosome to AND the Cell Membrane-PAUL J . LEIBOWITZ MOSELIOSCHAECHTER Regulation of the Lactose Operon in Escherichiu coli by c A M P - G . CARPENTER A N D B. H. SELLS Regulation of Microtubules in TetruhymenaNORMAN E. WILLIAMS Cellular Receptors and Mechanisms of Action of Steroid Hormones-SHUTSUNG LIAO A Cell Culture Approach to the Study of Anterior Pituitary Cells-A. TIXIER-VIDAL,D. GOURDJI, A N D C. TOUGARD Immunohistochemical Demonstration of Neurophysin in the Hypothalamoneurohypophysial System-W. B. WATKINS The Visual System of the Horseshoe Crab Limulus polyphemus-WOLF H. FAHRENBACH SUBJECT INDEX

Volume 42 Regulators of Cell Division: Endogenous Mitotic Inhibitors of Mammalian Ceh-BISMARCK B. Lozzro, CARMEN B. Lozzro. ELENAG. BAMA N D STEPHEN V. LAIR BERGER, Ultrastructure of Mammalian Chromosome AND WALTER Aberrations-B. R. BRINKLEY N. HITTELMAN Computer Processing of Electron Micrographs: A Nonmathematical Account-P. W. HAWKES Cyclic Changes in the Fine Structure of the Epithelial Cells of Human EndometriumMILDRED GORDON The Ultrastructure of the Organ of CortiROBERTS. KIMURA Endocrine Cells of the Gastric Mucosa-ENNco SOLCIA, CARL0 CAPELLA, GABRIELE VASSALLO, A N D ROBERTO BUFFA Membrane Transport of Purine and Pyrimidine Bases and Nucleosides in Animal CellsRICHARD D. BERLINA N D JANETM. OLIVER SUBJECT INDEX

Volume 43 The Evolutionary Origin of the Mitochondrion: A Nonsymbiotic Model-HENRY R. MAHLER A N D RUDOLFA. RAFF Biochemical Studies of Mitochondria1 Transcrip-

CONTENTS OF PREVIOUS VOLUMES tion and Translation-C. SACCONEA N D E. QUAGLIARIELLO The Evolution of the Mitotic Spindle-DONNA F. KUBAI Germ Plasma and the Differentiation of the Germ Cell Line-E. M. EDDY Gene Expression in Cultured Mammalian Cells-Row P. COX A N D JAMES c . KING Morphology and Cytology of the Accessory Sex Glands in Invertebrates-K. G . ADlYODl A N D R. G. ADIYODI SUBJECT INDEX

353

Small Lymphocyte and Transitional Cell Populations of the Bone Marrow; Their Role in the Mediation of Immune and Hemopoietic Progenitor Cell Functions-CORNELIUS ROSE The Structure and Properties of the Cell Surface Coat-J. H. LUFT Uptake and Transport Activity of the Median Eminence of the Hypothalamus-K. M. KNIGGE, S. A. JOSEPH,J. R. SLADEK,M. F. M. A. NOTTER,M. MORRIS,D. K. SUNDBERG, HOLZWARTH,G. E. HOFFMAN,AND L. O’BRIEN SUBJECT INDEX

Volume 44 The Nucleolar StNCtUre-slBDAS GHOSH The Function of the Nucleolus in the Expression of Genetic Information: Studies with Hybrid Animal Cells-E. SIDEBOTTOMA N D I. I. DEhK Phylogenetic Diversity of the Proteins Regulating Muscular Contraction-WILLIAM LEHMAN Cell Size and Nuclear DNA Content in Vertebrates-HENRYK SZARSKl Ultrastructural Localization of DNA in Ultrathin Tissue Sections-ALAlN GAUTIER Cytological Basis for Permanent Vaginal Changes in Mice Treated Neonatally with Steroid Hormones-NoBoRu TAKASUCI On the Morphogenesis of the Cell Wall of StaphylOCoCCi-PETER GIESBRECHT,J ~ R C WECKE, AND BERNHARD RElNlCKE Cyclic AMP and Cell Behavior in Cultured Ceh-MARK c. WILLINGHAM Recent Advances in the Morphology, Histochemistry, and Biochemistry of Steroid-Synthesizing Cellular Sites in the Nonmammalian Vertebrate OVV-SARDUL S. GURAYA SUBJECT INDEX

Volume 45 Approaches to the Analysis of Fidelity of DNA Repair in Mammalian Cells-MICHAEL W . LIEBERMAN The Variable Condition of Euchromatin and Heterochromatin-FRIEDRICH BACK Effects of 5-Bromodeoxyuridine on Tumorigenicity, Immunogenicity, Virus Production, Plasminogen Activator, and Melanogenesis of Mouse Melanoma Ceh-sELMA SILAGI Mitosis in Fungi-MELVIN S. FULLER

Volume 46 Neurosecretion by Exocytosis-TOM CHRISTIAN NORMANN Genetic and Morphogenetic Factors in Hemoglobin Synthesis during Higher Vertebrate Development: An Approach to Cell Differentiation Mechanisms-VICTOR N E O N AND JACQUELINE GODET Cytophysiology of Corpuscles of Stannius-V. G. KRISHNAMURTHY Ultrastructure of Human Bone Marrow Cell Maturation-J. BRETON-GORIUSAND F. REYES Evolution and Function of Calcium-Binding Proteins-R. H. KRETSINGER SUBJECT INDEX

Volume 47 Responses of Mammary Cells to Hormones-M. R. BANERJEE Recent Advances in the Morphology, Histochemistry, and Biochemistry of Steroid-Synthesizing Cellular Sites in the Testes of Nonmammalian Vertebrates-SARDUL S. GURAYA Epithelial-Stromal Interactions in Development of the Urogenital TTaCt-GERALD R. CUNHA Chemical Nature and Systematization of Substances Regulating Animal Tissue GrowthVICTORA. KONYSHEV Structure and Function of the Choroid Plexus and Other Sites of Cerebrospinal Fluid Formation- THOMAS H. MILHORAT The Control of Gene Expression in Somatic Cell Hybrids-H. P. BERNHARD Precursor Cells Of Mechanocytes-ALEXANDER J. FRIEDENSTEIN SUBJECT INDEX

3 54

CONTENTS OF PREVIOUS VOLUMES

Volume 48 Mechanisms of Chromatin Activation and ReAND VAUGHAN pression-NORMAN MACLEAN A. HILDER Origin and Ultrastructure of Cells in Vim-L. M. FRANKSA N D PATRICIAD. WILSON Electrophysiology of the Neurosecretory CellKINIIYAGIAND SHIZUKOIWASAKI Reparative Processes in Mammalian Wound Healing: The Role of Contractile PhenomA N D DENWMONena- GIULIOGABBIANI

New Aspects of the Ultrastructure of Frog Rod Outer Segments-JURGEN ROSENKRANZ Mechanisms of Morphogenesis in Cell CulAND I. M. GELFAND tures-J. M. VASILIEV Cell Polyploidy: Its Relation to Tissue Growth and Functions-W. YA. BRODSKY AND I. v . URYVAEVA Action of Testosterone on the Differentiation and Secretory Activity of aTarget Organ: The Submaxillary Gland of the Mouse-MoNIQUE CHRBTIEN SUBJECT INDEX

TANDON

Smooth Endoplasmic Reticulum in Rat Hepatocytes during Glycogen Deposition and DepletiOn-ROBERT R. CARDELL, JR. Potential and Limitations of Enzyme Cytochemistry: Studies of the Intracellular Digestive Apparatus of Cells in Tissue Culture-M. HUNDGEN

Uptake of Foreign Genetic Material by Plant Protoplasts-E. C. COCKING The Bursa of Fabricius and Immunoglobulin Synthesis-BRUCE GLICK SUBJECT INDEX

Volume 49 Cyclic Nucleotides, Calcium, and Cell DiviSion-LIoNEL I. REBHUN Spontaneous and Induced Sister Chromatid Exchanges as Revealed by the BUdR-Labeling Method-HATAo KATO Structural, Electrophysiological, Biochemical, and Pharmacological Properties of Neuroblastoma-Glioma Cell Hybrids in Cell Culture-B. HAMPRECHT Cellular Dynamics in Invertebrate Neurosecretory Systems-ALLAN BERLIND Cytophysiology of the Avian Adrenal MedullaASOKGHOSH Chloride Cells and Chloride Epithelia of Aquatic Insects-H. KOMNICK Cytosomes (Yellow Pigment Granules) of Molluscs as Cell Organelles of Anoxic Energy Production-IMRE ZS.-NAGY SUBJECT INDEX

Volume 50

Volume 51 Circulating Nucleic Acids in Higher Organisms-MAURICE STROUN, PHILIPPE ANKER, PIERREMAURICE,A N D PETERB. GAHAN Recent Advances in the Morphology, Histochemistry, and Biochemistry of the Developing Mammalian OVW-SARDUL s. GURAYA Morphological Modulations in Helical Muscles (Aschelminthes and Annelida)-GIuLIo LANZAVECCHIA Interrelations of the Proliferation and Differentiation Processes during Cardiac Myogenesis and Regeneration-PAVEL P. RUMYANTSEV The Kurloff Cell-PETER A. REVELL Circadian Rhythms in Unicellular Organisms: An Endeavor to Explain the Molecular Mechanism- HANS-GEORG SCHWEIGER AND MANFRED SCHWEIGER SUBJECT INDEX

Volume 52 Cytophysiology of Thyroid Parafollicular Cells-ELADIO A. NUNEZA N D MICHAEL D. GERSHON Cytophysiology of the Amphibian Thyroid Gland through Larval Development and Metamorphosis-ELIANE REGARD The Macrophage as a Secretory Cell-Roy C. PAGE,PHILIPDAVIES,AND A. C. ALLISON Biogenesis of the Photochemical ApparatusTIMOTHY TREFFRY Extrusive Organelles in Protists-KLAUS HAUSMANN

Cell Surface Enzymes: Effects on Mitotic Activkctins-JAY c. BROWNANDRICHARDC. HUNT ity and Cell Adhesion-H. BRUCEBOSMANN SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 53 Regular Arrays of Macromolecules on Bacterial Cell Walls: Structure, Chemistry, Assembly, and Function-UwE B. SLEYTR Cellular Adhesiveness and Extracellular Substrata-FREDERICK GRINNELL Chemosensory Responses of Swimming Algae AND D. c. and Protozoa-M. LEVANDOWSKY R. HAUSER Morphology, Biochemistry, and Genetics of Plastid Development in Euglena gracilis-V. NIWN AND P. HEIZMANN Plant Embryological Investigations and Fluorescence Microscopy: An Assessment of Integration-R. N. KAPIL A N D S . C. TIWAN The Cytochemical Approach to Hormone Assay-J. CHAYEN SUBJECT INDEX

Volume 54

355

The Isolated Mitotic Apparatus and Chromosome Motion-H. SAKAI Contact Inhibition of Locomotion: A KeapPraiSd-JOAN E. M. HEAYSMAN Morphological Correlates of Electrical and Other Interactions through Low-Resistance Pathways between Neurons of the Vertebrate Central Nervous System-C. SOTELOAND H. KORN Biological and Biochemical Effects of Phenylalanine Analogs-D. N. WHEATLEY Recent Advances in the Morphology, Histochemistry, Biochemistry, and Physiology of Interstitial Gland Cells of Mammalian OVW-SARDUL S. GURAYA Correlation of Morphometry and Stereology with Biochemical Analysis of Cell FractionsR. P. BOLENDER Cytophysiology of the Adrenal Zona FasCiCUlataAASTONE G . NUSSDORFER. GIUSEPPINA MAZZOCCHI, AND VIRGILIO MENEGHELLI SUBJECTINDEX

Microtubule Assembly and Nucleation-MARC W. KIRSCHNER Volume 56 The Mammalian Sperm Surface: Studies with K. Synapses of Cephalopods-COLLETTE DUCROS Specific Labeling Techniques-JAMES KOEHLER Scanning Electron Microscope Studies on the The Glutathione status Of CellS-NECHAMA s. Development of the Nervous System in Vivo KOSOWERA N D EDWARD M. KOSOWER and in Vitro-K. MELLER Cells and Senescence-ROBERT ROSEN Cytoplasmic Structure and Contractility in Immunocytology of Pituitary Cells from Teleost Amoeboid Cells-D. LANSINGTAYLOR AND Fishes-E. FOLL~NIUS, J . DOERR-SCHOTT, JOHN S. CONDEELIS A N D M. P. DUBOIS Methods of Measuring Intracellular CalciumFollicular Atresia in the Ovaries of NonmammaANTHONY H. CASWELL lian VertebratesSRINIvAs K. SAIDAPUR Electron Microscope Autoradiography of CalHypothalamic Neuroanatomy: Steroid Hormone cified Tissues-ROBERT M. FRANK Binding and Patterns of Axonal Pro- Some Aspects of Double-Stranded Hairpin StrucjeCtionsDoNALD w. PFAFF A N D LILYC. A. tures in Heterogeneous Nuclear RNA-HIROTO CONRAD NAOW Ancient Locomotion: Prokaryotic Motility Sys- Microchemistry of Microdissected Hypothalamic temS-LELENG P. T O A N D LYNNMARGULIS Nuclear Areas-M. PALKOWTS An Enzyme Profile of the Nuclear EnvelopeSUBJECT INDEX I. B. ZBARSKY SUBJECTINDEX Volume 57

Volume 55 Chromatin Structure and Gene Transcription: Nucleosomes Permit a New S y n t h e s i s THORU F’EDERSON

The Corpora Allata of hSeC&PlERRE CASSIER Kinetic Analysis of Cellular Populations by Means of the Quantitative Radioautography-J.-C. BISCONTE

356

CONTENTS OF PREVIOUS VOLUMES

Effects of Irradiation on Germ Cells and EmCellular Mechanisms of Insect Photoreceptionbryonic Development in Teleosts-Nosuo F. G.GRIBAKIN EGAMIA N D KEN-ICHIIim Oocyte Maturation-Yosmo MASUIAND HUGH Recent Advances in the Morphology, J. CLARKE Cytochemistry, and Function of Balbiani’s The Chmmaf!in and Chromaffin-likeCells in the Vitelline Body in Animal OOCY~~S-SARDUL Autonomic Nervous system-JACQUES TAXI S . GURAYA The Synapses of the Nervous System-A. A. Cultivation of Isolated Protoplasts and HybridiMANINA zation of Somatic Plant CellS-bISA G. SUBJECT INDEX BUTENKO SUBJECT INDEX Volume 58 Functional Aspects of Satellite DNA and Volume 60 Heterochromatin-BERNARD JOHN AND GEORGE L. GABORMIKLOS Determinationof Subcellular Elemental Concen- Transfer RNA-like Structures in Viral Genomes-TIMOTHY c. HALL tration through Ultrahigh Resolution Electron Microprobe Analysis-THOMAS E.HUTCHIN- Cytoplasmic and Cell Surface Deoxyribonucleic SON Acids with Consideration of Their The Chromaffin Granule and Possible MechJ. Oligin-BEVAN L. REIDAND ALEXANDER anisms of Exocytosis-HARVEY B. POLLARD, CHARLSON CHRISTOPHER J. PAZOLES, CARLE. CREUTZ, Biochemistry of the Mitotic SpindleAHRIsAND ORENZINDER TIAN PETZELT The Golgi Apparatus, the Plasma Membrane, and Alternatives to Classical Mitosis in HemopOKtic Functional Integration-W. G.WHALEY AND, Tissues of Vertebrates-VIBEKE E. ENGELMARIANNE DAUWALDER BERT Genetic Control of Meiosis-I. N. GOLUBOV- Fluidity of Cell Membranes-Current Concepts and Trends-M. SHINITZKY AND P. HENSKAYA KART Hypothalamic Neurons in Cell Culture-A. TIXIER-VIDAL A N D F. DE VITRY Macrophage-Lymphocyte Interactions in ImALAN The Subfornical Organ-H DIETERDELLMANN mune Induction-MARC FELDMANN, ROSENTHAL, A N D JOHNB. SIMPSON AND PETER ERB SUBJECT INDEX Immunohistochemishy of Luteinizing Hormone-Releasing Hormone-Producing Neurons of the Vertebrates-JULIEN BARRY Cell Reparation qf Non-DNA Injury-V. YA. Volume 59 ALEXANDROV Ultrastructure of the Carotid Body in the MamThe Control of Microtubule Assembly in thu-ELIZABETH c . RAFF mak-ALAIN VERNA Membrane-Coating Granules-A. F. HAYWARD The Cytology and Cytochemistry of the Wool Innervation of the Gastrointestinal TractFolkle-DONALD F. G . ORWIN GIORGIO GABELLA SUBJECT INDEX