INTERNATIONAL
REVIEW OF CYTOLOGY VOLUME56
ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN W. BERNHARD GARY G. BORISY ROBERT W. BRIGGS STANLEY COHEN RENE COUTEAUX MARIE A. DI BERARDINO N. B. EVERETT CHARLES J. FLICKINGER M. NELLY GOLARZ DE BOURNE K. KUROSUMI MARIAN0 LA VIA GIUSEPPE MILLONIG ALEXANDER L.
ARNOLD MITTELMAN DONALD G. MURPHY ROBERT G. E. MURRAY ANDREAS OKSCHE VLADIMIR R. PANTIC DARRYL C. REANNEY LIONEL I. REBHUN JEAN-PAUL REVEL WILFRED STEIN ELTON STUBBLEFIELD HEWSON SWIFT DENNIS L. TAYLOR TADASHI UTAKOJI ROY WIDDUS YUDIN
INTERNATIONAL
Review of Cytology EDITED BY
G. H. BOURNE
J. F. DANIELLI
S t . George's University School of Medicine St. George's, Grenada West Indies
Worcester Polytechnic Institute Worcester, Massachusetts
ASSISTANT EDITOR K . W. JEON Department of Zoology University of Tennessee Knoxville, Tennessee
VOLUME 56
ACADEMIC PRESS New York
San Francisco London
A Subsidiary of Harcourt Brace Jovanovich, Publishers
1979
COPYRIGHT @ 1979, 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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United Kingdom Edition published by ACADEMIC PRESS. INC. ( L O N D O N ) LTD. 24/28 Oval Road, London’NW17DX
LIBRARY OF CONGRESS CATALOG CARD NUMBER:52-5203 ISBN 0-12-364356-2 PRINTED IN THE UNITED STATES OF AMERICA
79808182
9 8 7 6 5 4 3 2 1
Contents LISTOF CONTRIBUTORS.
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vii
Synapses of Cephalopods COLETTE DUCROS I. II. 111. IV . V. VI .
Introduction . . . . . . . . . . . . . . . . . . . Anatomical Outlinesof the Nervous System in Cephalopods Cytological Features of Synapses in Cephalopod Ganglia . . Special Features in Peripheral Afferent Pathways . . . . Nerve Endings in Cephalopod Effectors . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
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Scanning Electron Microscope Studies on the Development of the Nervous System in Vivo and in Vitro K . MELLER I. I1 . III . IV .
Introduction . . . . . . . . . . . . . Development of the Nervous System in Vivo Development of the Nervous System in Vitro Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . .
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Cytoplasmic Structure and Contractility in Amoeboid Cells D . LANSING TAYLOR A N D JOHNS . CONDEELIS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Cytoplasmic Structure and Contractility: I n Vivo . . . . . . . . . . . . . 111. Cytoplasmic Structure and Contractility: In Virro . . . . . . . . . . . . IV . Actin. Myosin. and Associated Contractile Proteins . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .
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61 96 117 134
Methods of Measuring Intracellular Calcium ANTHONY H . CASWELL
I. 11. 111. IV . V. VI .
Introduction . . . . . . . Assay of Total Cellular Ca . . Metallochromic Dyes . . . . Luminescent Proteins-Aequorin Fluorescent Chelate Robe . . Fixation of Tissue for Cytology
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CONTENTS
VII . Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . VIII. Electron Probe Microanalysis . . . . . . . . . . . . . . . . . . . . IX. Concluding Comments . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .
168 173 178 179
Electron Microscope Autoradiography of Calcified Tissues ROBERTM . FRANK
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . 11. Methodology . . . . . . . . . . . . . . . . . . . . . . . . 111. Detection of Nucleic Acids in Calcified Tissues . . . . . . . . . . . IV . Synthesis of Organic Matrices of Calcified Tissues . . . . . . . . . . V . Transfer Routes of Inorganic Elements . . . . . . . . . . . . . . VI . Identification of Sensory Nerve Endings in Adult Dentin by Autoradiography VII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .
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183 185 188 188 226 246 247 248
Some Aspects of Double-Stranded Hairpin Structures in Heterogeneous Nuclear RNA HIROTONAORA I . Introduction . . . . . . . . . . . . . . . . . . . . . . . 11. Occurrence of Double-Stranded RNA in “Uninfected” Eukaryotic Cells 111. Characteristics of Isolated dsRNA . . . . . . . . . . . . . . IV . Some Comments on the HnRNA-mRNA Precursor-Product Relationship V . Double-Stranded Hairpin Structures in HnRNA . . . . . . . . . VI . Features of Eukaryotic mRNA . . . . . . . . . . . . . . . VII . A Model of Nuclear Processing of HnRNA . . . . . . . . . . VIII . Occurrence of Eukaryotic RNases Specific for dsRNA . . . . . . IX . Summary and Concluding Remarks . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .
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256 257 259 271 278 283 293 299 303 306
Microchemistry of Microdissected Hypothalamic Nuclear Areas M . PALKOVITS I. 11. 111. IV .
Introduction . . . . . . . . . . . . . . . Microdissection of the Hypothalamus . . . . . Microchemistry . . . . . . . . . . . . . . General Considerations . . . . . . . . . . . References . . . . . . . . . . . . . . .
SUBJECT INDEX . . . . . . . . . . . . CONTENTS OF PREVIOUS VOLUMES . . . . .
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List of Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin
ANTHONYH. CASWELL(149, Department of Pharmacology, University of Miami School of Medicine, Miami, Florida 33152 JOHNs. CONDEELIS (57), Department of Anatomy, Albert Einstein College of Medicine, Bronx, New York 10461 COLETTEDUCROS(l), Luboratoire de Cytologie, Bat. A , Universitk Pierre et Marie Curie, 4 Place Jussieu, 75230 Paris Cedex 05, France ROBERTM. FRANK(183), Groupe de Recherches Institut National de la Sante et de la Recherche Mkdicale U 157, Facultk de Chirurgie Dentaire, Universite Louis Pasteur, 1 Place de I'Hopital, 67000 Strasbourg, France
K. MELLER(23), Ruhr-Universitat Bochum, Institut f i r Anatornie, Arbeitsgruppe fur Experimentelle Cytologie, Bochum, Federal Republic of Germany HIROTONAORA (255), Molecular Biology Unit, Research School of Biological Sciences, The Australian National University, P.O. Box 475, Canberra A.C.T. 2601, Australia M. PALKOVITS (3 1 3 , First Department of Anatomy, Semmelweis University Medical School, 1450 Budapest, Tuzolto u 58, Hungary D. LANSINGTAYLOR(57), Cell and Developmental Biology, The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138
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INTERNATIONAL REWEW OF CYTOLlXiY, VOL. 56
Synapses of Cephalopods COLETTE DUCROS Laboratoire de Cytologie, Universite Pierre et Marie Curie, Paris, France
I. Introduction
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II. Anatomical Outlines of the Nervous System in Cephalopods. . .
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A. Receptors . . . . . . . . . . . . . . . . . . . B. Ganglia . . . . . . . . . . . . . . . . . . . . C. Effectors . . . . . . . . . . . . . . . . . . . D. The Squid Giant Fiber System . . . . . . . . . . . Cytological Features of Synapses in Cephalopod Ganglia . . . A. Examples of Octopus Brain Synapses . . . . . . . . . B. Synapses of the Squid Giant Fiber System . . . . . . . C. Distribution of Different Types of Presynaptic Elements in the Octopus Brain . . . . . . . . . . . . . . . . . D. Presumed Neurotransmitters in the Cephalopod Ganglia . . Special Features in Peripheral Afferent Pathways . . . . . . Nerve Endings in Cephalopod Effectors . . . . . . . . . A. Ultrastructural Observations . . . . . . . . . . . . . B. Biochemical and Cytochemical Data . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . ~
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I. Introduction Cephalopods are among the most highly organized invertebrates. Their nervous system is very well developed and contains several hundred million nerve cells. Extensive data exist on the anatomy of the cephalopod nervous system; while the giant nerve fibers of Loligo have long been used in the general study of nerve properties, the Octopus brain has proved to be of value in the study of memory. During the last decade, investigations on the ultrastructure, biochemistry, and cytochemistry of cephalopod nerve tissue have converged on the functional contacts (synapses) between neurons and between neurons and effector cells. Some of the main results dealing with cephalopod synapses are compared in this article in an attempt to present an overall view of some of the more general aspects of the functional organization of nerve fibers. 1 Copyright 0 1979 by Academic F’ress, Inc. All rights of reproductionin any form reserved. ISBN 0-12-3643562
2
COLETTE DUCROS
11. Anatomical Outlines of the Nervous System in Cephalopods
First, it is necessary to outline some basic features of the complex nervous system in cephalopods.
A. RECEPTORS The eyes are well known for their striking resemblance to vertebrate eyes. The retina contains highly specialized photoreceptors sending out axons to the optic lobe of the brain (Moody and Robertson, 1960; Young, 196Ob, 1962, 1964). Near the origin of the axon, collaterals are emitted from the perikaryon and contribute to a retinal plexus along with efferent nerve fibers. The epistellar body of octopods (Young, 1929, 1936b), as well as the paraolfactory vesicles of decapods (Boycott and Young, 1956), show cells resembling photoreceptors (Nishioka et al., 1962, 1966; Perrelet and Mauro, 1972; Mauro, 1977). The statocyst (Young, 1960a; Barber, 1965, 1966, 1968; Budelmann and Wolff, 1976; Budelmann, 1977) contains sensory hair cells and underlying neurons, and the processes of both cells contribute to a plexus that may also receive efferent fibers from the brain. The skin is thought to contain primary sensory neurons distributed diffusely in and under the epithelium, although sensory nerve cells are especially numerous in the suckers (more than 10,000 in each sucker of Octopus), where they have been thoroughly studied (Rossi and Graziadei, 1958; Graziadei, 1959, 1962, 1964, 1965a; Graziadei and Gagne, 1976a, b). The sensory nerve cells are of varying morphology, and some of them send axons to underlying neurons (Graziadei, 1962, 1965a; Graziadei and Gagne , 1976b). Multipolar nerve cells found in the muscle of the mantle (Alexandrowicz, 1960; Boyle, 1976), arms, and suckers (Graziadei, 196%; Santi and Graziadei, 1975) can be considered propnoceptive. Indeed, physiological methods have demonstrated the presence of mechanically excitable receptor units in the Octopus mantle (Gray, 1960; Boyle, 1976).
B. GANGLIA An anterior concentration of ganglia surrounding the esophagus forms a “brain” subdivided into lobes and protected by a capsule of cartilage. Other ganglia, often called peripheral ganglia, are the stellate ganglia, the nerve cords of the arms, and the ganglia supplying the digestive tract and other viscera (subradular, buccal, gastric, cardiac, fusiform, and branchial ganglia).
SYNAPSES OF CEPHALOPODS
3
The basic structure of ganglia is that of a multilayered cellular rind surrounding a central fibrous core. The rind is formed by the perikarya of neurons and intervening glial cells and blood vessels. Unipolar neurons dominate in cephalopods, and they send out processes to the core. The latter contains intricate nerve cell processes forming a neuropil where synaptic contacts occur, tracts of nerve fibers, glial cells, and blood vessels (Bogoraze and Cazal, 1944; Stephens and Young, 1969; Gray, 1969). Each ganglion, or brain lobe, is characterized by the nature of the rind (thin or thick, having large motoneurons or small granule cells) and by the texture of the neuropil which, in some instances, appears highly ordered (the plexiform zone in the cortex of the optic lobe, the “spine” of the peduncle lobe). Successful application of the classic neurohistological silver methods (Ramon y Cajal, 1917; Stephens, 1971) has greatly contributed to studies on ganglia structure and composition (Young, 1971, 1972, 1974, 1976, 1977a). Careful anatomical studies using selective stains, combined with degeneration experiments and electrical stimulation (Sereni and Young, 1932; Boycott and Young, 1950; Boycott, 1953, 1961; Young, 1965a, b, c, 1971, 1972), have added much to knowledge of the input and output systems in the brain and other ganglia, of regional functional differentiation, and of the connections between sets of neurons. Octopus capacities for both learning and surviving surgical procedures have been emphasized in studies correlating behavior and organization (Boycott and Young, 1950; Wells, 1962; Young, 1964, 1966). C. EFFECTORS Swimming or crawling cephalopods are very active animals in which a rich nerve supply to the muscles of arms, mantle, fins, and funnel ensures control of movement. Color changes also depend on nerve centers (Boycott, 1953) through innervation of the chromatophore muscle fibers (Hofmann, 1907; Sereni, 1930). Accommodation and pupillary changes (Alexandrowicz, 1927; Van Wee1 and Thore, 1936) occur by means of innervation of the eye muscles. The visceral organs, too, are profusely innervated, as are the digestive tract and glands, muscles of the ink sac, genital ducts, heart, and blood vessels (Alexandrowicz, 1928; Graziadei, 1960; Ducros , 1971a; Young, 1965a, 1967). Innervation of the luminescent organs present in several decapods has also been reported (Arnold and Young, 1974).
D. THESQUIDGIANTFIBERSYSTEM (PLATEI) Since the classic observations of Young (1934, 1936a, b, 1939), attention has been focused on the outstanding features of this system which provides jet
4
COLETTE DUCROS
I I
I I I1 I
I I I I
.,.. I
If
r,.
PLATEI: FIG. 1. Schematic representation of the giant fiber system, on one side, in Loligo. af, Giant accessory fiber; b, protoplasmic bridge; gcl and gc2, first- and second-order giant cells: gfl, gf2, and gf3, first-, second- and third-order giant fibers. FIG. 2. Synapses in the superior frontal lobe. FIG. 3. Synapses in the plexiform zone of the optic lobe. (After Young, 1971, modified.) FIG. 4. Synaptic terminal on gfl fiber. (After Froesch and Martin, 1972, modified.) FIG. 5 . gfl/ gf2 synapse. (After Froesch and Martin, 1972, modified.) FIG. 6. Serial synapses in the vertical lobe. (After Young, 1971, modified.) FIG. 7. Neuromuscular junction in the lip of Sepia. (m, muscle fiber.) FIG. 8. Neuromuscularjunctions in the Octopus posterior salivary gland. m, Muscle fiber; s, satellite cell. FIG. 9. Neuromuscular junction in the Octopus posterior salivary duct. m, Muscle fiber; s, satellite cell.
propulsion (Prosser and Young, 1937; Young, 1938; Wilson, 1960; Packard and Trueman, 1974). In Loligo, three sets of neurons with giant fibers (gfl, gf2, gf3) form a chain connected by two synapses (gflfgf2 in the brain; gf2igf3, the giant synapse, in the stellate ganglion). Most of the gf2 fibers run directly to the retractor muscles of the head and funnel, while on each side a single gf2 fiber extends to the stellate
SYNAPSES OF CEPHALOPODS
5
ganglion through the pallial nerve. The outgoing stellar nerves each contain one gf3 fiber (the largest reaching nearly 1 mm in diameter in Loligoforbesi) which ramifies with the nerve and thus distributes to the mantle muscles. The thirdorder giant fibers, gf3, result from the fused processes of nerve cells located in the rind of the stellate ganglion. Before fusion, these processes are connected by the terminal branches of a giant accessory fiber originating in the brain and forming proximal synapses on the gf3 fiber. After fusion, the resulting gf3 fiber lies side by side with a gf2 branch for 1 mm, and numerous short collaterals from gf3 contact gf2, forming the distal giant synapse in that area. Other remarkable features are: 1. The large cell bodies of the two first-order multipolar neurons (first-order giant cells), the soma and dendrites of which are covered with afferent terminals 2. The protoplasmic bridge formed by the fused decussate first-order giant fibers, which is thought to allow the backward bilateral spreading of impulses even when they are generated unilaterally on one of the giant cells (Young, 1939; Martin, 1965). Although there is no fusion in the chiasma of first-order giant axons in other decapod species (Martin, 1969), other arrangements (bifurcations, crossed collaterals) with specialized contact areas are thought to serve the same function as in Loligo (Martin, 1969).
III. Cytological Features of Synapses in Cephalopod Ganglia Since the neuropil of brain and ganglia contains numerous nerve endings (Plate 11, Fig. lo), it has been used in the ultrastructural study of cephalopod synapses. Thus the giant distal synapse of squid stellate ganglia (Hama, 1962) and various synapses in the optic lobe and other regions of the brain of Octopus and Eledone (Dilly et al., 1963; Gray and Young, 1964; Gray, 1970a; Case etal., 1972) have been described. A. EXAMPLES OF octopus BRAIN SYNAPSES The basic features of cephalopod synapses can be summed up from the observations of Gray and Young (1964) on the superior frontal lobe (Plate I, Fig. 2). There the presynaptic element contains mitochondria, and both clear and densecored vesicles (about 500 hi in diameter) are aggregated along the presynaptic membrane; the synaptic cleft contains extracellular material, and the clear, spinous postsynaptic element, devoid of vesicles, shows dense material along the cytoplasmic surface of the postsynaptic membrane. Thus this single two-element asymmetrical junction resembles the classic chemical synapses of vertebrates. The plexiform zone of the optic lobe cortex contains afferent optic axons
SYNAPSES OF CEPHALOPODS
7
(coming from retinal photoreceptors) forming radially oriented cylindrical dilatations (Plate I, Fig. 3) packed with numerous synaptic vesicles (400-700 6; in diameter); most of the latter are clear, though some are dense-cored. Numerous clear postsynaptic elements invaginate into the cylindrical presynaptic bags, while dense material outlines the pre- and postsynaptic membranes (Dilly et al., 1963; Case et al., 1972). In the Octopus vertical lobe, serial synapses have been identified (Gray and Young, 1964; Gray, 1970a) and a careful comparison between light and electron microscope results has allowed the lobe’s basic circuitry to be tentatively described (Gray, 1970a). In the vertical lobe, afferent fibers from the median superior frontal lobe show varicosities that form en passant synaptic contacts with the radial trunks from small amacrine cells of the vertical lobe rind (Plate I, Fig. 6). Presynaptic varicosities contain both clear and dense-cored vesicles (200-800 6; in diameter) crowded along the presynaptic membrane together with dense material (Plate 11, Fig. 11). The synaptic cleft, about 200 6; wide, contains dense, structured material, while the postsynaptic membrane is underlined by dense material slightly more conspicuous than that under the presynaptic membrane. The amacrine trunks are packed with agranular vesicles which show a bimodal repartition of diameters, with peaks at 300 and 900 A. The amacrine trunks in turn form synapses with clear postsynaptic processes (Plate 11, Fig. 12) containing a few vesicles and microtubules which are thought to be dendritic side branches of the trunks emitted by the rind’s large cells. Within the amacrine trunks (presynaptic with respect to the branches of the large cells), the clustered vesicles facing the synaptic cleft stand out from dense, somewhat granular ground material. An ultrastructural basis is thus provided for the existence of serial synapses in the Octopus brain, whose function remains speculative, although such an arrangement might be related to presynaptic inhibition (Eccles, 1961; Katz, 1966). In the Octopus vertical lobe, the trunks of.the large cells are also contacted by another type of afferent fibers (Gray, 1970a), the “pain” fibers, which contain
PLATE11: FIG. 10. Neuropil of Octopus superior buccal lobe. Varicosities with varied vesicles, and synapses (arrows). ~ 4 2 , 0 0 0FIG. . 1 1 . Octopus vertical lobe. Synaptic contact between affer. 12. Octopus vertical lobe. Synaptic contact ent fibers (SF)and amacrine trunk (A). ~ 8 0 , 0 0 0FIG. between amacrine trunk and a spinous process (SP). X60,OOO. FIG. 13. Octopus inferior buccal lobe. Fluorescent perikarya in the rind. X200. FIG. 14. Fluorescent sympathetic nerve of Octopus. X50. Frc. 15. Fluorescent nerve fibers in the chorion of the Octopus esophagus. E, Epithelium. ~ 1 3 0 .FIG. 16. Octopus superior buccal lobe. Fluorescent perikarya in the rind. ~ 1 3 0 . FIG.17. Transverse section of Octopus posterior salivary duct. Two fluorescent secretory trunks (S) and fluorescent varicose fibers are seen in the wall. L, Lumen of the duct. X75. FIG. 18. Eledone posterior salivary gland. Tangential section of a tubule (T) showing fluorescent varicose fibers. X200.
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COLETTE DUCROS
small, clear, flattened vesicles (15&300 bi in diameter). Convergence upon processes of the large cells of inputs from diverse sources can thus occur. The complex connections observed in the vertical lobe are in keeping with its general integrative function, although more information is needed concerning both the inhibitory and excitatory roles of the diverse sets of synapses, and the possible correlation between the types of synaptic vesicles and chemical transmitters involved. Another example of serial synapses has been found in the stellate ganglion (Young, 1971; Barlow et al., 1971) and was correlated with the presence of small cells (microneurons) in part of the wall of this ganglion.
OF B. SYNAPSES
THE
SQUIDGIANTFIBERSYSTEM
Varied and interesting morphological features have been observed in this system (Hama, 1962; Castejon and Villegas, 1964; Martin, 1969; Gervasio et al., 1971; Froesch and Martin, 1972; Martin and Miledi, 1975; Martin, 1977). Most parts of the soma and dendrites of the first-order giant cells are covered with synaptic terminals, and there is a cleft about 150 bi wide between the parallel and electron-dense membranes. Most of the presynaptic elements contain numerous small, clear vesicles (the majority are 370-440 bi in diameter) and larger, dense-cored vesicles. Some synaptic terminals with large, dense-cored vesicles (800 bi in diameter) and fewer small, clear vesicles are also present (Gervasio et al., 1971). The squid first-order giant axon is also in contact with numerous afferent synaptic endings along its course to the second-order giant fiber (Plate I, Fig. 1). There, too, a synaptic cleft 150 d; wide separates specialized dense membranes. However, synaptic vesicles are seen on both sides: small, clear vesicles (300-500 A in diameter) in the presynaptic endings, and larger, clear vesicles (350-500 6; in diameter) in the postsynaptic first-order giant fiber (Plate I, Fig. 4). In addition, a few larger, dense-cored vesicles are present in both vesicle populations (Gervasio et a l . , 1971). In the chiasma of Sepia and Illex, studied by Martin (1969), the giant firstorder fibers do not fuse but become directly apposed in “window” areas devoid of intervening glial processes. In Illex, specialized contacts occur at these sites, having clear vesicles 800 d; in diameter embedded in dense material clustered on both sides of dense membranes 150 6; apart. In Sepia, the window areas show several specialized ribbon-shaped contacts where a single row of clear vesicles, 500 d; in diameter and embedded in an electron-dense substance, lies on each side of the apposed axonal membranes separated by a fairly regular, 100-di wide cleft. Hence, in the Illex and Sepia chiasma, the specialized contacts between the unfused axons do not show any structural polarity, although aggregation of
SYNAPSES OF CEPHALOPODS
9
synaptic-like vesicles does occur. On morphological grounds, these contacts can be presumed to function like symmetrical synapses, where impulses can be transmitted in either direction, and to allow bilateral symmetrical spread from unilateral impulses, as in the Squid (Martin, 1969). The synaptic contacts between first- and second-order giant fibers have been studied by Gervasio et al. (1971). Direct apposition of the two fibers occurs in areas lacking glial investment, and there two types of specialized contacts are seen (Plate I, Fig. 5). Some have dense membranes separated by a cleft more than 100 A in width and clear vesicles clustered along the presynaptic membrane; others lack vesicle aggregation and show a closer apposition of membranes which are separated by a gap less than 50 di wide. A “mixed” synapse (according to the definition of Peters et al., 1970), with the morphological characteristics of both chemical and electrical synapses, is therefore observed at the junction of these two giant fibers. Ultrastructural characteristics of the giant synapse between the second- and third-order giant fibers in the squid stellate ganglion have been described by Hama (1962), Castejon and Villegas (1964), Martin (1969), and Martin and Miledi (1975). They have been reviewed by Martin (1977). Numerous short, lateral processes from the third-order giant axon cross the glial sheath and make contact with the afferent second-order giant fiber (Young, 1939; Hama, 1962; Martin, 1969). In other species (Castejon and Villegas, 1964; Martin, 1977), other situations occur, as the lateral processes are sent from the second- to the third-order giant fiber, or from both giant fibers. In each case, process endings and the opposite element possess electron-dense membranes separated by a synaptic cleft (100-200 di wide, depending on the species observed), while numerous clear vesicles are clustered along the presynaptic membrane (400-600 A in diameter in Loligo, 500-700 hi and 500-800 hi in diameter in other species). Some larger, dense-cored vesicles are mixed with the clear ones. An electron-dense substance resembling a synaptic ribbon has been observed at the presynaptic sites of the giant synapse of Sepioteuthys (Castejbn and Villegas, 1964). In Loligo, a complex of large lamellae and associated vesicles is frequently found at the second-order presynaptic terminals (Martin and Miledi, 1975). The proximal synapses show ultrastructural features (cleft; clustered, clear, synaptic vesicles) similar to those of the giant distal synapse (Castejon and Villegas, 1964; Martin, 1977). The proximal and giant distal synapses of the squid stellate ganglion have the structural characteristics of chemical synapses, although the transmitter involved is still unknown (Miledi, 1969). Zinc iodide-osmium tetroxide (ZIO) impregnation (Maillet, 1962) of aldehyde-fixed tissue has been used to analyze further differences between populations of clear vesicles observed at different synapses of the squid giant fiber system (Martin et al., 1969; Froesch and Martin, 1972). Thus the small,
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COLE'ITE DUCROS
clear vesicles of afferent terminals on first-order giant cells and axons are ZIOpositive, while the clear vesicles of the first- and second-order giant axons are ZIO-negative. Size and ZIO reactivity of vesicles allow one better to characterize synaptic contacts and to show they are always different in two successive links of the giant fiber system (Froesch and Martin, 1972).
C. DISTRIBUTION OF DIFFERENT TYPESOF PRESYNAPTIC ELEMENTS IN Octopus BRAIN
THE
Different types of vesicles are distinguished by their size, form, and reactivity following ZIO impregnation (Martin et al., 1969). This method, together with degeneration experiments, has permitted better identification of the vesicles contained in the afferent fibers of the posterior chromatophore lobe (Froesch, 1972). Diverse types of presynaptic elements in several lobes have been classified on the basis of the major types of vesicles they contain, and the characteristic frequencies defined for each lobe (Martin et al., 1969; Barlow and Martin, 1971). The differences in the relative distribution of the presynaptic types among the lobes are of interest when compared to the regional biochemical differences in presumed neurotransmitter content (Barlow and Martin, 1971).
D. PRESUMED NEUROTRANSMITTERS IN THE CEPHALOPoD GANGLIA With few exceptions (the gflIgf2 synapse in squid brain), the synaptic junctions in cephalopods show the morphological traits of chemical synapses, and electrophysiological evidence exists for chemical transmission at the squid giant synapse (Miledi, 1967; Katz and Miledi, 1969). Biochemical data on the presence of putative neurotransmitters in nerve tissue are thus of importance in the further characterization of cephalopod synapses. 1 . Biochemical Data
A high content of acetylcholine exists in Octopus, Sepia, and squid central ganglia (Bacq, 1935; Bacq and Mazza, 1935; Feldberg et al., 1951; Florey, 1963; Loe and Florey, 1966; Florey and Winesdorfer, 1968; Heilbronn et al., 197 l), from which synaptosomal fractions rich in acetylcholine have been separated (Florey and Winesdorfer, 1968; Jones, 1970; Welsch and Dettbarn, 1972). Here, too, the presence of choline acetylase and cholinesteraseshas been demonstrated (Bacq, 1937; Nachmanson and Meyerhof, 1941; Nachmanson and Weiss, 1948; h e and Florey, 1966; Welsch and Dettbarn, 1972; Barlow, 1977). Varied amines (dopamine, histamine, N-acetylhistamine, norepinephrine, octopamine, P-phenylethylamine, serotonin, p-tyramine, rn-tyramine) have been
SYNAPSES OF CEPHALOPODS
11
found in cephalopod ganglia (Florey and Florey, 1954; Welsch and Moorhead, 1960; Bertaccini, 1961; Lorenz et al., 1973; Cottrell, 1967; Roseghini and Ramorino, 1970; Barlow, 1971; Juorio and Molinoff, 1971, 1974; Juorio and Killick, 1972a, b; Juorio and Barlow, 1973, 1974, 1976; Juorio and Philips, 1975, 1976). Depletion of the amine concentration has been observed after reserpine administration (Piccinelli, 1958; Juorio, 1971; Juorio and Molinoff, 1971, 1974; Juorio and Killick, 1972b; Juorio and Philips, 1975), while increased amine concentration occurs after the administration of monoamine oxidase (MAO) inhibitors (Juorio and Killick, 1972a; Juorio and Molinoff, 1974; Juorio and Philips, 1975, 1976). M A 0 is present in cephalopod nerve tissue (Blaschko and Hawkins, 1952; Blaschko and Himms, 1954), as well as acid metabolites resulting from amine oxidation (Juorio and Killick, 1972a; Juorio and Barlow, 1973). Synthesis of catecholamines within the Octopus central ganglia, in vivo and in vitro, has been obtained from labeled precursors and suggests that the ganglia contain the enzymes required for hydroxylation and decarboxylation processes (Juorio and Barlow, 1973). The fact that the amine content shows characteristic regional, and even interspecies, differences in the ganglia and brain lobes is of great interest (Juorio, 1971; Juorio and Killick, 1972a; Juorio and Molinoff, 1974; Juorio and Barlow, 1974, 1976). The presence of y-aminobutyric acid has also been reported in the central ganglia of Eledone (Cory and Rose, 1969).
2. Histochemical and Cytochemical Investigations Although cholinesterase activity is not specific to cholinergic nerves, the parallel distribution of acetylcholine and acetylcholinesterase in the Octopus brain ( b e and Florey, 1966) may show that the histochemical localization of the latter is a valid method for localizing the presumed cholinergic endings in the central ganglia. The results obtained with various methods (Drukker and Schade, 1964, 1967; Barlow, 1971, 1977; Chichery and Chichery, 1974; Chichery, 1976) have been reviewed recently (Barlow, 1977). Acetylcholinesterase activity is generally absent from the neuronal perikarya, restricting itself to the neuropil, that is, the area of synapses. Some interspecies differences occur in the banding pattern of the plexiform zone of the optic lobe. Marked regional differences exist in the neuropil of the brain; for example, the reaction is increased in the optic and basal lobes and decreased in the vertical lobe. Direct histochemical localization of several endogenous amines (dopamine, norepinephrine, serotonin) is possible with the formaldehyde fluorescence method of Falck and Hillarp (Falck et a l . , 1962). In the Octopus brain, fluorescent perikarya (Plate II, Fig. 16) have been demonstrated in such a way in the superior buccal lobe (Martin and Barlow, 1972; Matus, 1973) that sends out fluorescent nerves (Plate 11, Fig. 17) to the posterior salivary gland. The localiza-
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tion of formaldehyde fluorescence in the neuropil and nerve fiber tracts of the supraesophageal ganglia correlates well with the known regional quantitative distribution of amines in these lobes (Matus, 1973) and suggests the presence of serotonin in the spine of the peduncle lobe (Matus, 1973). Fluorescence studies on other ganglia of Octopus and Eledone (Ducros and Arluison, 1977) show tangled varicose monoamine fibers in the neuropil of all ganglia examined (subradular, inferior buccal, gastric, stellate). In the stellate ganglion the fluorescent pattern of the neuropil correlates with the histological subdivision of this ganglion into two parts (Young, 1972); in addition, changes observed in the fluorescence after severing stellar or pallial nerves could be explained by the presence of peripheral aminergic fibers in the former (Ducros and Arluison, 1977). Fluorescent perikarya are present in the rind of the inferior buccal ganglion (Plate 11,Fig. 13) which sends out strongly fluorescent sympathetic nerves (Plate 11, Fig. 14) to the esophagus. Monoamine fluorescence in perikarya, neuropils, and outgrowing nerves of superior and inferior buccal ganglia is of particular interest, as the high monoamine content of these ganglia is well documented (Juorio, 1971; Juorio and Barlow, 1976). Selective uptake of labeled exogenous amines by aminergic nerve fibers is a valuable tool in localizing presumed aminergic nerve endings by autoradiotography. However, care must be taken that the specificity of uptake is closely related to the amine concentration used (Iversen, 1965, 1967; Shaskan and Snyder, 1970; Descarries and Lapierre, 1973; Descarries et al., 1975; Taxi, 1976; Calas and Segu, 1976). Furthermore, selective uptake shows oniy a property of cells or cell processes and does not indicate that they in fact contain amine stores. Comparison of the results of both formaldehyde fluorescence and autoradiography is therefore necessary to ensure the localization of aminergic endings. An autoradiographic study of theuptake of tritiated serotonin and norepinephrine in some lobes of the Octopus brain has been carried out (Martin and Barlow, 1972, 1977); in the superior buccal lobe, serotonin uptake is observed in some neuronal perikarya as well as in some large varicosities of the neuropil; the labeled varicosities contain numerous dense-cored vesicles and reticulum-like profiles; however, in the same lobe, norepinephrine labeling occurs on glial cells of both the rind and neuropil, and its distribution is unrelated to that of the monoamine-specific fluorescence. Light and electron microscope autoradiographic studies on the uptake of several tritiated catecholamines (dopamine, norepinephrine, octopamine, and tyramine at lop5 M) in all the ganglia of Eledone cirrhosa and Octopus vulgaris, where significant amounts of these amines have been demonstrated biochemically, have led to similar results (Ducros, unpublished observations); catecholamine labeling is mainly restricted to glial cells, to the endothelium, and even to the muscles of blood vessels and does not correspond to the localization of endogenous amines revealed by the formaldehyde
SYNAPSES OF CEPHALOPODS
13
fluorescence method; some experiments with lower amine concentrations (norepinephrineand dopamine at lo-' M ;C. Ducros, unpublished observations) were canied out and the ganglia processed for light microscope autoradiography; label retention nearly disappeared in nonneuronal cells, but nerve cell bodies and processes were no longer clearly labeled. The autoradiographic results of catecholamine labeling are very disappointing compared to those of serotonin. It may be thought that, at least in some species and organs of cephalopods, catecholamines are not sufficiently bound to macromolecules to be preserved by conventional fixation methods. An alternative explanation might be the absence of selective uptake into cephalopod catecholaminergicnerve terminals. Yet, glial cells in Octopus ganglia have special properties, since they can accumulate both norepinephrine and 6-hydroxydopamine (Martin and Barlow, 1977). Moreover, after 6-hydroxydopamine administration, the Octopus brain shows degeneration of glial cells, while no changes are observed in neurons, except in the plexiform zone of the optic lobe (Martin and Barlow, 1977).
IV. Special Features in Peripheral Afferent Pathways Most cephalopod receptors give off axons, hence are primary receptors, just like those of the suckers (Graziadei, 1962, 1964; Graziadei and Gagne, 1976b)or the retinal photoreceptors. Ultrastructural studies on the retinal plexus (Tonosaki, 1965; Gray, 1970b) have shown one type of nerve ending belonging to retinal cell collaterals and another belonging to efferent fibers. The two types of nerve terminals have synaptic membrane specializations and associated vesicle clusters (Gray, 1970b). The synapses of the retinal cell collaterals contain small, clear vesicles (300-500 6; in diameter), while those of efferent fibers have large, dense-cored vesicles (loo0 b; in diameter) mixed with clear ones. Synthesis of acetylcholine occurs in the retina of several cephalopods (Lam et al., 1974). The turnover of acetylcholine is greater in the retina and optic lobes (i.e., in areas containing terminals) than in the optic nerves (Barlow, 1977). The Octopus retina contains dopamine and serotonin (Jucrio and Killick, 1973) which, along with acetylcholine, may be involved in afferent or efferent pathways of the eyes. Unusual connections between primary receptors of the sucker and basal interneurons or underlying encapsulated nerve cells have been observed by Graziadei (1962, 1965a) and Graziadei and Gagne ( 1976b). These connections probably allow a drastic reduction in input between the numerous primary receptors of the epithelium and ganglia of the arm.Synaptic contacts with membrane specializations occur on both the soma and dendritic processes of encapsulated nerve cells and form axosomatic and axodendritic synapses. The presynaptic axon terminals contain vesicles 200-800 A in diameter, some of which are dense-cored (Graziadei, 1965a).
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Secondary receptors are thought to exist in the statocyst, for several sensory hair cells are without axons and have afferent synapses at their bases (Barber, 1966; Budelmann, 1977).
V. Nerve Endings in Cephalopod Effectors
A. ULTRASTRUCTURAL OBSERVATIONS The basic characteristics of the neuromuscular junctions have been traced by Graziadei (1965b, 1966), and Barber and Graziadei (1966, 1967) in the muscles of Octopus suckers, of the lips of Sepia, and of the wall of the Octopus cephalic aorta; the axons join the muscle fiber and often become invaginated into it, into a funnel that is still connected to the outer muscle surface by a mesaxon-like structure (Plate I, Fig. 7). The glial sheath of axons disappears as they run into the muscle fiber. Thickening of both pre- and postsynaptic membranes is apparent at the synaptic junctions; a well-defined synaptic cleft contains a dense, structured material. Clear vesicles aggregate along the presynaptic membrane which also shows dense projections. A single nerve fiber can contact two muscle fibers and, conversely, several synaptic endings can occur on a single muscle fiber. The surface of the muscle fibers does not expose any preferred areas for synaptic junctions. Ultrastructural observations have been made on the nerve fibers of the chromatophore muscle (Cloney and Florey, 1968; Florey, 1969; Weber, 1970), and the earlier observations of Hofmann (1907) on the pattern of innervation have been corroborated (Cloney and Florey, 1968). The nerve bundles of the chromatophore muscles contain many axons with small, clear vesicles (300 bi in diameter) and occasional axons with dense-cored vesicles (600 bi in diameter); synaptic junctions are characterized by the usual membrane specializations, and clumping of clear vesicles occurs along the presynaptic membranes (Weber, 1970). The occurrence of neuromuscular junctions has been demonstrated in the PLATE111: FIG. 19. Octopus posterior salivary duct. Neuromuscular junction on lamellar interlocking (arrow) of two muscle fibers (M). C, Collagenous fibers; A, axon. X50,OOO. FIG.20. Eledone posterior salivary gland. Synaptic contact, with enlarged cleft (arrows), between axon (A) and glandular cell. X68,OOO. FIG. 21. Octopus posterior salivary duct incubated for 20 minutes with M tritiated serotonin. A nerve bundle, in oblique section, showing labeled and unlabeled nerve fibers. A, Axons; C, collagenous fibers. X30,OOO. FIG. 22. Octopus posterior M tritiated sertonin. A heavily labeled fiber salivary duct incubated for 20 minutes with contacts the muscle (M)next to another fiber (A) with no more labeling than the background. Arrow shows lamellar interlocking between muscle fibers. C, Collagenous fibers. X60,OOO.
SYNAPSES OF CEPHALOPODS
15
16
COLEmE DUCROS
annular muscle fibers surrounding the tubules of the Octopus posterior salivary gland (Matus, 1971b; Ducros, 1972a) and in the muscles of the posterior salivary duct (Ducros, 1972a). As bundles of nerve fibers join the muscle, the intervening glial sheath disappears between the closely apposed axons and muscle; on the opposite side, axons are capped with a glial cell or a lamella projecting from the muscle fiber (Plate I, Fig. 8). Mesaxon-like processes from the outer muscle surface are uncommon therein. Presynaptic axons contain both clear and densecored vesicles; small, clear vesicles are clumped near the presynaptic membrane that is separated from the muscle by a cleft (200 A) wider than that in the next appositional zones. Synaptic contacts on the muscle of the posterior salivary duct are often observed close to interpenetrating lamellae from two neighboring muscle fibers (Plate I, Fig. 9, and Plate 111, Fig. 19; Ducros, 1972a). Although this interlocking seems to fulfill a mechanical function, it cannot be excluded that here the nerve terminal acts simultaneously upon two muscle fibers. Nerve sections with varied vesicles but without synaptic differentiations are often seen to contact the muscle of the posterior salivary duct. On purely morphological grounds, it cannot be said whether they are preterminal varicosities or whether they constitute a separate set of nerve fibers. The glandular tissue of the posterior salivary gland contains numerous nerve fibers (Matus, 1971b; Ducros, 1971a, 1972b; Martin and Barlow, 1972) running into mesaxon-like processes provided by the membranes of epithelial cells that line the glandular tubules. Neuroglandularjunctions (Ducros, 1972b) are characterized by small specialized areas with an enlarged synaptic cleft, thickening of pre- and postsynaptic membranes, and accumulation of small, clear vesicles along the presynaptic membrane (Plate Lu, Fig. 20). The presynaptic elements also contain larger, clear, dense-cored vesicles. Although there are few ultrastructural data compared to the number of effectors still unexplored, the known instances of nerve effector synapses of cephalopods show the morphological characteristics of chemical synapses.
B. BIOCHEMICAL AND CYTOCHEMICAL DATA Recent biochemical findings related to the presence of presumed neurotransmitters in peripheral organs are limited. Neither catecholamines nor serotonin were detected by Juorio and Killick (1973) in the skin, branchial and systemic hearts, stomach, and cecum of Octopus; yet they found dopamine in the intestine, and dopamine and norepinephrine in the esophagus and crop. In the esophagus, the fluorescence method reveals strongly fluorescent sympathetic nerves (Plate 11, Fig. 14) and plexuses of fluorescent fibers (Plate 11, Fig. 15) in the muscular coat and in the chorion (Ducros and Arluison, 1977). Consequently,
SYNAPSES OF CEPHALOPODS
17
aminergic nerve fibers exist in the esophagus and probably supply the muscle fibers; doparnine and/or norepinephrine are the presumed neurotransmitters. Large amounts of varied amines have long been known to exist in the octopod posterior salivary glands (Erspamer, 1952; Erspamer and Boretti, 1951); recent studies have corroborated the unusual concentrations of serotonin, octopamine, and tyramine in these organs (Juorio and Molinoff, 1971; Juorio and Killick, 1973; Juorio and Philips, 1975), which are mainly due to the presence of nonneuronal chromaffin and argentaffin cells (Verne, 1922; Lison, 1931; Vialli and Erspamer, 1938; Vialli, 1946; Ducros, 1971b; Matus, 1971a). Norepinephrine and serotonin are present in the posterior salivary duct and accumulate proximal to the ligation of the duct (Barlow et al., 1974). The formaldehyde fluorescence method has shown fluorescent aminergic fibers (Plate 11, Fig. 18) in the posterior salivary glands of Octopus and Eledone (Matus, 1971a), which are thought to be the nerve fibers supplying the glandular tubules (Martin and Barlow, 1972; Arluison and Ducros, 1976). The aminergic supply to the gland is provided by the branching of secretory nerves which originate in the superior buccal lobe of the brain. In the posterior salivary trunk, these secretory trunks show strong fluorescence (Plate 11, Fig. 17) which contrasts with that of neighboring motor trunks which contain only a few aminergic fibers; numerous fluorescent aminergic fibers (Plate 11, Fig. 17) are distributed within the wall of the duct (Arluison and Ducros, 1976). Thus aminergic nerve fibers exist in both duct and gland, and norepinephrine and/or serotonin may be involved in neurotransmission. The distribution of norepinephrine labeling in the Octopus posterior salivary duct and gland is completely unrelated to that of the formaldehyde fluorescence (C. Ducros, unpublished observations) and raises problems similar to those mentioned for exogenous catecholamine uptake in the ganglia. Degeneration of duct and gland nerves has not been observed after 6-hydroxydopamine administration (Martin and Barlow, 1975). Serotonin incorporation has been obtained in the posterior salivary duct and gland and studied with electron microscope autoradiography (Martin and Barlow, 1972; Ducros, 1975); serotonin-labeled fibers are seen in the glandular tubules (Martin and Barlow, 1972) where they coexist with unlabeled nerve fibers (Ducros, 1975). In the wall of the duct, bundles containing both labeled and unlabeled fibers (Plate 111, Fig. 21) are distributed among the muscles; the direct contact of labeled nerve fibers and muscle fibers (Plate 111, Fig. 22) without synaptic specializations has been observed (Ducros, 1975); last, the nerve fibers connected to the muscle by welldefined synapses were never labeled (Ducros, 1975). Thus some evidence exists for a heterogeneous supply to both glandular tubules and duct muscles. Dual innervation is possible, as in the case of the chromatophore muscles where the antagonistic effects of acetylcholine and serotonin upon the frequency and amplitude of miniature potentials have been demonstrated (Florey and Kriebel, 1969).
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VI. Conclusions As the anatomy of the nervous system of cephalopods is by far the best known among the invertebrates, owing to the extensive studies of Young and co-workers (Sereni and Young, 1932; Young, 1936b, 1939; Boycott, 1953, 1961; Boycott and Young, 1956; Young, 1960b, 1962, 1965a, 1967; Stephens and Young, 1969; Young, 1971; Barlow et al., 1971; Young, 1972; Hobbs and Young, 1973; Young, 1974, 1976, 1977a, b), a firm basis exists for further investigations. There is much to be learned about the circuitry of the diverse ganglia. Ultrastructural studies are therefore helpful in the localization and morphological characterization of synaptic endings and in the observation of changes following degeneration experiments. However, correct fixation of ganglia is often very difficult, as noted by Gray (1970a), and a good knowledge of the histological results obtained after selective staining is necessary in any attempt to analyze the intricate pattern of neuropil fibers. Moreover, the observation of serial sections would be helpful in obtaining a better understanding of the spatial arrangement of synapses, for example, in determining if varicosities rich in vesicles all show synaptic junctions with other fibers. Precise identification of the connections between elementary components in the central ganglia is closely related to study of the functional capacities of the brain and other ganglia. Both anatomical and behavioral studies are more advanced in the Octopus, because of rearing facilities, learning ability, and a high percentage of survival after surgical experiments. The sustained efforts and varied approaches of many skillful workers have already provided enough information to construct unitary explanations (Young, 1964, 1966), which in turn will stimulate further analysis of the neural basis of cephalopod brain functioning. In this investigation the Octopus brain is the best suited for study of the neural organization underlying memory capacity, the understanding of which would be of great general value. Another field of general interest involving cephalopod nerve tissue concerns the molecular events associated with nerve impulse conduction and transmission. The giant fiber system that has evolved in decapods for fast-escape responses has greatly contributed to the general knowledge of nerve functioning, and its value will increase with the identification of the neurotransmitters involved. Chemical transmission is dominant in cephalopod synapses, and accumulating biochemical information has shown that many of the presumed neurotransmitters are the same in cephalopod and in vertebrate nerve tissue, often with the advantage of a higher concentration in cephalopod ganglia. Other features that can be used for further analytical studies are the regional differences between ganglia and brain lobes and also the biochemical differences between species. Although considerable physiological and pharmacological evidence must be provided for the true iden-
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19
tification of neurotransmitters, histochemical and cytochemical results can offer valuable information on the actual neuronal localization of the putative neurotransmitters. Correlated morphological, histochemical, and cytochemical methods can also be used for better characterization of cephalopod synapses and to obtain a better understanding of some neuronal pathways. Although each of these methods has its own limitations and difficulties when applied to cephalopod nerve tissue, in general the first results obtained have not been disappointing. The immediate purpose of these methods might be to point out which organs, belonging to which cephalopod species, can serve as models in determination of the nature and role of neurotransmitters.
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Iversen, L. L. (1967). “The Uptake and Storage of Noradrenaline in Sympathetic Nerves.’’ Cambridge Univ. Press, London and New York. Jones, D. G. (1970). Brain Res. 17, 181-193. Juorio, A. V. (1971). J. Physiol. (London) 216, 21S226. Juorio, A. V., and Barlow, J. J. (1973). Experientia 29, 943-944. Juorio, A. V., and Barlow, J. I. (1974). Comp. Gen. Pharmacol. 5, 281-284. Juono, A. V., and Barlow, J . J. (1976). Brain Res. 104, 379-383. Juorio, A. V., and Killick, S. W.(1972a). Comp. Gen. Pharmacol. 3, 283-295. Juorio, A. V., and Killick, S. W. (1972b). Int. J. Neurosci. 4, 195-202. Juorio, A. V., and Killick, S. W.(1973). Comp. Biochem. Physiol. A 44, 1059-1067. Juorio, A. V., and Molinoff, P. B. (1971). Br. J . Pharmacot. 43, 438-439. Juorio, A. V., and Molinoff, P. B. (1974). J. Neurochern. 22, 271-280. Juorio, A. V., and Philips, S. R. (1975). Brain Res. 83, 180-184. Juorio, A. V., and Philips, S. R. (1976). Neurochem. Res. 82, 501-509. Katz, B. (1966). “Nerve, Muscle and Synapse.” McGraw-Hill, New York. Katz, B., and Miledi, R. (1969). J. Physiol. (London) 203, 459-487. Lam, D. K., Wiesel, T. N., and Kaneko, A. (1974). Brain Res. 82, 365-368. Lison, L. (1931). Arch. Biot. 41, 344436. Loe,P. R., and Florey, E. (1966). Comp. Biochem. Physiol. 17, 509-522. Lorenz, W., Matejka, E., Schmal, A., Seidel, W., Reimann, H. J., Uhlig, R., and Mann, G. (1973). Comp. Gen. Pharmacol. 4 , 229-250. Maillet, M. (1962). Trab. Inst. Cajal Invest. Biol. 54, 1-36. Martin, R. (1965). 2. Zellforsch. Mikrosk. Anat. 67, 77-85. Martin, R. (1969). Z. Zellforsch. Mikrosk. Anat. 97, 50-68. Martin, R. (1977). Symp. Zoof. Soc. London 38, 261-275. Martin, R., and Barlow, J . J. (1972). 2. Zellforsch. Mikrosk. Anat. 125, 16-30, Martin, R., and Barlow, J. J. (1975). J. Ultrastrucr. Res. 52, 167-178. Martin, R., and Barlow, J. J. (1977). Proc. R. Soc. London, Ser. B 196, 431-441. Martin, R., and Miledi, R. (1975). J. Neurocytol. 4, 121-129. Martin, R., Barlow, J., and Miralto, A. (1969). Brain Res. 15, 1-16. Matus, A. I. (1971a). Tissue & Cell 3, 389-394. Matus, A. I. (1971b). Z. Zellforsch. Mikrosk. Anat. 122, 111-121. Matus, A. I. (1973). Tissue & Cell 5, 591-601. Mauro, A. (1977). Symp. 2001.SOC.London 38, 287-308. Moody, M.F., and Robertson, 1. D. (1960). J. Biophys. Biochem. Cytol. 7 , 87-91. Miledi, R. (1967). J. Physiol. (London)192, 379-406. Miledi, R. (1969). Nature (London) 223, 1284-1286. Nachmanson, D., and Meyerhof, B. (1941). J. Neurophysiol. 4, 348-361. Nachmanson, D., and Weiss, M. S. (1948). J. B i d . Chem. 172, 677-687. Nishioka, R. S., Hagadorn, I. R., and Bern, H. A. (1962). Z . Zellforsch. Mikrosk. Anat. 57, 406-42 1. Nishioka, R. S., Yasumasu, I., Packard, A., Bern, H. A., and Young, J. Z. (1966). 2. Zellforsch. Mikrosk. Anat. 75, 301-316. Packard, A.. andTrueman, E. R. (1974). J. Exp. Biol. 61, 411-419. Perrelet, A., and Mauro, A. (1972). Brain Res. 37, 161-171. Peters, A,, Palay, S. L.,and Webster, H.F. (1970). “The Fine Structure of the Nervous System.” Harper, New York. Piccinelli, D. (1958). Arch. Int. Pharmacodyn. Ther. 117, 452-458. Prosser, C. L., and Young, J. Z. (1937). Biot. Butl. (Woods Hote, Mass.) 73, 237-241. Ram6n y Cajal, S. R. (1917). Trab. Lab. Invest. Biol. Univ. Madrid 15, 1-32.
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Roseghini, M., and Ramorino, L. M. (1970). J. Neurochem. 17, 48P-492. Rossi, F., and Graziadei, P. (1958). Acta Anat. 34,Suppl. 32, 1-79. Santi, P. A., and Graziadei, P. (1975). Tissue & Cell 7, 68S702. Sereni, E. (1930). Biol. Bull. (Woods Hole, Mass.) 59, 247-268. Sereni, E., and Young, J. Z. (1932). Pubbl. Sin. Zool. Napoli 12, 17S208. Shaskan, E. G. and Snyder, S. M. (1970). J . Pharmacol. Exp. Ther. 175, 404-418. Stephens, P. R. (1971). In ”The Anatomy of the Nervous System of Octopus vulgaris” ( J . Z. Young, ed.), pp. 646649. Oxford Univ. Press (Clarendon), London and New York. Stephens, P. R., and Young, J. Z. (1969). Philos. Trans. R. SOC.London, Ser. B 255, 1-12. Taxi, J. (1976). Bioi. Cell. 27, 243-248. Tonosaki, A. (1965). Z. Zellforsch. Mikrosk. Anat. 67, 521-532. Van Weel, P. B., and Thore, S. (1936). Z. Vergl. Physiol. 23, 2633. Verne, J. (1922). C. R. Assoc. Anat. 17, 309-320. Vialti, M. (1946). Boll. SOC.Ital. Biol. Sper. 22, 107-108. Vialli, M., and Erspamer, V. (1938). Mikrochemie, 24, 253-261. Weber, W. (1970). Z. Zellforsch. Mikrosk. Anat. 108, 446-456. Wells, M. J. (1962). “Brain and Behaviour in Cephalopods.” Stanford Univ. Press, Stanford, California. Welsch, F., and Dettbam, W. D. (1972). Brain Res. 39, 467-482. Welsch, J. H., and Moorhead, M. (1960). J. Neurochem. 6, 146-169. Wilson, D. M. (1960). J. Exp. Biol. 37, 57-72. Young, J. Z. (1929). Boll. SOC.Ital. Biol. Sper. 4, 741-744. Young, J. Z. (1934). J. Physiol. (London) 83, 27P-28P. Young, J. 2. (1936a). Proc. R. SOC.London, Ser. B 121, 31P-337. Young, J. Z. (1936b). Q. J. Microsc. Sci. 78, 367-386. Young, J. Z. (1938). J . Exp. Biol. 15, 17C185. Young, I. Z. (1939). Philos. Trans. R. SOC. London, Ser. B 229, 465-503. Young, J. Z. (1960a). Proc. R. SOC.London, Ser. B 152, 3-29. Young, J. Z. (1960b). Nature (London) 186, 836839. Young, J. Z. (1962). Philos. Trbns. R. SOC.London, Ser. B 245, 1-18. Young, J . Z. (1964). “A Model of the Brain.” Oxford Univ. Press (Clarendon), London and New York. Young, J. Z. (1965a). Philos. Trans. R. SOC.London, Ser. B 249, 27-44. Young, J. Z. (1965b). Philos. Trans. R. Soc. London, Ser. B 249, 45-67. Young, J. Z. (1965~).J . Exp. Biol. 43, 581-593. Young, J. Z. (1966). “The Memory System of the Brain.” Univ. of California Press, Berkeley. Young, J. Z. (1967). Philos. Trans. R. SOC.London, Ser. B 253, 1-22. Young, J. Z., ed. (1971). “The Anatomy of the Nervous System of Octopus vulgaris.” Oxford Univ. Press (Clarendon), London and New York. Young, J. Z. (1972). Philos. Trans. R. SOC.London, Ser. B 263,40%429. Young, J. Z. (1974). Philos. Trans. R. SOC.London, Ser. B 267, 26>302. Young, J. Z. (1976). Philos. Trans. R. SOC.London, Ser. B 274, 101-167. Young, J. Z. (1977a). Philos. Trans. R. Soc. London, Ser. B 274, 101-167. Young, J. Z. (1977b). Symp. Zool. SOC.London 38, 377-434.
l”AT1ONAL
REVIEW OF CYTOLOGY, VOL. 56
Scanning Electron Microscope Studies on the Development of the Nervous System in Vivo and in Vitro K. MELLER Ruhr-Universitat Bochum, Institut fitr Anatomie, Arbeitsgruppe f i r Experimentelle Cytologie, Bochum, Federal Republic of Germany
. . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Development of the Cerebral Cortex . . . . . . . . . .
I. Introduction
11. Development of the Nervous System in Vivo
B. Development of the Retina . . . . . . . . . 111. Development of the Nervous System in Vitro . . . . The Morphology of Brain and Retinal Cell Cultures IV. Conclusions . . . . . . . . . . . . . . . . . The Columnar Organization . . . . . . . . . References . . . . . . . . . . . . . . . . .
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I. Introduction The neurons and glial cells of the nervous system originate in a zone of neuroepithelial precursor cells lying close to the ventricular surface. This proliferative zone is characterized by the presence of numerous mitotic figures (Watterson et al., 1956; Sauer and Walker, 1959; Kallen, 1962; Sidman and Angevine, 1962; Fujita, 1963, 1965). The daughter cells of the germinative, matrix, or ventricular cells migrate variable distances toward the periphery of the nervous system before becoming localized in determined regions to form specific neuron centers, for example, the brain cortex. These two phases of neurogenesis, proliferation and migration, represent early stages of development in the nervous system and have been the subject of classic light microscope, particularly Golgi, studies (Morest, 1968; 1969a,b, 1970a,b). During the past two decades histoautoradiographic (Sidman et al., 1959; Sidman, 1961; Smart, 1961; Angevine and Sidman, 1961; Altman, 1962a,b, 1963; 1966a,b, 1967; Fujita, 1964, 1965, 1966; Altman and Das, 1965a,b; Berry and Rogers, 1965; Fujita et al., 1966; Hinds, 1968) and transmission electron microscope (TEM) (Bellairs, 1959; Tennyson, 1962, 1970; Fujita and Fujita, 1963; Pappas and Purpura, 1964; Meller et aE., 1966; Wechsler and Meller, 1967; Hinds and Ruffett, 1971) investigations 23
Copynght 0 1979 by Academic Press, Inc All nghts of reproduction In any form reserved
ISBN 0-12-361356-2
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have contributed extensive data on these processes. An abundance of data and observations has been compiled in numerous reviews (Langman, 1968; Angevine, 1970; Sidman, 1970, 1974; Sidman and Rakic, 1973; Berry, 1974). Proliferation of the germinal cells gives rise to three principal cell types: (1) macroneurons (e.g., Purkinje cells, pyramidal cells of the cortex, ganglion cells of the retina), (2) glial cells, and (3) microneurons (Diagram I). In many verte-
DIAGRAM I. Schematic representation of neurogenesis. (1) A population of undifferentiated rnatrix cells divides to give rise to all cell types that form the nervous system. (2) The daughter cells migrate in different waves from the ventricular zone to the periphery. The macroneurons (M) are the first cell population to migrate. (3) Glia cells (G)originate from subventricular cells called glioblasts. (4) The migration of microneurons (m) continues into the postnatal period.
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Diagram I1 illustrates examples of the different modes of migration in the cortex plate, retina, and cerebellum. All ventricular cells, when examined with the Golgi method or the TEM, have similar morphological features. This observation, however, cannot be regarded as support for the hypothesis that these cells possess the same potentialities. Golgi impregnations reveal the mono- or bipolar shape of the matrix cells. They possess an external or basal process which terminates beneath the pia or, as in the retina, near the vitreal surface, and an internal one which extends to the free surface of the embryonic ventricle where the adjacent cells are connected by terminal bars. These cells show ultrastructural features such as poorly developed endoplasmic reticulum and abundant free ribosomes, which are also common to other embryonic cells (Meller et al., 1966; Meller, 1968; Tennyson, 1970). A to-and-fro movement of the nuclei of the ventricular cells has been reported during the mitotic cycle. Prior to mitosis the cell rounds up, and its nucleus approaches the ventricular surface. Subsequent to mitosis, at the ventricular surface, the daughter cell nuclei move outward and the cells recover their elongated shape. Large translocation movements occur during the migration phase. The structure and intercellular relationships of such migrating cells have been the subject of several light and electron microscope investigations. The interpretations of the observations, however, differ to such an extent that several different theories as to the mechanisms of migration have been proposed. According to Morest (1970b), the neurons do not migrate, they simply translocate their nuclei from the ventricular zone outward in the cylindrically shaped cytoplasm and then detach their prolongations from the inner or outer surfaces of the brain wall. Rakic (1972) claims the existence of guiding structures, glial fibers, along which the cells migrate. These contradictory interpretations have their origin in the difficulties in obtaining a three-dimensional representation of the nervous structures imposed by conventional serial sectioning techniques. The scanning electron microscope (SEM) allows us to approach this problem in a new way. Particularly in the field of biology, the SEM has proved to be a valuable tool in the examination of free surfaces. The ventricular surface of the nervous system is a convenient subject for SEM observation, and in this case the preservation of topographical relationships is optimal (Scott et al., 1974). FIGS. 1-3. Sagittal fracture of the frontal region of the cerebral hemisphere of a 10-day mouse embryo. FIG. 1. The wall of the cerebral vesicle is composed of a pseudostratified epithelium covered by early pial connective tissue. v, Lateral ventricle of the cerebral vesicle. FIG.2. A portion of Fig. 1. The arrow marks a round mitotic cell at the ventricular surface. v, Lateral ventricle. FIG.3. A portion of Fig. I . High magnification of the neuroepithelium.End-feet-like prolongations of the cerebral wall. Numerous fibrils of the pial connective tissue insert on the basal lamina which covers these prolongations (arrows).
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By fracturing the cerebral vesicles, the retina, or the neural tube of an embryo, it is possible to expose and scan large areas of neural tissue. This allows observation of the arrangement of the neuroepithelial cells and of the migration of the daughter cells. In this regard, the SEM data provide information which fills the gap between the classic Golgi work and the fine-structural data supplied by transmission electron microscopy.
11. Development of the Nervous System in Vivo A. DEVELOPMENT OF THE CEREBRAL CORTEX
1. The Neuroepithelium
A saggital fracture of the brain of 10- to 12-day mouse embryos exposes a large portion of the hemisphere wall. All cells of the neuroepithelium extend from the inner (ventricular) to the outer (pial) surface of the brain. These ventricular cells have an irregular cylindrical shape, depending on the variable position of the cell nucleus. Their morphological features are, however, very similar (Figs. 1-3). His (1904) postulated that neuroblasts originate from germinal cells and migrate to the embryonic cortex. The frontal region of the hemisphere in Fig. 7 contains closely packed cells and shows an advanced stage of development. Many cell types with diverse morphological features can be identified. However, the old problem of identifying cell types in the developing nervous system has not yet been satisfactorily solved. The classically described cell types (His, 1904; Ramon y Cajal, 1960), the spongioblast, the germinal cell, and the neuroblast, cannot be distinguished infallibly on the basis of nuclear or cytoplasmic characteristics. Furthermore, neuroblasts in the early stages of development cannot be impregnated with the aid of the Golgi technique. As a consequence of these limitations, the description and identification of embryonic nervous cells are influenced by subjective and speculative interpretations. The introduction of the SEM technique in the study of the early stages of development of the neuroepithelium merely provides an approximate or simplified solution to this problem. Hattori and Fujita (1974) FIGS.4-6. Sagittal fracture of the parietwccipital region of the cerebral hemisphere of a 10-day embryo. FIG. 4. The neuroepithelium consists of a layer of cylindrically shaped cells. Mitotic cells reveal the characteristic round-to-ovoid form and are situated at the ventricular surface (arrow). FIG. 5 . The inner or ventricular part of the neuroepithelial cells. The ventricular surface consists of a mosaic of irregular spherical cell apexes which protrude into the ventricle. FIG. 6. Basal portion of the neuroepithelium. Interdigitations of cellular processes protrude into an enlarged extracellular space or channel system beneath the pia (arrows).
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described as matrix cells those cells which have a spindle-shaped soma and whose inner processes always reach the ventricular surface. Another cell type, probably the neuroblast, is bipolar and has a thin outer process and a short, thin inner process that does not reach the ventricular surface. Seymour and Berry (1975), who also used the SEM, described in great detail the appearance of mitotic neuroepithelial cells. These investigators recognized three classes of differently shaped mitotic cells: (1) pyriform cells, with very long, fine, basal processes, probably in prophase or prometaphase, (2) conical cells with many microvilli, considered to be in prometaphase and early anaphase, and (3) globular cells without microvilli, representing cells in metaphase and late anaphase (Figs. 2 and 4). Obviously, the SEM technique allows a clear distinction of all of the cell shapes which are images of the cell membranes “frozen” at a particular moment. Thus changes in the cell surface which occur during movement, such as translocation of the nucleus in matrix cells, migration of daughter cells after mitosis, and growth and retraction of prolongations of pre- and postmitotic cells can be studied. The existence of ruffling membranes and the ameboid movement of cell processes during the entire developmental period of the matrix cells probably give a single type of cell a varying morphological appearance. It follows that characterization of germinative and daughter cells by morphological criteria alone is impossible. Matrix cells and cells emigrating from the ventricular zone are morphologically very similar. Three cell types found in the pseudostratified neuroepithelium have been described (Meller and Tetzlaff, 1976): 1. Elongated cells, probably glioependymal cells. Their processes span the whole extent of the brain wall. Their nuclei are frequently positioned near the ventricular zone. The surfaces of these cells are irregular and bear pseudopodia which, in later stages, become large and interdigitate with adjacent cells (Figs. 5 and 6). 2 . The second type is also bipolar but in contact only with the ventricular surface. 3. The third type of cell is in contact only with the external pial surface. The distal prolongation of the first and third cell types (Figs. 3 and 6) form the external pial surface of the nervous system. Consequently, it is very probable that these elements are glial in nature. Type 2 are matrix cells or young premigratory neurons (Fig. 7). The ventricular surface of the cerebral vesicles shows a mosaic-like arrangement of cell surfaces whose protrusions extend into the ventricle. It is very difficult to identify these protrusions as cilia or microvilli with the SEM. These ventricular surfaces show regional differences in later developmental stages, but they have not yet been investigated in detail. For the regional changes occurring during maturation of the ependymal surfaces of the third ventricle, the report on hypothalamus development by Mestres (1976) may be consulted.
FIG. 7. Fracture of the frontal region of the cerebral hemisphere of a 10-day mouse embryo. Most of the cells display varying elongated shapes, depending on the position of the nucleus. V , Lateral ventricle.
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The external or pial surface of the cerebral vesicles is covered by a basal lamina, and consequently the cell boundaries are unrecognizable. In an earlier study (Meller and Tetzlaff, 1975) we reported that, beneath the pial surface, the cells have fine prolongations which interdigitate with those of adjacent cells. As a result, a complicated extracellular channel system is formed under the pial surface. This organization of the marginal zone has been found in cerebral vesicles but cannot be seen in the retina or spinal cord (Figs. 4 and 6).
2 . The Process of Migration Studies on the processes of cell migration were conducted in the 1960s with the aid of histoautoradiography. The injection of thymidinePH and subsequent histoautoradiographic analysis of animals surviving for different periods of time provide evidence of cell migration during the histogenesis of the cortex. Cerebral cortex cells migrate pre- and postnatally, and their migratory patterns are characteristic for each species. This cell migration from the ventricular zone to the early cortex occurs in successive waves and continues into the postnatal period (Angevine and Sidman, 1961; Smart, 1961; Smart and Leblond, 1961; Berry and Rogers, 1965; Shimada and Langman, 1970; Butler and Caley , 1972). Migrating cells move through the intermediate zone to the cortical plate, and it can be assumed that they must pass many obstacles before they reach their final destination. Figures 8 and 10 show the morphological features in the early phase of migration. As a consequence of active cell proliferation, the wall of the cerebral vesicle increases in thickness. We can distinguish between a ventricular, a subventricular, and a small peripheral or marginal zone. The most important morphological feature is that all the cells, both the matrix and the migrating cells, are arranged in columns. Lorente de NO (1949) first suggested an elementary unit of neocortical organization based on vertical chains of neurons. The investigations of Hubel and Wiesel (1962) demonstrated that a functional columnar organization exists, and Colonnier (1966) reaffirmed the concept that the basic structural units are radially oriented columns. We have shown that during embryogenesis the basic columnar unit is a constant component in the organization of the total nervous system. Our SEM studies were limited to the prenatal stage. The cell columns are formed by a
FIG. 8. Sagittal fracture of the cerebral hemisphere of an 11-day mouse embryo. Note the marginal zone which is composed of numerous cell prolongations. The ventricular and migrating cells are arranged in columns. FIG.9. Golgi impregnation of the glioependymal cells (radial fibers) in an embryonic cerebral hemisphere. FIG. 10. Columnar arrangement of the ventricular and migrating cells in the cerebral hemisphere of a 12-day mouse embryo. These cell cords extend from the ventricle (bottom of the micrograph) to the marginal zone.
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variable number of cells framed lengthwise between or grouped around the glial fibers (Fig. 9). The cells are closely packed, and most of them are bipolar. The apical portions of the migrating cells loose their attachments to the ventricular surface. Near the ventricle these cells are monopolar, whereas near the periphery they become bipolar. The cell somata are irregularly ovoid or trapezoid; their general configuration seems to depend on the way in which groups of cells are packed together. Stensaas and Stensaas (1968) reconstructed, with the aid of serial sections, models of cells very similar to those seen in SEM micrographs. As seen with the SEM, the morphological aspects of the cell surface lack appreciable differentiation. The shape of the cells that divide or migrate changes continuously. For these reasons, the identification of a classically determined type of cell such as a spongioblast, neuroblast, glioblast matrix cell, or germinal cell is in most cases speculative. Histoautoradiographic (Fujita, 1962, 1963) and electron microscope studies (Wechsler and Meller, 1967) have demonstrated that morphologically the germinal cells are a homogeneous population. Therefore it is not surprising that scanning microscopy of these cells does not reveal any significant differences. Several years ago, with the aid of the TEM, clear differences between glial and neuron-like cells were found to appear for the first time during formation of the cortical plate (Meller et al., 1966). Figure 10 shows the typical columnar arrangement of ventricular and migrating cells in a 12-day-old cerebral vesicle. These cell columns are diversely packed, and the cells located at the periphery have typical membrane boundaries that may be recognized as ameboid features. The glioependymal cells are identifiable in well-preserved preparations. We can summarize the main features of the migration process as follows: (1) All migrating cells have similar morphological aspects, although the mono- or bipolarity of a cell seems to change during migration. (2) The distal process, especially in the outermost marginal zone, is identifiable as ameboid (growth cones). (3) The cells are densely packed in columns. The arrangement of the migrating cells becomes radial to the pia and they move toward the periphery without breaking their adhesion to other migrating cells. The cellular cord or column extending to the cortical plate is associated with the radial fibers. We assumed (Meller and Tetzlaff, 1975) that cell-to-cell guidance is present during the formation of the intermediate zone and the cortical plate. Later, the columnar organization becomes obscured by the developing cell processes oriented in many directions (Fig. 12). FIG. 1 1 . Sagittal fracture of the cerebral hemisphere of a 14-day mouse embryo. The thickness of the hemisphere has increased, and a ventricular zone (VZ), intermediate zone (IZ), and cortical plate (CP) have become discernible. Note that the continuity of the cell columns has not been interrupted by the appearance of various processes in the intermediate zone (arrows).
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3 . The Formation of the Early Cortical Plate In classic descriptions, formation of the cortical plate is considered to be the consequence of a lengthy emigration of cells which must overcome many obstacles (vessels, many types of cell prolongations) in the intermediate zone. Electron microscope studies on migrating cells and on the primitive cortical plate reveal that these cells possess the usual organelles: mitochondria, Golgi apparatus, smooth endoplasmic reticulum, some rough endoplasmic reticulum, microtubules, and numerous free ribosomes. When the cells arrive in the cortical plate, an increase in the formation of endoplasmic reticulum takes place, and consequently for the first time their identification as young neurons and distinction from glioblasts are possible (Meller et al., 1966). Figure 11 shows that all the cells emigrating from the germinal zone through the intermediate zone to the early cortex plate do not lose the cell-to-cell contacts with the other elements of a given column. The columnar arrangement is uninterrupted from the ventricular surface to the marginal zone. The growth of the basal and the laterally oriented prolongations and the growth of cortical fibers in the intermediate zone is one reason why the columnar arrangement of the cell somata becomes obscured in the following stage of cortex formation. According to histoautoradiographicstudies, cells which lead during emigration can be found in the deeper layers of the cortex, whereas the cells which follow form the upper layers. Hicks and D’Amato (1968) claimed that migrating cells move to the developing cortex along the curved, oblique paths of pallial fibers. The duration of migration seems to differ for the various neurons from 3 to 10 days. SEM observations show that the persisting columnar organization provides the guideline formation for later migration waves, including the migration of glial cells to the cortex during the postnatal period, as had already been postulated by some investigators (Altman, 1962a,b, 1966a,b; Vaughn, 1969). At the end of prenatal life, the following features of the cerebral vesicle can be observed (Fig. 14): 1. The ventricular zone is composed of compact columns of matrix cells. 2 . In the subventricular zone the cells are closely’associated, but their arrangement is not clear. Smart (1961) described the formation of the subependymal layer as an essential step in the development of the cerebral cortex. The cells of the subependymal layer are still undifferentiated, mitotically active, and, at least in rats and mice, retain their ability to form nerve cells. Histoautoradiographic studies by the same investigator demonstrated that these cells produced FIG. 12. Fracture of the cerebral hemisphere of a 14-day mouse embryo. VZ, Ventricular zone; IZ, intermediate zone; CP, cortical plate. FIG. 13. Young neurons in the cortical plate of a 14-day mouse embryo. Note their bipolarity and the branching of their apical prolongations, that is, the apical dendrites which extend to the marginal zone.
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neurons and neuroglia cells after birth. Other workers claim that the mitotic activity of neuroglia continues during the animal’s entire lifetime (Dalton et al., 1968). SEM micrographs of the subependymal cells provide little information about their nature. The cell somata are round to ovoid and display short processes. Most cells form clusters, but spatial orientation is lacking. The morphological features of these cells, in contrast to those of the migrating neuroblasts, support the suggestion that they are glial precursors. 3. The intermediate zone contains tangentially oriented fibers. Groups of cells, oriented in the same direction, are clearly visible between these fibers. Fractures of the fiber bundles, as seen in Fig. 14, are very irregular, so that glia-fiber relationships cannot be observed without artefacts. 4. In the cortex plate the columnar organization is still preserved (Fig. 14). Some cells are in an advanced stage of differentiation (Fig. 13). These pyriformshaped young cortical neurons, each of which possesses a thin basal process, can be observed in Golgi preparations (Meller et al., 1968). The process which leaves the basal pole of the cell and extends toward the intermediate zone is identifiable as the axon. The thin, irregular, apical prolongation, which is the developing apical dendrite, extends to the outermost portion of the cerebral wall and branches directly beneath the pia (Figs. 13 and 15). The growth of basal prolongations (basal dendrites) and efferent and afferent fibers masks the columnar organization of the cortex and is the principal morphological transformation during maturation of the cortical nerve cells in the first postnatal week. Numerous TEM studies have contributed more information about the next step of development, the establishment of specific contacts (Noback and Purpura, 1961; Van der Loos, 1963; Voeller et al., 1963; Molliver and Van der Loos, 1970; Adinolfi, 1971; Purpura, 1971; Foelix and Oppenheim, 1974; Konig et al., 1975). Present possibilities for use of the SEM technique in the study of synaptogenesis are still very limited.
B. DEVELOPMENT OF THE RETINA The retina is a favorable region of the central nervous system for the analysis of neurogenesis. Previous Golgi studies (Ramon y Cajal, 1894), histoautoradioFIG. 14. Sagittal fracture of the cerebral hemisphere of an 18-day mouse fetus. Between the ventricular and intermediate zones, a subventricular zone (SZ) has developed, which contains multipolar cells. In the cortical plate the deeper-lyingcells are at a more advanced stage of differentiation than those in the upper zone of the cortical plate. The columnar organization of its cells is still evident. FIG. 15. Young neurons in the cortical plate of a newborn mouse. The branched apical dendrites and axon hillocks (arrows) can be seen.
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graphic (Sidman, 1961) and TEM investigations (Meller, 1964, 1968; Olney, 1968; Morest, 1970b; Sheffield and Fischman, 1970; Kuwabara and Weidman, 1974) provide a solid basis for the interpretation of SEM observations. As in the other regions of the nervous system, development of the retina is a highly coordinated process of cellular division, migration, and differentiation. Furthermore, the primitive optic vesicle invaginates, forming the so-called optic cup and, as a consequence of this process, the retina can be divided into the neural retina and the pigment epithelium. The presence of pigment granules indicates the early differentiationof the pigment epithelium which is closely apposed to the neural retina after obliteration of the optic ventricle. It is probable that these two tissues become metabolically linked in the adult. We can summarize the principal characteristics of retinal development as follows: (1) The ganglion cells are the first cells which migrate from the retinal germinal layer. In general, the distances across which the other cell types (bipolar cells and amacrine cells) migrate are reduced. (2) The Muller cells, comparable to the radial fibers of the cortex, also persist in the adult. (3) The primitive ventricular cells of the optic vesicle differentiate into receptor cells whose morphological and functional properties are totally different from those of ependymal cells. Morphologically, neuroblasts and primitive ventricular cells of the optic vesicle do not have any features in common (Morest, 1970b). TEM and SEM studies on the developing retina (Meller, 1968; Meller and Tetzlaff, 1976; Olson, 1977) are confined to chick embryos whose retinas are fully developed at the time of hatching and are avascular. However, a comparison of the principal processes involved in chick retina neurogenesis with retinal development in other vertebrates and in humans is possible (Mann, 1964; O’Rahilly, 1966; Raviola and Raviola, 1967; Fisher and Jacobson, 1970 Sheffield and Fischman, 1970; Caley et al., 1972; Magalhaes and Coimbra, 1972; Spira and Hollenberg, 1973; Uza and Smelser, 1973; Hinds and Hinds, 1974). Here again, the SEM technique provides insight into the three-dimensional features appearing during neurogenesis in the retina (Figs. 16-19). 1. The Matrix Cells The close apposition of the pigment epithelium and neural retinal cells on the fourth day of incubation is shown in Fig. 16. The pigment cells are arranged in a FIGS.16-19. Sagittal fractures of chick embryo retinas at different stages of development. FIG. 16. The retina of a 4-day chick embryo consists of a pseudostratified epithelium. FIG. 17. After 5 days of incubation, the thickness of the retina has increased and the retinal cells reveal a columnar arrangement. FIG. 18. After 6 days of incubation, the thickness is still increasing as a consequence of cell proliferation, and the columnar organization prevails. FIG.19. After 8 days of incubation the cell columns are interrupted by the appearance of the inner plexiform layer which is formed by the growth of dendritic and axonal processes of the ganglion and bipolar cells (arrows).
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simple cuboid epithelium, whereas the retinal cells form a pseudostratified epithelium. The morphological aspects and organization of the retinal epithelium is very similar to that found in other regions of the neural tube. All cells are tightly packed, and the fracture shows that they are organized in cell columns. Three cell types can be recognized: 1. Cells, attached only to the ventricular surface, with an ovoid, cylindrical, or spherical shape. These are matrix cells in different stages of mitosis. The precise description of Seymour and Berry (1975) of the pre- and postmitotic shapes of cells in the developing cortex (see above) might also apply to the matrix cells in the developing retina. Mitotic figures can be found during the first week of incubation. 2. Elongated cells, radial Miiller “fibers.” They are also identifiable during the first week of incubation, but their maturation process lasts until the end of incubation (Meller and Glees, 1965; Meller, 1968). In SEM preparations, these cells are recognizable by their processes which span the whole extent of the retina, from the “membrana limitans externa” to the “membrana limitans interna.” In the first week of incubation, their nuclei are found in variable positions, frequently near the vitreal surface. During the following weeks, the cells elongate, their processes branch, and their nuclei are finally located in the inner granular layer. The “end feet” of their prolongations form the vitreal surface of the retina. The shape of the Miiller cells becomes more and more incompletely revealed by the fracture technique after the first week of incubation. It becomes necessary to consult Golgi preparations for a more precise comparison. TEM observations (Meller and Glees, 1965; Meller, 1968) show that Miiller cells possess a characteristic ultrastructure which can easily be distinguished from that of other cell types. 3. Mono- or bipolar-shaped cells. These cells are the neuroblasts of the retina and are the precursors of several types of neurons. In the retina it is more difficult than in the cortex to distinguish between these cells and matrix cells on the basis of morphological criteria alone. The free basal prolongations at the distal ends resemble axonal growth cones and suffice to identify these cells as young neurons. FIGS.20-25. Ventricular surface on the neural retina of the chick embryo at different stages in the development of the receptor cells. FIG.20. Irregular cell protrusions are the characteristic feature of the ventricular surface of the neural retina after 8 days of incubation. FIG.21. After 10 days of incubation, spherical protrusions, the early inner segments of the receptor cells, can be seen among a large number of microvilli belonging to the Miiller cells. FIGS.22 -25. The inner segments display an increasing number of microvilli during the following days of incubation. FIG.22. Eleven-day chick embryo. FIG.23. Twelve-day chick embryo. FIG.24. Fourteen-day chick embryo. FIG.25. Fifteen-day chick embryo.
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2 . The Migration Process in the Retina The process of cell migration in the retina is very direct. We believe that in the retina the period in which proliferation occurs is short and that the matrix cells give rise to nearly all cell types simultaneously. The distance across which the cells migrate is, in comparison to that of the cerebral elements, shorter. The SEM shows that the features and arrangement of the migrating cells are the same as in the brain; as a consequence of active cell proliferation, the thickness of the retina increases and the columnar organization becomes apparent (Figs. 17- 19). At the end of the first week of incubation large cells have arrived at the vitreal surface. Some of them develop numerous apical dendrites and, as a result of the branching of these and other processes, a neuropil-rich layer, the inner plexiform layer, appears (Fig. 19). The migrating cells are oriented radially to the vitreal surface and move without breaking their adhesions to each other. At this stage the migration processes in the retina seem to end, although slight displacements of all cell types can still be observed as a result of the development of many neuronal processes (dendrites and axons) and the above-mentioned Muller cell differentiation. The layers of the retina are a product of the growth and arborization of the cellular processes, beginning with the dendrites of the ganglion cells and the axons of the bipolar cells forming the inner plexiform layer. The formation of the outer plexiform layer occurs nearly a week later (Melier, 1968; Meller and Tetzlaff, 1976) as a consequence of the branching of the bipolar cell dendrites and growth of basal prolongations of the receptor cells.
3 . The Development of the Receptor Cells The pigment epithelium can easily be removed from the neural retina during the development of the eye. This allows the study of the outer surface of the neuronal retina and with it observation of the diverse stages of receptor cell development (Figs. 20-25). Concretely we can observe only the growth of the apical portions of these cells, that is, the formation of their inner and outer segments. During the first days of incubation the ventricular surface of the neuronal retina is morphologically comparable with other embryonic ependymal surfaces (Fig. 20). The free surface of the neuroepithelium resembles a mosaic of tightly packed cell areas. The apical portions of the receptor cell membranes have irregular boundaries and rough surfaces and possess a centrally located prolongation, probably a short cilium. FIG. 26. The inner segments of the receptor cells are elongated after 16 days of incubation, and most of them possess a conical outer segment at their tips. FIG. 27. The inner (IS) and outer (0s)segments of the chick retina after 20 days of incubation. Note the irregular, rough surface of the outer segment membrane. The connection between the inner and outer segments, the optic stalk, is not visible, because a corona of calycal processes (arrows) surrounds the basis of the outer segment.
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A precise morphological identification of cell types (mitotic cells, receptors) is not possible at this stage. The first morphological change in differentiating receptor cells is the growth of a spherical protrusion from the apical surface into the obliterated optical ventricle between the neuronal retina and the pigment layer. Reconstructions of large areas of this external surface of the retina show that the differentiation and growth of receptors does not occur in complete synchrony, as was assumed earlier (TEM studies, Meller, 1968). Development of the inner segment is the principal process during the second half of incubation. The spherical protrusions of the inner segments change the features of the apical surface. The number of microvilli increases, and the spherical protrusion elongates to become cylindrical. The development of the outer segments is generalized on the sixteenth day of incubation (Figs. 26 and 27). In this connection a SEM study without TEM controls may lead to incorrect conclusions, because the first stage of development of the outer segment as the differentiation of a ciliary structure is difficult to analyze in SEM micrographs only. Microvilli of the inner segment surround the outer segment as so-called calycal processes and partially obscure the ciliary stalks. The outer segment membranes show irregular surfaces through which the stacked membrane disks are not clearly discernible. All outer segments interdigitate with the long microvilli of the pigment cells at the end of incubation. Removing the pigment cells in the adult stage produces distortion or damage of the outer segments.
111. Development of the Nervous System in Vitro Today cell and tissue cultures of the nervous system are essential for the experimental study of neurocytological differentiation processes (Boyde et al., 1968; Ebendal, 1974; Hill et al., 1974; Shahar et al., 1977). The SEM is a useful tool for obtaining a three-dimensional view of the features and organization of cell cultures and facilitates rapid morphological control of in vitro experiments. Isolated nerve and glial cells obtained by tissue trypsinization are capable of aggregating and forming histotypical patterns of nervous tissue. Garber and Moscona (1972a,b) demonstrated that cell aggregates of diverse regions of the nervous system are characteristic in size and shape according to the region from which they are derived. FIGS.2%32. Light micrographs of trypsin-isolated and aggregated rat brain cells cultured from 24 hours to 14 days. FIG.33. Scanning micrograph of a typical mesenchymal cell with numerous filopodia. FIG.34. Scanning micrograph of portion of a neuronal prolongation with several growth cones. FIG.35. Scanning micrograph of a I0-day-old culture of brain cells.
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THEMORPHOLOGY OF BRAINAND RETINAL CELLCULTURES
A few hours after trypsin isolation, the cells in suspension aggregate in small groups. In flask cultures, the aggregates attach to the glass or plastic surfaces during the first 24 hours of cultivation. A comparison between Figs. 28-32 and Figs. 36-38 shows that the cerebral aggregates are larger than the retinal ones. However, they stretch out and flatten after the first day of cultivation. Mesenchymal cells of brain cultures quickly attach to the plastic surfaces and form a monolayer under the brain cell aggregates. On the second day of incubation, large cellular prolongations grow out, linking the aggregates to each other and forming a network of prolongations between them. The differentiation of the ultrastructural features of the aggregates into histotypical nervous patterns involves the formation of synapses. A proliferation of glial cells surrounding the aggregates is commonly observed at the end of the first week. This morphological organization of aggregates remains unchanged over long periods of time (Meller et al., 1969). If the above-described cultures undergo renewed txypsinization and the cell suspension is cultivated with fresh medium, reaggregation takes place. The morphological aspects of cultures after the second aggregation are similar to those after the first. After several passages the aggregates show a lack of proliferation of neurites, and the cell clumps probably consist only of glial cells. Retinal aggregates have a tendency to confluence (Figs. 37-39). The result is the formation of large islands of different-sized cells. Figure 35 shows a lateral view of a small conglomerate during the first week of cultivation. These aggregates (clumps) consist of 20 to 40 cells. The neurons are localized in the basal portion and are almost covered by glial cells. From these neurons, bundles of axons grow out to the neighboring aggregates. These connections seem to provide guidelines for the migration of cells between the clumps. Figure 40 shows the organization of a large aggregate. The innermost part of the aggregate is occupied by densely packed cells covered by a thick layer of glial cells-a feature that is also visible in small aggregates. In all aggregates three types of cells can be distinguished, mesenchymal cells, glial cells, and nerve cells. Mesenchymal cells proliferate and form a confluent monolayer during the first week of cultivation. SEM micrographs show them to be flattened cells whose surfaces are covered with microvilli and which have characteristic ruffling membranes. The nucleus is so flattened that it can be localized only with difficulty. Macrophages are often present in great numbers in brain cultures. Their cell surfaces are covered with numerous microvilli, blebs, and membrane protrusions (Fig. 33). Nerve cells in FIGS.36-38. Light micrographs of trypsin-isolated retinal cells derived from a 7-day chick embryo. Cultured from 1 hour to 4 days. FIG. 39. Scanning micrograph of a monolayer of retinal cells. Cultured 4 days.
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FIG. 40.
K . MELLER
Scanning micrograph of a portion of a brain cell aggregate. Six days in virro
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vitro observed with the SEM technique show several morphological forms and sizes. The axons can easily be identified, especially the growth cones (Fig. 34). A feature common to all nerve cells is the smoothness of the membrane surface of the cell soma. Spines of dendritic processes are still lacking. All neurons occupy a central position in the aggregates. The glial cells surround the neurons as a cellular corona, forming a network of prolongations among them. The observation of cell cultures with the SEM can be summarized: Isolated and reaggregated cells reconstitute histotypical patterns in which all cells possess positional spatial information and recognize each other. Typical in vitro arrangements seem to reproduce the cellular relationships which exist in vivo. In this particular aspect of the experimental study of neurogenesis, the SEM technique will continue to provide information, contributing to a more detailed understanding of the process of cell movement and cell recognition that occurs during neurogenesis.
IV. Conclusions These results reveal two limitations of the SEM technique when applied to the study of neurogenesis. The powers of optic resolution are limited, and breakage artefacts are produced by fracture procedures. The low resolution of the SEM as compared with the TEM is not the principal limitation. More important are the conditions of preparation of the biological specimens. The internal organization of a tissue can be exposed only when the material is fractured. The fracture surface can be obtained in different ways. Both blunt dissection and freezefracturing allow only limited preservation of cell structure. Long cell prolongations or the characteristic cell arborizations of neural elements are irregularly amputated. In this case, similar to an incomplete Golgi impregnation, a comparative morphological analysis of similar cell types is very difficult. Consequently SEM studies in neurogenesis can be fruitful only in connection with background experience with Golgi and TEM techniques. Nevertheless, the possibility of examining the three-dimensional organization of (different) cell groups and the continuously varying aspects of cell surfaces of differentiating and moving cells, and the ease with which larger areas of a specimen can be scanned and compared, render the SEM an indispensable tool for morphogenetic studies.
ORGANIZATION THECOLUMNAR The investigations described here allow general speculation on the organization of nerve structures in the developing central nervous system. The idea has already been accepted that development of the nervous system is an interaction
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balanced between the expression of genetic information and the influences of the external environment. In this connection the existence of two essentially different neuron types has been proposed. The first type are the macroneurons (Altman, 1970), also called class I neurons (Jacobson, 1974), whose structure and function are constrained genetically and whose functions are invariant and unmodifiable. A malformation in the development of these neurons is not compatible with life. Class I1 neurons or the microneurons of Altman, however, develop in later stages, including the postnatal stages (e.g., the granule cells of the cerebellum), and are more susceptible to changes in environment (hormones, radiation, sensory stimulations). The site at which a nerve cell is generated differs from the site at which it will later reside, and migration takes place in continuous waves of cell production. It seems that the first cells to migrate are of great importance as leaders in the migration of cells subsequently generated. Previous SEM studies (Meller and Tetzlaff, 1975, 1976) and the results dis-
DIAGRAM 111. Three-dimensional representation of the mosaic of columns in the developing nervous system. Columnar units are formed by groups of elongated cells and their processes, surrounded by radial fibers.
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cussed here indicate a columnar organization to be the basic arrangement in the early structure of the developing nervous system. This agrees with Rakic’s hypothesis (1972) which states that the columnar organization is a consequence of the radial disposition of leading structures such as the radial fibers. However, it is of fundamental importance that the cells migrating within a given column maintain continuous cell-cell contact. One can suppose that the cell-cell recognition phenomena, as seen in in vitro experiments, provide spatial orientation for the cells during the migratory process. Thus the cell-cell contacts in a given column, the number of cells in a column, or the variable ratio of macro- to microneuron types characterizes the ‘‘individuality” or the specific composition of groups or columns of migrating cells. Cortical columns differ from those of the retina. In this way a mosaic of similar (in cell number and arrangement) columns could be the basis of a specific area of the nervous system. Diagram I11 shows such a hypothetical mosaic of columns of migrating cells; they are bounded by glial fibers. The cell composition and the spatial orientation of cell columns would contribute to form a framework of organized areas with particular functional characteristics. These different columnar groups would contain the information for emigrating fibers to establish specific connections between different functional nervous areas. Therefore, despite the large number of facts which have appeared in the literature over almost a century, one unanswered question remains: How do members of an immense population of neurons unerringly take their places and establish connections at the right time during neurogenesis.
ACKNOWLEDGMENTS The data summarized in this chapter were obtained in collaborative studies carried out with M. Waelsch and W. Tetzlaff. I am deeply indebted to P. Mestres for helpful criticism and discussion. I wish to express my gratitude to R. Maeder for technical assistance, K. Donberg for preparing the photographs, K. Rascher for help with the English translation, and C. Bloch for typing the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Me 276/6 and Me 27617).
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INTERNATIONAL REVIEW Of CYTOLOGY. VOL. 56
Cytoplasmic Structure and Contractility in Amoeboid Cells D. LANSING TAYLOR A N D JOHNs. CONDEELIS' Cell and Developmental Biology, The Biological Laboratories, Harvard University, Cambridge, Massachusetts
1. Introduction
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A. Aim of This Article . . . . . . . . . . . . . . B. Amoeboid Movement . . . . . . . . . . . . . C. Categories of Cells . . . . . . . . . . . . . . 11. Cytoplasmic Structure and Contractility: In Vivo . . . . A. Historical Sketch . . . . . . . . . . . . . . . B. Terminology Used to Describe Cytoplasmic Structure . C. Cytoplasmic Contractions: In Vivo . . . . . . . . D. Physical Evidence for Cytoplasmic Structure . . . . . E. Light and Electron Microscope Evidence of Cytoplasmic Structure and Contractility . . . . . , . . . . . F. Environmental Effects on Cytoplasmic Structure and Contractility . . . . . . . . . . . . . . . . 111. Cytoplasmic Structure and Contractility: In Vitro . . . . A. Early Model Systems of Contractility . . . . . . . B. Models of Cytoplasmic Structure and Contractility . . IV. Actin, Myosin, and Associated Contractile Proteins . . . A.Actin.. . . . . . . . . . . . . . . . . . B. Myosin . . . . . . . . . . . . . . . . . . C. Regulation . . . . . . . . . . . . . . . . . D. Other Contractile and/or Cytoskeletal Proteins . . . . E. Summary and Future Challenges . . . . . . . . . References . . . . . . . . . . . . . . . . . .
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62 63 65 67 74 91 96 97 100 117 118 123 128 132 132 134
I. Introduction
A. AIMOF THISARTICLE Rapid advances in the understanding of vertebrate striated muscle contraction were made possible by integrating information obtained at three separate and 'Present address: Department of Anatomy, Albert Einstein College of Medicine, Bronx, New York. 57 Copyright 0 1979 by Academic Press, Inc All rights of reproduction in any form reserved ISBN 0-12-364356-2
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D. LANSING TAYLOR AND JOHN S . CONDEELIS
distinct levels of organization via different research approaches: ( I ) physiological and structural characteristics of whole cells, (2) chemical and structural dynamics of motile model systems, and (3) the properties and interactions of purified contractile and regulatory proteins (Mannherz and Goody, 1976; Heilmeyer et al., 1976; Lehman, 1976). In contrast, the molecular concepts of nonmuscle cell movements have been difficult to identify, since they include a more diverse collection of possible structures and movements than vertebrate striated muscle. Instead of integration of information at different levels of organization in a few cell types, the field of nonmuscle cell motility has been characterized by the collection of specific facts about a large number of different cell types. This process has allowed the identification of a diversity of movements and possible motile mechanisms (Pollard and Weihing, 1973; Clarke and Spudich, 1977; Weihing, 1978). Because information is available on the physiology, motile model systems, and biochemistry of several cell types, it is presently feasible to correlate these three levels of analysis for defined groups of cells. Although this process will require more specific information at each level of organization before a completely cohesive picture emerges, it is appropriate at this time to begin the integration. In this article a select group of cell types is analyzed with the hope that some basic relationships between cytoplasmic structure and contractility will emerge. We are not seeking a unifying theory or mechanism of cell movement, since present knowledge does not support this notion. However, common types of structures, structural dynamics, proteins, and regulatory mechanisms might be operable in certain cell types. The role of actin in both cytoskeletal and contractile events is emphasized. Previous reviews and symposia discuss both general characteristics of nonmuscle cell motility (R. Goldman et al., 1976; Inoue and Stephens, 1975; Clarke and Spudich, 1977; Pollard and Weihing, 1973; Pollard, 1977b; Taylor, 1977b; Tilney, 1977; Allen and Kamiya, 1964) and specific types of amoeboid movement (Komnick et d., 1973; Allen, 1961a, 1972b; Allen and Allen, 1978) and tissue culture cell movement (Ciba Foundation Symposium, 1973; Vasiliev and Gelfand, 1977). An exhaustive review of all the historical facts discussed in such reviews is not attempted here. However, selected studies are discussed in order to integrate information on cytoplasmic structure and contractility at the three levels of analysis. Detailed information on each of the types of cells can be obtained from the literature cited here and in previous reviews and symposia.
B. AMOEBOID MOVEMENT Amoeboid movement has been defined mechanistically as cell movement occurring in one of two possible ways: by the use of pseudopodia or by cytoplasmic
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streaming (Allen, 1961a, 1972a; Komnick et a l . , 1973; DeBruyn, 1947). This definition includes many types of cells that exhibit dramatically different forms of movement. The Sarcodina, in particular the Amoebaea, contain the classic amoeboid cells for which the general movements are named (Allen, 1961a; Bovee and Jahn, 1972). In addition, this definition includes the varied motile forms of tissue culture cells (see Ciba Foundation Symposium, 1973), macrophages (Mudd et al., 1934), leukocytes (DeBruyn, 1946; McCutcheon, 1946; Senda et al., 1975), and some developmental stages of most if not all multicellular animals (Allen, 1961a; Cloney, 1966; Trinkaus and Lentz, 1967; Trinkaus, 1973). In fact “amoeboid-like motions” have been demonstrated in the cytoplasm of some marine eggs that do not exhibit overt locomotion, suggesting the significance of amoeboid movement in early development (Kitching and Moser, 1940; Rebhun, 1975). The shuttle streaming exhibited in acellular slime molds (i.e., Physarum polycephalum) is also discussed, since many of the pioneering studies on nonmuscle cell movements have been performed on this organism. C. CATEGORIES OF CELLS The amoeboid cells discussed in this article have been separated into three categories based on the nature and extent of changes in cytoplasmic structure and contractility during movement and not on any proposed mechanism of movement (Fig. 1). In addition, several specific cellular motile events are discussed which might be related to amoeboid movement. 1. Class 1 (Lobopodia and Cytoplasmic Streaming) The first category consists of cells exhibiting cyclic and extensive changes in cytoplasmic structure and contractility correlated with amoeboid-like movement. Rapid cytoplasmic streaming characterizes this type of movement (Fig. 1A and B). The cell types include the giant amoebas Chaos carolinensis and Amoeba proteus, the acellular slime mold P. polycephalum, and leukocytes in which lobopodia (large cylindrical pseudopods) are predominant. These organisms and cells have been extensivkly investigated at the physiological level and therefore make up the bulk of the section on cytoplasmic structure and contractility in vivo (Section 11). 2. Class I1 (Filopodia and Lobopodia) The second category consists of amoeboid cells in which filopodia (long slender pseudopods) and small lobopodia predominate and in which cyclic but more localized changes in cytoplasmic structure and possibly contractility occur. Movement is characterized by a combination of relatively slow cytoplasmic
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D. LANSING TAYLOR AND JOHN S. CONDEELIS
FIG. 1. Composite light microscope images of some of the cells discussed in this article. (A) Amoeba proteus (class 1). Scale = 100 p m . (B) Microplasmodium of P. polycephalum (class I). Scale = 100 p m . (C) Human platelets (class 111). Scale = 5 p m . (Courtesy of R. D. Allen.) (D) Ameboid stage of D . discoideum (class I). Scale = 1 0 p m . (E) Acanthumoeba castellanii (class 11). Scale = 10 p m . (F) Rat embryo cell (class 11). Scale = 10 p m . (Courtesy of R. D. Goldman.)
streaming and the extension and retraction of filopodia (Fig. ID and E). This class of cells includes Acanthamoeba castellanii, Dictyostelium discoideum, Hyalodiscus simplex, and rnacrophages. 3 . Class I l l (Pharopodia and Filopodia) The third category of cells includes the various mammalian cells grown in culture and cells that exhibit cell sorting during embryogenesis. Pharopodia are broad, flat pseudopods (veils or lamellipodia)that exhibit a “ruffling” activity at
61
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
the leading edge of most of these cells. Motility is characterized by very slow movements of the leading edge of the cells (pharopodia) sometimes coupled with retraction of the “tail” andlor extension and retraction of filopodia (Fig. 1F). In addition to the three basic categories of amoeboid cells, we discuss several special motile events not necessarily performed by amoeboid cells. These events include formation of the acrosomal process in echinoderm sperm, reorganization of the cortex in echinoderm eggs following fertilization and during cytokinesis, and activation of human platelets (Fig. 1C). The changes in cytoplasmic organization are quite dramatic in these special motile events and may involve basic molecular processes that can be correlated with the structural and contractile processes involved in amoeboid movement. For example, the extension of filopodia in some amoebas may result from molecular events similar to the rapid extension of filopodia in activated platelets, the formation of acrosomal processes in certain sperm or the rapid extension of microvilli on marine eggs. Furthermore, the amoeboid cells discussed in this article undergo cytokinesis during cell division which may utilize some of the motile machinery involved in amoeboid movement. Interestingly, some amoeboid cells such as leukocytes exhibit “constricting rings” in the ectoplasm during movement, which are reminiscent of cleavage furrows.
11. CYTOPLASMIC STRUCTURE AND CONTRACTILITY: In Vivo It was four fundamentalists to learning much inclined, Who went to see the Protoplast (though all of them were blind) That each its structure might observe to satisfy his mind. The first advancing hurriedly and happening to fall Right through its soft interior at once began to bawl “God bless me! But the Protoplast is very like a sol.” The second poked the animal and felt his staff repel Its tough and springy cortex, so he began to yell ’Tis evident the Protoplast is very like a gel. ” “
The third approaching gingerly did not only pinch and squeeze its slippery oleaginous hide when he began to wheeze “It seems to me the Protoplast is just a lump of grease.” The fourth man, having punched and probed and proved its plastic state, Watery yet indissoluble, did thus asserverate “The Protoplast is a compound, complex co-a-cerv-ate. ” And so these fundamentalists disputed loud and long Each in his own opinion exceeding stiff and strong, Though each was partly in the right and all of them were wrong. G. W. SCARTH 1942
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D. LANSING TAYLOR AND JOHN S. CONDEELIS
A. HISTORICAL SKETCH Historically, development of the concepts of cell structure and contractility has progressed in a discontinuous fashion. The existence of a viscous substance within living cells was recognized as early as the last part of the seventeenth century, when Malpighi made his discovery of the cytoplasmic structure of plant cells. The term protoplasm was suggested by von Mohl in 1846 for the “slimy, granular, semifluid” constituents of plant cells that were distinct from the cell wall and nucleus. However, Dujardin (1835) had earlier identified the “living substance” in various protozoans and coined the short-lived term sarcode which was defined as a “living jelly, glutinous and transparent, insoluble in water and capable of contracting into globular masses and of adhering to dissecting needles so that it can be drawn out like mucus.” Dujardin coined the term sarcode because he believed that the substance possessed the inherent properties of life, which included muscle-like contractility. He was the first to suggest a contractile mechanism of amoeboid movement that required the contraction of extended sarcode, which caused movement by pulling the cells forward. The concept of contractility was universally accepted as a general property of living substances only after Schultze (1861, 1863) equated sarcode with protoplasm of both plant and animal cells. Once it was realized that protoplasm was the seat of many life processes, speculative reasoning and a few observations cast some doubt on the possibility that it was completely fluid. In addition, the frequent detection of structure in protoplasm suggested the existence of at least some solid components. These few ideas and even fewer observations led to the reticular and fibrillar theories of protoplasmic structure (Heitzmann, 1873; Fromann, 1880). Heitzmann’s theory (1873) of the structure of protoplasm was an attempt to explain contractility on a morphological basis. He viewed protoplasm as a living three-dimensional contractile reticulum embedded in an inactive fluid. The homogeneous appearance of parts of the protoplasm was explained as the extreme stretching of the framework that made it “invisible.” The use of coagulating fixing agents strengthened these early structural theories. Many early cytologists maintained the belief that protoplasm was essentially fluid. Berthold (1886) and Butschi (1892) suggested that the filamentous networks observed in fixed protoplasm could be due to artifacts. In fact, it was demonstrated that even egg albumen exhibited a reticular structure when it was coagulated with fixatives. Therefore theories of cell movement not involving contractions gained popularity for an extended period of time (Allen, 1961b; Chambers, 1924). The relationship between protoplasmic structure and contractility suggested by Dujardin (1835) and Heitzmann (1873) was ultimately recognized by Hyman (1917), Pantin (1923), and Mast (1926) and was developed in their contractile
STRUCTURE AND CONTRACTIUTY IN AMOEBOID CELLS
63
theories of amoeboid movement. Probably the first to suggest changes in the consistency of protoplasm was von Mohl (1846), who demonstrated that the viscosity of plant protoplasm increased with age. Subsequently, Hyman ( 1917) described differences in the consistency of the protoplasm of amoebas in distinct colloidal terms. She suggested that the cell cortex (ectoplasm) had the properties of a gel, while the central protoplasm (endoplasm) was a sol of varying viscosity. Her studies indicated that protoplasm varied both spatially and temporally in structure during cell movement. Thus physicochemical characterizationsbecame necessary to define the structural nature of protoplasm. The term protoplasm was ultimately replaced with the term cytoplasm which is defined as the protoplasm surrounding the nucleus and karyoplasm or the protoplasm of the nucleus. Allen and his colleagues (see Allen and Allen, 1978, for a review) demonstrated that the streaming cytoplasm or endoplasm of the giant amoeba Chaos was not a simple fluid but a viscoelastic substance. Therefore the endoplasm could not be automatically dismissed as a passive medium. In fact, Allen (1960) developed the frontal contraction theory of amoeboid movement to explain his observations. For a more complete historical development of the theories of amoeboid movement the reader is directed to earlier reviews (Schaeffer, 1920; DeBruyn, 1947; Allen, 1961a; Allen and Allen, 1978).
B. TERMINOLOGY USED
TO
DESCRIBECYTOPLASMIC STRUCTURE
DeBruyn (1947) aptly pointed out in his review that “the various theories of amoeboid movement have been greatly influenced by the prevailing concepts of protoplasmic structure. ” Furthermore, the terminology used to describe cytoplasm has also varied historically depending on the prevailing notion of the structure of cytoplasm. The term consistency has been used in a loose sense to describe the “structure” of cytoplasm by most investigators. However, Allen (1961a) referred to the definition of consistency proposed by the Society of Rheologyi “. . . that property of a material by which it resists permanent change of shape, and is defined by the complete force-flow relation. ” This definition of consistency suggests the parameter of viscosity which has been discussed in detail by Allen (1961a) and in several books on rheology (see Reiner, 1949; Scott Blair, 1938). In fact, the early presumption that cytoplasm was a simple fluid induced early investigators to attempt measurements of cytoplasmic viscosity. Viscosity is defined as that property of liquids which causes them to resist flow. The force-flow relationship can be understood by considering a model system of two plates separated by the fluid whose consistency is being studied (Allen, 1961a). One plate is moved by a force, and the fluid is deformed as adjacent
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D. LANSING TAYLOR AND JOHN S. CONDEELIS
lamellae of fluid slide relative to one another. The rate of deformation or the velocity gradient of the fluid is defined as the velocity of the fluid divided by the distance separating the plates. The force part of the force-flow relationship is the shear stress or force required to maintain the velocity gradient (forcelarea). The flow part of the relationship is the velocity gradient (dvldx). In many fluids the shearing stress (forcehea) necessary to set up a given relative movement is found to be proportional to the rate of shear or velocity gradient.
F A
dv
-’dr
where F = force applied, A = area of moving plate, dvldx = velocity gradient between plates, r ) = coefficient of viscosity. Fluids in which q is a constant independent of the shearing rate are known as Newtonian fluids. In contrast, some fluids such as cytoplasm exhibit variable viscosities r ) at different rates of shear and are called non-Newtonian fluids. Plastic flow is a special property of a substance which is characterized by the presence of a yield value of shear stress below which no motion occurs. Plastic flow occurs when taffy or plastic is stretched. The viscosity of some non-Newtonian fluids such as blood and cytoplasm decreases when they are sheared by flowing or stirring. This special property is called thixotropy and involves a delayed restoration to the original structure. Therefore the “history” or time course of observations must be considered when assaying the consistency of substances. In 1865 Isaac Newton described his law of fluid flow which he never tested. It remained for Jean Poiseuille to prove and extend Newton’s law in the form of Poiseuille’s formula of fluid flow in tubes which has been discussed by Seifriz (1936) and Allen (1961a): P, r4tl.r
’
=
81v
wherer) = viscosity, p = pressure head, g = acceleration of gravity, 1 = length of tube in which flow occurs, r = radius of tube, t = time of flow, v = volume of tube. Cytoplasmic streaming of endoplasm within lobopodia (i.e., Physarum, Carolinensis, and A . proteus) has been compared to the flow of water through tubes (Allen, 1961a) (Section 11, D). Once the simple Newtonian fluid nature of cytoplasm was rejected, physical terms describing three-dimensional solids were employed to describe cytoplasmic consistency. Elasticity is defined as the property of materials which causes them to return to their original shape following deformation. An ideal elastic element returns to its original shape immediately and completely upon release of the deforming force. This restoration is also totally independent of time. These substances have straight length-tension curves up to the elastic limit (White,
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
65
1974). This property of cytoplasm can be observed by stretching and then releasing cytoplasm from cells between two microneedles (Chambers and Chambers, 1961). However, viscoelastic materials oppose external shearing by combined viscous and elastic forces. Unlike purely elastic materials viscoelastic substances exhibit hysteresis (i.e., the length-tension curves are different for increasing than for decreasing lengths). In fact, viscoelastic substances do not necessarily return to their original shape when a shearing stress is released (Scott Blair, 1938). Spatial and temporal changes in cytoplasmic consistency have been described with extensive variation from the extreme of being almost fluid to being almost solid. In fact, Hyman (1917) used the colloidal terms sol and gel to describe local cytoplasmic structure. By definition, colloidal substances can exist in fluid and solid states which are called sols and gels, respectively. Freundlich (1937) suggested that the equilibrium between sols and gels fell into three basic categories based on volume changes that occur during gelation. Freundlich group 111 gels exhibit small increases in volume and absorb heat during gelation. Marsland and Brown (1936) demonstrated that cytoplasmic gels (ectoplasm) and in vitro biological gel preparations were Freundlich group 111 gels. The application of hydrostatic pressure favored solation in biological gels as described by LaChatelier’s principle. In contrast, gelatin and agar are Freundlich type I1 gels, since they decrease in volume during gelation and heat is evolved. The phenomenon of thixotropy is a property of gels as well as viscoelastic fluids. A thixotropic gel solates upon agitation and gelates again when at rest (Freundlich, 1942). The above terms have been used historically to describe cytoplasmic consistency based largely on qualitative and some quantitative studies. It has become apparent that cytoplasm has complex rheological properties including viscosity, elasticity, plasticity, and viscoelasticity. At the present time there is no complete rheological model of cytoplasm, although several investigators have offered some simplified models (Allen, 1972b; Hiramoto, 1968). It is necessary to identify all the cellular components responsible for the gross rheological properties identified in the cytoplasm of intact cells (Section 11, D) and cell models (Section III, B) before a complete understanding of cytoplasmic rheology can be achieved. Therefore the rheology of cytoplasm and cell models is correlated with the present understanding of the ultrastructure and biochemical constituents of the amoeboid cells discussed in this article. In Vivo C. CYTOPLASMIC CONTRACTIONS:
The contractile basis of cell movements has been inferred for the most part from contractions induced in motile models (Section 111). However, cytoplasmic contractions defined critically as the generation of force have actually been measured in a few types of nonmuscle cells. For comparative purposes, the maxi-
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D. LANSING TAYLOR AND JOHN S. CONDEELIS
mum tension exerted by frog sartorius muscle is about 1.5 kg/cm2 (1.47 X lo6 dynes/cm2) (Huxley, 1960), while actomyosin threads exert a maximum tension of about 0.25 kg/cm2 (0.25 x lo6 dynes/cm2) (Portzehl, 1951). Crooks and Cooke (1977) recently developed an elegant tension transducer that allowed detailed physical and chemical analyses during the contraction of actomyosinthreads. Isometric and isotonic contractions have been studied in P. polycephalum. Physarum exhibits a contractiowrelaxation cycle believed to cause cytoplasmic streaming, which alternates direction rhythmically. Kamiya has characterized the motive force responsible for cytoplasmic streaming using his ingenious double chamber (Kamiya, 1959). The positive pressure required to “balance” or stop endoplasmic streaming was ca. 6-15 cm positive pressure of water and is probably related to the contractile tension produced by contractions in the ectoplasm. Direct measurements of tension were made on single plasmodial strands which were hung vertically in a moist chamber while still exhibiting motility (Kamiya and Seifriz, 1954). Subsequently, a tension transducer was developed to measure both isometric and isotonic contractions (Kamiya, 1970). The slime mold developed isotonic tensions between ca. 0.018 and 0.05 k g / c d (ca. 0.05 X 106 dynes/cm2)during the relaxationfcontraction cycle. Furthermore, isometric contractions exhibited maximum tension when the plasmodial strands were stretched (N. Kamiya et al., 1972; Wohlfarth-Botterman and Fleischer, 1976). The tension developed by the transient contractile ring during cytokinesis has also been measured. Rappaport (1967) determined the average tension generated by echinoderm cleavage furrows as ca. 0.078 X 106 to 0.25 X 106 dynes/cm2 using calibrated, flexible glass needles (Rappaport, 1971). Hiramoto (1976) has also reported values within a factor of 2 of those described by Rappaport (1967). More recently, Rappaport (1977) demonstrated that the isometric contraction of the cleavage furrow was not length-dependent, in contrast to that in striated muscle and smooth muscle. The site of active tension in dividing cells has recently been correlated with the presence of both actin (see Schroeder, 1975) and myosin (Fujiwara and Pollard, 1976) (Section 11, E), which strengthens the idea that cytokinesis involves a muscle-like contraction. Isometric contractions have been measured in human platelet-rich plasma clots (platelets connected by fibrin fibers) activated by thrombin within glass tubes (Cohen and de Vries 1973). The clot was attached to a tension transducer, and the calcium requirement for maximum tension (ca. 1.2 mg/cm2) was identified. It was found that the tension was independent of the length as shown for cleavage furrows. Contractile events defined loosely by the shortening or condensation of cytoplasm have been identified in many cells. Large decreases in cytoplasmic volume were described as contractions in A. proteus microinjected with 2.0% ATP (ca. 33 mM) (Goldacre and Lorch, 1950). However, the presence of contaminating calcium ions as well as the high ionic strength were probably responsible for the
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
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contractions (Allen and Taylor, 1975). More recently, calcium and/or pH regulated contractions were identified in C. carolinensis by the microinjection of defined contraction solutions (Taylor, 1977a) (Fig. 2). An increasing gradient of both cytoplasmic consistency and contractility was described in this latter study, extending from the posterior endoplasm to the anterior endoplasm and then into the ectoplasm. Furthermore, when the cell was injected with contraction solution at ]/lo its volume, all the cytoplasm contracted reversibly into a central mass. Actin has been shown to concentrate in these contracted regions by quantitation of the amount of exogenous fluorescently labeled actin in the contracted mass (Taylor and Wang, 1978). Marsland and Brown (1936) (reviewed by Marsland, 1956) originally demonstrated that amoeba cytoplasm condensed or contracted when hydrostatic pressure was applied (ca. 6000 psi) and then suddenly released. These cells soon recovered and exhibited normal motility very similar to the recovery described for cells injected with contraction solutions (Taylor, 1977a). The recovery of artificially contracted cells probably involved the return to precontraction ionic conditions. Contractions have also been demonstrated when either A. proteus or C . carolinensis was placed in an electric field (Mast, 1931; D. L. Taylor, unpublished). When the current was raised up to ca. 0.5 V/cm, the anodal side of the cell visibly condensed in a fashion visually identical to the response of cells microinjected with a contraction solution. In addition, the retraction of filopodia on many amoeboid cells may represent active contractions (Albrecht-Buehlerand Lancaster, 1976; R. D. Goldman et al., 1976; Chen, personal communication, 1977). In fact, some filopodia actually exert tension on the substrate, as demonstrated by the “puckering” of an elastic substrate during the shortening of the filopodia (Izzard, 1974). However, the activity of filopodia must be demonstrated quantitatively using force generation assays before they can be described critically as contractions. Contractions in the cells discussed here have been correlated with changes in cytoplasmic structure and, as we shall see in Section 11, E, a rearrangement of cytoplasmic microfilaments. The coupling of cytoplasmic contractions to force generation relative to the cell’s substrate probably involves complex interactions between the contractile proteins, the cell membrane, and the cell surface. This exciting area of research is relatively new, and little information is available.
D. PHYSICAL EVIDENCE FOR CYTOPLASMIC STRUCTURE Physical evidence supporting the various notions of cytoplasmic structure in living cells has been variable both in quality and methodology. However, the application of novel in vivo techniques has permitted at least the semiquantitative
68
D. LANSING TAYLOR AND JOHN S. CONDEELIS
FIG.2. The cytoplasm in living specimens of C. carolinensis can be reversibly contracted by injecting greater than '/lo the cell volume with contraction solution (B) and relaxed by injecting greater than '/TO the cell volume with relaxation or stabilization solution (A). (Taylor, 1977a, with permission.)
STRUCTURE AND CONTRACTILJTY IN AMOEBOID CELLS
69
description of cytoplasm as a viscoelastic, thixotropic gel of variable structure. A historical view of the techniques applied to studying cytoplasmic structure demonstrates the gradual development of the concept of cytoplasmic consistency and its possible relationship to contractility. 1. Brownian Motion Measurements of Brownian motion as measures of viscosity were one of the first assays used to detect variations in cytoplasmic consistency in living cells. Baas-Becking et al. (1927) analyzed the Brownian movement of particles in Spirogyra and found that the cytoplasmic consistency varied over a large range within a small area. They concluded that cytoplasm had a large number of viscosities. Stewart and Stewart (1959) found no difference in the viscosity of endoplasm and ectoplasm in P . polycephalum, which suggests that they measured the microviscosity rather than the macroviscosity of the cytoplasm. Seifriz (1936) observed that the number of particles in motion within quiescent amoebas was small and that the amplitudes of movement were small. The movements of carmine particles in glycerin appeared to match the movements of similar-sized particles in amoebas, so the viscosity of cytoplasm was judged to be equal to that of glycerin (ca. 1500 cP). Quantitative descriptions of Brownian movement were also used by Lewis (1939) in an attempt to characterize the variations in cytoplasmic consistency in various tissue culture cells. Therefore at least class I and class 111 cells exhibited large ranges of presumed viscosity (see Heilbrunn, 1958, for review). There are several problems with using Brownian motions as probes of cytoplasmic consistency, as pointed out by Seifriz (1936) and Allen (1961a). First, the quantitation of viscosity based on Brownian movements assumes that the medium is both homogeneous and Newtonian. However, the historical studies and data discussed in Sections I1 and III have indicated that it is likely that neither of these conditions is met in cells. Second, Brownian (random, short-range) motions must be differentiated from saltatory (directed, indefinitely long) movements of inclusions. Rebhun (1972) has written a review on saltatory movements and has compared them to Brownian movements. Saltatory movements are apparently the result of mechanochemical processes, whereas Brownian motions are caused by thermal agitation. Finally, the observed particles may either be suspended in the cytoplasm or entrapped in local environments such as vacuoles. Therefore it is difficult to identify the absolute environment. 2 . Velocity Profiles
Assuming a uniform viscosity, the velocity profile across the longitudinal section of a capillary containing a streaming fluid can indirectly characterize the consistency of the fluid. Velocity profiles can be quantitated in class I cells such
70
D. LANSING TAYLOR AND JOHN S. CONDEELIS
as the giant amoeba and Physarum, because they exhibit the streaming of endoplasm through the more rigid ectoplasmic tube (Allen, 1961a). Kamiya (1950) was the first investigator to measure the velocity profile across the endoplasmic stream within the plasmodium of P . polycephalum. Assuming that the flow of cytoplasm was caused by hydrostatic pressure, this investigator attempted to analyze the endoplasmic consistency. This rationale was reasonable for Physarum (assuming uniform viscosity), since Kamiya developed evidence suggesting that endoplasmic flow resulted from hydrostatic pressure caused by rhythmic contractions of the ectoplasm (Kamiya, 1959). By cinematographic analysis it was determined that the velocity of flow was the greatest in the central region of the streaming endoplasm and decreased toward the inner walls of the ectoplasmic tube. However, a substantial portion of the inner cytoplasm exhibited the same velocity. The form of the velocity distribution in an optical section through the long axis of the ectoplasmic tube was represented by a truncated parabola (Kamiya, 1950). Since non-Newtonian fluids also exhibit truncated parabolic velocity profiles when forced through a capillary under pressure, Kamiya suggested that endoplasm was non-Newtonian. Newton et al. (1976) have analyzed the streaming velocities of both stationary and moving light scatterers in Physarum with photon correlation spectroscopy. The results extended Kamiya’s earlier observations. Allen and Roslansky (1959) measured the velocity profile in amoebas, also based on the assumption that hydrostatic pressure was responsible for flow. The velocity profile of the anterior half of the endoplasm was a flattened truncated parabola (plug flow) similar to the profile reported for Physarum (Kamiya, 1950). In contrast, the tail region had a more pointed velocity profile which was indicative of Newtonian fluids. However, only relative information on cytoplasmic consistency could be derived from these measurements, since the mechanism of motive force was questioned. However, this study indicated the nonNewtonian behavior of at least part of the endoplasm. The major flaw in the use of velocity profiles to ascertain cytoplasmic structure has been the necessity to assume that the viscosity of cytoplasm is uniform. 3 . Centrifugation Centrifugation of whole cells has been utilized in measuring the viscosity of cytoplasm. Stokes’ law has been applied to measurements of cytoplasmic viscosity based on the centrifugal force required to displace cytoplasmic inclusions. For the simple case of rigid spheres Stokes’ law reduces to
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
r
71
where V = rate of sedimentation, = radius of particle, p = density of particle, pm = density of medium, a = acceleration due to gravity, 77 = viscosity. The originator of the centrifugation method was apparently Lyon (1907) who detected localized dense regions in marine eggs. Moore (1933) attempted to use this method on the cytoplasm of the slime mold Physarum but found that centrifugation injured the cytoplasm and retarded subsequent growth. Heilbmnn (1929a,b) assumed that the cytoplasm of Amoeba dubia and A . proteus was Newtonian and calculated the cytoplasmic viscosity by determining the force and time required to sediment cytoplasmic crystals or particles. The calculated maximum viscosity of amoeba cytoplasm was twice that of water or ca. 2 cP. Heilbmnn (1921) also demonstrated that the consistency of cytoplasm in echinoderm eggs decreased shortly before cleavage, only to rise again following cleavage. However, Heilbrunn's measurements indicated that cytoplasm was a Newtonian fluid with values of viscosity lower or a little greater than that of water. In contrast, Hiramoto (1967) demonstrated that the resistance of sea urchin eggs to deformation by centrifugal force was greatest just before cleavage but decreased as cleavage began. Hiramoto suggested the presence of a cytoplasmic meshwork to explain the apparent large viscosity before cleavage. The contraction of the cleavage furrow was immediately preceded by, or resulted in, a decrease in the overall rigidity of the gelled cortex. Development of the centrifuge microscope (Harvey and Loomis, 1930) permitted direct observation of the effects of centrifugation. Using the centrifuge microscope, Harvey and Marsland (1930) found that particles in A . dubia and A . proteus did not fall at a constant velocity but moved as if caught up in a reticular network. Allen (1960) confirmed and extended Harvey and Marsland's observation with C. carolinensis. Allen (1960) also showed that endoplasm could transport particles against forces up to 170 X g, which supported his view that endoplasm possessed some rigid or gel structure. Furthermore, inclusions in the posterior half of Chaos were displaced at forces lower than those required in the anterior half, suggesting a gradient of cytoplasmic consistency. In fact, recent observations by Kalisz and Korohoda (1976) on centrifuged fragments of A . proteus demonstrated the presence of cytoplasmic fibrils in the hyalin caps. Interestingly, Wilson (195 1) reported that less centrifugal force was required to disrupt the granules in the cortex of marine eggs just prior to and during cytokinesis, implying a decrease in viscosity during cell division. However, centrifugation methods are of limited quantitative value for several reasons. First, the shear stress applied may be aphysiological and may alter the cytoplasmic structure. Second, the variation in the density of inclusions makes quantitative measures difficult, since the densities of both particles and medium are necessary facts for accurate calculations of viscosity [see Eq. (3)]. Third, the presence of Newtonian fluids must be assumed in these measurements, but
72
D. LANSING TAYLOR AND JOHN S. CONDEELIS
cytoplasm may contain the extremes of Newtonian fluids and thixotropic gels. In fact, the results of Harvey and Marsland (1930), Allen (1960), and Wilson (195 1) indicated that endoplasm was not a simple Newtonian fluid, in contrast to the results of Heilbrunn (1929a,b). 4. Movements of Heavy Particles
Heilbronn (1922) developed a technique whereby cells were forced to ingest iron particles and the particles were moved by an electromagnet mounted on a microscope stage. He applied this method in analyzing cytoplasmic consistency with only limited success in Physarum. Yagi (1961) modified Heilbronn’s method and measured the response of ingested nickel particles to magnetic forces in A. proteus-type amoebas. He detected no sign of elasticity or gellike consistency in the endoplasm but did not specify the region of the endoplasm tested. However, Yagi identified viscoelasticity in the ectoplasm, which increased with an anterior-posterior gradient. In contrast to Yagi’s (1961) findings, Allen (1961b) demonstrated that iron, gold, or mercury spheres fell by gravity through amoeba endoplasm in a “halting” manner that suggested a partial gel structure. Instead of falling straight through the center of the endoplasm as expected in a fluid, the heavy particles fell through the shear zone between the ectoplasm and endoplasm. Cultured chick embryo cells (class III) were probed for cytoplasmic structure by Crick and Hughes (1949), employing a magnetic particle method similar to that used on the larger cells. They determined that the cytoplasm was a thixotropic gel with weak elastic properties. This study indicated that the cytoplasm of some of class III cells possessed structure similar to that of class I cells. Hiramoto (1968) applied the heavy particle method to marine eggs and identified cytoplasmic viscoelasticity which was maximal at the cell cortex and the central region of the aster. He suggested the presence of fibrous elements forming a network in the cytoplasm to explain the observed viscoelasticity. The heavy particle experiments were valuable, since they represented a direct and localized quantitative method for assaying cytoplasmic consistency. However, the specific intracellular locations of assays were not always described. 5. Micromanipulation of Cells
The evidence that cytoplasm consisted of a variable gel structure led to the use of direct micromanipulation to assay local cytoplasmic consistency. Historically, microneedles inserted into various regions of cells have suggested a complex cytoplasmic structure. Chambers and Chambers (1961) reviewed earlier studies describing the highly qualitative differences between the consistency of endoplasm and ectoplasm in cell types ranging from class I to marine eggs. These early studies were clever but unquantitative indicators of Cytoplasmic structure. Re-
73
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
cently, this method was used in conjunction with a strain birefringence assay to demonstrate quantitatively an increasing gradient of viscoelasticity from the posterior endoplasm to the anterior endoplasm, which extended into the anterior ectoplasm in C. carolinensis (Allen and Taylor, 1975; Taylor, 1977a). These latter results proved that a gradient of cytoplasmic consistency existed in motile amoebas. Francis and Allen (1971) originally developed the cellular strain birefringence assay to measure induced birefringence in endoplasm strained by negative pressure applied to pseudopodia by closely fitting capillaries. The application of constant negative pressure caused an increase in endoplasmic birefringence which gradually decreased following release of the pressure. However, the birefringence did not return to the original value, suggesting positive deformation of a viscoelastic medium. Furthermore, the increased birefringence followed the amount of induced strain rather than the velocity gradient, which thus identified the phenomenon as strain birefringence and not flow birefringence (Fig. 3). Kanno (1964a) measured the elasticity of the surface structure and cell cortex of A. proteus using a microcapillary suction method first employed by Mitchison and Swann (1954) to measure the elasticity of the cortex of marine eggs. The anterior, middle, and posterior regions of A. proteus were assayed for the length of ectoplasm that could be drawn into a capillary under various negative pressures. The anterior region of the cells was the least elastic, while the posterior ectoplasm-membrane complex was the most elastic. Observation of this anterior-
+ Plane of polarization Red illuminator
r io-
-->2
00
into cylinder
5
Ib
I;
20
2'5 3'0 SECONDS
i5
60
45
&I
FIG.3. Changes in endoplasmic birefringence in C. cardinensis caused by suction applied to a pseudopodium (see inset). Retardation (in angstrom units) is measured by phase-modulation photometry. (Francis and Allen, 1972, with permission.)
74
D . LANSING TAYLOR AND JOHN S. CONDEELIS
posterior gradient of increasing cytoplasmic consistency was similar to the results obtained by Yagi with the metal particle measurements and by Taylor using the direct manipulation technique. Thus three different physical probes of cytoplasm structure suggested similar results in this class I cell. Mitchison and Swam (1954) demonstrated that the amount of suction on a microneedle needed to cause a hemispherical bulge in an egg surface was maximal just prior to and during the early stages of cleavage. However, the resistance to deformation decreased during the later stages of cytokinesis, indicating a decrease in the rigidity of the cortex during division. Similar results have been reported by Hiramoto (1969), which suggests that the gel structure of the egg cortex breaks down during contraction of the cleavage furrow. This interpretation was supported by measuring the stiffness of the egg surface with calibrated microneedles, The stiffness changes measured by compression exhibited a sharp rise and peaked just before cleavage, followed by a decrease during furrowing (Hiramoto, 1963a,b). Therefore the local cytoplasmic structure changes in preparation for or in response to contraction. Micromanipulative probes of cytoplasmic consistency can also be criticized, since these procedures are as disruptive as heavy particle assays. There is a risk that the direct physical probes will create artifacts. For example, Chambers (1919) observed that cytoplasmic consistency was apparently decreased by mechanically agitating cytoplasm with a microneedle which is indicative of thixotropic gels. However, these methods are the most direct and have succeeded in characterizing the viscoelastic properties of both endoplasm and ectoplasm. This section has demonstrated that the cytoplasm of cells possesses variable structure which can be correlated in some cases with the state of contraction. These semiquantitative descriptions of cytoplasm in vivo require more definitive and quantitative analyses in order to characterize the structures responsible for the complex rheological properties. In addition, the components responsible for the structure and contractility must be studied quantitatively in model systems (Section 111) and with reconstituted purified proteins (Section IV). E. LIGHTA N D ELECTRON MICROSCOPE EVIDENCE OF CYTOPLASMIC STRUCTURE AND CONTRACTILITY The morphological basis for cytoplasmic structure and contractility has been investigated with both light and electron microscopes.
1. Light Microscope Observations and Measurements a. Birefringence. The detection and measurement of ordered linear elements is possible in living cells with the polarizing microscope (Inoue, 1953; Allen et al., 1966; Taylor and Zeh, 1977). For example, complex and fluctuating patterns of birefringence have been identified in P. polycephalum (Nakajima and
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
75
Allen, 1965). Birefringent fibrils were identified in the plasmodial ectoplasm oriented either parallel to the long axis or circularly around the plasmodial strands, where contractile structures were suggested to exist (Kamiya, 1959; Wohlfarth-Bottermann, 1962). The birefringence of ectoplasmic fibrils was on and disappeared when the fibrils disappeared. This the order of +3.0 X observation confirmed the presence of linear elements organized in a manner that could produce forces for streaming. Birefringence was not detected in the cytoplasm of the giant free-living amoeba C. carolinensis (class I) (Mitchison, 1950) until Allen (1958) measured weak (ca. +2.0 X lW5) pseudopodial birefringence with a simple photometric method. More recently, Allen (1972a) characterized the pattern of birefringence in Chaos after the majority of light-scattering inclusions were removed micrurgically. The streaming endoplasm possessed diffuse positive axial birefringence (ca. l(r5),while transient patches of negative birefringence were identified in the ectoplasm at the tips of advancing pseudopods (Fig. 4). Retracting pseudopods exhibited declining birefringence during the retracting process, and no discrete bundles were observed. Allen (1972a) has stated that the presence of endoplasmic birefringence indicated the presence of oriented structures which have not been adequately demonstrated by electron microscopy (see Section 111, E). The conversion from positive axial birefringence in the endoplasm to patches of negative birefringence in the ectoplasm at the tips of advancing pseudopods has been explained as the result of compressive forces of contractions (Allen, 1972a). However, the negative birefringence has recently been interpreted as the formation of plasmagel sheets (Taylor, 1977a). Positive birefringence has been identified in the cytoplasm of other cells, including cultured fibroblasts (class III). Birefringent fibers developed in BHK 21 cells during the transition from the rounded cell shape to the elongated polarized form during cell spreading (Goldman and Knipe, 1972). These birefringent fibers, termed stress fibers, have also been identified in many other cell types, including human ENSON cells (R. D. Goldman el al., 1976b) and mouse 3T3 cells (Goldman ef al., 1975), and have been identified as actin-containing contractile fibers (Isenberg et al., 1976). The birefringence of the sea urchin egg cortex generally increases just before the onset of cleavage and then decreases during cytokinesis (Inoue and Dan, 1951). This observation is consistent with the formation of oriented structures during the onset of cleavage, which become disoriented during the contractile process. Measurements of birefringence have been a valuable nonperturbing method of identifying the regions of changing organization in living cells. The correlation of birefringence with electron microscope images has been particularly valuable (Section 11, 2). b. Refractive Index. Interference microscopy and phase-contrast micros-
+
76
D.LANSING TAYLOR AND JOHN S. CONDEELIS
FIG. 4. Three views of a large C. carolinensis with a compound leading pseudopodium in polarized light. (A and a) Tail region; (B and b) middle; (C and c) advancing pseudopodium. MF, Membrane folds; CR, contrast reversal due to orthogonal orientations; HC, hyalin cap; S+, streaming direction. (Allen, 1972a, with permission.)
copy have been used to measure or detect, respectively, refractive index differences in various regions of motile cells which can be related to the local protein concentration (Allen et al., 1962). Interference microscopy was used to identify an anterior-posterior gradient in the refractive index in flattened A. proteus (Allen and Roslansky, 1959). Subsequently, Allen et al. (1962) demonstrated that the endoplasm had a lower refractive index than the ectoplasmic tube, which indicated a higher protein concentration in the ectoplasm. The hyaline caps at the tips of advancing pseudopods were shown to have the lowest refractive index, suggesting ca. 1.0% dry matter. However, Korohoda and Stockem (1975) identified dense microfilament networks in one type of hyaline cap, which
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
77
could explain the absence of other cytoplasmic structures. Phase-dense, gelled (rigid, nonmotile) regions have been demonstrated within the endoplasmic stream of A. proteus by AM (1961, 1962) with the phase-contrast microscope. The gelled regions in the endoplasm formed and disappeared over a short interval of time. Phase-dense fibers have also been identified in many fibroblasts, which coincided with birefringent stress fibers (Goldman and Knipe, 1972; R. D. Goldman et al., 1976b; R. D. Goldman et al., 1975; Buckley and Porter, 1975). Hiramoto (1957) estimated the thickness of the cortical layer of marine eggs by measuring the refractive index prior to and during cytokinesis. Following fertilization, the cortex increased uniformly in thickness around the egg but decreased in thickness during cleavage, further suggesting a decrease in the overall structure of the cortex during contraction. c. Znterjerence Reflection. Motile cells have dynamic points of substrate attachment that are theoretically required to convert the tension generated by cytoplasmic contractions into motive forces applied to the substrate, thus producing movement. The points of attachment have been demonstrated quantitatively in several types of fibroblasts with interference reflection microscopy as first described by Curtis (1964). The cell-to-substrate separation distance has been measured recently in chick heart fibroblasts using this technique (Izzard and Lockner, 1976). Focal contacts were demonstrated in both moving and stationary cells coincident with cytoplasmic fibrils. Furthermore, close contact was observed under the leading lamellae of spreading cells. Pharopodia or lamellipodia were observed ca. 10.0 nm above the substrate at the leading edge of the cells. The fibrils were thus in a position to generate tension relative to the substrate, as well as to form stable attachment sites. The location of attachment sites corresponded to the contact points identified by micromanipulation (Harris, 1973). Heath and Dunn (1978) recently confirmed the results of Izzard and Lockner and correlated the interference reflection images with high-voltage electron microscope images. The results were consistent with the hypothesis that microfilament bundles form at the sites of focal adhesions in chick heart fibroblasts. The physical interaction of potentially contractile fibers with points of contact between the cell and substrate would explain the coupling of cytoplasmic contraction with cell movement.
2. Electron Microscopy The ultrastructuralbasis of the birefringence, refractive index, and viscoelastic properties of cytoplasm has been investigated for many years. In fact, some of the structures detected in vivo with light optical and mechanical methods can be correlated with ultrastructural images. The relevant reviews deal with class I cells (Komnick et al., 1973), class I1 cells, Filopodia and Lobopodia (Komnick et al., 1973), class 111 cells (Ciba Foundation Symposium, 1973), and special motile
78
D. LANSING TAYLOR AND JOHN S. CONDEELIS
events ( h o d and Stephens, 1975; Behnke et al., 1971). A few representative papers are discussed in this section. Class I cells are characterized by the presence of microfilaments (actin) with variable structures but very few if any cytoplasmic microtubules or 10.0-nm filaments. In fact, a range of cytoplasmic structures composed of actincontaining microfilamentshas been identified within all the cells discussed in this article. These structures range from filament bundles to “meshworks” or “feltworks” of filaments (Fig. 5). Furthermore, other regions of cells have been described as being devoid of filaments. Therefore alterations in the supramolecular structure of microfilaments have been suggested by direct electron microscope observations. However, it is important to note that many other amoeboid cells also contain both cytoplasmic microtubules and 10.0-nm filaments; they are discussed below. A significant advance in the ultrastructural analysis of nonmuscle cells was made by Ishikawa et al. (1969) who applied Huxley’s (1963) heavy meromyosin (HMM) labeling technique for actin to nonmuscle cells. Using the specific binding of HMM to actin filaments these workers identified and localized actin in a wide variety of cells. One potential pitfall of this technique is the fact that HMM can induce actin to polymerize. Therefore filaments can be “formed” in locations where no filaments exist in vivo. Filamentous and fibrillar differentiations visible in the cytoplasm of Physarum have been interpreted as constituting the morphological basis of cytoplasmic streaming (Wohlfarth-Botterrnann, 1962, 1964a,b). Cytoplasmic fibrils were found only in the ectoplasmic region and attained the maximum size when the plasmodial strands were hung vertically and the lower regions of the strand had to increase the tension required to cause cytoplasmic streaming (WohlfarthBottennann, 1964b). The formation of these large fibrillar structures was transitory, thus matching the formation of birefringent fibers and coinciding in time and location with increased generation of motive force. Nagai et al. (1975) correlated structural changes in the ectoplasmic tube of Physarum with the cyclic contraction-relaxation cycle. By using a vertical tension transducer either isometric tension or relaxation phases were identified, and fixation was completed while monitoring the physiological state. During the contraction phase the ectoplasmic fibrils were maximally developed and oriented parallel to the long axis of the plasmodial strand. The fibrils consisted of ca. 6- to 7-nm filaments that had previously been identified as actin by HMM labeling (Allera et al., 1971). In addition, thicker filaments were spaced sporadically FIG.5 . (A) Light micrograph of C.carolinensis showing the hyalin ectoplasm (HE) and granular ectoplasm (GE). (B) An isolated membrane-ectoplasm complex from C.carofinensis, demonstrating the association of actin filaments with the membrane. The actin exists in the form of meshworks and free filaments. (Taylor et al., 1976a, with permission.)
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D. LANSING TAYLOR AND JOHN S. CONDEELIS
between the actin filanents and have been thought ,o represent myosin aggregates. These ectoplasmic actin fibers were converteJ into “feltlike networks” at the end of the contraction phase. Thus there appeared to be a cycle of changes or “transformations” of the actin-containing structures during the contractionrelaxation cycle. There also appeared to be changes in the supramolecular form of actin during the endoplasm-to-ectoplasm transition in Physarum, since no actin filaments could be observed in the endoplasm, but the latter contained soluble actin as demonstrated by Hinssen (1972). The combined results of Nagai et al. (1975), Hinssen (1972), and Isenberg and Wohlfarth-Botterman (1976) suggested the possibility of a polymerization step in the transition from endoplasm to ectoplasm, followed by the lateral assembly of actin filaments in concert with myosin to form contractile fibrils in the ectoplasm of Physarum. However, the physiological role of the feltlike network has not yet been identified. In contrast, Nagai and Kato (1975) have suggested that the reversible lateral association of actin filaments occurs in the absence of cyclic polymerization-depolymerization. This question has not been answered adequately in Physarum and most other cells, but Edds (1977) has good evidence that reversible lateral associations of actin filaments are responsible for dramatic changes in cell shape and movement in the amebocytes from sea urchins (Fig. 6). Thick and thin filaments have been identified in both l(le endoplasm and ectoplasm of giant free-living amoebas by several investigators (WohlfarthBottermann, 1960; Nachmias, 1964, 1968; Daneel, 1964; Moore, 1975). The ca. 6- to 8-nm filaments were originally identified as actin by the HMM labeling method by Pollard and Korn (1971). Furthermore, Comly (1973) also demonstrated membrane association of the actin filaments in C. carolinensis using the HMM labeling method applied to purified membranes of A. castellanii (Pollard and Korn, 1973). In addition, the ca. OS-/.~m-longthick filaments (Nachmias, 1968; Comly, 1973) have been identified morphologically as myosin aggregates (Taylor et al., 1973; Moore et al., 1973). Both thick and thin filaments have also been demonstrated in dense arrays in the cell cortex of A. proteus following physical damage. Active contractions have been postulated to close these cellular wounds (Jeon and Jeon, 1975). It is important to emphasize that thick and thifi filaments have been the only linear elements observed that couId account for the viscoelastic and birefringent properties of amoebas described earlier. However, ultrastructural studies on giant amoebas have been hindered by the injury response of these cells to fixatives. Therefore caution in interpretation has been suggested by Allen (1972a). Bruce and Christiansen (1965) demonstrated that a medium 20% saturated with diethyl ether or 10% saturated with halothane caused the giant amoeba C. carolinensis to reversibly cease movement and form three distinct layers of cytoplasm: a central, granular, and peripheral clear zone. The peripheral clear
FIG.6 . A low-magnification view of a portion of a partially transformed coelomocyte of the sea urchin Strongylocentrotus droebachiensis. The filament bunales in the peripheral regions of the cell (top) fuse to form still larger bundles, whereas in the innermost cytoplasm (bottom) some individual filaments are still apparent. Scale = 5 p m ~ 4 0 1 2 . (Edds, 1977, with permission.)
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D. LANSING TAYLOR AND JOHN S. CONDEELIS
zone remained intact even after the cell was ruptured with a probe which indicated that it did not represent a syneretic fluid. Electron microscopy indicated that the peripheral clear zone excluded membranous organelles and consisted of a homogeneous granular material. Bruce and Christiansen suggested the presence of a “meshwork” in the ectoplasm. The anesthetics apparently caused a marked increase in cross-linking of the cell cortex forming a dense gel. Taylor et al. (1976a) recently isolated the plasmalemma and ectoplasm from single C. carolinensis by microsurgical techniques and have identified a possible transformation of the actin-rich ectoplasm from a relatively more amorphous state (feltlike meshwork) to an organized filamentous state during the initiation of contraction (Fig. 5 ) . The two distinct states induced in the amoeba ectoplasm can be correlated with the earlier results of Nachmias (1964) who described a close association of distinct fibrils with a “network region” close to the membrane. Furthermore, J. A. Rhodes and D. L. Taylor (unpublished) observed both filamentous regions and more granular meshworks in the ectoplasm of Chaos, while no free cytoplasmic microtubules or intermediate filaments were seen. The demonstration of actin in the form of filaments, small filament bundles, and meshworks, and the apparent absence or at least low concentration of cytoplasmic microtubules and 10.0-nm filaments, suggest that both the rheological properties and the light optical characteristics of giant amoebas and Physarum are the result of primarily an actin-based system. The transition from the streaming endoplasm to the ectoplasm, and vice versa, as well as contractions, appear to involve the reorganization of actin-containing structures. A large amount of ultrastructural information has been generated on the cells described in this article as class 111. Porter and his colleagues demonstrated very early in the development of electron microscopy that tissue culture cells were ideal model systems for investigating cytoplasm because of their minimal thickness (Porter, 1976). The major ultrastructural difference between average class I1 and class I11 cells and Physarum and giant amoebas (class I cells) is the presence of variable numbers of cytoplasmic microtubules andor 10.0-nm filaments in many of the Class I1 and I11 cell types (Goldman, 1971; Buckley and Porter, 1967). The fine structure of the microfilaments (actin) in the cytoplasm of most tissue culture cells is differiented into several forms. Filament bundles or birefringent stress fibers (Buckley and Porter, 1967) later identified as actin bundles (Ishikawa et al., 1969) form the backbone of many cells running along the lower margin of the cells near the substrate and also forming the core of filopodia (Goldman et al., 1973). Furthermore, meshworks (Buckley, 1974) or “finemesh networks formed by diversely oriented interconnected short filaments” (Buckley, 1975) have also been identified in the cortex of many cells. The cortex has also been described in terms of a combination of granular and filamentous materials (Buckley, 1974).
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
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High-voltage electron microscopy has permitted whole critical point dried cultured cells to be studied in detail. Many cultured cells exhibit interconnected networks of microfilaments, microtubules, endoplasmic reticulum, and unidentified filaments (Buckley and Porter, 1976) (Fig. 7). This complex cytoplasmic network has been designated the microtrabeculae (Wolosewick and Porter, 1976) (Fig. 8). An alternative ultrastructural approach was used by Brown et al. (1977) who prepared Triton X- 100 extracts of whole chick embryo fibroblasts which left the cytoskeletal structures attached to the tissue culture dish. These cytoskeletons consisted of microfilament bundles and intermediate or 10.0-nm filaments. Gel electrophoresis of these cytoskeletons revealed the presence of three main proteins: a 42,000-molecular-weight component that comigrated with muscle actin, as well as 52,000- and 230,000-molecular-weight components. This ultrastructural method is significant, because it should permit correlation of the structures with specific proteins. Actin has been “mapped” in many types of glycerinated or fixed dehydrated whole cells by light-microscope techniques, including the use of fluorescently labeled HMM by Aronson (1965), Sanger (1975), and Schloss et al. (1977). These investigators suggested that actin changed patterns of localization depending on the phase of the cell cycle. More recently, immunofluorescence methods have been used to characterize the distribution of actin (Lazarides and Weber, 1974; Lazarides, 1976), myosin (Weber et al., 1975; Fujiwara and Pollard, 1976; Fujiwara and Pollard, 1978), tubulin (Weber et al., 1975; Brinkley et al., 1975; Eckert and Snyder, 1978), filamin (Wang etal., 1975), calcium dependent regulatory protein (CDR) (Welsh et al., 1978) and other structural andore regulatory proteins. A complete discussion of these methods is beyond the scope of this review and the reader is directed to Cell Motility (R. Goldman et al., 1976) for the details of this recent approach. Several investigators attempted to correlate the phase-contrast, polarized light, electron microscope, and fluorescent antibody images of cytoplasmic structures in the same cell types. These studies demonstrated that the results of all these methods could be correlated, and the fibrous structures were identified as actin bundles located primarily on the attached sides of cells (R. D. Goldman et al., 1975; R . D. Goldman etal., 1976a) (Fig. 10). More recently, R. D. Goldman et al. (1976~)demonstrated that the fluorescent antibody methods of localizing actin did not always correspond to the ultrastructural images. This latter work of Goldman and his colleagues emphasized the importance of combining light and electron optical methods in characterizing cytoplasmic structures. In addition, the more specific nature of direct labeling of antigens with fluorescently labeled antibodies demands that future antibody studies utilize this method (Fujiwara and Pollard, 1977). Many class I11 and some class 11 cells contain a complex array of microfila-
D. LANSING TAYLOR AND JOHN S. CONDEELIS
FIG. 7. High-voltage eiectron micrograph of the periphery of a whole critical-point-dried rat embryo cell. Several bundles of filaments (F) can be seen surrounding a mitochondrion (M). Filament meshworks are observable throughout, especially at the cell periphery. X23.600. (Compliments of K. R. Porter.)
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
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ments (actin), microtubules, and 10.0-nm filaments. The complex rheological properties of class III cells identified by Crick and Hughes (1949)may be an expression of the total microtrabeculae described by Porter and his colleagues. Therefore the thixotmpic structure of the cytoplasm of these cells can be explained in part by the actin based structures. Class I1 cells have an ultrastructural appearance somewhere between the class I and class III cells. There are fewer filament bundles in the form of stress fibers and there are variable numbers of microtubules and possibly 10.0-nmfilaments (Eckert et al., 1977;Pollard and Korn, 1973). Clarke et al. (1976)utilized a clever cell shearing technique developed by Mazia et al. (1975)to observe the cytoplasmic surface of membranes in amoebas of Dictyostelium. Scanning and transmission electron microscopy showed that the cytoplasmic surface of the exposed membrane was covered with fibers consisting of actin-containing filaments. This work extended earlier observations which indicated that actin was associated with the plasma membrane (see review by Tilney, 1977). The cell shearing technique was recently utilized by Taylor et al. (1977)to demonstrate that the supramolecularform of actin-containing structures depended in part on the ionic environment. Meshworks were observed when the membrane patches of D . discoideum were formed in the presence of a relaxation solution, while distinct F-actin filaments or filament bundles were maximized in the presence of a contraction solution (Fig. 9). The ultrastructural aspects of single event processes, such as cytokinesis, mentioned in this article have been discussed thoroughly (Inoue and Stephens
FIG. 8 . A model of the cytoplasmic ground substance (microtrabeculae) showing a lattice and contained microtubules, microfilaments,ribosomes, and elements of the endoplasmic reticulum. The actin filament bundles are depicted here as part of the cytoplasmic cortex, but it is doubtful that this is uniformly the case. This is Porter’s current view of the thixotropic gel that occupies the space between the upper and lower surfaces of a thinly spread cell. X43,500. (Porter, 1976, with permission.)
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D. LANSING TAYLOR AND JOHN S. CONDEELIS
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
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FIG. 10. A well-spread 3T3 cell that has been fixed with formaldehyde, extracted with acetone, and treated with actin antibody for indirect immunofluorescence. (A) Phase-contrast optics; (B) dark-field fluorescence optics. Note the 1:1 relationship between phase-dense and fluorescent fibers. X520. (Goldman et al., 1975, with permission.) FIG. 9. Plasmalemm&ectoplasm fragments of D. discoideurn isolated on poly-L-lysine-coated electron microscope grids exhibited many amorphous aggregates or meshworks and relatively few separate F-actin filaments when prepared in the presence of the relaxation solution (A) and many F-actin filaments when prepared in the presence of the contraction solution (B). X26,775. (Taylor et al . , 1977, with permission.)
88
D. LANSING TAYLOR AND JOHN S. CONDEELIS
1975) and are not considered in detail here. Fertilization of sea urchin eggs initiates a dramatic elongation of microvilli which contain a core of actin filaments (Burgess and Schroeder, 1977). Subsequently, a structural “transformation’’ of actin occurs in the cell cortex from optically isotropic (not birefringent) meshworks to actin bundles during cytokinesis (Schroeder, 1975). Finally, the cortex of various eggs have been shown to be actively contractile based on the localized wound healing responses following injury (Bluemink, 1972; Gingel, 1970). The filaments in the cortex actively “contracted” forming a dense array or an “intracellular clot” reminiscent of the wound healing observed in amoeboid cells (Jeon and Jeon, 1975). The structural alterations of actin during the activation of acrosomal processes from various sperm have been reviewed (Tilney, 1975). Also, Behnke et al. (1971) described the transformation of actin from a less structured but uncharacterized state to F-actin filaments and bundles during the activation of human platelets. More recently, Nachmias et al. (1977) demonstrated that human platelets could be reversibly inhibited from activation (extending microspikes) by treatment with lidocaine. The inhibited platelets, subsequently lysed with Triton X-100 and negatively stained, exhibited very few filaments but contained meshworks of granular material. In contrast, the activated platelets contained bundles of actin filaments. One characteristic similar to all the special motile events was the transformation of actin from a less organized state to readily identifiable F-actin, usually with distinct polarity. The resultant contractions or motions were always along the axis of the actin bundles. However, it has not been adequately determined whether the unorganized state of actin (meshworks) in all the cells discussed consisted of randomly oriented F-actin filaments or various other possible forms of actin includiw complex aggregates of G-actin with “associated” proteins (Tihey, 1976a) (Fig. 11) (see also Taylor et al., 1976a; Condeelis and Taylor, 1977). The molecular form or state of actin in living cells is a fundamental question and has not been resolved. This question probably will not be answered by electron microscopy or biochemical methods alone. In fact, recent evidence suggests that one fixation artifact may be the destruction of actin filaments (Pollard, 1976b; Maupin-Szamier and Pollard, 1978). Therefore, all the ultrastructural information relating to the different possible structures containing actin must be interpreted with caution. A recent method of incorporating fluorescently labeled G-actin into living cells may ultimately help identify the molecular form of actin in connection with microspectrofluorometricand polarized light techniques applied to labeled cells (Taylor and Wang, 1978). For example, changes in fluorescence parameters during polymerization could be used as an indication of the state of assembly. Actin can be labeled and incorporated into the functional actin pool by direct microinjection. In Physarum, birefringent actin bundles in the ectoplasm have
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FIG. 11. Thin section through a mature Thyone sperm. Lying within an indentation in the nucleus (N) is the spherical acrosomal vacuole (A) and beneath and lateral to it is the periacrosomal material (P). X72,200. (Courtesy of L. G . Tilney.)
become distinctly fluorescent following the microinjection of labeled actin into the cytoplasm (Fig. 12). In contrast, distinct birefringent and fluorescent bundles can be observed only in plasmagel sheets of Chaos following the microinjection of labeled actin. Therefore, fluorescent bundles form in regions exhibiting actin bundles &;ring movement and do not form in regions where no actin bundles
have been detected.
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FIG. 12. Region of a single living microplasmodium of P . polycephalum injected with labeled actin containing a birefringement bundle (A) which is distinctly fluorescent (B). X365. (Taylor and Wang, 1978, with permission.)
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Dramatic changes in the distribution of fluorescently labeled actin have also been demonstrated in the sea urchin egg, L. pictus, following fertilization. A rapid increase in peripheral fluorescence has been observed upon fertilization which may be explained by the elongation of microvilli. Cytokinesis was characterized in some eggs by the formation of fluorescent streaks perpendicular to the cleavage furrow (Wang and Taylor, 1979). Future studies on the distribution of actin in living cells during the cell cycle will be valuable. A major obstacle to the structural studies on nonmuscle cells is the apparent dynamic structural changes that precede or accompany motility. However, the new concept of molecular cytochemistry in vivo (Taylor and Wang, 1978) may overcome the limitations of electron microscopy in defining the structural dynamics of cell movements and can be applied to most of the cell types mentioned in this article.
F. ENVIRONMENTAL EFFECTS ON CYTOPLASMIC STRUCTURE AND CONTRACTILITY Environmental effects on cytoplasmic consistency and contractility have been outlined previously (Allen 1961a; Jahn and Bovee, 1969, 1971; Bovee and Jahn, 1972; Chambers and Chambers, 1961; Taylor, 1977b). The intent of this section is to choose some of the environmental stimuli studied over the years and to explore the relationship between alterations in the environment and changes in cytoplasmic consistency and contractility. The cellular responses to environmental stimuli are subsequently compared directly with the same environmental stimuli applied to motile model systems and purified proteins in Sections I11 and IV. Furthermore, we limit our discussion to the following stimuli: (1) ionic, (2) hydrostatic pressure and temperature, and (3) mechanical. 1. Ionic Ionic effects on the consistency of cytoplasm have been studied either by immersing the cells in different salt solutions or by microinjecting solutions directly into the cytoplasm. Studies using immersion and microinjection have often produced conflicting results, making it difficult to interpret the specific ionic effects. Immersion studies are not discussed in this article, since we have no means of knowing whether the observed effects on the cells were caused by the entrance of some ions, surface changes, or the movement of other ions out of the cells. Chambers and Chambers (1961) have discussed the problems of interpretation and have reviewed the results of this method (see also Heilbrunn and Daugherty, 1931, 1932; Kanno, 1964; Jahn and Bovee, 1969; Mast and Prosser, 1932; Pitts and Mast, 1933; Kriszat, 1950, 1951). Early studies demonstrated that direct microinjection of ions had a profound
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effect on cytoplasmic consistency and contractility in a wide variety of cells. The cells that have been most widely investigated are amoebas and echinoderm eggs. Monovalent salts (0.01-2.0 M) have been shown to disperse (decrease cytoplasmic consistency as judged qualitatively with a micropipete), while calcium or magnesium (0.005-2.0 M ) have been shown to “coagulate” the cytoplasmic matrix in amoebas (Chambers and Reznikoff, 1926), Physarum (Chambers, 1943), and echinoderm eggs (Heilbrunn, 1930). These studies were purely qualitative and cannot be interpreted directly. Calcium chelating agents such as sodium alizarin (Pollack, 1928), potassium oxalate, and EGTA (Taylor, 1977a) caused cells to cease motility and to round up. Very similar results have been described in Physarum and echinoderm egg cytoplasm (Chambers and Chambers, 1961). In contrast, Goldacre and Lorch (1950) microinjected 2.0% ATP (33 mM) directly into A. proteus and induced local contractions. However, slight contamination with calcium was probable, since no calcium chelators were available at that time. a. fntracellularpCa. The use of aequorin luminescence as a measure of the intracellular free calcium ion concentration has been applied to many cells (Shimomura and Johnson, 1976; Blinks, 1977). The change in intracellular calcium during cellular processes including motility (Ridgeway and Durham, 1976) and even the fertilization of eggs (Gilkey et al., 1978) appears to range from ca. 10-8-10-6
M.
The free calcium ion concentration has been estimated in several cell types discussed in this article. Ridgeway and Durham (1976) demonstrated that this concentration fluctuated sinusoidally with a slight phase advance relative to the reversal of the streaming cycle in Physarum, using the aequorin luminescence method of calcium detection. The results are consistent with calcium induced contraction of the ectoplasm, since the cytoplasm streamed away from the transient rise in free calcium. Taylor et al. (1975) originally failed to observe spontaneous luminescence in single specimens of C. carolinensis injected with aequorin. However, electrical stimulation induced directed movements simultaneously with luminescence. More recently, Taylor et al. (1978) identified weak spontaneous luminescence emanating from the uroids or tails of Chaos. This luminescence represented submicromolar concentrations of free calcium. Nuccitelli et al. (1977) have identified complex electric currents in streaming C. carolinensis, and the evidence indicates that calcium is primarily responsible, but protons can partially replace calcium. Chaos exhibited apparent calcium currents in the tail or uroid and sporadic currents in the tips of advancing pseudopods. These complex results were consistent with the aequorin results reported by Taylor et al. (1975) during electrical stimulation and in part with the results with spontaneous luminescence (Taylor et al., 1978). Wick et al. (1978) measured the influx and efflux of 45Cain D. discoideum upon stimulation with CAMP. CAMP caused an increased rate of 45Ca influx
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within 10 seconds of stimulation. These results indicated that chemotaxis caused by CAMP may increase the local free calcium ion concentration which may in turn regulate motile activity. Future studies must differentiate between an influx of ions and merely an association of ions with the cell surface or membrane. b. Intracellular p H . The hydrogen ion concentration has been estimated in several cells from all three classes discussed in this article by microinjecting pH indicators and comparing colors with standards (see Chambers and Chambers, 1961). Average pH values in the cytoplasm of single cells have been detennined for freshwater amoebas (pH 7.0 f 0.4) (Chambers et al., 1927; Spek and Chambers, 1933; Needham and Needham, 1925), various echinoderm eggs (pH 6.8 2 0.2) (Needham and Needham, 1925), gastric epithelium (pH 6.7 2 0.2) (Chambers, 1933), human sarcoma cells (pH 6.8 f 0.1) (Chambers and Ludford, 1932) and many other cells (see Chambers and Chambers, 1961). These studies were based on the qualitative comparison of cells with standards, so that the absolute values may not be accurate. Furthermore, pH measurements have been made directly with pH microelectrodes inserted in the cytoplasm of cells, including Physarum (Gerson and Burton, 1977). In this latter study, it was demonstrated that the average intracellular pH varied from ca. 6.6 to ca. 6.0 during the cell cycle. This study was significant since it may explain the range of pH values measured in other cells. That is, the pH could change during cellular activity including movement. In fact, Taylor et al. (1978) identified a lower pH in the tail of Chaos compared to that in the pseudopods, using a ratio photometer and the dye phenol red. Recently, an increase in intracellular pH was demonstrated directly in sea urchin eggs during fertilization (Shen and Steinhardt, 1978). pH sensitive microelectrodes indicated that the pH increased from ca. 6.9 to ca. 7.4 during fertilization and remained at the higher value for over 20 minutes. These results differ from previous reports (Johnson et al., 1976) which suggested initial acidification. The absolute changes in pH will require further verification. Measurement of extracellular pH in unbuffered cell suspensions have indirectly identified changes in pH during cellular activity. Malchow et al. (1978) have identified a decrease in the extracellular pH of cell suspensions of D . discoideum treated with nanomolar concentrations of CAMP. Interestingly, removal of extracelldar calcium increased the two observed proton peaks up to 10-fold. These indirect measurements suggested that the cytoplasmic p H increases when movement is stimulated by CAMP. The control of gelation and contraction of cell extracts from D . discoideum by pH as well as calcium (Section 111, B) supports the hypothesis that transient changes in pH could play a regulatory role in cell movement. Tilney (1977) and Tilney et al. (1978) have demonstrated that several echinoderm sperm rapidly exhibit the acrosomal reaction when the intracellular pH is increased. Measurements of extracellular pH in a suspension of cells and
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utilization of proton ionophores demonstrated that the polymerization of actin in the acrosomal process required the elevation of intracellular pH. These investigators suggested that a two step change in intracellular ionic conditions was required for the completion of the acrosomal reaction. Calcium was required for fusion of the acrosomal vacuole, while an increase in pH was necessary for the polymerization of actin. The ionic modulation of interactions between actin and other cytoskeletal proteins is discussed further in Section 111, B. The intracellular cytoplasmic pH has also been shown to change in cells when damaged or when microinjected with divalent cations. Cells exhibited an “acid of injury” when damaged, since the pH dropped as low as pH 5.0 when amoebae or echinoderm eggs were injured (Chambers and Chambers, 1961). In addition, the microinjection of small volumes of 5.0 mM calcium induced reversible and dramatic decreases in pH in amoebae. However, the cytoplasmic pH increased to the normal level during recovery. This latter observation suggests that intracellular pH and pCa might be interrelated (Taylor et al., 1976; Condeelis and Taylor 1977; Hellewell and Taylor, 1979). The possible relationship between the changes in free calcium ion concentration and pH must be studied in more detail since the regulation of both cytoplasmic structure and contractility could involve changes in both of these parameters. c. Studies of Ionic Manipulations in Chaos. Allen and Taylor (1975) and Taylor ( 1977a) microinjected contraction and relaxation solutions into single specimens of Chaos in order to ascertain the effect of calcium, ATP, and pH on the endoplasm and ectoplasm of living cells. These solutions were designed to meet the known ionic requirements of giant amoebas (Taylor et al., 1973; Bruce and Marshall, 1965; Friz, 1971). Anterior endoplasm as well as all of the ectoplasm contracted in response to the microinjection of l/10 the cell volume of a threshold calcium ion concentration. It was determined that the more viscoelastic anterior endoplasm and ectoplasm were the most contractile regions, suggesting a relationship between cytoplasmic consistency and contractility. Either raising the calcium ion concentration above threshold or increasing the volume of the contraction solution caused reversibly contracted regions to form in the injected amoebas. The same results were obtained when a low calcium relaxation solution at an elevated pH (pH greater than 7.0) was microinjected. Therefore either micromolar calcium or local changes in pH were observed to induce dramatic transitions from the gelled viscoelastic ectoplasm or the viscoelastic anterior endoplasm to actively contracting regions. It is probably significant that the cells recovering from contractions were not able to form distinct ectoplasm for several minutes (Taylor 1977a). Therefore conditions that induced contractions also appeared to inhibit subsequent formation of the rigid ectoplasmic tube. This phenomenon has also been observed in cell models (Section 111, B). In contrast, either the relaxation solution at pH 7.0 or pH buffers below pH 7.0
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caused loss of the differentiation between endoplasm and ectoplasm in the amoeba. Furthermore, the streaming in intact cells could be arrested by “relaxing” the cells with a solution containing MgATP and a calcium ion concentration less than ca. 1OP6M.It was apparent that changes in the free calcium concentration and/or pH could be involved in regulating cytoplasmic consistency and contractility (Taylor, 1977b).
2 . Pressure and Temperature Marsland and Brown (1936) demonstrated that cytoplasmic gels were Freundlich group I11 gels, since the application of high pressure (up to 10,OOO psi) and/or low temperatures (down to 4°C) favored solation (see Section I). Pressure effects on biological structures have been reviewed extensively (Zimmerman, 1971; Marsland, 1970; Allen, 1961a). Low temperatures and high pressures caused amoeba pseudopodia to retract and the cells to round up according to Marsland and Brown (1936) and Landau et al. (1954). These investigators established that ectoplasmic gel characteristics were reversibly reduced at high pressure (up to 10,OOO psi) and newly formed ectoplasm at the tips of advancing pseudopods were the most susceptible to the solation effects of pressure. High pressure and low temperature also inhibited cytokinesis in marine eggs (Marsland, 1938). Pressure-centrifuge studies have indicated that high pressures and low temperatures tend to weaken the cortical cytoplasm (ectoplasm) of dividing marine eggs. Furthermore, human cells in primary or continuous culture and fibroblasts of embryonic chick heart tissue also lose their normal irregular shape and become spherical under high pressure and reduced temperature (Landau, 1960; Landau and Peabody, 1963). The results of decompression are the same for all the cells described above (Taylor, 1977b). Rapid decompression from pressures applied to cause solation of ectoplasm always resulted in extensive contraction of the cytoplasm. In addition, there were fewer filaments and microtubules evident in the above cells when they were fixed at elevated pressures and/or lower temperatures (Zimmerman, 1971). The reduction in the cytoplasmic consistency of the cell cortex or ectoplasm always resulted in contractions when the temperature and/or pressure was returned to normal. One could speculate that the breakdown of the rigid ectoplasm was necessary for contractions to occur. The rigid ectoplasm in some cells might be a cytoskeleton which must be reduced quantitatively to permit extensive cytoplasmic contractions (Taylor et al., 1977; Condeelis and Taylor, 1977; Taylor, 1977b; Hellewell and Taylor, 1979). 3. Mechanical Stimulation Mechanical stimulation has a complex effect on many amoeboid cells. Lowfrequency vibrations appear to attract amoebas of the proteus type (Christiansen
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D. LANSING TAYLOR AND JOHN S. CONDEELIS
and Marshall, 1965; Bovee, 1960; Kolle-Kralik and Ruff; 1967). In contrast, direct physical contact causes amoeboid cells to stop and reverse direction (Mast, 1932; Folger, 1926). In addition, Angerer (1936) measured a decrease in cytoplasmic consistency after agitating A. proteus by shaking the cultures. It was found that the cytoplasm contracted during the recovery from agitation. The rigid wall of the cleavage furrow of marine eggs was “solated” and the furrow disappeared when the furrow region was mechanically agitated by probing with a microneedle. This breakdown of the cleavage furrow could be localized to one half of the cell, thus inducing one-sided furrows. However, when the agitation ceased, the cell cortex stiffened and cleavage continued normally (Chambers, 1919). Cytoplasm appears to exist in states ranging from a weakly viscoelastic fluid to a highly viscoelastic, thixotropic solid. The states of structure and contractility both appear to be sensitive to environmental stimulation. In addition, a relationship between the consistency and state of contraction also seems to exist in the cells discussed. It was evident from data from many of the cells that a breakdown (solation) of the gel or ectoplasm precedes or at least accompanies contraction. The possible functional relationship between solation and contraction is discussed in Section 111. There are many other interesting environmental effects on the consistency and motility of amoeboid cells that are not discussed in this article. Effects of light, enucleation, drugs, and electrical stimulation have been studied and could yield useful information at the molecular level (Jeon and Danielli, 1971; Lorch, 1972; Jahn and Bovee, 1971; Allen, 1972b). Three chemicals that have a profound effect on actin are worthy of special reference. The cyclic peptide phalloidin, one of the toxic components of Arnanita phalloides stabilizes actin filaments even in the presence of 0.6 M KI and also increases the rate of polymerization of G-actin to F-actin (Dancker er al., 1975; Lijw et al., 1975). Polyamines have recently been demonstrated to induce actin polymerization without losing the characteristic actin activation of myosin ATPase. A linear relationship was shown between the yield of actin polymerization and the chain length of the polyamine (Oriol-Audit, 1978). DNAase I has been shown to have the opposite effect on actin (Lazarides and Lindberg, 1974; see Hitchcock, 1977, for review). The DNAase I binds tightly to actin and blocks the polymerization of G-actin or causes depolymerization of F-actin. These three experimental probes should be valuable in dissecting the state of actin polymerization in whole cells and cell models.
111. Cytoplasmic Structure and Contractility: In Vitro We see how quickly through the colander The wines will flow; on the other hand
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The sluggish olive/oil delays: no doubt, Because 'tis wrought of elements more large, Or else more crook'ed and intertangled. LUCRETIUS
In vitro model systems of cell structure and/or contractility have been investigated in order to identify the basic molecular components, structure, and ionic regulation involved in the complex processes responsible for the cell structure and contractility discussed in Section 11.
A. EARLYMODELSYSTEMS OF CONTRACTILITY 1. Glycerol-Extracted Cell Models Treatment of muscle fibers or bundles of muscle fibers with glycerol causes the sarcolemmas to become partially disrupted. Albert Szent-Gyorgyi (1951) demonstrated that bundles of vertebrate striated muscle could be glycerinated, removing soluble components while maintaining the contractile machinery. The addition of Mg2+ and ATP induced contractions similar to those observed in intact muscle cells. Glycerination methods were extended to motile nonmuscle cells originally by Hoffman-Berling and Weber (1953) and Hoffmann-Berling (1954a,b), reviewed by Arronet (1973) and Seravin (1967) who demonstrated contractile events both in amoeboid interphase fibroblasts and telophase fibroblasts exhibiting active cleavage furrows after glycerination. Glycerol-extracted models have subsequently been prepared from A. proteus (Hoffmann-Berling, 1955; Simard-Duquesne and Couillard, 1962a), C. carolinensis (Rinaldi et al., 1975; Taylor et al., 1976b; Opas and Rinaldi, 1976), leukocytes (Norberg, 1970), P. polycephalum (Kamiya and Kuroda, 1965; Komnick et al., 1970), D . discoideum (Eckert et al., 1977), and tissue culture cells (R. D. Goldman et al., 1976b; Weber et al., 1976; see Arronet, 1973, for review). The usual response of glycerinated cells treated with Mg2+ and ATP was to exhibit small isodiametric contractions that seldom mimicked motile events in vivo. Furthermore, most of the glycerinated models exhibited incomplete or no calcium regulation, which has been discussed by Taylor et al. (1976b). However, the most important characteristic of glycerinated cells is the definite shortening or contractions induced by Mg2+ and ATP, which is reminiscent of the contraction of glycerinated striated muscles. In fact, contractions defined by the generation of tension have been measured in glycerinated fibroblasts. The tension amounted to about %oo that of skeletal muscle on the basis of cross-sectional area (Hoffman-Berling, 1956). The greatest number of physiological contractile events was induced in the highly polarized fibroblasts, where the addition of Mg2+ and ATP caused the thin tails to contract toward the main cell body in a fashion similar to the events in motile living cells (R. D. Goldman et al., 1976b). The ultrastructural basis of isodiametric contractions was demonstrated in
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several of the early glycerinated models. Schiifer-Daneel(l967) identified thick and thin filaments in glycerinated amoebas. However, the filament sizes did not correspond to the thick and thin filaments observed in normal cells (Nachmias, 1964, 1968; Comly, 1973). The most important observation was that the filamentous material condensed after contractions were induced with Mg2+ and ATP. It was further suggested that filaments assembled from a less stmctured form during contractions (Holberton and Preston, 1970). The thin (ca. 50-70 A) filaments have been identified as actin in glycerinated models of many nonmuscle cells (Ishikawa et al., 1969; Komnick et al., 1970; Comly, 1973; Goldman et al., 1976; Eckert et al., 1977; see R. Goldman et al., 1975, for review). Furthermore, thick filaments similar to those identified in intact cells and thought to be myosin aggregates have also been detected in some glycerinated models (Holberton and Preston, 1970; Comly, 1973; Rinaldi et al., 1975). Many actin filaments have been observed in close proximity to the plasmalemma in numerous glycerinated cells, suggesting an anchorage of actin to the membrane (Pollard and Korn, 1973b; Comly, 1973; Komnick et al., 1970; see Tilney, 1977, for a review of actin-membrane interactions). Tilney and co-workers have utilized Triton X-100, which solubilizes membranes rather than “punching” holes like glycerination, to prepare models of the acrosomal reaction in Thyone sperm. These model acrosomal caps were accessible to ionic manipulation and exhibited limited actin polymerization in the presence of Mg2+, ATP, and Ca2+.However, polymerization was incomplete. Interestingly, raising the pH to ca. pH 8.0, caused the actin to solubilize from the acrosomal caps (Tilney, 1976b, 1977). Actin was localized in glycerinated tissue culture cells by fluorescent antibody labeling (Weber et al., 1976). This study showed that the actin-containing fibers (bundles) shortened and thickened during ATP-induced contractions. These results confirmed the presence of a condensing or contractile process in the activated models. Furthermore, cytochalasin B had no effect on the contraction of glycerinated cells but did alter the distribution of actin in cells prior to glycerination. Apparently, cytochalasin B altered the interaction between actin monomers or other actin-associated proteins other than myosin (Section 111, B). Glycerinated models have been valuable, since they have demonstrated contractile events involving actin and thick filaments (at least in the giant amoeba) upon the addition of a Mg-ATP energy source, which strengthens the notion of actin-myosin contractile processes in nonmuscle cells. However, the value of glycerinated models has been limited, since glycerination produces a rigor model (myosin rigidly cross-linked to actin) while stabilizingonly filamentous actin and myosin. Furthermore, the calcium regulation observed in some living cells is abolished in most glycerinated models. The reduction in calcium regulation may occur as a result of the removal of specific soluble proteins and/or alteration in the supramolecular form of the proteins involved in regulation (Taylor et al.,
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1976b). Therefore these models are valuable for studying single-event contractions but not for studying cycled motile events involving filament assembly or cytoskeletal transformations. 2. Superprecipitation of High-Salt Extracts Weber and Portzehl (1952) demonstrated that actin and myosin could be extracted from muscle at a high ionic strength (0.6 M KCl). The extract exhibited a fall in viscosity upon the addition of ATP as the result of dissociating myosin from actin. In contrast, superprecipitation, considered to be a biochemical analog of contraction, occurred when the ionic strength of the extract was lowered to 0.1 M KCl in the presence of ATP. High ionic strength extracts were prepared subsequently from P . polycephalum by Loewy (1952). The demonstration that ATP dissociated the presumptive “actomyosin” prompted Loewy to suggest that muscle contraction and cytoplasmic streaming in P . polycephalum were based on similar biochemical constituents. In addition, Bettex-Galland and Luscher (1965) extracted a crude actomyosin from human platelets which they called thrombosthenin. Within a period of a few years, crude actomyosin-like extracts were prepared from a variety of cells (see Pollard and Weihing, 1973, for review). The effects of ATP on the actomyosin extracts from cells suggested that actinand myosin-like proteins might be present in a wide variety of cells and that the interaction of these proteins could be responsible for such diverse motile events as cytoplasmic streaming and direct contractions. However, no calcium regulation was detected in these early models. “Actomyosin threads” were formed when the ionic strength of high-salt extracts was lowered rapidly by squirting the solutions through hypodermic needles into low-salt solutions. These models of muscle (Szent-Gyorgyi, 1951; Portzehl, 1951) and Physarum (Beck et a l . , 1969) consisted of a three-dimensional network of actin and myosin filaments that contracted to approximately half the original volume after the application of ATP. It has been suggested that the actomyosin threads isolated from Physarum react the same as the fibrils in vivo (Komnick et al., 1973; Hinssen and D’Haese, 1976). Matsumura and Hatano (1978) controlled the formation of actomyosin bundles and myosin filaments by varying the ATP concentration in synthetic actomyosin from Physarum. At physiological ATP concentrations (ca. lov4M )the myosin filaments were ca. 0.2 pm in length and interacted with actin as judged by the ATPase activity. In addition, the reversibility of superprecipitation was correlated directly with the shortness of myosin filaments. Actin bundles formed when the ATP concentration was lower than ca. lop6M. Therefore contractions of these bundles as judged by the ATPase activity would require an elevation of the ATP concentration. The possible relationship between the bundles formed in vitro and those detected in living Physarum (Section 11, E) will require more
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extensive investigation. This study is interesting since modulation of the intracellular ATP concentration could play a role in controlling the cytoskeletal and contractile state. Oplatka and his co-workers investigated models of cytoplasmic streaming (Oplatka and Tirosh, 1973). They observed streaming motions in solutions of partially purified actomyosin, purified actin, and myosin or myosin subfragments from rabbit psoas muscle and P. polycephalum and postulated that the motion was generted by the reversible interactions of myosin with actin, which propelled actin through the solution. Because HMM or S1 subfragments of myosin were observed to cause the same type of streaming activity, superprecipitation, and contraction of glycerinated muscle models as observed with whole myosin molecules (Oplatka et al., 1974, 1975), it was suggested that the tail portion of the myosin molecules was not required for movement. The interpretation of these experiments has been challenged by Matsutake et al. (1975) who suggested that the streaming activity observed in microcapillaries was not the result of actomyosin activity but some uncharacterized microscopic fluid properties. This interesting motile model system has not been analyzed in enough detail to warrant acceptance or rejection of the proposed interpretations.
OF CYTOPLASMIC STRUCTURE AND CONTRACTILITY B. MODELS
1. Single-Cell Models
Motile models prepared from single cells have permitted investigations of the consistency and contractility of cytoplasm under suitable physiological conditions. Furthermore, the effects of ionic environment, temperature, and mechanical agitation have been assayed in membraneless systems. Allen et al. (1960) initiated this experimental approach by rupturing single C. carolinensis inside quartz capillaries. The cytoplasm, which was maintained in the normal ionic milieu in the capillaries, exhibited patterns of cytoplasmic streaming reminescent of the fountain pattern observed in intact cells. This important observation indicated that the motile force for movement was inherent at least partially in the cytoplasm and that a membrane-closed system was not required for cytoplasmic streaming. Gicquand and Couillard (1970) subsequently attempted to maintain streaming in cytoplasmic droplets obtained from A. proteus. Streaming and the appearance of contracting fibrils was maximized in the presence of Mg2+, EGTA, and ATP, while Ca2+ was suggested to inhibit motility. However, the duration of streaming was short compared to the original results of Allen et al. (1960). It is likely, in retrospect, that the calcium actually caused rapid and irreversible contractions rather than inhibition. Single-cell models were also utilized to determine the ionic regulation of cytoplasmic structure and contractility. Taylor et al. (1973) designed several
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different physiological solutions for amoeba cytoplasm, which mimicked the conditions for contraction, relaxation, and rigor of vertebrate striated muscle. At pH 7.0 calcium ions regulated both cytoplasmic structure and contractility. Just M free calcium the cytoplasm was viscoelastic and below ca. 7.0 X nonmotile (gel), while a threshold concentration of calcium ca. 7.0 X lo-’ M induced force-generating contractions as the overall cytoplasmic structure decreased. Actin and myosin filaments were identified morphologically, and ATP was identified as the energy source. In addition, fibrils were observed both in the low-calcium viscoelastic state, when the cytoplasm was oriented with a micropipet, and in the streaming loops that erupted from the cytoplasmic droglets in the presence of a threshold contraction solution (Fig. 13). The streaming cytoplasm formed loops that extended into the medium and returned to the main cytoplasmic mass. The outward extending cytoplasm exhibited a similar rate of streaming and refractive index as endoplasm, while the returning cytoplasm exhibited a similar rate of streaming and refractive index as ectoplasm in intact cells (Taylor et al., 1973) (Fig. 13). The gradient of cytoplasmic consistency was suggested to involve the interaction between actin and myosin. Finally, the stabilized cytoplasm (a viscoelastic gel) contracted without streaming when the temperature was shifted from 25” to 4°C and then to 25”C, or when the cytoplasm was agitated with a micropipet. These single-cell experiments on C. carolinensis demonstrated a calciumregulated, ATP-dependent contractile basis of movement which involved variations in cytoplasmic consistency. The experimental method of isolating cytoplasm from single cells was subsequently applied with success to Acrinosphaerium (Edds, 1975) and mammalian fibroblasts (Izzard and Izzard, 1975), where calcium-regulated contractions were also identified. Calcium-regulatedstreaming was first identified in plasmodial fragments of P. pofycephalum prepared by treating plasmodia with caffeine, which presumably causes membranes to become leaky to calcium (Hatano and Oosawa, 1971). The membrane-bound plasmodial fragments responded subsequently to the extracellular calcium ion concentrations. At free calcium ion concentrations below ca. A4 the cytoplasm was quiescent, while contractions and streaming were initiated by raising the free calcium ion concentration to ca. Wohlfarth-Bottermann and his colleagues (see Isenberg and WohlfarthBotterman, 1976, for review) prepared droplets of endoplasm from Physamm by rupturing individual plasmodial veins. During resorption of the endoplasmic drops by the vein the cytoplasm exhibited dramatic changes in cytoplasmic structure and contractility. The membrane was first reformed locally, followed by the appearance of actin filaments and filament bundles. The parallel use of an electrobalance demonstrated that the endoplasm increased in consistency during the “aging” process. Subsequently, contractions were monitored in the resorbing droplets with a tension transducer. Thus it is evident that a sequence of
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changes in the cytoplasmic consistency preceded movement (Isenberg and Wohlfarth-Botterman, 1976) (Fig. 14). Stossel and his colleagues (Davies and Stossel, 1978; Hartwig et al., 1978) prepared a plasmalemma-hyalin ectoplasm model from rabbit lung macrophages which they termed podosomes. The podosomes were enriched in actin, myosin, and actin binding protein (250,000 MW). Simultaneous analyses of cell bodies and isolated podosomes demonstrated that most of the cytoskeletal and contractile proteins were concentrated at the cell periphery where they are used in motility and phagocytosis. This creative approach to cell fractionation should be applied to other types of cells. A very clever method of demonstrating contractility of cytoplasmic fibrils in tissue culture cells (class 111) has been described by Isenberg et al. (1976). The stress fibers from single rat mammary adenocarcinoma cells have been isolated directly by laser microbeam dissection and induced to contract upon the addition of ATP. Actin was identified as the major constituent of the fibrils, and the fibrils were monitored before and after dissection with polarized light and Nomarski optics. Therefore the fibers described in living cells as stress fibers are capable of contracting, at least in this particular cell type. The above single-cell models have yielded valuable information correlating specific cytoplasmic structures with motile events. The cytoplasmic consistency and contractility observed or measured in living cells (Section 11) have become accessible to quantitative analysis under controlled conditions. The viscoelastic structure of cytoplasm observed in living cells (Section 11, D) has also been detected in model systems. Furthermore, the structural transformations of cytoskeletal andor contractile proteins have been described. These physiological investigations have identified some of the important concepts which require more detailed molecular and biochemical approaches. These concepts include (1) variation of cytoplasmic consistency during contraction, (2) apparent calcium regulation of cytoplasmic structure and contractility, (3) the relationship between the cytoskeletal and contractile roles of actin in nonmuscle cells. 2 . Bulk Cell-Free Extracts The elegant demonstration that naked cytoplasm from single cells retained the capacity to stream and contract (Allen et al., 1960) prompted Thompson and
~~
FIG. 13. Composite of single-cell models of cell structure and motility of C. carolinensis. (A) Cytoplasm isolated from single cells in the stabilization solution (low calcium) and oriented with a micropipet to demonstrate fibrils. X88. (B) Longitudinal sections of fibrils demonstrating parallel bundles of actin filaments and a thick filament (arrow). (C) A membraneless cytoplasmic droplet from a single cell in the threshold contraction solution (Flare solution). Fibrillar pseudopods extend into the solution and actually cause the droplet to locomote. X 124. (Taylor et at., 1973, with permission .)
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0
I
1
I
I
10
20
30
LO
rnin
FIG.14. Tensiometer curve of the contraction behavior of an endoplasmic drop from P. polycephalurn. Ordinate: Tension force in millipoises; abscissa: age of endoplasmic drop in minutes. The curve shows a strong volume contraction of the drop from the age of 30 seconds to the age of 9 minutes. This period is followed by a volume increase and subsequently by the start of cycling radial contraction activity typical of protoplasmic drops which remain on the vein from which they have been protruded. (Isenberg and Wohlfarth-Botterman, 1976, with permission.)
Wolpert (1963) to devise a method of bulk isolation of cytoplasm for biochemical studies. These investigations succeeded in isolating a crude motile extract from A. proteus by differential centrifugation of homogenized cells to remove nuclei and heavy inclusions. When warmed to room temperature in the presence of ATP, the extracts appeared to gel as observed in the light microscope and contracted with streaming patterns similarly to the single-cell model described by Allen et al. (1960). The contracted mass exhibited filaments 9.0-12.0 nm wide which extended from networks of “spongy” material (Wolpert et aE., 1964). These studies indicated that conversion from the cold fluid extract to the warmed, gelled and then the contracting state might involve the interconversion of cytoplasmic elements from a less structured state to filamentousbundles. Furthermore, ATP was implicated in the formation of both filaments and streaming movements. However, calcium concentrations of 1.O-5.0 mM inhibited motility irreversibly by causing the cytoplasm to “clump.” The classic description of the coagulation of cytoplasm in intact cells after the application of calcium (Section II, F) and the clumping process described in these experiments might have actually represented massive calcium-elicited contractions. Subsequent to these experiments Morgan et al. (1967) demonstrated that ethylenediaminetetraacetic acid (EDTA) caused lateral aggregation of thin fila-
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
105
ments (ca. 4.0 nm) into fibrils, and the major protein was compared with actin (Morgan, 1971). Pollard and Ito (1970) and Pollard and Korn (1971) confrmed and extended the observations on cell extracts from A. proteus (class I). Electron microscopy of the extracts in the cold sol state, the gelled state, and the contracted state showed a dramatic transformation from an unstructured form to a random meshwork of filamentous material and finally to bundles of readily identifiable thin filaments. The ca. 5.0- to 7.0-nm filaments were identified as actin filaments, and the assembly of actin into filaments was suggested to be the major structural transformation during the warming process. The extract was fractionated by differential centrifugation, and movements were observed only when thin actin filaments were present with thicker filaments (Fig. 15). In agreement with Thompson and Wolpert (1963) the motility of the extracts did not require calcium ions, however, calcium chelators were not used during the isolation procedure.
FIG.1 5 . Thin section of an extract from A . proreus undergoing streaming and contraction 8 minutes after warming to 22°C with ATP. Thin filaments 50-70 A wide (later identified as actin) radiate from a cluster of 160-A-wide thick filaments (later identified as myosin). X68,OOO. (Pollard and Ito, 1970, with permission.)
106
D. LANSING TAYLOR AND JOHN S. CONDEELIS
The results from investigations on cytoplasmic extracts from A. proteus coincided with many of the observations made on intact cells and single-cell models. The dramatic structural transformations of actin during the apparent “sol-gelcontract” sequence was the most striking feature and possibly forms the basis of the rheological changes detected in living cells and single-cell models (Section II, A and F). However, the absence of calcium regulation in these bulk extracts could not be reconciled with the calcium regulation observed in single-cell models. Therefore Taylor et al. (1976a) investigated the possible calcium regulation of bulk extracts of A. proteus. Calcium control was maintained by preparing A. proteus extracts in the stabilization (rigor-like) solution used in the original single-cell studies (Taylor et al., 1973). The structure and contractility were demonstrated to be dependent primarily on the pH, ATP and calcium ion concentrations. Below pH 7.0 the cytoplasm remained fluid (sol), and calcium-regulated contractions were inhibited. At pH 7.0 the extract gelled at calcium concentrations below ca. l(r6 M. Very few distinct filaments formed during warming in the presence of low calcium and ATP concentrations at a pH below pH 7.0. In contrast, raising the Mg-ATP concentration to 1.O mM caused the formation of some actin filaments without inducing contractions. However, large arrays of free F-actin filaments and many myosin aggregates were detected when the free calcium ion concentraM in the presence of 1.0 mM Mg-ATP, which also tion was raised to ca. induced contractions. These results implicated changes in calcium ad ATP concentrations as well as pH in the regulation of cytoplasmic structure and motility. Furthermore, the transitions from the sol to the contracted state observed in the extracts were equated to similar transitions detected in living cells (Taylor, 1977a). Kane (1975, 1976) prepared low ionic strength extracts from sea urchin eggs in the presence of glycerol and EGTA. Subsequent additions of 1.O mM ATP and 20.0 mM KC1 with warming to 40°C induced the extract to form a solid gel, which prompted Kane to revive the classic terminology of sols and gels. The gel was described as a birefringent network of laterally aggregated F-actin filaments (Fig. 16). Gelation required two proteins with molecular weights of 58,000 and 220,000, in addition to actin in the presence of ATP and KC1. The reconstituted gel consisted of laterally aggregated F-actin filaments (bundles or fibrils) and was not temperature sensitive. These experiments suggested a possible explanation for the cytoplasmic structure of the cortex in marine eggs, particularly the transition from the high consistency precleavage state to the contracting state during cleavage (Section 11, D). The technique of image reconstruction has recently been applied to the paracrystalline bundles of actin and associated proteins in an effort to define the cross-linking structure (DeRosier et al., 1977). However, the calcium regulation of the gelation observed in the crude extract has not yet been reconstituted from purified proteins. Pollard (1976a) prepared a 136,OOO X g supernatant from extracts of A.
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
107
FIG. 16. Paracrystalline structure formed by the recombination of actin 58,000- and 220,ooOmolecular-weight componentsfrom sea urchin eggs. These proteins interact to form a highly birefringent gel network. Negatively stained after 24 hours at 0°C. Scale = 0.1 ptn. X 158,920.
castellunii (class 11) in the presence of 0.34 M sucrose, 1.0 mM ATP, and 1.0 mM EGTA. Upon warming the extract to room temperature a solid gel formed and then contracted (condensed) anywhere from 30 minutes to 2 hours after warming (Fig. 17). The rates of gelation and subsequent contraction were temperature-dependent, suggestive of a Freundlich type 111 gel, and required the presence of ATP and M$+. Although there was no complete regulation of the gelation-contraction cycle, micromolar concentrations of Ca2+ accelerated the contractile stage of this process. The major proteins of the gel were assumed to be identified after compacting the gel by centrifugation. The possible effect of centrifugation on cytoplasmic gels is discussed later. Pollard suggested that the partially purified actin (0.7 mg/ml) would gel in the presence of ATP, MgCh ,and KC1. A dual role for actin was proposed in the cell: Actin could (1) generate forces required for movement and (2) form a cytoskeleton (gel) to determine cell shape. The ultrastructure of the gel was also investigated, and the meshwork appearance of actin was attributed to OsO, destruction of actin filaments. However, this interpretation may be only partially correct since a meshwork appearance is usually identified in gelled extracts and intact cells. Stossel and his co-workers identified a high-molecular-weight protein (ca. 250,000) from rabbit pulmonary macrophages (class 11) that associated with actin in extracts prepared in the presence of 0.34 M sucrose, 0.5 mM ATP, and 1.0 mM EDTA (Hartwig and Stossel, 1975; Stossel and Hartwig, 1975). This pro-
108
D. LANSING TAYLOR AND JOHN S. CONDEELIS
FIG. 17. Gelation and contraction of Acunrhumoeba extracts. When a clear 136,000 X g supernatant is warmed to room temperature, it gels in 20-30 minutes (middle). Upon further standing the gel contracts and expresses soluble components (right). Samples of the gelled extract and contracted gel were centrifuged at 100,000 X g for 60 minutes, and the proteins in the resulting Supernatants (S) and pellets (P) analyzed by gel electrophoresisin SDS. Actin (A) is the chief component of the gel, and it becomes associated with myosin 11 (M)and an unidentified 50,000-molecular-weightprotein in the contracted gel. (Pollard, 1975b, with permission.)
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
109
tein was originally compared with spectrin from red blood cells but has more recently been characterized as a distinct “actin-binding protein” (Stossel and Hartwig, 1976). Stossel and Hartwig (1976) demonstrated the gelation-contraction sequence in the high-speed supernatant fraction of macrophage extracts. ATP and Mg2+ were required for the sequential steps of gelation and contraction. However, there was an apparent calcium inhibition of the transition from the sol to the contracted state. It is possible that this apparent calcium inhibition of contraction actually was a calcium inhibition of gelation, which has been described in D. discoideum extracts (Taylor et al., 1977; Condeelis and Taylor, 1977) sea urchin egg extracts (Kane, 1976), and recently in Xenopus oocyte extracts (Clark and Merriam, 1978). Stossel and Hartwig further demonstrated that purified actin (ca. 2.0 mg/ml) plus actin-binding protein (ca. 0.4 mg/ml) reconstituted a gelled state. The additional presence of purified macrophage myosin and a crude mixture of components called cofactor and 2.0 mM MgCA induced a contraction (Fig. 18). These important experiments suggested that an agent other than myosin could cross-link actin, allowing myosin to move randomly oriented filaments without elaborate mechanical or polarity restrictions on the actin filaments. These same investigators discussed interesting speculations on the molecular dynamics involved in movement and phagocytosis. A subsequent study demonstrated that cytochalasin B caused inhibition or breakdown of the gel (Hartwig and Stossel, 1976), which has also been observed in extracts from HeLa cells (Weihing, 1976a,c), Acanthamoeba (Pollard, 1976b), and D. discoideum (Condeelis and Taylor, 1977). Taylor and co-workers (Condeelis and Taylor, 1977; Hellewell and Taylor, 1979) investigated the calcium-regulated sol-gel-contract cycle in extracts prepared from A. proteus (class I) and particularly D . discoideum (class 11). These extracts are prepared in the absence of sucrose or glycerol and are characterized by the presence of slightly modified stabilization or relaxation solutions originally designed for single amoeboid cells (Taylor et al., 1973; Taylor, 1977a; Condeelis and Taylor, 1977). Most of the studies have been performed on D. discoideum, since large volumes of extract can be prepared easily (Taylor et al., 1977; Condeelis and Taylor, 1977). The extract gels upon warming to 25°C and contracts in response to micromolar Ca”+ or pH in excess of 7.0. Optimal gelation occurs in the presence of a solution essentidly equivalent to the relaxation solution used in single-cell models of C. carolinensis (Taylor et al., 1973) and intact cells (Taylor, 1977a) (2.5 m M (PIPES) buffer, 2.5 mM EGTA, 1.0 mM MgCA, 1.O mM ATP, and 20 mM KCl at pH 7.0) (Condeelis and Taylor, 1977). All experimental conditions that caused the gel to solate also initiated contractions during the solation. The parameters that induced the simultaneous decrease in gel structure and the initiation of contraction included micromolar free calcium at pH 7.0, high myosin concentrations, cytochalasin B, KCl con-
110
D. LANSING TAYLOR AND JOHN S. CONDEELIS
\
\
‘W
myosin
\
--
i
FIG. 18. SDS (5%) gel electrophoresis of gelled supernatant extract of rabbit pulmonary macrophages and of purified macrophage actin, myosin, and actin-binding protein. Right: Morphology of macrophage actin, myosin, and actin-binding protein stained with uranyl acetate and viewed in the electron microscope. X57,785. Inset: X91,OOO. (Stossel and Hartwig, 1975, with permission.)
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
111
centrations 240 mM and 6 1 5 0 mM, MgC12 concentrations ca. 4.0mM, and sucrose, as well as the physical parameters high pressure, cold, and mechanical stress (Condeelis and Taylor, 1977). The solation of gels by high pressure has been demonstrated clearly by attempting to sediment the gelled extract from D . discoideum. The forces required to sediment the gel caused at least a partial solation and contraction when myosin was present (Condeelis and Taylor, 1977). This experiment suggests that caution must be exercised when centrifugation is used to collect and characterize the components in cytoplasmic gels. The contractile effect of all these parameters had previously been demonstrated in intact cells (Section 11, F) (Taylor, 1977b). Gelation was demonstrated to occur in extracts from D . discoideum even in the absence of myosin, while contraction required the presence of myosin (Condeelis and Taylor, 1977). Transition from the initial sol state to the gel state involves a large increase in turbidity at 350 nm, which is larger than the increase in turbidity of pure actin at the same concentration, suggesting supramolecular interactions. In addition, transition from the initial sol state to the gelled state does not increase the optical anisotropy, although strain birefringence can be induced in the gel as it can be in intact cell cytoplasm (Fig. 19). However, the ultrastructure of the gel has been difficult to characterize, since both the initial sol and the gel are optically isotropic. The optical isotropy of the gel from D . discoideum represents a distinct difference from the optically anisotropic (bire801-
GE L AT I 0 N
1.0
2.o
30
4.0
Time ( m i d FIG. 19. Strain birefringence assay for gelation in extracts of D . discoideum. The phase retardation r was monitored versus time and the applicationof lO-p,m stretches by a micropipet inserted into the extract (bars). No strain birefringence could be induced until the extract gelled. (Condeelis and Taylor, 1977, with permission.)
112
D. LANSING TAYLOR AND JOHN S. CONDEELIS
fringent) gels from Acanthamoeba (Pollard, 1976) and sea urchin eggs (Kane, 1975). Furthermore, both negative staining and thin-sectioningprocedures on the D. discoideum gel reveal a complex aggregate structure characterized by the absence of free readily identifiable F-actin filaments, although these aggregates may be composed of highly cross-linked and disorganized F-actin. In contrast, transition from the gelled to the contracting state was characterized by a large increase in turbidity (Taylor et al., 1977) and a dramatic increase in the number of apparently free readily identifiable F-actin filaments, using the same ultrastructural preparative procedures applied to the gel (Condeelis and Taylor, 1977) (Fig. 20). The apparent change in actin morphology may arise most simply by the dissociation of F-actin from the complex aggregate gel state to form free F-actin filaments. However, our present ultrastructural methods cannot rule out a more complicated transformation of actin including polymerization-depolymerization and changes in actin filament structure. Since the supramolecular structure of actin could not be adequately defined during the sol-gel-contract cycle, the qualitative differences were described as actin transformations. In addition, the breakdown of the myosin-free gels under the same ionic conditions that promoted contraction in gels containing myosin suggested that gel breakdown and contraction occurred concurrently or sequentially. Since the breakdown or solation of myosinless gel occurs under exactly the same conditions as the contraction of a myosin-containing gel, the sequence of steps (sol-gel-contract) has been named the sol-gel-gel breakdown and contraction cycle (Taylor et al., 1978). The calcium and/or pH regulation of the sol-gel-gel breakdown and contraction cycle was suggested as a potential regulatory mechanism, in which the gel inhibited or at least minimized actin-myosin shortening. The protein(s) involved in gelation have not been identified specifically, but a partially purified preparation from D. discoideum containing actin, 250,000; 95 ,000; 90,000; and 75,OOO molecular weight, and several low-molecular-weight components, exhibited calcium- and pH-regulated gelation (Condeelis and Taylor, 1977). More recent results (Hellewell and Taylor, 1979; Taylor et al., 1978) have indicated that an intact high-molecular-weight protein (250,000) may not be required for calcium- and pH-sensitive gelation. This recent model system was derived from the contracted pellets of D. discoideum cell free extracts. Therefore, the calciii- a.nd pH regulated gelation and contraction observed in this partially purified preparation depended on proteins that remained associated with actin andlor myosin during contraction. The interaction of the regulatory proteins with the contractile cytoskeleton in living cells is an important unanswered question. Future work should identify the calcium- and pH-regulated gelation factors, as well as the relationship between gelation and the low-calcium inhibition of contraction. The results with extracts from D. discoideum match very well the single-cell models of amoeboid movement (Taylor et al., 1973) and investigations on intact cells (Section II).
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
113
FIG. 20. Electron microscope observations on gelled (A and C) and contracted (B and D) extracts of D. discoideum. (A) Aggregates and short, thin filaments are observed after negatively staining the S3 gelled in relaxation solution at pH 7.0 in uranyl acetate. (B)S3 in relaxation solution at pH 7.0 after contraction with CdEGTA = 0.4 demonstrates an increase in the number of thin filaments measuring 6-8 nm in diameter; these filaments contain actin as demonstrated by the HMM binding technique. (C) A thin section of gelled 53 as in (A). (D) A thin section of contracted gel as in (B). Scale = 0.1 pm. x52,975. (Condeelis and Taylor, 1977, with permission.)
114
D. LANSING TAYLOR AND JOHN S. CONDEELIS
Changes in cytoplasmic structure and contractility appear to be distinct but integrated events. A simple model of the relationship between gelatiofi and contraction has been proposed (Taylor et al., 1977; Condeelis and Taylor, 1977) and further discussed by Taylor et al. (1978) (Fig. 21). Gelation has also been demonstrated in extracts from HeLa cells (class 111) prepared in a buffer containing sucrose, ATP, EGTA, imidazole, and Triton X-100at pH 7.0 (Weihing, 1976a,c) Very little HeLa cell myosin remained in the extract supernatant, reminiscent of the sedimentability of myosin in A. prof e w extracts (Taylor et d.,1976a; Condeelis, 1977a), and contractions were possible only upon the addition of exogenous myosin. Similar to the results for
A
GEL
/
CONTRACT
\
GEL
-
Bv
FIG. 21. (A) A highly schematic diagram of a gel containing actin cross-linked by as yet incompletely characterized actin-binding protein(s). The physical state of the actin has not been adequately characterized. (B) A highly schematic diagram of the gel breakdown and contraction induced by raising the pH or by raising the free calcium ion concentration to ca. 1 .O p m . The affinity of the actin-binding protein(s) is diminished, and the myosin actively pulls the free ends of F-actin filaments. Bipolar arrows, Myosin; spheres, unidentified actin-binding protein(s); lines, actin of unknown supramolecular state. (Taylor et al., 1978, with permission.)
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
115
D . discoideum extracts (Condeelis and Taylor, 1977), myosin or myosin subfragments inhibited the formation of a gel. Interestingly, the addition of HMM or myosin decreased the cosedimentation of actin and the high-molecular weight protein (ca. 280,000) suggested but not identified as the gelation factor. The 280,000-molecular weight protein was previously shown to be an enriched component in plasma membrane fractions of HeLa cells (Weihing 1976a). Weihing has suggested that dynamic changes in the interaction of actin with myosin and the 280,000-molecular weight protein at the cell membrane interface might determine motile events. Maruta and Kom (1977a) recently isolated five proteins from extracts of Acanthamoeba, which separately induced the gelation of actin. The four most active proteins had subunit molecular weights of ca. 23,000; 28,000; 32,000 and 38,000. These results indicated that proteins other than the high molecular weight components described in macrophages (Stossel and Hartwig, 1976), HeLa cells (Weihing, 1976a,c), marine eggs (Kane, 1976), Acanthamoeba (Pollard, 1976), Dictyostelium (Taylor et al., 1977; Condeelis and Taylor, 1977) and A . proteus (Taylor et al., 1976a) should be investigated in more detail. However, the low-molecular-weight fractions may be active proteolytic products of the high-molecular-weight proteins (Hellewell and Taylor, 1979). In contrast to the results with D. discoideum extracts (Taylor et al., 1977; Condeelis and Taylor, 1977; Hellewell and Taylor, 1979) and sea urchin egg extracts (Kane, 1976), high concentrations of divalent cations (2.0-5.0 mM MgZ+ or C 2 + ) were required for the gelation of purified Acanthamoeba models (Marutaand Kom, 1977a). Furthermore, gelation assays on proteins from Acanthamoeba were performed at pH 8.0. The presence of divalent cations and a pH greater than 7.0 during gelation cannot be reconciled with the solation effects of these ionic conditions on extracts from D . discoideum. Care should be exercised in the future when determining the ionic conditions for gelation assays. The physiological relevance of Maruta and Korn ’s observations are questionable in view of the extensive studies on ionic regulation in other cell extracts. In the same set of experiments discussed above Maruta and Kom identified a second myosin protein from Acanthamoeba characterized by a high C$+- and low (K-EDTA-ATPase with an apparent heavy chain molecular weight of 170,000 in sodium dodecyl sulfate (SDS). The physical properties of this “new” myosin have recently been reported (Section IV). A sol-gel-gel breakdown and contraction cycle has been observed in other cell types including leukocytes (Class I) from leukemic patients (Boxer and Stossel, 1976), human platelets (special motile events) (Lucas et a l . , 1976) P . polycephalurn (Heiple, 1977; V . T. Nachmias, personal communication, 1977), Ehrlich tumor cells (Mimura and Asano, 1978) and Xenopus oocytes (Clark and Merriam, 1978; Memam and Clark, 1978). The distinct phenomenon of gelation must be investigated in more detail using more quantitative analyses. To date most of the assays have been qualitative,
116
D. LANSING TAYLOR AND JOHN S. CONDEELIS
assaying structure by slowly inverting test tubes. Measurements of such properties as strain birefringence (Francis and Allen, 1971; Taylor, 1977a; Condeelis and Taylor, 1977; Hellewell and Taylor, 1979) must be utilized in characterizing the cytoplasmic consistency. Methods that could be applied both in vitro and in vivo; such as the heavy particle method (Section 11, D, 3) and strain birefringence, would yield particularly important information. In addition, the concentration of actin and any accessory proteins must be kept within a range believed to be physiologically relevant. Furthermore, the ionic environment must be regulated critically in order to ascertain the exact ionic requirements for gelation in each model system. One of the major problems in comparing the gelled states from different systems is that several different gel or structured states can exist, differing from one another by the form of actin that is cross-linked and by the cross-linking protein@)involved (Taylor et al., 1977; Condeelis and Taylor, 1977). Some of the possibilities include: 1. Gelation may not require the formation of F-actin but may result from the formation of a flexible polymer of actin produced by warming G-actin in the presence of actin-binding proteins. Such a polymer of actin has been described by Hatano (1972), and the protein responsible for this behavior has been isolated (Hatano and Owaribe, 1976). Formation of the polymer required the presence of millimolar Mg2+ and ATP and was transformed to F-actin by the addition of 50 mM KCl. A similar process has recently been described for purified rabbit actin, and the troponin-tropomyosin complex (Ishiwata, 1973, 1978; Ishiwata and Kondo, 1978). These latter studies indicate that gelation can occur in a possibly physiological process. 2. Gelation may result from the binding of G-actin to accessory proteins. Evidence for such a structure was demonstrated with electron microscopy by Tilney (1976b) for Thyone acrosomes. 3. Gelation may arise from the random cross-linking of F-actin filaments by actin-binding proteins, forming an isotropic F-actin network. 4. Gelation may result from the precise cross-linking of F-actin into parallel bundles by actin-binding proteins. Birefringent gels would result and have been identified in sea urchin egg extracts and reconstituted models (Kane, 1976). Similar actin bundles have been identified in the acrosomal process of Lirnulus sperm (Tilney, 1975), extracts from A. proteus (Pollard and Ito, 1971; Taylor et al., 1976a), isolated cytoplasm from C. carolinensis (Taylor et al., 1973, and partially purified actin in response to high concentrations of Mg2+ and Ca2+ (Spudich and Cooke, 1975). 5. Gelation may also develop as a result of a rigor-like linkage between actin and myosin.
Therefore it has become important to define the high-consistency, nonmotile states of cytoplasm and models reconstituted with purified proteins with regard to
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
117
proteins involved, ionic conditions, and ultrastructure. The general and nonspecific term gel must be replaced in the future with more definite terms describing the type of gel and the molecular basis of its formation. The same statement can be made for the term sol, which might vary over a considerable range as a result of contained structures. Furthermore, another basic problem has been the description of gelation as a simple, uniform process. However, at the level of the cell it is possible that some actin-binding proteins exist in specific locations within cells, such as on the membrane, while other actin-binding proteins are free to organize actin within the cytoplasm. Therefore the preparation of model systems from bulk extracts could artifactually produce a homogeneous system. The future interpretation of biochemical events must be correlated with protein localization studies in intact cells. In short, the phenomenology of crude extracts and interactions of purified proteins must be related to the organization and phenomenology measured in living cells. Model systems of cytoplasmic structure and contractility have demonstrated that amoeboid cells in class I, class 11, and class In, as well as several special motile events, possess the ability to form rigid gels that can contract. The grossstructural properties of the gels are reminiscent of those of the ectoplasm or cortex of the individual cells described in Section 11. The possible close correlation between cytoplasmic structure (consistency) and contraction in vivo (Section II) has been demonstrated in vitro (Taylor et al., 1977, 1978; Condeelis and Taylor, 1977; Hellewell and Taylor, 1978). The absolute relationship between cytoplasmic structure and contraction has not yet been defined, but the model systems should stimulate rapid advances. The next major step in the investigation of cytoplasmic structure and contraction is identification of the proteins involved in gelation and contraction and then the reconstitution of a fully regulated model from purified proteins. Section IV summarizes some of the basic biochemical facts concerning actin, myosin, and the associated cytoskeletal and/or regulatory proteins. Most of the data are presented in the form of tables in order to compare the molecular components from the different classes of cells that exhibit similar phenomena. The remainder of Section IV is devoted to exploring some of the purely biochemical characteristics of actin and myosin that could ultimately explain the possible supramolecular forms.
IV. Actin, Myosin, and Associated Contractile Proteins The isolation, purification, and characterization of actin and myosin, as well as cytoskeletal and regulatory proteins, have begun to advance our knowledge concerning how these proteins interact to determine cell structure and to cause and regulate cell motility. A large body of information is now available on the
118
D. LANSING TAYLOR AND JOHN S. CONDEELIS
contractile and associated proteins from vertebrate striated muscle (see reviews by Heilmeyer et a l . , 1976; Mannherz and Goody, 1976; Lowey, 1971; Oosawa and Kasai, 1971; Squire, 1975; Cohen, 1975; Weber and Murray, 1973; Gergely, 1976; k h a n , 1976) and vertebrate smooth muscle (Shoenberg and Needham, 1976), as well as some nonmuscle sources (Pollard and Weihing, 1973; R. D. Goldman et a l . , 1976a, Hitchcock, 1977; Clarke and Spudich, 1977). Because the nonmuscle contractile proteins have been compared in detail in these reviews, we limit the following discussion to the cells that have been studied at the physiological and biochemical levels (see Sections I1 and 111). Vertebrate striated muscle and vertebrate smooth muscle have been used as sources in the comparison of contractile systems.
A. ACTIN 1. Basic Characteristics Actin is one of the most ubiquitous proteins and appears to be highly conserved in its general properties (Tables I and 11). It is also present in many nonmuscle cells (Pollard, 1977b; Weihing, 1976b) in very large amounts, constituting between 5 and 15% of the total cell protein, which is remarkable considering the amount of actin found in skeletal muscle (Table I). The ca. 43,000-molecular-weight globular protein, actin, has been highly conserved, and there is very little difference in the amino acid sequences between vertebrate striated muscle actin and several nonmuscle actins (Elzinga and Lu, 1976). For example, there is variance between vertebrate striated muscle actin and Acanthamoeba actin at only about 6.0% of the amino acid positions (Elzinga and Lu, 1976). However, these slight differences may be important structurally and functionally. In fact, it has been shown that actin from different tissues is the product of distinct structural genes (Lu and Elzinga, 1976; Storti and Rich, 1976). A detailed structural analysis of different actins by x-ray diffraction will be possible in the future, since crystals of actin have been prepared (Carlsson et a l . , 1976; Lindberg et al., 1976). Many of the properties of actin prepared from muscle and nonmuscle sources are very similar, as shown in Table II. Purified actin can exist in two forms: G-actin or monomeric actin at low ionic strength, and F-actin or polymeric actin at 0.1 M ionic strength. In skeletal muscle actin is found primarily as F-actin. However, in nonmuscle cell cytoplasm, actin may exist in several additional forms (Table I): profilamentous, actin bundles, actin networks, and apparently amorphous complexes.
2 . Possible Reasons for Variable Actin Structures At present there are two possibilities under consideration as to how actin can assume these additional forms in nonmuscle cytoplasm: (1) actin is heterogene-
TABLE I PROPERTIES OF ACTIN
Polymerization, 11 KED (dYgm)
Cell protein Sourced
(%) ~____
Rabbit skeletal muscle Acanthamoeba
25 10-15
CJNDER PHYSIOLOGICAL CONDITIONS
0.1 M KCl
2 mM Mg
KCI plus Mg
Actin + HMM ATPase HMM ATPase
Components associated with actin‘
Supramolecular forms of actin under physiological conditionsa
6.1 9
-
20-50 I
100 (actinin) 280, 170, 160,93, 68, 50, 35, 38, 32, 28, 23 280, 180, 95, 48, 32
F-actin (0.1 M KCI, pH 7.0) F-actin, gelled actinb, actin bundlesb, G-actin, amorphous aggregate G-actin, F-actin, gelled actinb, actin bundlesb, amorphous aggregate G-actin, F-actin, gelled actinb, actin bundles” amorphous aggregate G-actin, F-actin, gelled actinb, actin bundlesb, amorphous aggregate G-actin, F-actin, MgZ+polyme+, amorphous aggregate G-actin, F-actin, gelled actin*, (specifically requires the 280,000 component) G-actin, F-actin, actin bundlesb G-actin, F-actin G-actin, F-actin, gelled actinb, (requires the 280,000 component), actin bundlesb (requires the 58,000cornponent) (continued)
~
6.1 9.2
Amoeba proteus
8
Chaos carolinensis
8
3.5
-
2. I
5
250, 95, 75, 50, 38, 30, 28, 25, 18
2-4
5.6-10.9
10.4
0.56
4
45 (actinin)
9.5
2.3
-
10-15
-
-
-
-
10-13
2.1
0.29 0.91
Dictyostelium
5
Physarum
Macrophage
Platelets Fibroblast Sea urchin egg (echinoderm)
-
40
12 -
15 8
-
-
280, 90
?
Actinin 220, 58
TABLE I (continued) Cell protein Source Thyone acrosome (echinoderm)
(%)
Polymerization, 1)RED (dYgm) 0.1 M KC1
20
3
2 m M Mg
-
KCl plus Mg
-
Actin + HMM ATPase HMM ATPase
-
~
a Physiological conditions are here defined as the ionic conditions that approximate those in vivo,
Components associated Supramolecular forms of actin with actin" under physiological conditions" 250, 230, 17
F-actin, gelled actin* (250,000, 230,000, and 17,000 components appear to be involved), actin bundlesb, amorphous aggregate (same as gelled actin, here also called profilamentous actin) ~~
for example, 0.1 M KC1,l mM ATP, 1-4 mM MgCI2, 10-7-10-5M
CaClb52, pH ca. 7.0. "ormation of this structure under physiological conditions requires actin-associated proteins. CComponentsassociated with actin other than tropomyosin, troponin and myosin identified as molecular weight in SDS x lW3. dReferences:Rabbit skeletal muscle (Gordon etal., 1976a; R. Kamiya etal., 1972; Elzinga er al., 1973; Eisenberg and Moos, 1968; Hayashi and Tonomura, 1970; Cohen, 1966; Rees and Young, 1967). Acanthamoeba (Weihing and Korn, 1971; Gordon et al., 1976b; Pollard and Korn, 1973a,b; Maruta and Korn; 1977a, Pollard, 1976a). Amoeba proteus (Condeelis, 1975, 1976; Taylor et al., 1977). C h s carolinensis (Taylor et al., 1973, 1976; Condeelis, 1975). Dictyostelium (Spudich and Cooke, 1975; Wooley, 1972; Taylor et al., 1977; Condeelis and Taylor, 1977; Hellewell and Taylor, 1979). Physarum (Hatano and Owaribe, 1976; Jacobson et al., 1976; Adelman and Taylor, 1969a,b; Totsuka and Hatano, 1970). Macrophages (Hartwig and Stossel, 1975; Stossel and Hartwig, 1975, 1976). Platelets (Probst and Luscher, 1972; Pollard ef al., 1974; Adelstein and Conti, 1973). Fibroblasts (Yang and Perdue, 1972; Schollmeyer et al., 1976). Sea urchin egg (Miki-Noumura and Oosawa, 1969; Hatano et al., 1969; Miki-Noumura and Kondo, 1970). Thyone (Tilney et al., 1973; Tilney, 1976b).
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
121
ous; that is, minor modification of the amino acid structure of otherwise highly conserved actin might endow actin in a single cell type or in different cells and tissues with the ability to assume different forms and/or functions. (2) The association of actin with various actin-bindingproteins accounts for the multitude of forms. a. Heterogeneous Actin. It is now established that various forms of actin, differing from one another by small changes in the primary structure of the protein, exist in different cells and tissues and within a given cell. Gruenstein and Rich (1975) demonstrated different tryptic peptide maps of chicken brain and muscle actins. There is evidence for amino acid substitution differences in human muscle and platelet actin (Elzinga et al., 1973) and in bovine brain; skeletal muscle and cardiac muscle actin (Lu and Elzinga, 1976). Furthermore, actin prepared from 3T3 fibroblasts and HeLa cells was found to differ from chicken muscle actin by several tryptic peptides (Gruenstein et al., 1975). These differences may not be merely due to interspecies changes, since tryptic peptide analysis has failed to show differences in actin prepared from chicken and from mammalian muscle (Carsten and Katz, 1964). Multiple forms of actin have been identified by several laboratories, using two-dimensional gel electrophoresis techniques (Garrels and Gibson, 1976; Whalen et al., 1976; Rubenstein and Spudich, 1977). These investigations demonstrated three forms of actin that were distinguishable on the basis of their isoelectric points. The three forms are called a , p , and y . a-Actin is the most acidic, while y-actin is the most basic. a-Actin appears to be the major form found in muscle tissues containing sarcomeres, while p - and y -actin are found primarily in nonmuscle cells. These different forms of actin probably do not result from posttranslational modification, since evidence has been advanced (Storti and Rich, 1976) that the different forms of actin are synthesized in vitro using mRNAs from embryonic thigh muscle. This suggests that cytoplasmic @3 and y ) and muscle (a) type actins may be coded for by different structural genes. This may have important implications for the regulation of actin synthesis during the cell cycle and development. In addition to the a , p , and y forms of actin, Garrels and Gibson (1976) have presented evidence for two unstable forms of actin in B35 nerve cells, which are more basic than y . The lifetime in the cell of these forms of actin was estimated to be <2 hours, suggesting a metabolic relationship with one or more of the major forms. The significance of these various forms of actin remains unknown. Future studies will require that differences in the isoelectric point and peptide maps of actin be correlated with the physical and enzymic properties of the intact protein under physiological conditions. Differences in these properties may be anticipated, since there is evidence for two different forms of actin with different
122
D. LANSING TAYLOR AND JOHN S. CONDEELIS TABLE 11.
PRoPERTrEs OF
Polymerization ?RED (dYgm)
Sourcea Rabbit skeletal muscle Acunthumoeba Physarum
Activation of rabbit muscle HMM Molecular (micromoles Pi per second weight 0.1 M KC1 2 mM MgCI, per micromole HMM) 41,785 42,000 45,000
6.7, 10.0 9.2, 3.9 10.9
6.7 9, 7.9 10.4
12 12
-
"References: Rabbit skeletal muscle (Gordon e t a l . , 1976a; Elzinga et al., 1973; R . Kamiya erul., 1972; Tsuboi, 1972; Tsuboi, 1968; Adelstein and Kuehl, 1970; Hatano and Owaribe, 1976). Acanrhamoeba (Gordon et al., 1976b; Weihing and Korn, 1971). Physarum (Hatano and Owaribe, 1976; Jacobson et al., 1976; Lestourgeon, 1975; Hatano and Totsuka, 1972; Hatano and Oosawa, 1966).
polymerization properties in platelets (Abromowitz el al., 1975), although this difference has been explained recently as only a ionic-strength-dependentchange in the critical concentration (Korn, 1978). Finally, as work on the amino acid sequence (Lu and Elzinga, 1976) and crystallization (Lindberg et al., 1976) of actin proceeds toward a complete three-dimensional structure of the protein, it should be possible to identify the significance of the amino acid substitutions giving rise to the various forms of actin. b. Modification of the Behavior of Actin by Associated Proteins. Many different proteins appear to remain associated with actin at various stages of purification. These proteins range from very low to high molecular weight, and only a few have been characterized in detail (Table I). The presence of one or more of these contaminant or associated proteins even at low concentrations affects the polymerizability, supramolecular structure, and interaction with myosin under various ionic conditions (Table I) (Section 111). High molecular weight proteins ca. 250,000 molecular weight have been purified from macrophages (Hartwig and Stossel, 1975), and smooth muscle (Wang et al., 1975; Shizuta et al., 1976). Cyclic AMP-dependent phosphorylation of the high molecular weight proteins has recently been demonstrated in mammalian smooth muscle (Wallach et al., 1978). A low-molecular-weight protein (ca. 15,00020,000) (profilin) has also been identified in association with actin from calf spleens, which affects the state of actin polymerization (Carlsson et al., 1976; Hitchcock et al., 1976).
3 . Well-Characterized Actins Actin from the acellular slime mold P. polycephalum and A . castellanii has been purified completely and compared with vertebrate striated muscle actin.
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
123
HIGHLY PURIFIED ACTIN
Bound HMM Bound or S1 KAPPb nucleotide ( p M ) (moleshole) (moles/mole) 8 22
1.02 1.02
-
-
~~~
1 .0 0.96 0.74
Bound metal
3-Methylhistidine N-Methyllysines (moleshole) (moles/mole)
1.1
0.93 (Ca2+)
~
1.05 1 1.42
0 1 0 ~
~~~
*Concentration of actin required to give half-maximal activation.
The most obvious difference between cytoplasmic’and muscle actin is the kinetics of actin activation of myosin ATPase (Table 11). Also, the quantitative aspects of polymerization (Gordon et al., 1976a,b) and interactions with striated muscle myosin (Gordon et al., 1976a) and tropomyosin (Yang et al., 1977) have been reported as being different in vertebrate striated muscle and Acanthamoeba actin (Table 11). The fundamental properties of nonmuscle actins are at least quantitatively similar to the muscle counterparts. (1) Actin can assemble into ca. 6.0-nmdiameter filaments with distinct polarity that (2) activate myosin Mg-ATPase.
B . MYOSIN Myosin has now been isolated from a large number of nonmuscle cells (summarized by Pollard, 1977a,b; Weihing, 1976b; also Table 111) and is believed to be almost as ubiquitous as actin. According to current ideas of motive force production, myosin acts as the mechanochemical transducer while actin is the major structural protein in the force-producing event. However, myosin is present in much smaller quantities compared to actin in nonmuscle cells, usually accounting for less than 2.0% of the total cell protein (Table 111). This disparity in the myosin and actin contents of nonmuscle cells has led to the suggestion that actin might have an additional function as part of the cytoskeleton (Section 111). As shown in Table 111, the properties of myosin are quite variable depending on source. Such interspecies differences in both the enzymic and physical properties might account for much of the variety of motility observed in nature. In addition to these interspecies differences there is evidence for several isozymic forms of myosin in several cells.
TABLE u1. ~~
~~
Subunit composition, number and molecular weight
Stokes' radius (nm)
Sedimentation coefficient
(S)
(%)
K-EDTA
Rabbit skeletal muscle
470.000
Two (200,000); one (20,000); two (18,000); one (16,500)
17.5
6.4
55
Smooth muscle
470,000
Two (200,000): two (20,000); one (L7.000)
17.5
-
Acanrhmoebo Myosin I
180,000
One (140,000); one
55
So-'
Myosin I1
4GO.000
(16,000); one (14.0001 Two (175,000); one (17,500); two
Total cell protein
ATPase (&moles P,per minutes per milligram)
Native molecular weight
C$'
Mgf
1.5
0.19
0.002 25
-
1.5
0.8
0.01
37
8
0.3
1.0
0 12
0.01
29
13.7
5.9
1.0
0.14
0.3
0.01
30
"C
(16,500)
Amoeba proreus
-
225.000
17.4
-
-
0.01
0 14
002
22
Chaos coralinensis
-
225,000
17.4
-
-
0.01
0.01
0.03
22
Dicryosrelium discoideum
-
Two (210,000, 18.000: 16,000)
-
-
0.5
0.01
009
0.01
25
Two (250,000240.000); four (21 ,000); two
17.1
6.4
1-2
0.03
I0
0.03
22
Two (200,000); two (19,000); two (16.000)
19
-
0.7-1.5
0.56
0.56
0.05
37
Two (200.000); two (19,000); two
19
6.2
1.5
0.55
044
0.01
37
Physorum
458,000
(17,000)
Macrophages
Platelets
-
460,000540.000
(16.000)
Fibroblasts
-
Echinoderm egg 500,000
200.000: 20,000
19
-
-
0.43
0.5
0.01
37
Two (ZlO,000); two (20,000); two
19.5
6.3
-
0.3
0.45
0.01
25
02
0.01
25
(17,000)
Echinoderm Sperm
(Thyone)
-
Two (210,000); two (20,000); one (17.000)
"Rcferences:RabtitsLeletalmuscle(Lowey e r d . . I%%Gershman erol., 1%9;Gazitherol.. 1970;GodfreyandHarrington. 1979FranlrandWads. 1974;bweyandRisby. 1971;SsrLareral. 1971; W e e d s a n d b w e y , 1971;KaminerandBeU, 1966). Smooth muscle (Sobieszek and Small, 1 9 n . chscko er d.,1977; Kendrifk-lones, 1973; Adelstein and Conti, 1975; Cooke, 1975: Kaminer, 1969). Acanrhamoeba (Pollard and Korn, 1973b; T. D. PoUard, 1975b; Maruta and Korn, 1977a.b; R. T.PoUard, 1978). A m eba protern (Condeelis, 19na.b; Hinssen, 1972). Chaos corolinemis (Condeelis, 1977a.b).
124
F'RO€€RTlES OF
MYOSIN
Components required for maximum actin activation of myosin ATPax None required
20.030 molecular weight
Polymerization Activation (fold)
r/2
pH
Metal
Length (nm)
20-50
0.1
6.5
-
I800
22
0.1
7.5
-
400
14
0.1
6.0-7.5
-
m
-
0.15 7.0-8.6
-
I030
-
LO
myosin light chain kinase; C f ' and tropomyosin
Width (nm)
Morphology Spindle-shaped bipolar filaments Spindle-shapd bipolar filaments Twisted bipolar filaments Bipolar filaments
15
-
-
-
-
-
None identified
1
01
68
-
205
6.6
None ideniified
6
0.06
7.0
0.01 mM or I mM Ci' I mM Mgz+ 2mMEGTA
352
13.9
Multipolar aggregated filaments
425 289
16.3
Bipolar filaments Multipolar aggregated filaments
0.01 mM
352
13.9
Multipolar aggregated filaments
425 289
16.3 12.7
Bipolar filaments Multipolar aggregated filaments
700
Cofactor (kinase)
None identified
6
0.06
7.0
12.7
01
I mM Cd' 1 mM Mg"
2 m M EGTA None identified
None identified
9
16
0.1
0.05
7.5
7.0
IOmMMg'+ 0.1 mM EDTA
I mM EGTA 5 mM Mg"
-
-
-
Bipolar filaments Small, "loose" filaments
-
-
No filaments Bipolar twisted filaments Bipolar filaments Aggegated filaments head to head Aggregated filaments head to bead
IOOO-2500
+OlmM Ci' Cofac1or
83,000molecular-weight for 20.000 molecular weight myosin light chain
I0
28
0.1
02
7.0
7.0
1 mM EDTA I mM C P
300 300
-
I mM MG'+
300
-
I mM EDTA
271 359 350
8.6 13 I I .6
Bipolar filaments Bipolar filaments Bipolar filaments
200
10
Bipolnra~tnks
260
13
Bipolar filaments Large head-to-head aggregates of thick filaments
I mM Cf*
I mM Mg*+
None identified
9
0.1
7.0
None identified
7
0.3 0.25
7.0 7.0
0.35 0.2
7.0 7.0
-
Globular, soluble under all conditions Bipolar filaments
-
-
-
260
-
-
-
-
-
Bipolar filaments Large aBgregates of thick filaments
Dictyosreliw (Clarke and Spudich, 1974). Physmrn (Nachmias, 1974; Adelman ud Taylor, 1969a.b). Macrophages (Hartwig and Stossel, 1975;Stossel and Hartwig, 1975). Platelets (Pollprd et al.. 1974; Adelstein era!., 1971; Booyse et at., 1971; Niedemmn and P o U d , 1975; Daniel and Adelstein, 1976; Adelstein and Conti, 1975). Fibroblasts (Adelstein er 01.. 1972). Echinoderm egg (Mabuchi, 1976a). Thyone (Mabuchi, 1976b).
125
126
D. LANSING TAYLOR AND JOHN S. CONDEELIS
1, Interspecies Differences All the nonmuscle cell myosins characterized to date (excpt for both the Acanthamoeba myosins (MW 190,000 and 400,000) (Korn, 1978) are large proteins with native molecular weights in excess of 450,000 and are composed of two heavy chains and two classes of light chains. These are filamentous molecules with a long tail and two globular heads at one end (Lowey, 1971; Clarke and Spudich, 1974; Hatano and Takahashi, 1971). Substantial interspecies differences in the heavy-chain molecular weights in SDS have been reported, while the light chains usually consist of an 18,000-20,000 class and a 15,000- 17,000 class (Table 111). The light-chain composition of nonmuscle cell myosins is similar to that of smooth muscle myosin and does not resemble that of skeletal muscle myosin. Since CaZ+ regulation of several forms of vertebrate smooth muscle actomyosin (Bremel, 1974; Sobieszek and Small, 1977) appears to be myosin-linked, regulation of nonmuscle cells might well reside in part in the myosin. There are also substantial differences in the ATPase activities of the various myosins (the so-called K-EDTA-, C$+- and MP-ATPases). Since these assays are performed under aphysiological conditions (high ionic strength and the absence of actin), the physiological significance of these ATPase activities remains unknown. However, under physiological conditions in the presence of actin most nonmuscle myosins demonstrate substantial Mg2+-ATPase activity which is diminished upon the removal of actin (Table 111). This activity is believed to be the significant ATPase in force production and is used to identify the presence of myosin. The differences in the actin-activated M$+-ATPase shown in Table I11 may reflect differences in the assay conditions, the presence of contaminants, or the basic enzymic properties of the myosin. Two myosins have been shown to require a crude protein fraction called cofactor for maximum activity (Hartwig and Stossel, 1975; Pollard and Korn, 1973b), while in other cases improvement in the myosin isolation procedure has resulted in a substantial increase in actin activation (Nachmias, 1974; Pollard et af., 1974; Adelstein et al., 1971). Some of the most striking interspecies differences among myosins are differences in the ability to form thick filaments. With the exception of the original Acanthamoeba myosin (myosin I) (Pollard and Kom, 1973b) all nonmuscle cell myosins (including the second Acanthamoeba myosin-myosin 11) (Maruta and Korn, 1977b; Pollard et al., 1978) form bipolar filaments under physiological conditions (Table 111). In several cases (Condeelis, 1977a; Hartwig and Stossel, 1975; Niederman and Pollard, 1975) it has been demonstrated that divalent cations are not required for filament formation, while in the case of Physarum myosin (Nachmias, 1974) millimolar Mg2+ is required for compact myosin filament formation. There is also wide variation in the size and morphology of the myosin filaments formed under identical conditions. However, the differences
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
127
(presumably in the tail) producing these effects are not substantial, since myosin from different sources can be copolymerized into well-formed bipolar filaments (Pollard, 1975a). The significance of myosin filaments in nonmuscle cell movements remains unknown. In analogy with skeletal muscle it has been suggested that the thick filament is required for mechanochemical force transduction in nonmuscle cells. However, since there is evidence both for (Hanson and Huxley, 1955) and against (Oplatka et al., 1974) this proposal, the precise role of the thick filament in nonmuscle cell movement has not been established. Since myosin might cross-link actin in nonmuscle cell cytoplasm (Taylor et al., 1973) and change the consistency of the cytoplasm, it has been suggested that the physical form of myosin in the nonmuscle cell determines the mode of movement observed (Condeelis, 1977a; Taylor et al., 1976a,b).
2 . Intraspecies Differences The most substantial intraspecies difference observed is that between Acanthamoeba myosins I and 11, as described by Pollard and Kom (1973b), Maruta and Korn (1977b), and Pollard et al. (1978), and all other muscle and nonmuscle myosins. Acanthamoeba myosin I (Pollard and Korn, 1973b) is globular in its native form at physiological ionic strength with a native molecular weight of 180,000 and a heavy-chain weight of 140,000 in SDS. However, evidence has been accumulating that another form of myosin exists in Acantharnoeba. Maruta and Kom (1977a) observed a myosin-like ATPase distinct from that previously described (myosin 11) that binds to actin and is apparently required for the contraction of cell extracts. Pollard et al. (1978) found that myosin I1 had a short tail and two heads and that it was capable of forming bipolar filaments at physiological ionic strength. These results indicated that two very different forms of myosin were isolated from Acanthamoeba (Table 111). More information regarding the properties of the new filamentous Acanthamoebu myosin will have to be gathered before the significance of two myosins can be determined. Burridge (1976), using the cyanylation technique on myosins prepared from chicken tissue, recently demonstrated the presence of at least six different types of myosin heavy chains. Two of these corresponded to the nonmuscle tissues, brain and platelets, whereas others corresponded to smooth, cardiac, and skeletal muscle. The sixth type demonstrated a difference between chicken leg and breast muscle myosins, which may reflect a different ratio of myosin isoenzymes within these different muscles. The significance of these intraspecies differences is unknown, but since most of the large fragments analyzed to determine heterogeneity probably came from the rod region of myosin, it may indicate variations in the capacity of these myosins to form filaments (Bumdge, 1976).
128
D. LANSING TAYLOR AND JOHN S. CONDEELIS
C. REGULATION It has been suggested that the interaction of actin and myosin can generate the forces required for amoeboid movement, as well as for other muscle and nonmuscle systems. However, the mechanisms of regulating actin and myosin interactions remain a major unsolved problem for cell movements, except for striated muscle (Cohen, 1975; Kendrick-Jones et al., 1970; Weber and Murray, 1973; Hitchcock, 1977; Gergeley, 1976; Lehman, 1976; Szent-Gyorgyi, 1975). Vertebrate striated muscle has been studied in detail, and it appears that the calcium regulation of contraction is mediated by tropomyosin and three subunits of troponin bound to the actin filaments. This mechanism was identified initially by Ebashi and has been reviewed by Weber and Murray (1973), Cohen (1975), and Gergeley (1976). In short, every seven actin monomers within an actin filament are associated with one tropomyosin molecule and one troponin molecule. The troponin consists of three separate subunits: troponin C (calciumbinding component), troponin I (inhibitory component), and troponin T (tropomyosin-bindingcomponent). At low calcium concentrations (ca. l(r7 M) the troponin, via the tropomyosin, sterically blocks the myosin-binding sites along the seven actin monomers covered by each tropomyosin (seven actin monomers or ca. 40 nm). At micromolar calcium concentrations the troponin binds calcium, and the troponin in conjunction with the tropomyosin removes this steric interference (Table IV). In molluscan muscles the myosin molecule regulates its own interaction with actin (Table IV). These muscles contain tropomyosin but not troponin, so that regulation cannot be based on the actin filament. The myosin molecule is possibly inhibited from binding to actin at low calcium ion concentrations by a steric block formed by the light chains. This block is removed when the myosin binds calcium. Furthermore, some muscles have both actin-linked and myosin-linked regulation at the same time (Lehman, 1976). The regulation of vertebrate smooth muscle is also linked to myosin, but the mechanism appears to be based on a calcium-dependent phosphorylation (Table IV). The kinase responsible for smooth muscle myosin phosphorylation increases the actin activation of myosin MgZ+-ATPaseby four- to eightfold. In addition, the covalent phosphorylation of the light chain endows the actin-myosin interaction with C$+ regulation of smooth muscle in some laboratories (Sobieszek and Small, 1977). A light-chain kinase of nonmuscle sources can also specifically phosphorylate the 20,000-molecular-weightlight chain of platelet myosin (Adelstein and Conti, 1973; Daniel and Adelstein, 1976) and murine astrocyte myosin (Scordilis et al., 1977) but differs from that in smooth muscle in two ways: (1) It does not require CaZ+ for activity; and (2) the platelet myosin does not require C$+ for actin
TABLE IV REGULATORY SYSTEMS-CALCIUM-MODULATED
Source“ Vertebrate striated muscle Molluscan muscle
Vertebrate smooth muscle (seetext)
Regulatory protein and molecular weight Tropomyosin (35,000);troponin C (18,000); troponinI (23,OOO); troponin T (37,000) EDTA light chains (18,000); DTNB light chains (18,000) Myosin light chain (20,000) Myosin light chain kinase
Dictyosrelium discoideum
Crude fraction containing proteins of molecular weight 30,000 and 15,000 and many other proteins at small concentrations Crude fraction containing actin, myosin, and proteins of molecular weight 250,000, 95,000; 75,000,38,000,
Proposed mechanism Troponin and tropomyosin regulate actin by sterically blocking myosinbinding sites on actin in the absence of C 2 + ; the binding of C 2 + by troponin removes the steric blocking of tropomyosin The EDTA and DTNB light chains of myosin regulate the myosin by sterically blocking the myosin binding site for actin in the absence of C 2 + ; the binding of C 2 + by the light chains removes the steric blocking The myosin light chains are unphosphorylated in the absence of CaZ+which minimizes the actin activation of myosin ATPase A specific myosin light chain kinase phosphorylates the light chain in the presence of C 2 + and the actin activation of myosin ATPase increases in reponse to C 2 + Actin-linked, but Dictyostelium myosin required for calcium-sensitive ATPase
Actin-linked; one or more of these proteins binds actin in a “gel”complex at low CaZ+concentrations which inhibit force generation; the gel complex is broken at ca. M Ca2+,permitting free F-actin filaments to interact with myosin
32,000;28,000;20,000 Physarum polycephalum
Crude precipitate containing several proteins Crude precipitate containing proteins of molecular weight 38,000;35,000;24,000 and other proteins
Actin-linked; but dependent on myosin in an undefined way Actin-linked; similar to the troponin-tropomyosin mechanism
(continued)
TABLE IV (continued)
Source ’
Regulatory protein and molecular weight Presumptive pyrophosphohydrolase (PPH) (55,000)
Human platelets
Tropomyosin (30,000) and other proteins of molecular weight 36,000; 18,000; 14,000 Tropomyosin (30,000), troponin C, and troponin I
Proposed mechanism PPH binds to actin and produces 5‘-AMP which increases actin polymerization in the presence of C 2 + ; the polymerized actin can then interact with myosin Actin linked; tropomyosin-based system similar to vertebrate striated muscle, but no troponin was identified Actin linked; tropomyosin-troponin system
Vertebrate striated muscle (Szent-Gyorgyi, 1975; Lehman and Szent-Gyorgyi, 1975; Weber and Murray, 1973). Molluscan muscle (Szent-Gyorgyi, 1975; Lehman and Szent-Gyorgyi, 1975; Kendrick-Jones et al., 1970). Vertebrate smooth muscle (Sobieszek and Small, 1977; Chacko et al., 1977). Dicryostelium (Mockrin and Spudich, 1976; Condeelis and Taylor, 1977; Hellewell and Taylor, 1979). Physarum (Nachmias and Asch, 1974; Nachmias and Asch, 1976; Kato and Tonomura, 1975a; Kato and Tonomura, 1975b; Tanaka and Hatano, 1972; Nachmias and Asch, 1976). Human platelets (Cohen and Cohen, 1972; Cohen et al., 1973; Puszkin et al., 1975).
STRUCTURE AND CONTRACTILITY IN AMOEBOID CELLS
131
activation of its Mg2+-ATPase. Hence light-chain phosphorylation in the nonmuscle cells studied proceeds without apparent calcium regulation (Table V). A calcium dependent regulatory (CDR) protein has been identified in striated muscle and several nonmuscle cells whose structure and function is related to muscle troponin C (Kuo and Coffee, 1976a,b; Watterson et al., 1976; Amphlett et al., 1976; Dedman et al., 1977). This modulator protein has recently been identified as the activator protein of striated muscle myosin light chain kinase (Yagi et al., 1978). The (CDR) protein should be actively sought in nonmuscle cells since it has been shown to exhibit multiple calcium dependent, regulatory functions (i.e., some phosphodiesterase, adenylate cyclase and C$+ and Mf+ATPases). It may play a pivotal role in cellular regulation. The molecular mechanisms for regulation have not been defined in complete detail in nonmuscle systems, and it appears that multiple mechanisms might operate within the same cell (Tables IV and V). Furthermore, the interrelationship between the regulation of cytoplasmic structure and contractility has presently been observed in only a few types of cells (Taylor et a l . , 1977; Condeelis and Taylor, 1977; Taylor, 1977b). The possible regulatory mechanisms in nonmuscle cells can be separated into two basic categories: (1) calcium-modulated (Table IV) and (2) calcium-independent(Table V). Calcium ions appear to be the molecular ‘‘switches in calcium-modulated systems, while diverse biochemical ”
TABLE V POSSIBLE REGULATORY SYSTEMS-CALCIUM-INDEPENDENT
Source“
Protein molecular weight
Proposed mechanism
Acanthamoeba castetlanii
Cofactor, 97,000
The presence of this protein increases the actin-activated MgZf-ATPase of myosin I by an unknown mechanism (apparently a kinase)
Rabbit alveolar macrophages
Cofactor, 90,000
The presence of this protein increases the actin-activated Mg2+-ATPaseof myosin by an unknown mechanism
Human blwd platelets
Myosin lightchain, 20,000; myosin lightchain kinase, 83,000
Myosin-linked; the actin-activated MgZ+-ATPase of myosin is low when the myosin light chains are unphosphorylated; the myosin light chain kinase phosphorylates the myosin light chains, which increases the actin-activated Mg2+-ATPase
Dictyostelium
Myosin heavy chain kinase
CAMP stimulates the phosphorylation of the myosin heavy chain. No second messenger has been identified
“Acanthamoeba (Pollard and Kom, 1973b; Maruta and Kom, 1977b). Macrophages (Stossel and Hartwig, 1975). Platelets (Adelstein and Conti, 1975; Daniel and Adelstein, 1976). Dicfyostelium (Rahmsdorf et al., 1978).
132
D. LANSING TAYLOR AND JOHN S. CONDEELIS
alterations are involved in calcium-independentprocesses. Furthermore, the role of hydrogen ion gradients causing localized changes in pH has not yet been investigated in sufficient detail to assess their importance in motility.
D. OTHERCONTRACTILE AND/OR CYTOSKELETAL PROTEINS Class I cells have been shown to contain actin, myosin, and as yet incompletely defined associated proteins as discussed in Section IV, A to C. However, many class I1 and class III cells contain cytoplasmic microtubules and/or 10.0nm intermediate filaments which complicate the correlation between cytoplasmic consistency and motility. Furthermore, many “minor” proteins have been identified in specific cell types, and their general importance must await further investigation. The reader should refer to reviews on microtubules (Snyder and McIntosh, 1976; Stephens and Edds, 1976) and 10.0-nm filaments (Gaskin and Shelanski, 1976). No specific interactions between 10.0-nm filaments, microtubules, and actin or myosin have been demonstrated, but this possibility requires detailed study (Lehto et al., 1978).
E. SUMMARY AND FUTURE CHALLENGES At the present time it is apparent that cytoplasmic consistency and contractility are at least partially dependent on the reversible interactions among actin, myosin, and protein@)that cross-link actin into supramolecular structures. Furthermore, the cytoskeletal state of actin appears to be antagonistic to contraction, so that contractions do not occur in the highly structured (gel) cytoskeletal regions without simultaneously decreasing the local cytoplasmic consistency. This process has been observed in both intact cells and in model systems. A good example of this possible interrelationship between cytoplasmic consistency and contractility is readily observed in the cortex of dividing marine eggs (Section 11, C). Several different physical probes have indicated that the structure of the cortex diminishes during formation and contraction of the cleavage furrow. The easiest explanation is the transformation of actin from the cytoskeletal state in the predivision egg cortex to organized F-actin filaments with interspersed myosin in the cleavage ring. In order for shortening to occur with a minimum of resistance the gel structure is diminished in the cleavage region, permitting contractile forces to cut the cell into two pieces. It is reasonable to suggest that the regulation of gelation (the cytoskeletal state) is the same as the regulation of contraction. In fact, the gelated state may represent a form of contractile regulation (Section 111,
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B). However, a separate actomyosin regulatory mechanism may also exist (Taylor et al., 1977; Condeelis and Taylor, 1977; Taylor, 1977b). Another specific example of the interrelationship between cytoplasmic consistency and contractility has been demonstrated clearly in giant free-living amoebas. The streaming cycle of the less structured endoplasm into the highly structured ectoplasm and finally back to the endoplasm is a major process during amoeboid locomotion. Calcium appears to regulate both cytoplasmic consistency and contractility, as observed in single-cell microinjection experiments (Section 11, E), single-cell models (Section 111, C), and bulk cell extracts (Section III,D). The cell appears to be capable of regulating the intracellular free calcium ion concentration as well as the local pH which in turn appears to regulate both motility and cytoplasmic structure. The sol-gel-gel breakdown and contraction cycle of events seems to be due to cyclic interactions between actin and actinbinding protein(s) and myosin. The mechanism($ of regulation have not been demonstrated and may involve multiple events in cellular intermediary metabolism. The main characteristic is the close correlation between cytoplasmic consistency and contractility. It is not presently known whether all nonmuscle cell movements involve a dynamic antagonistic interplay between the cytoskeletal states and motile states, using the same regulatory trigger. However, future investigations will shed light on this question. A simple working hypothesis for proposed sol-gel-gel breakdown and contraction processes has been presented (Taylor et al., 1977; Condeelis and Taylor, 1977) and subsequently discussed in detail (Taylor et al., 1978). It is hoped that this working hypothesis can be refined ultimately to explain events occurring in specific regions within living cells. This article has focused on the correlation between cell structure and contractility and the role that actin plays in both processes. We have not discussed several important concepts that are vital links in the chain of events involved in cell motility. Future studies must focus on topics such as: (1) the calcium regulatory system that varies the intracellular free calcium ion concentration; this system could involve a specialized intracellular membrane system or other organelles, or free proteins; (2) the role of the cell surface and membrane in transmitting information resulting from nutrient or signal gradients (Zigmond, 1978) ligand binding or cell-cell contacts; (3) the cause-and-effect relationship between the variation in cytoplasmic consistency and motility and the cell cycle and development; (4) the possible structural interaction between actin, microtubules, and 10.0-nm filaments; and (5) the presence and function of actin in nucleii and its relationship to cytoplasmic activity (Goldstein, Rubin, and KO, 1977; Clark and Merriam, 1977; Jockusch, Becker, Hindennach, and Jockusch, 1974). Answers to the questions discussed in this article and the proposed new frontiers will require further physiological, biochemical, and ultrastructural in-
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vestigation. Continued integration of the results from studies on cell physiology, ultrastructure, and biochemistry will ultimately allow a cohesive molecular picture of cell structure and motility to emerge.
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INTERNATIONALREVIEW OF CYTOLOGY,VOL. 56
Methods of Measuring Intracellular Calcium ANTHONY H. CASWELL Department of Pharmacology University of Miami School of Medicine Miami. Florida
I. Introduction . . . . . . . . . . . . . . . . . . . . II. Assay of Total Cellular Ca . . . . . . . . . . . . . . 111. Metallochromic Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Murexide B. ArsenazoIII . . . . . . . . . . . . . . . . . . IV. Luminescent Proteins-Aequorin . . . . . . . . . . . . V. Fluorescent Chelate Probe . . . . . . . . . . . . . . . VI. Fixation of Tissue for Cytology . . . . . . . . . . . . . A. Chemical Fixation . . . . . . . . . . . . . . . . B. Freeze-Drying . . . . . . . . . . . . . . . . . . VII. Autoradiography . . . . . . . . . . . . . . . . . . VIII. Electron Probe Microanalysis . . . . . . . . . . . . . . IX. Concluding Comments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .
145 147 151 151 152 153 159 165 165 167 168 173 178 179
I. Introduction The original discovery of the role of cytoplasmic C 3 + in initiating contraction of skeletal muscle has been followed by the observation that intracellular C$+ serves as a trigger for a wide range of physiological and biochemical functions. It is now apparent that Ca2+ acts as a modulator in almost the ubiquitous fashion that cyclic AMP (CAMP)does. CgZ+exists normally in cells in a concentration range from ca. 1W8to ca. lW5M. A physiological event is triggered in the region lW7-1W6M. The ion is almost ideal for the purpose of initiating rapid physiological events such as release of neurotransmitter at the neuromuscular junction or contraction of skeletal muscle. C3+ chelates to unpaired electrons on oxygen or nitrogen atoms of a wide range of molecules. This chelation involves exchange of the coordination sphere of surrounding water molecules for those of the chelating molecule. The rate at which chelation takes place varies materially with different ions in the same group in the periodic table. Thus for C3+ the exchange rate for the water of hydration is 5 x 108 sec-' , while that for Mf+ is 145 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved ISBN 0-12-364356-2
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lo5sec-' (Eigen, 1963). Hence the binding of Ca2+to a molecule which initiates a physiological process is much faster than the binding of Mg2+ and, similarly, release is faster. While Na+ and K+ have binding rates similar to that of C d + , they have in general a lower binding affinity. The requirement for a trigger is that it bind rapidly and at low concentration to the receptor which initiates the physiological process. Thus C 2 + is favored over Mg2+ in its rapidity of binding, and over alkali ions in the high affinity of its complexes. The intense interest in physiological processes triggered by Ca2+ has stimulated the development of a wide variety of techniques for measuring this ion. This article concentrates on techniques which are appropriate for the study of C2+ in whole-cell systems. The use of techniques for studying C$+ movements in isolated organelles is considered only in describing the properties of reagents and probes used in intact cells. The intent of this article is to discuss the advantages and limitations of various measurement assays, but no attempt is made to be exhaustive in describing the employment of these methods. The choice of measurement technique depends critically on the type of information required about intracellular C 2 + . Considerations which affect the choice are whether detailed distribution of the cation is required, whether dynamic or static information is sought, and whether quantitative data are required. It is clear from the role of Ca2+ in physiological processes that its intracellular distribution is uneven. Much emphasis has been placed on free cytoplasmic Ca2+ as the determinant of physiological triggering. However, it will be apparent that the ultimate source of the trigger arises elsewhere. The event which may cause cytoplasmic C2+ to alter may in certain cells be an influx of C2+ from the extracellular space, or a pumping of C2+ to the outside. However, in all muscle systems and in many nonmuscle systems a complex reorganization of C$+ binding and sequestration causes cytoplasmic C2+ to alter. We may thus distinguish three types of intracellular C 2 + . Expressed simply they are (1) cytoplasmic free Ca2+, (2) Ca2+ bound to membrane surfaces or intracellular chelating molecules, and (3) Ca2+sequestered inside subcellular organelles. Thus estimates of total intracellular C 2 + are a sum of these three sources and reflect only indirectly any changes in cytoplasmic C 2 + . Tine techniques which measure cytoplasmic free C 2 + may therefore be preferable to those which measure total intracellular C 2 + , since they reduce the complexity of the interpretation, especially if the role of cytoplasmic C 2 + in triggering a physiological event is being investigated. However, once it is established that free cytoplasmic C2+ triggers an event such as contraction, the source of this C 2 + must be investigated. In theory it is preferable to employ a method which gives not only the C2' concentration but also its detailed distribution. The only techniques which can currently approach this aim are those involving electron microscopy. However, even electron microscopy has limits of resolution which may restrict the usefulness of the information obtainable. Thus to determine whether a membrane binding site for
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Ca2+ is on the inside or outside of the membrane is beyond the resolution of current techniques. Moreover, it is frequently difficult to extrapolate quantitatively microscopic data to concerted multicellular processes or measurements. Therefore several factors limit the resolution and utility of C$+ assays to below the ideal, and we do not currently have the means to explore in intact cells all the events associated with triggering and physiological response. The second major factor which concerns the physiologist is the time sequence of events mediated by Ca2+ movements. It has already been stated that C$+ has the property of initiating very rapid events such as muscle contraction. Thus the time course of many processes is on the order of a few milliseconds. This imposes special requirements on monitoring Ca2+ movement in order that the time domain of the event may be followed. Here a fundamental distinction should be made between techniques which monitor C$+ on-line and without destruction of the cell system and techniques which require the reaction to be stopped and assayed under conditions which destroy the integrity of the system. This distinction is not based solely on speed of response, but rather is associated with the particular difficulties of statistical evaluation and averaging which occur when each specimen must be destroyed in order to make a measurement. Thus techniques which involve nondestructive on-line measurement have proved of particular value in studying many cellular processes. The great diversity of techniques and approaches to the measurement of Ca creates a problem for the reviewer in terms of comparison of the merits and limitations of different techniques. Thus sensitivity and resolution may be expressed in different units for each specific technique. For example, the limits of resolution for Ca measurements cannot adequately be compared for electron probe microanalysis and for aequorin luminescence. Thus estimates of quantitative parameters have been related to each technique individually without any attempt to compare the procedures directly.
11. Assay of Total Cellular Ca
The earliest method for evaluating the Ca content of cells and Ca fluxes across the plasma membrane was total tissue analysis. However, Ca fluxes associated with physiological perturbations such as hormone or electrical stimulation frequently proved to be too small to be assayed accurately by this method. In a few cells, such as squid axon or skeletal muscle fibers, Ca fluxes may be assayed directly, since these cells do not contain extensive extracellular Ca-sequestering stores, hence accurate estimates of intracellular Ca can be made directly. However, more general application of tissue analysis has required the prior removal of extracellular Ca-binding stores. Hodgkin and Keynes (1957) assayed cytoplasmic Ca2+ of the squid axon by
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extruding the axoplasm from the fiber using a roller. This technique is clearly not of general appIication. Bianchi and Shanes (1959) measured the Ca2+ influx into frog sartorius fibers by a short exposure of the fiber to 45Ca. The muscle was washed by frequent changes of the unlabeled medium for a period of 90 minutes in order to remove the external Ca from the medium. The fibers were then ashed and counted. These investigators detected an enhancement of Ca influx when the fiber was stimulated electrically. In nerve fibers and skeletal muscle the rate of Ca movement across the plasma membrane is low, and the fibers have a relatively large intracellular space in comparison to the plasma membrane area. These fibers also have a relatively low amount of extracellular binding proteins. Even under the most favorable circumstances it was necessary to wash the fiber extensively in order to deplete extracellular Ca binding. In many cells, however, Ca fluxes are high and extracellular Ca binding to connective tissue and membranes is extensive. For these reasons many of the early experiments on Ca fluxes proved equivocal. This problem has been particularly pronounced in studies on smooth muscle in which investigators were unable to observe consistent Ca fluxes under conditions in which muscle contraction occurred (van Breemen and Daniel, 1966; Krejci and Daniel, 1970; Lammel and Golenhofen, 1971). For these reasons techniques were devised to diminish the contribution of extracellular C$+ binding to the total C$+ measurements. Any method which reduces extracellular binding sites should display one of two properties. It should either (1) act without in any way influencing Ca fluxes across the plasma membrane so that the physiological fluxes are unaltered or (2) cause complete cessation of Ca movement so that the cell may be subsequently analyzed for Ca without Ca loss during washing and preparation for analysis. The first approach has not been successfully exploited, but the second has been utilized by employing Ld+ both to remove extracellular Ca-binding sites and to inhibit Ca fluxes. Weiss and Goodman (1969) observed that 1.5 mM Id+inhibited the contraction of ileal smooth muscle under the influence of both high K+ and acetylcholine. They recorded a decrease in 45Caaccumulation by the muscle and little change in the 45 Ca efflux, provided that the extracellular concentration of Ca2+ was normal. These workers demonstrated that both slowly exchangeable and rapidly exchangeable Ca was diminished by Ld+ treatment. They argued that Ld+ displaced C$+ from the rapidly exchangeable surface sites and also exerted a stabilizing action on the membrane, which inhibited C$+ fluxes across the plasmalemma. Goodman and Weiss (1971) further observed a similar displacement of superficial Ca-binding sites by Ld+ in uterine muscle. Van Breemen (1969) showed that La3+ exerted a differential blocking action on the contraction of aorta by norepinephrine and by K+. He related these actions to a blockage of Ca2+ influx by La3+. Subsequently van Breemen and
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McNaughton (1970) proposed the employment of La3+ as a means of distinguishing extracellular from intracellular C 2 + , hence as a tool for the assay of Ca content of cells. These investigators argued that La3+ displaces Ca2+ from rapidly exchangeable binding sites and at the same time blocks C$+ fluxes. They demonstrated a small residual Ca2+ in aortic strips after prolonged La3+ treatment and observed a higher Ca content in the cells when the strips had been pretreated with 160 mM Li+ or K + in place of Na+ in order to depolarize the muscle. Thus they successfully correlated apparent Ca content of smooth muscle with contraction. It is emphasized that the La displacement technique for assaying intracellular Ca requires (1) displacement of extracellular C 3 + but not of intracellular Ca2+ by L 2 + , (2) inhibition of C 2 + influx into the cell, and (3) inhibition of Ca2+ efflux from the cell. The evidence that intracellular Ca2+ is not displaced is partly the empirical evidence that the measured Ca2+ content correlates with estimates under conditionsin which Ca2+ fluxes are known or can be predicted, for example, from measurements of contraction of muscle. Also, when cardiac muscle is perfused with high concentrations of La3+ and thin sections made for electron microscopy, the La3' is apparently confined to the extracellular space and cell junctional region (Smith et al., 1972). Devine and Somlyo (1970) reported that sarcolemmal vesicles were accessible to La3+ but not sarcoplasmic reticulum of vascular muscle. Since high concentrations of La3+ were employed in these experiments and electron density observations were rather insensitive, this evidence is confirmatory rather than conclusive. Hodgson et al. (1972) showed that 140La was slowly accumulated by uterine muscle, suggesting the possibility of La-induced Ca2+ extrusion. However, provided La3+ blocks Ca2+ efflux, the penetration of La3+ into the cell should not cause the release of intracellular C 2 + . Considerable evidence of inhibition of Ca2+ influx by La3+ has been obtained in a variety of cells. It has been observed in smooth muscle cells (Weiss and Goodman, 1969; van Breemen, 1969; Burton and Godfraind, 1974; Godfraind, 1976), in heart (Palmer and van Breemen, 1970), and in polymorphonuclear leukocytes (Boucek and Snyderman, 1976). In all cases 0.5 mM La3+ proved adequate to inhibit most Ca2+ influx. Freeman and Daniel (1973) reported 80-90% inhibition of Ca2+ influx into vascular muscle by 2 mM L$+. The inhibition by L$+ of Ca2+ efflux from cells appears to be variable. Van Breemen et al. (1977) emphasize that several efflux mechanisms may exist in a cell, each of which is variably responsive to extracellular W+.Van Breemen and de Weer (1970) reported the inhibition by 5 mM La3+ of Ca2+ efflux from squid axons to be greater than 80%.Inhibition of efflux of Ca2+ from aortic strips in the presence of 2 mM La3+ was almost complete when the muscle was poisoned with 2,4-dinitrophenol and iodoacetic acid (van Breemen et al., 1973). However, van Breemen et al. (1977) report that when the muscle was incubated in a physiological medium, 10 mM La3+ inhibited efflux only partially such that
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1 hour of incubation was associated with a loss of intracellular Ca2+ of 36%. Similarly, Freeman and Daniel (1973) report that incubation of uterine muscle in 2 mM La3+ caused a 50% loss of cellular C 2 + in 1-3 hours. Since prolonged incubation in La3+ is necessary to remove all extracellular Ca2+, the estimates of cellular CaZ+ after a La3+ wash represent a material underestimate in certain cells. This can be overcome by extrapolating the linear portion of the curve of log[Ca2+]versus time after La3+ addition back to zero time (van Breemen et al., 1977). An alternative approach has been to cool the tissue after La3+ addition in order to diminish further the rate of C 2 + efflux (van Breemen et al., 1977; Palaty , 1977). The La approach has been employed to estimate intracellular CaZ+ in several cells. Van Breemen et al. (1973) studied the Ca2+ fluxes associated with K+ depolarization and norepinephrine-inducedcontraction of vascular muscle. They found that high K+ caused the entry of Ca into the cytoplasm from the extracellular space, while only the slow component of norepinephrine-induced contraction was associated with Ca influx. Godfraind (1976) similarly showed enhanced Ca uptake induced by norepinephrine in aorta. He demonstrated that the hormone also enhanced Ca efflux from the cell by preincubating the muscle in 45Ca, transferring it to 40Ca medium, and subsequently adding norepinephrine. The cells were then washed with La3+ and assayed for 45Ca. Van Breemen ef al. (1973) showed that conditions which increased intracellular Na+ in taenia coli, such as ouabain treatment, did not cause a similar increase in intracellular C$+. They argued that a Na-Ca exchange carrier was not primarily responsible for the high C 2 + gradient across the plasma membrane. Burton and Godfraind (1974) demonstrated an influence of intracellular Na+ on intracellular C 2 + in ileum muscle, however, they were unable to demonstrate this dependence effectively when they assayed Ca2+ content using La3+ to release extracellular C 2 + . Marshall and Kroeger (1973) reported an increased Cd+ content in uterine muscle when the muscle was treated with KCl, which was reversed by the relaxants isoproterenol, dibutyryl-CAMP, and papaverine, but not by the relaxant D600. However, Hodgson and Daniel (1973) did not find an increase in C$+ content of uterine muscle on treatment with KCl, while LiCl did increase intracellular C 2 + . These investigators also were unable to observe a decrease in intracellular Ca2+ when a Lit contracture was followed by administration of the relaxant, isoproterenol or papaverine. Boucek and Snyderman (1976) employed La3+ to determine the C$+ content of neutrophils before and after stimulation of chemotaxis by activated serum. They found an enhanced accumulation of Ca2+ within cells after stimulation of chemotaxis, which was effectively inhibited by simultaneous treatment with L$+. Concentrations of La3+ adequate to prevent Ca2+ ingress were equally effective in inhibiting chemotaxis.
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111. Metallochromic Dyes A. MUREXIDE Murexide is an ion-binding dye which has been successfully employed in observing Ca2+ movements of isolated organelles (Mela and Chance, 1968; Inesi and Scarpa, 1972). It has a response time for Ca of less than 5 psec (von Geier, 1968), hence may be employed for monitoring Ca2+ transients of rapid physiological processes. The dye binds weakly to divalent cations, and therefore does not effectively alter the concentration of the free ion. It binds considerably more strongly to Ca2+ than to M$+, hence is suitable for use in studying Ca2+ concentration in environments containing M g + . The dye is a polar molecule which can be expected to cross membranes slowly, if at all. The major drawback to the employment of this agent in studies of intracellular Ca2+ lies in the low absorbance change of the molecule at the physiological Ca2+ concentration. However, the dye has been used occasionally (Jobsis and O'Connor, 1966; Le Breton and Feinberg, 1974) and therefore is discussed. Murexide is a dye which binds to divalent cations, giving a chelate with an altered molecular conformation and absorbance and absorption wavelength. The millimolar extinction coefficient of free murexide is 1.25 at pH 6.8. The addition of Ca2+ causes the absorption maximum to shift from 520 nm to 505 nm with a decrease in€ to 1.18 m W ' - cm-' (Ohnishi and Ebashi, 1963). Mela and Chance (1968) have estimated the AE associated with subtraction of the absorbance at wavelength 510 nm from that at 540 nm using a dual-wavelength spectrophotometer. This gives a value for AE (€540 - €510) caused by 375 p M C$+ of 1.0 mM-' . cm-' at pH 7.4. This value of AE for the difference spectrum is comparable to the absorption of murexide and implies a significant alteration of the spectrum when murexide chelates to C$+. The dissociation constant of the Ca-murexide complex at pH 7.85 is 1.6 mM. The affinity varies only slightly between pH 4.6 and 8.2 (von Schwarzenbach and Gysling, 1946). Thus under physiological conditions in the cytoplasm, in which a change in C 2 + may range from l(r* to l(r5 M after a physiological stimulus, the change in absorption of the dye caused by Ca binding is less than 1% of the maximum absorbance change at high Ca2+ concentration. This renders observation of the intracellular C2+ problematical. In situations in which the expected absorbance change of the dye is small a major problem arises as a result of light-scattering artifacts which are especially compounded in muscle where movement is extensive even under isometric conditions. Jobsis and O'Connor (1966) attempted to reduce this in their study of Ca2+ transients associated with skeletal muscle contraction by recording the absorbance changes at different wavelengths and subtracting the absorbance at an
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isobestic point such as 505 nm from that of a peak (470 nm) or trough (540 nm). Although light scattering is also a function of wavelength, this technique reduced interference from light-scattering artifacts to low levels. Murexide is a polar molecule which does not penetrate membranes readily. Therefore special techniques are necessary to introduce it intracellularly. Jobsis and O’Connor (1966) and Le Breton and Feinberg (1974) introduced the molecule, employing dimethyl sulfoxide (DMSO) as a vehicle. This compound is known to facilitate the transfer of polar molecules across membranes. However, it introduces the possibility of artifactual alterations of the cell membrane. A second problem may arise if the vehicle permits murexide to penetrate within intracellular organelles, since the distribution of C2+ in cells is not uniform and the response of intracellular organelles to a physiological stimulus may be different from that of the cytoplasm. Jobsis and O’Connor (1966) employed murexide to monitor Ca2+movementsin toad (Bufo marinus) sartorius muscle during contraction. These workers injected murexide in a solution of DMSO intraperitoneally into the live animal for 2 days prior to use. They found a change in absorbance of the murexide in the muscle on stimulation which corresponded to increased Ca-murexide complex formation, hence increased cytoplasmic C 2 + . The absorbance at 470 nm first decreased and then increased such that the maximum absorbance change occurred before full tension development. This result is consistent with the finding of Rude1 and Taylor (1973) who employed aequorin luminescence to observe an increase in cytoplasmic C 2 + which took place before full tension occurred. The technique of murexide absorbance in monitoring intracellular Ca2+ has not found general favor, probably owing to its low absorbance difference at physiological concentrations of intracellular C 2 + .
B. ARSENAZO I11 Recently other metallochromic dyes have been described which may prove more convenient than murexide for studying intracellular C 2 + . Appropriate indicators should have a higher affinity for C2+ than murexide and should show considerable specificity for C 2 + over M$+. Use of the dye arsenazo I11 as an indicator has recently been reported (Vallieres et al., 1975; DiPolo et al., 1976). This dye binds both M$+ and C2+ but shows different absorption changes when chelated to the two cations. At pH 7.0 the K D for dissociation of the Ca complex varies with ionic strength from 20pM at 0.1 M KCl to 55 p M at 0.5 M KC1, representing a materially higher affhity for C2+ than murexide (KD= 1.6 mM). Formation of the Ca complex causes a difference spectrum compared with the free dye that has peaks at 600 and 660 nm and a trough at 530 nm. In the region of 665-685 nm the absorption change registers the binding of dye to C2+ with a selectivity over M$+ of nearly 3000.
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Vallieres et al. (1975) employed a dual-wavelength spectrophotometer set at wavelengths of 665 and 685 nm to monitor CaZ+ accumulation by mitochondria. DiPolo et al. (1976) employed arsenazo I11 to monitor the C$+ content of an internally dialyzed squid axon. Approximately 1 mM of the dye was microinjected into the fiber, and the absorption was monitored using a dual-wavelength spectrophotometerand wavelengths of either 570400 nm or 660485 nm. Light pipes were employed to pass the light from the rotating filter of the dualwavelength source to the axon and from the axon to the photomultiplier (Brinley and Scarpa, 1975). These workers estimated the axoplasmic CaZ' activity to be 50 nM. Thus this indicator technique is highly sensitive and has the advantage that accurate quantitative estimates of cytoplasmic [Ca] may be made. However, arsenazo 111, like murexide, is not lipid-soluble and may be difficult to introduce into many cells.
IV. Luminescent Proteins-Aequorin One of the most powerful tools in the study of intracellular CaZ+ has been the monitoring of aequorin chemiluminescence. The properties and use of aequorin have been reviewed in detail by Blinks et al. (1976). Aequorin is a protein extracted from the jellyfish Aequora aequorea. This protein has the property of emitting a quantum of light in the presence of C2'. It may be microinjected into a cell, and the endogenous C$+ determined from its luminescence. This technique has a potential spatial resolution of approximately the wavelength of the emitted light, although this may not be achieved in practice. Time resolution is in the millisecond range, and the sensitivity is at least down to lO-' M C$+. Cation selectivity is probably for Ca2+and Sr2+. Aequorea and other luminescent jellyfish belong to the class Hydromedusae. Alexander von Humboldt, during his journey through South and Central America, discovered that the luminescence of the jellyfish could be excited electrically. Modem investigations of this jellyfish followed the burgeoning interest in biological chemiluminescence associated with the isolation of luciferase and luciferin. Shimomura et al. (1962) first isolated aequorin from Aequorea and have defined the chemical properties of this protein in detail. One of the factors which has restricted employment of this reagent has been its limited availability. The protein is now obtainable commercially in an impure form (Sigma Chemical Company), and Johnson and Shimomura (1972) have described the procedure for extraction and purification of the active form. While several different species exhibit Ca2+-induced luminescence, Shimomura has found Aequorea to be the most abundant and practical source. The purified protein is stable in a solution containing lop4M ethylenediaminetetraacetic acid (EDTA) at neutral pH at low temperatures, but stability is reduced at temperatures above about 30°C (Shimomura et al., 1962). However,
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even low amounts of Ca2+ in the EDTA solution-such as might be leached from glass or quartz-cause significant luminescence and inactivation of the protein (Shimomura and Johnson, 1969). This occurs because the reaction of C$+ with aequorin is an irreversible process which gives rise to an inactive protein product. A more useful method of storing aequorin is in solutions containing a chelating resin such as Chelex. In this way the aequorin solution which is injected can be readily separated from the chelating matrix immediately prior to use (Blinks et al., 1969). Aequorin has an absorption spectrum with a peak at 280 nm and shoulders at 290 and 310 nm (Shimomura et al., 1962) and a very weak peak at 465 nm (Shimomura and Johnson, 1969). On incubation with C$+ , it emits light with a spectral peak at 466 nm. The inactive protein which is the product of the interaction with C$+ has identical absorption peaks at 280 and 290 nm,but the shoulder at 310 nm is replaced by a maximum at 333 nm. While native aequorin has low fluorescence, the inactive product is highly fluorescent with an excitation peak at 333 nm and emission at 465 nm. Shimomura et al. (1974) have studied the chemical transformations associated with the reaction of Ca2+ with the protein. The chromophore of the native molecule undergoes an intramolecular interaction with a peroxide group and a second chromophore to yield a peroxidized form of the first chromophore. All these reactions take place within the aequorin molecule and need concern the experimentalist only to the extent of recognition of the fact that C$+ inactivates the molecule and the inactivated aequorin binds to C$+ . Thus in the presence of C$+ the molecule has a limited lifetime, and it is necessary to limit the access of free Ca2+ rigorously until the functional reaction is observed. For most purposes aequorin has been injected into cells which have typical cytoplasmic C$+ concentrations of less than l C 7 M .The initiation of a C$+-stimulated reaction may involve increasing C$+ to = l C 5 M, for example, in the stimulation of muscle contraction. The ion selectivity of aequorin has been the subject of controversy but appears to be limited to Ca2+ and lanthanides with a much lower sensitivity f w light emission in the presence of Sr (Shimomura and Johnson, 1973). Mg2+ does not initiate luminescence but does influence the luminescence induced by C$+ . The number of Ca-binding sites required for light emission is open to question. Moisescu et al. (1975) have proposed two sites, while Allen et at. (1977) have suggested three or four. Similarly the number of Ca sites bound to the reacted inactivated molecule is not known. Shimomura and Johnson (1970) have proposed three binding sites, one of which has a KD of 1.4 X lW7 M, while the other two probably have a higher affinity. The interpretation of their data has been questioned by Blinks et al. (1976). Stopped-flow kinetic experiments have shown that the rise in luminescence on mixing aequorin with C$+ is pseudo-first order with a rate constant at 20°C of 100 sec-' (Hastings et al., 1969). This corresponds to a half rise time of 7 msec.
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However, h s c h e n and Chance (1971) demonstrated a Ca dependence of the rate constant of luminescence rise. The limit of time resolution of aequorin luminescence is on the order of 10 msec. This may impose some limitations on measuring the fastest physiological processes such as skeletal muscle excitation. The signal decays exponentially when Ca2+ is present continually in the aequorin solution. The maximum rate of decay occurs at or above lW4 M C$+ with a rate constant of 1.4 sec-’ or a half-time of 0.5 seconds (van Leeuwen and Blinks, 1969). This decay occurs as a result of aequorin utilization in the reaction with CaZ+ and not as a result of Ca2+ utilization. If aequorin is mixed with CaZ+ and subsequently mixed with EDTA or 1,2-di(aminoethoxy)ethane-N,N,N’,N’ = tetracetic acid EGTA, the decay in luminescence in the presence of the chelating molecule occurs with a rate constant of 100 sec-’ or a half-time of 7 msec. Thus the rate of light response to a drop in Ca2+ is approximately the same as the response to an increase in Ca2+.Hastings et al., (1969) have drawn a scheme for the kinetics of the aequorin reaction: L,
A
+ CrP+ k.,
=
100 m-’
= fast
k, = 1 sec-l
ACa-
ks
X
=
100 sec-‘
k4 = fast
Y*
-+ hv
This reaction is inhibited competitively by Mg2+. Thus 10 mM M$+ increases the Ca2+ required for a similar light output by a factor of 10. In theory it should be possible to estimate the Ca2+concentration from the rate of luminescence decrease. In practice the calculation depends on knowing the concentration of all factors such as Mg2+ which may inhibit the reaction, and on being able to ensure that CaZ+ remains constant for sufficient time to estimate a tt of decay. Mg2+ may frequently be estimated, but there are only a few experimental situations in which CaZ+ remains constant over a period of minutes to allow the calculation to be applied effectively. The alternative approach, determining the amount of aequorin present by activation of total luminescence with C$+ and integration of the light output at the end of the experiment, has been employed by Baker et al. (1971). Nevertheless, the difficulties of quantitating cellular CaZ+ with aequorin are considerable. Baker et al. found the light output of aequorin in extruded axoplasm to be lower than in an equivalent Ca-buffered solution. Spatial resolution of CaZ+ using the aequorin technique is theoretically limited by the wavelength of the emitted l i g h t 4 6 6 nm. However, practical situations may materially reduce this resolution. In practice few workers have attempted to achieve substantial spatial resolution. This may reflect in part the embryonic stage of the use of this reagent, in part the technical difficulties associated with measuring low light output, and in part the normal assumption that C 2 + is evenly diffused throughout the cytoplasm. However, Rose and hewenstein (1976) employed a microscope attached to an image intensifier to observe the distribution of Ca2+ in Chironomus salivary gland cells. The image from the image intensifier was monitored with a television camera. They reported a resolution of
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the image intensifier and camera system of 1-2pm and of the sharp Ca boundary of the cell of 1-2pm, while the direct optical visualization of the cell membrane had a resolution of ca. 4 p m . These values are probably close to the best resolution obtainable. It should be emphasized, however, that these workers injected Ca into the cell. At normal cytoplasmic Ca concentrations, high resolution depends on integration of the light output over a considerable period of time. In experimental circumstances the spatial resolution may be limited by statistical fluctuations when low light output is measured. Most workers have obtained concentrations of aequorin inside the cell in the range of 1(r6-l(r5 M. Higher concentrations may be difficult to achieve in practice, since it is desirable not to alter the intracellular milieu excessively by injection of a large volume of protein. One may calculate that with a volume of (5p my and a concentration of aequorin of l(r5 M the number of protein molecules is 7 X 105. If l(r6 of the total number of molecules decay in 1 second at physiological C P activities and if 50% of the quanta of light that are released is recorded, the number of light quanta observed is 0.35 per second. It may be seen therefore that a resolution of a few micrometers is a likely limitation for aequorin at physiological Ca2+ levels. In addition, further loss of resolution may occur if the depth of the preparation being observed with the microscope is in excess of a few micrometers, owing to limitations of focus of the microscope. The mode of introduction of aequorin into the cell has been injection through a microsyringe. Ashley and Ridgway (1970) employed a Hamilton microliter syringe with a glass capillary. They used a solution containing 2.5 X l(r4 M aequorin and injected 0.4 p1 into a fiber of the giant barnacle Balanus nubilis. They estimate the final concentration of aequorin in the cytoplasm to be ca. 1(r6 M. They state that 20-30 minutes is sufficient time to permit even distribution of the protein in the fiber after injection. Hallett and Carbone (1972) injected 1p 1 of 5 x l(r4 M aequorin into the squid giant axon from a capillary. For work with smaller cells the mode of injection has been different. Brown and Blinks (1974) employed a micropipet of 4-Mn impedance. They injected aequorin into Limulus ventral photoreceptors by applying a 5 to 20-psi pressure at the thick end of the micropipet. They estimated the volume injected by simultaneous injection of H235 SO, to be 2.5 X low5p 1and the concentration of aequorin in the cell to be l(r5 M. Llinas et af. (1972) injected 2.5 X l(r4p1 of 0.5 mM aequorin into the squid giant synapse. These experiments show that cells have to be sufficiently large that a micropipet may conveniently be implanted in them. Thus the Limulus photoreceptor cell has a cell body of 200 X 60 p m , while the giant barnacle muscle fiber has a length in centimeters. A judicial choice of cell type is essential for successful employment of the aequorin technique. Thus Brown and Blinks (1974) observed that injection of as little as 2 X l(r4 p1 of aequorin caused lowering of resting membrane potential in Limulus photoreceptors. Neverthe-
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less, adequate light output was obtainable from mole of aequorin to observe Ca2+ transients following a single flash of light. Aequorin has now been employed in the observation of C$+ transients of several cells. Early applications were those of Ashley and Ridgway (1968, 1970) in which aequorin was injected into giant barnacle muscle. The C 2 + transients associated with excitation-contraction coupling were observed. These workers recorded membrane potential with an electrode inserted into the fiber through a cannula and an external electrode; they monitored aequorin luminescence and the isometric force generated by the contraction. They observed that the first event following electrical stimulation was depolarizationof the fiber. The aequorin light output increased after the initial depolarization, and finally contraction followed the initial light increase by a delay of 200 msec. After the initial rise there was a fall in aequorin light output having a time constant T of 70 msec. The maximal contractile force occurred when the light output had already dropped close to the original baseline. Increasing stimulus strength in this muscle-which has a graded contractile response to variations in stimulation intensity-also increased the aequorin luminescence. Increasing stimulus duration caused an increased duration of the aequorin light output. Ridgway and Gordon (1975) found that muscle length affected the release of Ca2+by sarcoplasmic reticulum. When the muscle was stretched, greater aequorin luminescence occurred, but when it was shortened, the C2+ transient was lower. They found that the force generated was proportional to the C 2 + transient. Rude1 and Taylor (1973) carried out similar experiments in semitendinosus muscle from Rana pipiens and iliofibularis muscle of the toad Xenopus laevis. They observed a similar rapid increase in luminescence on stimulation of the muscle, as had been observed for the barnacle. The light level had declined almost to the baseline by the time maximum force was generated. The low cytoplasmic C$+ at the time of maximum force is an intriguing phenomenon which has not yet been adequately explained by the classic requirement of C$+ for the initiation of myofibrillar cross-bridge formation. Baker et al. (1971) monitored C$+ movements during stimulation of giant squid axons. A train of impulses caused an increase in aequorin output up to a steady level. Cessation of stimulation caused a decay of light with a time constant of 10-15 seconds. After a considerable period of stimulation, the light output started to increase again. Tetrodotoxin abolished the light response to short voltage-clamped depolarization but was less effective in inhibiting C2+ changes caused by trains of pulses of duration longer than 300 p sec. These investigators consider that the early C$+ influx is associated with passage of the ion through the Na channel, while the later C$+ flux is apparently not associated with opening of the Kf channel, since tetraethylammonium did not block the Ca2' flux while MrP did. Hallett and Carbone (1972) employed higher aequorin
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concentrations (5 X mole) in studying squid axons and observed C$+ transients associated with single impulses using a computer-averagingtechnique. The light output changes are too slow to be suitable for registering the time domains of the C2+ transient. DiPolo et al. ( 1976) employed internal dialysis of squid axons and confined the aequorin inside the dialysis tube in order to subject it to a defined environment. They reported an intracellular C P activity measured by aequorin luminescence of 2 X l(rs M . Llinas et al. (1972) injected aequorin into the giant squid synapse. They found a progressive inactivation of aequorin, implying a high resting C2+ concentration. Stimulation of luminescence was observed during impulse propagation. However, the luminescence increase was slow compared with the rate of transmitter release, possibly reflecting the slow aequorin response to changes in C 2 + . These workers discussed possible artifacts, such as the rapid depletion of aequorin in the region of the terminal membrane, which might account for difficulties in observing C$+ transients. Subsequently, Llinas and Nicholson (1975) prolonged the action potential by inhibiting the K+ repolarization and observed a luminescence response on excitation, which paralleled the release of transmitter. Stinnakre and Tauc (1973) observed similar luminescence increases in aequorin when neurones of Aplysia were injected with the photopTotein. Thus the luminescence increase in aequorin corresponded to conditions under which C$+ influx occurred during excitation of the neurone. Kusano et at. (1975) injected the postganglionic axon of the giant synapse of the squid with aequorin and observed a light transient initiated by presynaptic stimulation. Tetanic pulses induced luminescence when the stimulation was presynaptic but not when antidromic stimulation of the postsynaptic axon was applied. These workers reported that extrapolation of a plot of luminescence versus membrane potential implied that C2+ influx vanished at a potential of +140 mV. Brown and Blinks (1974) investigated CaZ+ transients in Limulus photoreceptor cells after they had been stimulated by light. They injected aequorin by pressure and determined that under optimal conditions the volume injected was 25 pl and was equal to 10% of the cell volume, while higher volumes caused large unstimulated aequorin signals. The final concentration of aequorin was l(r5 M . The half-time for the aequorin luminescence rise after stimulation was 35 msec. Stimulus-induced luminescence changes were not abolished by low external C$+ or by clamping the potential of the cell at the reversal potential of the light-induced current. These investigators found that the value of [CaJ did not correlate exactly with light adaptation of the photoreceptor cell and suggested that the light adaptation occurred when Ca2+ bound to an internal site in a reaction which was not linearly related to [Cail. Ridgway and Durham (1976) investigated C$+ changes in cells of the slime
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mold Physarum polycephalum during cytoplasmic streaming. Rhythmic fluctuations of aequorin luminescence were correlated with streaming activity as measured with an electroplasmograph. These workers argue that cytoplasmic C 2 + changes are responsible for cytoplasmic streaming through Ca-activated actomyosin. Rose and Loewenstein (1976) observed the association between intracellular Ca2+ concentration and cellular junctional communication in Chironomus salivary gland. They found that the presence of high Ca2+ at the cell junction was associated with uncoupling and loss of cell communication. When Ca2+ was high at sites distal to the junction, there was no uncoupling. They dissociated plasma membrane depolarization from uncoupling. From these data they argued that intracellular C 2 + at the junction is the effector of intercell uncoupling. The wide range of experiments employing aequorin to monitor intracellular Ca is indicative of the power of this technique in studying many cellular processes. The extensive employment of this reagent has revealed some of its drawbacks. Problems arise in introducing the reagent into small cells and in estimating the intracellular Ca2+ level quantitatively. The time course of the response to C 2 + is slightly below that of the fastest physiological processes.
V. Fluorescent Chelate Probe The antibiotic chlorotetracycline has been employed in monitoring C2+ movements in biological systems. The quantum yield of chlorotetracycline fluorescence increases when it binds to divalent cations, and this antibiotic has been employed extensively in studies of isolated organelles (Caswell, 1972; Caswell and Warren, 1972). Relatively little systematic work with the reagent in studying intracellular C2+ has been carried out. However, its employment in studying C 2 + transients in intact cells has increased recently. In principle, the antibiotic is capable of giving information on Ca2+ transients with excellent time response and good space resolution. Chlorotetracycline is one of a family of tetracycline antibiotics, all of which bind to divalent cations. However, they differ markedly in their fluorescence properties, and only chlorotetracycline has proved suitable as a fluorescent chelate probe (Caswell and Hutchison, 1971a). Chlorotetracycline binds to divalent and trivalent cations in aqueous solution. The dissociation constant of the chlorotetracycline-cation complex is 2.7 X l(r4 M for Mg2+ and 4.4 X l(r4 M for C2+ at pH 7.4 (Caswell and Hutchison, 1971a). The complexation of antibiotic with divalent cations causes a shift in the absorption spectrum and enhancement of the fluorescence of the compound. In less polar solvents the affinity for the cation is greater, and the fluorescence of the complex is more intense. In apolar media the order of affinity constants favors C 2 + over Mg2+ with a KD
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for Cd+ of 9 pA4 and for M g + of 25 p M in 70% methanol (Caswell and Hutchison, 1971b). Hallett et al. (1972) report a higher fluorescence for the Ca chelate of chlorotetracycline than for the Mg chelate by 65% in the presence of red cell membranes. Hallett et al. found that the fluorescence of tetracycline antibiotics in the presence of Ca2+ and red cell membranes was chlorotetracycline > demeclocycline > tetracycline > oxytetracycline, while chlorotetracycline had the lowest fluorescence of the four in the absence of Cd+ . Chlorotetracycline has a pK of an acidic group of 7.4 (Stephens et al., 1954), being uncharged at the more alkaline pH. The molecule is sufficiently lipophilic at neutral pH to be able to cross membranes. There is no evidence that the divalent cation chelate, which has a charge of 1, can cross membranes. Experiments using chlorotetracycline fluorescence to observe divalent cation movements in biological membranes have shown that the most readily observed divalent cation is that which lies on the membrane surface. Free Cd+ or MgZ+ attracted to the membrane Stem layer by electrostatic charge forces is capable of complexing with chlorotetracycline. The chelate has a high fluorescence efficiency, since the polarity of the membrane surface environment is low. Thus fluorescence of chlorotetracycline in the presence of divalent cation and biological membranes such as erythrocytes, mitochondria, and sarcoplasmic reticulum is high by reason of the migration of chlorotetracyclineprobe and divalent cation to the membrane surface (Caswell and Hutchison, 1971a; Caswell, 1972; Hallett et al., 1972). Many subcellular organelles are able to accumulate Cd+ by energy-linked transport. In these cases the divalent cation accumulates to high concentrations within the organelle and forms an electric double layer on the internal membrane surface. The chlorotetracycline is able to migrate passively across the membrane and complex with the internal divalent action on the membrane surface. This accumulation of Cd+ is reflected in an increase in chlorotetracycline fluorescence. When chlorotetracycline is employed in whole-cell studies, it can be expected that the antibiotic will penetrate the cell membrane and migrate to subcellular organelles which contain high C 2 + concentrations. Thus it would be predicted that mitochondria and sarcoplasmic reticulum would show fluorescence which would be. dependent on the Cd+ concentrations of the organelles. DuBuy and Showacre (1961) showed selective fluorescence of mitochondria in monkey kidney cells, thus supporting this concept. The selectivity for chlorotetracycline fluorescence is for Ca2+ and Mg2+. While the antibiotic fluoresces from MgZ+ on membrane surfaces, several factors determine its primary response to Ca2+: (1) The affinity of chlorotetracycline for C d + on membrane surfaces is three times higher than for Mg2'; (2) the fluorescence of the chelate of CaZ+ is higher than that of the Mg chelate by 65%; (3) fluorescence occurs at membrane sites of divalent cation binding; Cd+ shows a pronounced tendency for accumulation in subcellular organelles, hence binds to
+
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the inner membrane surface of the organelle, giving an enhanced chlorotetracycline fluorescence signal, while Mg2+ is not known to be actively accumulated in several organelles; and (4) the fluorescence spectra of the Mg and Ca chelates are sufficiently different to permit distinction between these two ions (Caswell, 1972). The time resolution of the chlorotetracyclinetechnique appears to be excellent. Hallett et al. (1972) have reported alterations of chlorotetracycline fluorescence when the antibiotic is administered either externally or directly into the axoplasm of squid giant axons and lobster nerve fibers. The fluorescence change responds during the time course of the action potential. However, this may not apply universally, since it is possible that the fluorescence response to depolarizationof the axon represents an alteration of the Ca2+ environment in the membrane, giving rise to a change in the fluorescence quantum yield. Under the circumstances of Ca2+ translocation from the membrane or organelle, the time resolution may be slower. When Ca2+ is accumulated by an organelle, the limit of time resolution may be that of chlorotetracycline permeation into the organelle, while the response to Ca2+ release from an organelle may be that of dissociation of the complex with C$+. However, the kinetics of chlorotetracycline have not been investigated. The spatial resolution of chlorotetracycline fluorescence is limited theoretically to the wavelength of the emitted light-530 nm. DuBuy and Showacre (1961) observed fluorescence from individual mitochondria in hepatocytes. In the observation of Ca2+ transients, the spatial resolution may be further limited by the low intensity of light emission during the time period of the observation. This can be increased by increasing the intensity of light excitation. The practical limits of resolution have not been investigated. The sensitivity of the fluorescent chelate probe technique is influenced by the time and space resolution required and by optical limits of the assaying device. For work on bulk preparations, for example in cell suspensions, the sensitivity is such that physiological CaZ+ transients can readily be detected. However, when the subcellular sites of CaZ+ storage are being observed directly, a fluorescence microscope is employed. Under these circumstances the fluorescence is low. The intensity of incident light may not be raised beyond a certain level, since the light causes photoxidation of the chlorotetracycline and consequent loss of Ca-induced fluorescence and cell death. Thus we attempted to monitor chlorotetracycline in tissue-cultured myoblast cells. Even when high-aperture lenses were employed, the fluorescence could barely be observed at incident light levels which did not destroy the cell. However, this problem should be resolved by employing an image intensifier detection system which enhances the optical image by several orders of magnitude. These attachments are no longer prohibitively expensive, and the sensitivity problem in directly observing chlorotetracycline fluorescence
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at subcellular sites in whole cells is potentially resolvable and should give valuable information on the subcellular sources which secrete Ca2+ during physiological events. One of the difficulties in employing chlorotetracycline in monitoring internal Ca2+ concentrations concerns interpretation of the data. Fluorescence intensity is highly sensitive to the physical environment of the probe, and the probe fluoresces primarily at the surface of the membrane where the environment is highly anisotropic. Thus any change in fluorescence in the probe may reflect either movement of Ca2+ into or away from the membrane, or it may reflect movement of the Ca-chlorotetracycline complex to a more polar or less polar region of the membrane. Expressed in another way, a change in fluorescence may reflect a change in quantum yield of the fluorescing molecules rather than bulk migration of C 2 + . Two approaches may resolve this problem: The first approach is an empirical one-interpreting the data in conjunction with other signals of CaZ+ movement or comparing the fluorescent chelate probe signal with the signal of other probes which are sensitive to membrane environments (Le Breton et al., 1976a; Chandler and Williams, 1977). Thus the chlorotetracycline technique may be employed to observe the time course of Ca2+ transients in conjunction with electron microscope techniques of C3+ localization which can be employed to confirm the interpretation of the fluorescence signals. The second more direct approach is to measure the quantum yield of fluorescence employing fluorescence lifetime measurements, since fluorescence lifetime is proportional to quantum yield. However, the situation is likely to be complicated in wholecell systems if there are several intracellular sites of CaZ+ from which chlorotetracycline fluoresces. Thus caution should be employed in interpreting changes in probe fluorescence unless the fluorescence proceeds from a single well-defined subcellular site. Some limitations on the employment of chlorotetracycline in whole-cell systems occur as a direct consequence of the Ca-binding properties of the molecule. Le Breton et al. (1976b) and Behn et al. (1977) have described the influences of chlorotetracycline on the physiology of cells. These workers employed millimolar concentrations of the probe. At lower doses, below 1 0 0 p M , no direct influence of chlorotetracycline on cell physiology has been reported. The effects of high antibiotic levels probably occur as a consequence of chelation of a material portion of the intracellular C 2 + . A second limitation of this technique involves the necessity to incubate most cells in C$+- or Mg+-containing media. Since in most cases the extracellular medium is present in large excess, the extracellular Ca2+ and Mg2+ may chelate most of the chlorotetracycline and either prevent accumulation or cause release (Behn et al., 1974). This problem may be diminished by employing a lower amount of divalent cation in the medium. Also, the rate of chlorotetracycline uptake and release is sufficiently slow that observa-
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tion of stimulus-induced Ca2+ movement may be made in normal media after the antibiotic has been allowed to accumulate (Chandler and Williams, 1977; Le Breton et al., 1976a). Although chlorotetracycline has not been extensively employed in observing Ca2+ transients in intact cells, it has now been studied sufficiently often to give an indication of its potential. Hallett et al. studied the influence of nerve action potentials on chlorotetracycline fluorescence. The probe was either perfused internally through the squid axon at a concentration of 100 1.1M or injected in a bolus of 1 mM probe. Signal averaging of 2600 action potentials was employed to increase the signal-to-noise ratio. The fluorescence change during the action potential matched the membrane potential change but required external Ca2+ to produce a signal. Voltage-clamp experiments showed that the fluorescence change was proportional to the membrane potential change. The response of the chlorotetracycline fluorescence was within milliseconds of the potential change. It is not clear whether the dye responds primarily to C$+ migration within the membrane or directly to the membrane potential. Taljedal (1974) employed chlorotetracycline to monitor Ca2+ movements in islet of Langerhan cells microdissected from pancreatic glands of mice. He incubated the cells with lC5M chlorotetracycline for 40 minutes prior to observation in a fluorescent microscope with a photometer attachment. He showed that either removal of Ca2+ from the incubation medium or increasing M$+ to 15 mM caused suppression of fluorescence. When Na+ was replaced in the bathing medium by choline, there was considerable fluorescence enhancement. Since Na+ inhibited insulin release from islet cells, he argued that the data implied a change in intracellular Ca2+ caused by changing extracellular Na+ and that an increase in intracellular Ca2+ could be responsible for triggering insulin release from the cells. Chandler and Williams (1977) and Chandler (1976) investigated Ca2+ transients in exocrine acinar cells of the pancreas. They preincubated the cells in l(r4 M chlorotetracycline and then observed them in a medium devoid of antibiotic using a spectrofluorometer. A slow decrease in fluorescence with time could be caused by chlorotetracyclinepassing out of the cell as a new distribution of antibiotic was approached. Administration of bethanechol caused an immediate sustained loss of fluorescence. This correlated with an enhanced release of the pancreatic enzyme amylase from the cells. These workers correlated the ffuorescence decrease with diminished CaZ+secretion within subcellular organelles. chlorotetracycline binds to both CaZ+and Mg2+ but with slightly different excitation and emission spectra. Chandler and Williams found that the initial fluorescence showed properties of Ca complexation, while the residual fluorescence after cholinergic stimulation correlated with Mg complexation, implying the selective release of C2+ from its C 3 + reservoir sites. A control experiment
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showed that stimulation of secretion did not affect the fluorescence of another fluorescent probe which responded to altered membrane environments without chelating to divalent cations. Le Breton et al. (1976a) employed chlorotetracycline to monitor Ca2+ movements associated with the stimulation of platelet aggregation. Platelets were preincubated in chlorotetracycline and then observed in a microspectrofluorometer. The cells accumulated chlorotetracycline over a period of 20 minutes, and the fluorescence emission spectrum of the accumulated antibiotic was that of a Ca chelate. The administration of ADP, or of the divalent cation ionophore A23187, caused a decrease in fluorescence which correlated with the lightscattering change which normally precedes platelet aggregation. When ATP was added exogenously, ADP was unable to initiate the light-scattering change or the decrease in fluorescence. The employment of 1-anilinonaphthalene-8-sulfonate as a control for nonspecific alterations of membrane properties showed no fluorescence change in this probe associated with thk triggering of aggregation. Le Breton et al. (1976b) have also described the inhibition of aggregation induced by 500 p M chlorotetracycline. They argue that these high doses cause chelation of intracellular CaZ+ such that the physiological response mediated through cytoplasmic free CaZ+ is inhibited. Thus when chlorotetracycline is employed as an indicator, it should not be used above approximately 100 p M , since higher levels may influence the response being monitored. Peterson and Freund (1976) carried out fluorescence experiments with human sperm cells in the presence of 25 p M chlorotetracyclinein a spectrofluorometer. In most experiments the external medium did not contain added CaZ+ or Mg+,so that the fluorescence of the chelate in the external medium was negligible. The cells slowly accumulated chlorotetracycline. Caffeine and theophylline, both of which increase sperm motility, both caused decreases in chlorotetracycline fluorescence. This was not associated with CaZ+ accumulation by the cells but appeared to reflect a redistribution of intracellular CaZ+ . Propranolol increased fluorescence, and this effect was inhibited by caffeine. Behn et al. (1974) studied the influence of the molting hormone of arthropods, ecdysone, on CaZ+ transients in salivary glands of Chironomus thummi. They employed fluorescence microscopy in the presence of 5 p A4 chlorotetracycline and used the mitochondrial uncoupling agent coumarin to determine that mitochondria contributed significantly to cell fluorescence. They found that ecdysone increased the fluorescence and concluded that the hormone acted on mitochondrial CaZ+ accumulation. The experiments on whole-cell systems suggest that chlorotetracycline has potential as a dynamic nondestructive probe of intracellular C$+. It has the advantages over aequorin that it can be employed in small cells and, provided its potential is realized, may provide information on the subcellular locus of the
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stored C 2 + which passes into the cytoplasm when a physiological event is triggered. The disadvantages of the system include a less complete cation selectivity than that of aequorin and the improbability that the probe can be employed for quantitative information on C 2 + concentrations in intact cells.
VI. Fixation of Tissue for Cytology Microscopic techniques have been widely employed in observing and assaying intracellular C$+. However, a preliminary to histological observation is to fix the C 2 + so that it will not migrate from its physiological site subsequent to the experimental protocol and before observation. Standard fixatives for membranes such as glutaraldehyde and OsO, are not suitable for preventing migration of Ca, hence specific procedures have been developed to retain the ion at its original in vivo site. Some workers have not employed a specific C2+ fixative and report little or no migration, although in general migration of C 2 + may be expected during preparation of the specimen unless specifically prevented. Two main procedures of fixation have been employed: (1) precipitation of C 2 + with a complexing molecule such as pyroantimonate or oxalate iron, and (2) freezesubstitution or freeze-drying in the presence or absence of a fixing ion.
A. CHEMICAL FIXATION Pyroantimonate was originally employed to precipitate Na+ in thin sections in order to locate intracellular Na+ . However, it became apparent that precipitation occurred with several other ions, including C 2 + , and pyroantimonate has recently been employed specifically in order to precipitate and observe C 2 + . Klein et al. (1972) gave values of cations concentration which caused precipitation with pyroantimonate: l(r6 M for C 2 + , l(r5 M for M$+, and < 1 t 2 M for N 2 + . Thus it is apparent that the appearance of a precipitate of pyroantimonate in a section is not diagnostic of the presence of either C$+ or Na+ . However, the precipitation of C$+ by pyroantimonate can prevent further migration of the ion, hence provides a means to fix it. The pyroantimonate ion serves the purpose in these studies of both precipitating the Ca2+ and also of increasing the electron density, owing to the presence of the dense antimony atom in the precipitate so that the precipitates may be observed readily with electron microscopy. The pyroantimonate ion has the property that it can cross biological membranes, hence can be employed to fix C 2 + at all sites in biological tissue. Some workers have sought to determine the nature of the pyroantimonate precipitate by observing the effect of modifying the incubation medium on for-
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mation of the precipitate. Thus Yarom and Meiri (1973) and Legato and Langer (1969) both observed pyroantimonate precipitates in muscle in the myofibril region, in the terminal cisternae, and on the plasma membrane. They found that, when muscle was extensively incubated in the presence of EGTA or EDTA, the precipitate in the myofibrillary region was absent from electron micrographs, while the plasma membrane precipitate was still present. However, the plasma membrane precipitate was removed when the incubation contained low Na+ in place of normal Ringers’ solution. They argued that the precipitate on the plasma membrane was a Na precipitate, while that in the terminal cisternae and myofibrils was a Ca precipitate. However, these techniques may not be of general application, since (1) they may disturb the physiological integrity of the system and (2) they depend on a complete exchange of the ions in the cell with the extracellular incubation medium. Thus Yarom and Meiri (1973) found that prolonged incubation of skeletal muscle with EGTA was necessary in order to remove the precipitate within the terminal cisternae, although this precipitate almost certainly contained C$+. A very slow exchange of extracellular C$+ with that of the lumen of the terminal cisternae in skeletal muscle accounts for this observation. The more limited goal of employing pyroantimonate to fix the ion appears to be more appropriate. The formation of a precipitate with pyroantimonatedoes not necessarily fix the C$+ at its original site under physiological conditions and at its original physiological concentration. Some artifacts may occur through migration of Ca2+ during the initial perturbation of the physiological medium by the most commonly employed fixative which has contained OsQ as well as K pyroantimonate . A second fixative for C$+ which has been widely employed is oxalate ion. This ion has much lower solubility as the Ca salt than for other physiological cations. Thus the solubilities of the salts are 0.28 M for Na+, 0.97 M for K+,4.8 mM for Mg2+,42pM for Ca2+,and 260pM for Sr2+.Thus an oxalate precipitate in cell tissue is most likely to be that of the Ca salt, despite the frequently much higher concentration of K+ and Na+ in the cell. Oxalate ion generally cannot permeate biological membranes, hence it is unsuitable as a fixative under conditions in which the C$+ is sequestered within a cell or organelle. A notable exception to this is the sarcoplasmicreticulum of skeletal muscle, which displays ready permeability to oxalate ion. Thus Podolsky et al. (1970) and Diculescu and Popescu (1973) treated skinned or broken muscle fiber with oxalate in order to fix the C$+ in the cytoplasm and in the sarcoplasmic reticulum. There is a considerable likelihood that oxalate enhances C$+ accumulation by sarcoplasmic reticulum in vivo, as it does in vitro, hence causes an artifactually high [Ca] in the sarcoplasmic reticulum. The limited ability of the oxalate ion to permeate most membranes restricts its usefulness as an initial fiative, though it can be beneficial in preventing Ca2+ movement during preparation of the material for
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sectioning. Thus Coleman and Terepka (1972a) employed 1% Na oxalate in their acrolein fixative and throughout fixation and dehydration.
B. FREEZE-DRYING
A second basic approach to the fixation of tissue for Ca2+ analysis has been freeze-substitution or freeze-drying. With this technique the tissue is rapidly frozen in a liquid such as Freon or isopentane in order to halt any reaction or any solute migration. The water is then withdrawn from the specimen either by freeze-drying, in which the ice is sublimed at low temperature, or by freezesubstitution, in which the tissue is treated with ethanol in order to substitute ethanol for ice. The tissue is subsequently dried. Several variations in this protocol have been employed. The simultaneous administration of a fixative or precipitating agent during freeze-substitution may diminish the translocation of Ca2+ during the prolonged ethanol substitution. Some attempts have been made to determine the Ca2+ loss from the preparation. However, little evidence exists concerning C 2 + migration within the organ. It is likely that the simultaneous use of a fixative or precipitating agent will diminish Ca2+ migration, at least to the extent that the ion is contained within the organelle in which it was situated. Geyer et al. (1974) described the employment of 1% oxalic acid in ethanol at -80°C for 2 weeks in the freeze-substitution of erythrocytes. They reported a total Ca2+ loss during the procedure of 0.02% of 45Ca. They observed an even distribution of Ca2+ within the lumen of the erythrocytes and argued that major ion translocation did not occur during fixation. Ingram et al. (1972) employed freeze-drying in order to remove water. They found serious tissue damage when the drying was carried out at -3O"C, but when the drying was performed at -60°C tissue damage was minimized. Winegrad (1965a) employed freezesubstitution at -75"C, in which the ethanol contained 1% Os04 to fix the muscle. He reported that less than 5% of the 45Cawas lost from the muscle during fixation and dehydration. Winegrad (1968) evaluated the degree of Ca2+ translocation during the preparation of the specimen with a control in which 45Cahad not been given time to pass into the cytoplasm of the muscle. He found a considerably greater grain density in the extracellular space than within the cell. However, some migration of Ca2+ appeared to have occurred, caused by distortion of the preparation during sectioning. This problem was resolved by employing a harder embedding matrix. These various techniques for fixation of Ca2+ have represented attempts to prevent Ca2+ migration during preparation of the specimen for electron microscopy. Empirical evidence suggests that precipitation of C2+ or freezesubstitution prevents substantial C$+ translocation. Nevertheless these techniques may interpose perturbations in the cells between the time of the physiolog-
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ical event being monitored and the final micrograph, and the optimal fixation technique has not been investigated systematically.
VII. Autoradiography Although it might appear that autoradiography could solve many of the problems inherent in measuring intracellular C 2 + , this technique has been employed only on a restricted scale and has several limitations. Autoradiography may be used either with the light microscope or with electron microscopy. The limits of resolution with the two techniques are dependent on different factors. The major advantage of electron microscopy is the clarity of the image obtained of the specimen overlying the photographic emulsion rather than a dramatic improvement of resolution of the radioactive source. The principle of the technique is to introduce the C 3 + into the cell as its radioactive isotope, 45Caor 47Ca.The cell is then fixed to prevent redistribution of the isotope and cut into sections. A section is placed in direct contact with a thin, fine-grain photographic emulsion and exposed for a sufficient period so that an appropriate number of developed spots caused by radioactive decay may be seen. The emulsion is developed, and the section stained for observation. Thus microscopic examination reveals both the morphology of the stained preparation itself and the appearance of dense silver grains from the exposed emulsion. Autoradiography can reveal both the amount and distribution of intracellular C 2 + . Car0 (1962) and Car0 et al. (1962) have given a detailed description of the theoretical and practical determinants of the resolution of autoradiography. However, most discussions have centered around isotopes giving low-energy emission, while both radioisotopes of Ca have high energy such that p particles emitted by these isotopes penetrate a considerable distance through matter. The spatial resolution of the technique is therefore primarily influenced by geometric considerations. If the section which contains the isotope is sufficiently thin and if the underlying emulsion is in close contact with the section and is also thin, the resolution is much improved. This depends on the fact thatp particles are emitted on the average equally in all directions in space. However, the section and emulsions are planar, so that the region of the emulsion immediately underlying the radioactive source covers a greater solid angle from the source than the distal regions do. Several factors limit the thinness of the section and emulsion. These have been discussed in detail by Winegrad (1965a). The emulsion is composed of fine, approximately spherical grains of silver halide. For an individual grain to be exposed, it is necessary that the p particle transfer a certain amount of its energy to the grain. Less energy is transferred by high-energy /3 particles than by low-energy particles. Pelc et a2. (1961) estimated that p particles from 14C require a grain size of 0.1 p m in order to obtain
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exposure, and Winegrad (1965a) extrapolated this value to estimate a required grain size of 0.14 p m for 45Ca. Thus the minimum thickness of the emulsion is approximately 0.14 p m. Hence the limit of resolution of an exposed spot on an emulsion is not less than the size of the grain, since the grain is the unit which is developed. In addition, the thickness of the section must be added to that of the emulsion in estimating the resolution, since this further influences the distance between the source and the exposed grain. The geometric factor relating the distribution of p particles from a source impinging on a grain in the plane of the emulsion is described by the solid angle of the grain from the source. The solid angle is equal to n-r 2 / x 2at far distances, where r is the radius of the grain andx is the distance from the source. In practice the spatial resolution of the technique is similar to the grain size (Caro, 1962). Normal sections for electron microscopy are on the order of 0.1 p m , while sections for light microscopy are generally thicker in order to obtain good contrast. Winegrad, however, used a O.l+m section for light microscopy autoradiography of 45Cain skeletal muscle. Winegrad has also discussed other factors which influence resolution. Backscatter of electrons from emulsion grains occurs readily when the angle of incidence on the emulsion is high. High angles of incidence occur when the electron impinges on a distal grain of emulsion, hence backscatter improves resolution, since electrons which are scattered away from the emulsion do not expose any grains. Using these arguments and a simple geometric description of emulsion and section, Winegrad estimated that the number of exposed grains from a source declined to 40% at a 0.2-pm separation from the point immediately under the source and was close to 0 at 0.5 p m from the source. He assumed a section thickness of 0.1 p m and an emulsion thickness of 0.14 p m . In light microscopy, a further limit on resolution is associated with a blurring of the photographic image of the section. The ultimate resolution of the light microscope is that of the wavelength of light, and this is ca. 0.5 p m . However, it is seen that electron microscopy can improve the autoradiographic resolution only to about 0.2 p m , although the electron microscope can dramatically enhance the resolution of the stained section overlying the emulsion. The ultimate limit of sensitivity of autoradiography is dependent on the specific activity of the labeled c$+and on the ability to average grain distribution over a substantial area of the micrograph. If carrier-free Ca2+ is introduced into the cell, at a concentration of 2 X 1W6 M C$+ there will be an average of one molecule in each (0.1pmY space. However, several factors reduce the number of grains of emulsion exposed. These include (1) geometric limitations caused by /3 particles emitted which do not penetrate the emulsion, (2) sensitivity of the film to the isotope, and (3) the fact that not all isotope molecules decay during the period of exposure of the emulsion. Thus 45Cahas a half-life of 167 days, and a normal period of exposure of the emulsion might be 4 months, so that half the isotope molecules will not have decayed in this time. Also, it is not likely that the
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cell will be free of unlabeled isotope. Even if carrier-free 45Ca2+is introduced into the cell, it will be diluted with endogenous unlabeled ion. Winegrad (1965a) further estimated that geometric factors and the limifed sensitivity of the film resulted in one out of three disintegrations causing exposure of the grain. The sensitivity of the technique is further dependent on the extent of the region from which exposed spots are employed to average the density of C 2 + in a particular intracellular environment. The time resolution of the technique is clearly slow, being dependent on the rapidity with which the material may be removed from its incubation medium, prepared for fixation, and finally fixed. Winegrad (1968) employed rapid freezing in liquid Freon and assayed C 2 + distribution in muscle frozen 20 seconds after termination of tetanic stimulation. The choice of Ca radioisotope is limited to the two commercially available, 45Ca and 47Ca. Both isotopes are /3 emitters of high energy. 47Caalso gives y rays. The half-lives of the two isotopes are 165 and 4.5 days, respectively. A disadvantage of ,'Ca is that the energy of the /3 particles is very high, but for neither isotope is the energy sufficiently low materially to limit the path length of the /3 particle in the section. The short half-life of 47Ca reduces the time of exposure of the emulsion to a few days, while for optimal exposure of 45Cathe exposure must be about 1 year. However, the much greater availability of 45Ca and its considerably lower cost have caused it to be employed in the vast majority of autoradiographic studies on Ca. Winegrad (1965a,b, 1968, 1970) has described in detail the application of autoradiography and light microscopy to the study of skeletal muscle contraction. He employed three separate procedures to fix the muscle. In one type of experiment the extensor longus digiti muscle of frog was incubated for several hours in 45Ca and then washed with unlabeled C$+. The muscle was fixed initially in 0.01% OsO, in a solution containing 2 mM oxalate. Fixation was continued in more concentrated solutions of OsO, . The muscles were subsequently dehydrated in ethanol and embedded in methacrylate. Winegrad found that the initial fixation in Os04 caused some contraction of the muscle. In studies on relaxed muscle he employed procedures such as prolonged depolarization in 50 mM KCl in order to prevent contraction during fixing. He also observed that contraction in the fixative was reduced by lowering the temperature to 4°C. Some 45Cawas lost from the muscle during the fixation procedure. The second type of fixation employed by Winegrad (1968) was rapid freezing. The muscle was incubated in 45Caand then rapidly washed and partially dried by allowing fluid to soak onto a filter paper. It was then dipped into Freon at - 160", sublimated to dryness at -6O"C, and fixed in OsO, by exposure to the vapor of the fixative at -40°C. This was followed by embedding in methyl methacrylate. The third experimental protocol was identical to the second except that, following dehydration of the frozen muscle, it was treated with 1mM oxalk acid
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and OsO, in ethanol in order to effect a freeze-substitution of ethanol for water. When freeze-substitution was employed in fixation, sections were cut into a solution of acetone, and Winegrad recorded the loss of some radioactivity into the suspension each time a section was cut. By employing the technique of light microscope autoradiography and by establishing a theoretical description of the position of an exposed spot on the film with respect to the position of the radioactive source, Winegrad defined the position of Ca2+ within the sarcomere of the myofibrils in muscle at rest and under tension. In analyzing the CaZ+ distribution in autoradiogram, he divided the sarmmere transversely into O.1-pm sections in order to obtain the C 3 + distribution within the sarcomere. He further divided the muscle longitudinally between the myofibrillar space and the interfibrillar space. The latter was assumed to consist mainly of sarcoplasmic reticulum. Since the sarcoplasmic reticulum is subdivided within the sarcomere into the longitudinal reticulum and the terminal cisternae, it was possible to distinguish Ca2+ localization within these organelles. A statistical analysis was further carried out to estimate possible red distributions of Ca2+ within the sarcomere which would give rise to the observed distribution of exposed grains. The longitudinal distribution of grains in muscles which had relaxed after K+ depolarization had the highest Ca2+ density in the I-band region of the muscle, while in muscles contracted in OsOl the grains were located primarily over the A band (Winegrad, 1965a). Transverse distribution of grains showed that in the contracted muscle most of the A-band CaZ+ was associated with the myofibrils, while most of the I-band grains were located in the interfibrillar space, hence were presumably associated with the terminal cisternae (Winegrad, 1965b). Winegrad (1968) observed the distribution of CaZ+ during the relaxation phase following tetanic stimulation. The Ca2+ in the interfibrillar space rose first in the region of the longitudinal reticulum and subsequently in the terminal cisternae, implying that initial accumulation of Ca2+ occurred primarily at the longitudinal reticulum, and this was followed by luminal translocation of C$+ toward the triad. Further analysis employing freeze-drying techniques showed that C 3 + in tetanically contracted muscle was located primarily in the I band of the muscle where troponin was located, while in relaxed muscle CaZ+ was present in the terminal cisternae (Winegrad, 1970). Winegrad estimated a tt of 9 seconds for the migration of CaZ+ from the longitudinal reticulum to terminal cisternae during muscle relaxation. This work well illustrates both the experimental complexity of autoradiography and the degree of precision obtainable by this method. Maeda and Maeda (1973) employed autoradiography and light microscopy to monitor Ca2+ movements associated with stalk formation of the slime mold Dictyostelium discoideurn. They fixed tissue by placing it in ice-cold ethanol and then dehydrated and embedded it in Paraplast. During stalk formation Ca was located in the anterior prestalk region in preference to the posterior prespore
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region. This differentiation of Cd+ content within the cell slug was observed to be more marked in the late stage of cell migration associated with spore formation, and Cd+ content was highest in differentiating stalk cells. Thus these workers were able to argue that Cd+ played an important role in cell differentiation and stalk formation of the slime mold. Geyer et al. (1974) described a technique for fixing Cd+ by freezesubstitution in 1% oxalic acid in ethanol at -79°C. Human erythrocytes were examined in this way by electron microscopy. Autoradiographic spots were observed in the cytoplasm of the erythrocyte, and a high concentration was seen in the region of the cell membrane, indicating that a portion of the Cd+ was bound to the cell surface. Ishida and Yoneda ( 1974) employed electron microscopy and autoradiography to examine the C d + content of neurohypophysis axon terminals. The cells were fixed in OsO, and dehydrated in ethanol which contained Ca oxalate. These workers found a higher density of grains in fibers which had been stimulated electrically or by K-depolarization. They located the majority of the exposed grains within or associated with the neurosecretory granules with low concentrations in the mitochondria or plasma membrane. A novel variant of autoradiography has been devised by Langer et al. (1969) and Langer and Frank (1972) who employed scintillation autoradiography to monitor Cd+ fluxes in tissue-cultured myoblast cells. The labeled C d + is assayed through the production of light pulses by a glass phosphor. The myoblasts are grown directly on the phosphor, so that Cd+ attached to the cells is in immediate proximity to the scintillator. Thus the technique is directly comparable to that of emulsion autoradiography in which the same principles of resolution apply, except that in this case the Cd+ is assayed directly and without fixation. The spatial resolution of this approach is limited, since the only restriction on the production of a light pulse is the path length of a /3 particle from Ca2+. The p particle has a path length of 30 p m in water. Therefore light pulses are produced not only by the cultured cells but also by incubation medium in the immediate vicinity of the cells. By subtracting the light pulses from a phosphor lacking the cells it is possible to estimate the Cd+ content of the cells. This method does not permit localization of C d + within the cell but has the significant merit that observations may be made without modification of the cells. The time resolution and sensitivity are dependent on the specific activity of the isotope and on the frequency of sampling the counts, but the resolution is in any case on the order of seconds. These investigators observed an inhibition of Cd+ flux in heart cells on administration of 0.5 mM L$+, and at the same time surface Cd+ was displaced. The rate of washout of labeled C d + gave two experimental decays with rate constants of 0.77 and 0.023 min-'. These two rates accounted for the full C d + content of the cells. After La3+ treatment the fast phase of the washout disappeared.
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VIII. Electron Probe Microanalysis Electron probe microanalysis is one of several microanalytical spectroscopic techniques devised recently for identifying and quantitating elements in a section. The other techniques include ion probe, proton probe, x-ray probe, and laser probe analysis. However, electron probe analysis has been the most widely employed, since it requires only slight modifications of current electron microscopes and has adequate sensitivity and resolution. Only electron probe microanalysis is discussed here. Hall (1971) and Lechene and Warner (1977) have described the theory and practice of electron probe microanalysis. The technique depends on the ability of a specimen to release x rays when a beam of high-energy electrons impinges on it, hence is a natural corollary of electron microscopy. The emitted x ray has an energy and frequency which are characteristic of the atom from which it was emitted and are virtually independent of the chemical status of the atom. Thus the technique is suitable for elemental analysis of the specimen for all atoms of atomic number greater than 5. Two different modes of electron microanalysis are currently available. The microprobe may be employed in a scanning electron mode, so that the x ray output from a sample is monitored as the electron beam scans a specimen. The x ray output is passed through a diffracting crystal or semiconductor spectrometer, and the output at a particular wavelength is recorded on an oscilloscope which scans synchronously with the electron beam. The alternative mode is analytical electron microscopy using a transmission electron microscope. In this mode the impinging electrons are focused on small portions of the specimen by an accessory minilens. The electrons transmitted through a thin section are viewed in the normal way with focusing electron lenses and a fluorescent screen. However, scattered x rays are collected and passed through an x ray spectrometer as described above. While the electron probe is basically identical in the two modes, the complementary structure of the specimen is observed in different ways, so that the microenvironment of the probe is visualized differently. When the microprobe is used in conjunction with a transmission electron microscope, the former is normally employed in a static mode to analyze a fixed region of a specimen, although scanning facilities are sometimes available. However, use of the microprobe in conjunction with the scanning electron microscope is basically a scanning technique which records element density in a two-dimensional image. The limit of sensitivity of the electron probe technique is primarily defined by two factors. There is an ultimate limit of sensitivity of a specimen containing a single pure element, which is influenced by the number of x rays emitted by the sample and the proportion of these collected by the spectrometer. Two different detector systems are employed, which vary considerably in sensitivity. The crystal diffracting spectrometer provides a very high resolution of the energy
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spectrum, hence the atomic source of the x ray, but has an efficiency of collection of approximately lW4 (Hall, 1971).The other common mode of detection is the use of a solid-state detector which collects a considerable proportion of the emitted x rays. Discrimination is obtained by pulse height analysis of the electric current output of the detector. The efficiency of this system is approximately 1W2, but the discrimination between elements is lower. In particular this detector cannot discriminate between Ca and Sb, so that the pyroanthnonate precipitation procedure for Ca cannot effectively be combined with the microprobe assay of c2+. A more restricting limit of sensitivity is caused by the emission of white x rays by a specimen. These x rays are emitted by all elements according to the mass of the sample and interfere with the spectral lines of x rays from the element being investigated. Thus the lower the concentration of the element being investigated in a sample, the greater the proportion of background noise caused by the emission of white x rays by all elements of the specimen. This limit of sensitivity may be expressed as a weight fraction limit of detectability and is in the region 0.1% when a diffracting crystal is employed. However, it is emphasized that this applies to the final specimen after dehydration, staining, and embedding and does not apply to the original sample. The situation is less satisfactory for nondiffracting detectors, since the spread of detectable x ray energy associated with these detectors causes an increased proportion of white x rays to be recorded, hence the signal-to-noise ratio is lower. The limits of detectability with this detector are approximately 1%. Moreover, discrimination between elements is not total, hence a minor element may not be detectable in the presence of an interfering element. The spatial limits of resolution of the electron probe technique are primarily concerned with two factors: (1)the minimum size of the probe and (2) the experimental limits of detection of the system. Probe diameters as low as 0.1p m are available. In order to obtain a sufficiently large volume for adequate sensitivity, the sections are normally cut to a 0. l-pm thickness or greater. Calculation shows that, if the sensitivity of the technique is on the order of 1@l8 gm, the probe diameter is 0.1 p m , and the section has a density of 1.2 and a thickness of 0.1p m , Ca can be detected at 24pmoles/gm tissue. Greater sensitivities may be attained by long x ray collection times, though with some loss of resolution caused by destruction of the sample by the electron beam. While this estimate ignores changes in composition during preparation of the specimen, it reveals that for the majority of analyses the resolution is unlikely to exceed 0.1 p m and that only elements present at reasonably high concentration are detected. A corollary of the spatial resolution of the probe is the spatial resolution of the electron micrograph. If a transmission electron microscope is employed, resolution should be the same as that of conventional electron microscopy, except that the relatively thick sections frequently employed for analysis cause some loss of detail.
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Analytical electron microscopy may be employed to quantitate the elements present in a section. However, this has proved successful only for the estimation of relative abundance. The accuracy of quantitation is affected by the following considerations: (1) loss or gain of element during processing of the specimen, (2) loss or gain of mass of the specimen during processing, (3) need to estimate local thickness and density of the specimen, and (4) need to calibrate the detector. The alterations of specimen composition during fixation, dehydration, and embedding are the most intractable source of inaccuracy in estimating element abundance. Hall (1972) described the use of the background white x ray intensity in order to estimate the thickness of the section, since this is proportional to the mass thickness of the specimen. The calibration of the spectral x rays is readily effected by placing a calibrated sample in the path of the microprobe. The employment of the electron microprobe to detect intracellular Ca2+ has been mainly confined to the observation of sites of high Ca2+ concentration, since the device has limited ability to detect low C2+ concentrations. A widespread use of the microprobe has been in the analysis of a variety of intracellular granules. Hillman and Llinas (1974) and Oschman et al. (1974) analyzed granules found as dense plaques lining the internal axonal surface of squid axons and ganglia. Hillman and Llinas (1974) used conventional fixation and staining procedures for electron microscopy of ganglia. They employed a transmission electron microscope with a scanning attachment and an energy-dispersive analyzer and used 0.5j~m-thickspecimens with x ray analysis to detect Ca and P in the granules. Oschman et al. (1974) used similar procedures to obtain 0.2p m-thick specimens which they examined in a transmission electron microscope with an electron microprobe attachment. The spots were probed for periods up to 100 seconds. These investigators were able to quantitate the ratio of elements in the granule by dividing the specific signal for Ca or P obtained from a crystal spectrometer by the white background signal obtained by a slight offset of the spectrometer and obtained a C d P atomic ratio of 7:lO. These workers also recorded C 2 + signals from mitochondria. Mitochondria1granules within chondrocytes have been monitored by Sutfin et al. (1971). They used a scanning transmission electron microscope with a resolution of 200 A together with a silicon x ray detector for elemental analysis. The counting period was 500 seconds. A deposit tended to build on the sample, which obscured the x ray signal until they improved the vacuum. With this instrument they could detect elements with atomic numbers greater than that of Na. Chondrocytes from the costal epiphyseal plate were fixed in glutaraldehyde and RuO, . Ca and P were readily detected in the mitochondrial granules and were absent in the matrix. Somlyo et al. (1974) loaded a portal anterior mesenteric vein with Ba or Sr and analyzed the mitochondrial granules that were formed. They cut O.l-pm sections from conventionally fixed muscle and analyzed both by crystal spectrometry and energy dispersion using an electron microprobe attached to a transmission electron microscope. The diameter of the probe was 0.2-0.3 p m ,
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and the time of x ray collection was 300 seconds. In addition to the Ba or Sr observed in these mitochondrial granules they also detected Ca. Coleman et al. (1972, 1973a,b) detected Ca in granules of Tetrahymena pyriformis and of Amoeba proteus which had been rapidly heat-fixed. The cells were observed directly in the probe without embedding or sectioning. They employed a combination of reflection light microscopy and the sample current of the electron microprobe to visualize the specimen. A probe size of 0.2-0.3 p m was employed, and the x ray output was detected with a crystal diffracting spectrometer. These workers located a range of elements in the granules, including P, Ca, K, and Mg. They determined the source of the signals by scanning techniques and obtained quantitative estimates of the relative abundance of these elements. Quantitation was achieved by comparing the x ray output with that of standard specimens of known composition and calculating for different attenuation of the signal in the matrix of the sample and the standard. Several studies have been carried out in monitoring Ca in muscle fibers. Podolsky et al. (1970) and Diculescu and Popescu (1973) used the technique of forming Ca oxalate precipitates in muscle in which the plasma membrane was broken or absent. Podolsky et al. perfused a skinned fiber of frog semitendinosus muscle in 10 mM oxalate and then fixed it in glutaraldehydeand OsO, containing oxalate. Sections were analyzed in a transmission electron microscope containing a microanalyzer. The probe diameter was 0.2-0.3 p m , and crystal diffraction was employed in detection. Precipitates in terminal cistemae contained Ca at a weight fraction of 1%. Diculescu and Popescu perfused muscle fragments with oxalate. Precipitates in mitochondria were detected by transmission electron microscopy and analyzed by scanning the mitochondrial region. Sections 0.2 p m thick were employed, and the probe diameter was less than 1 p m . A crystal diffractometer was used for detection. The mitochondrial precipitates were shown to contain Ca. Yarom and Chandler (1974) employed the technique of fixing intact frog sartorius muscle in a solution containing pyroantimonate and OsO, . Gold sections were examined in transmission electron microscope with a microprobe attachment. Energy-dispersion spectra of pyroantimonate precipitates were analyzed for elements in an x ray detector. The probe diameter was 0.2 p m , and the time of examination was either 20 or 400 seconds. Since the Ca and Sb signals were almost superimposed, they were unable to estimate Ca by energy dispersion but employed a crystal spectrometer to estimate the Ca content of the sarcolemma, triad junction, I band, A band, and nucleus. The 20-second count gave very low counts in all regions of the cell, with a consequent high standard deviation. Somlyo et al. (1977) made comparative studies of the composition of sarcoplasmic reticulum and cytoplasm of the skeletal muscle of the toadfish Opsanus tau. They employed a transmission microscope with an energydispersive microprobe detector and reported a probe diameter of 50- 100 nm. The swim bladder muscle was rapidly frozen, dried, and sectioned, and then observed
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in the electron microscope. These workers found a higher Ca level in the sarcoplasmic reticulum than in the cytoplasm, while other elements such as C1, Na, K, and Mg were similar in concentration in the two regions. Yarom et al. (1972) employed electron probe microanalysis in estimates of quantitative alterations of the Ca content of heart cells under the influence of the inotropic catecholamine, isoproterenol. The heart was fixed in pyroantimonate by perfusion through the ventricle. Ca was analyzed using a crystal spectrometer. These workers found a drop in the content of C 2 + in the heart 2 hours after an isoproterenol injection and an increase in C$+ 4 hours after the injection. Atsumi and Sugi (1976) examined the distribution of C$+ in the retractor muscle of the mollusc Mytilus edulis, employing pyroantimonate as a Caprecipitating agent. Sections 0.2-0.3 p m thick were examined in a scanning electron microscope with an energy-dispersive microprobe. F'yroantimonate precipitates were observed along the plasma membrane, mitochondrial membrane, nucleus, and Golgi apparatus. When the muscle was stimulated with acetylcholine, precipitates were observed in the cytoplasm. The microprobe analysis revealed two Sb-Ca peaks, but the Sb signal could not be separated from that of Ca. Sugi and Daimon (1977) have similarly observed pyroantimonate precipitates in mitochondrial, sarcoplasmic reticulum, nuclear, and plasma membranes of taenia coli from guinea pig. They showed the Sb-Ca peak in these precipitates by an energy-dispersive microprobe but did not distinguish the superimposed peaks. Coleman and Terepka (1972a,b) carried out a detailed analysis of the embryonic chick chorioallantoic membrane with a view to estimating the opportunities and limitations of the microanalytic technique. They employed phase microscopy and the sample current of the electron probe to visualize sections of thickness between 0.25 and 2 p m and a crystal spectrometer for the analysis. Membranes were fixed in acrolein containing oxalate and stained and dehydrated in oxalate-containingsolutions, and thick sections were prepared. These workers reported a resolution of 0.5 p m in imaging with the sample current, but that the x ray resolution was on the order of 2 p m and that the diameter of the electron probe was not necessarily indicative of the actual x ray resolution. They carried out a detailed analysis of potential artifacts of preparation. They found the Ca signal to be localized in cytoplasmic arms of capillary-covering cells in the ectodermal cell layer. They suggested that C 2 + accumulation in these sites occurred through endocytosis. Tandler et al. (1970) used pyroantimonate as a fixative in the absence of OsO,, followed by formaldehyde. In some experiments the fixed tissue was heated in K pyroantimonate in order to wash out any residual precipitated K pyroantimonate from the material. Sections 1 p m thick were observed by electron microscopy and separately by microprobe analysis. Calcium was found to be concentrated in the nuclei of maize root and rat liver. Timurian et al. (1974)
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studied the Ca and P distribution in fertilized urchin eggs. The eggs were fixed in mercuric chloride and then acetic acid, frozen, and sectioned. Combined light microscopy and microanalysis was employed with 8-pm sections and a 2-pm beam diameter. They found Ca and P to be more concentrated in the mitotic centers than in the cytoplasm during both metaphase and anaphase. Robison et al. (1971) employed microanalysis to detect Ca, P, and I in normal and pathological human thyroid glands. Sections 12 p m thick were cut from frozen glands and lyophilized. A 1- to 2-pm probe beam and crystal diffraction spectrometry were employed in the scans. These investigators found I to be distributed in the colloid areas of the follicles. P was distributed in the epithelial cell lining, and Ca was found both in the colloid and in the epithelia..Herman et al. (1973) employed a transmission electron microscope with a microprobe attachment in the analysis of sections of isolated islets of Langerhans cells. The islets were isolated by digestion of collagen and removal of the endocrine cells using a microscope. The cells were fixed in pyroantimonate and OsO, and sectioned for analysis by crystal diffraction spectrometry. High levels of Ca were associated with the secretory granules.
IX. Concluding Comments The techniques described offer a wide range of approaches for assaying intracellular C d + . The variety of problems involved in monitoring in vivo Ca diffusion between compartments in the short period of the physiological event present peculiar technical challenges. In general techniques have kept pace with the problems, and at the same time the problems have stimulated development of the techniques. Thus optical methods for following rapid C d + transients have been developed over the last few years, and the search continues for more sensitive and selective dyes. Some of the future trends in physiological research on Ca may be predicted. The emphasis on the location of C d + may change from the assay of cytoplasmic Cd+ toward determination of the source of C d + which causes alteration of cytoplasmic Ca2+and toward the binding site of Ca2+which serves to produce the response. In some cells this development has already occurred. The technical problems in monitoring C d + fluxes within organelles of cells may be considerable, although some progress has already been made. Advances in the microscopic observation of C d + may be expected. The development of the electron microprobe has been accompanied by developments in other microprobes such as the proton microprobe and ion microprobe. Technical advances may render these more useful for general assay. These techniques may have advantages of increased sensitivity which may be accompanied by increased
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resolution. There is still a need for a high-resolution, high-sensitivity assay for
c2+.
In the more distant future interest may center on the location and fluxes of C2+ in individual cells of multicellular networks in which Ca2+ serves as an agent in control of concerted processes. An example of such a process is communication between mons of nerve cell aggregates such as ganglia and the central nervous system. The technical problems in making such observations may indeed be considerable. The future offers technical challenges in exploring the role of Ca2+ in living organisms.
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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 56
Electron Microscope Autoradiography of Calcified Tissues ROBERTM. FRANK Groupe de Recherches Institut National de la Sante et de la Recherche Medicale U 157, Faculte de Chirurgie Dentaire, Universite Louis Pasteur. Strasbourg, France
Introduction . . . . . . . . . . . . . . . . . . . . Methodology . . . . . . . . . . . . . . . . . . . Detection of Nucleic Acids in Calcified Tissues . . . . . . . Synthesis of Organic Matrices of Calcified Tissues . . . . . . A. Elaboration of Bone Matrix . . . . . . . . . . . . . B. Elaboration of Dentin Matrix . . . . . . . . . . . . C. Elaboration of Enamel Matrix . . . . . . . . . . . . V. Transfer Routes of Inorganic Elements . . . . . . . . . . A. Electron Microscope Autoradiography of 45Ca during Osteogenesis . . . . . . . . . . . . . . . . . . B. Electron Microscope Autoradiography of 45Ca during Dentinogenesis . . . . . . . . . . . . . . . . . C. Electron Microscope Autoradiography of 45Caduring Amelogenesis . . . . . . . . . . . . . . . . . . VI. Identification of Sensory Nerve Endings in Adult Dentin by Autoradiography . . . . . . . . . . . . . . . . . . VII. Conclusion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . I. 11. 111. IV.
183 185 188 188 1 89 202 214 226 228 233 242
246 247 248
I. Introduction When Becquerel (1896) discovered natural radioactivity in uranium ore, he used a photographic film and was therefore the first to employ autoradiography. Biologists soon became interested in the possibility of following metabolic processes by determining the fate of radioisotope-labeled molecules. Morphologists made extensive use of autoradiography, whereas biochemists, utilizing cell fractionation techniques, turned to radioactive tracers detectable by scintillation counting methods. Autoradiography has an advantage over usual biochemical methods in that it gives information pertaining to the site of the radioactivity, within the limit of resolution of the technique of course. Light microscope autoradiography pro183 Copyright @ 1979 by Academic Press, Inc All rights of reproduction in any form reserved. ISBN 0-12-3613%-2
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vides information at the cellular level, while electron microscope autoradiography allows localization at the subcellular level. The history of autoradiography during the past half-century is one of progressive refinement of techniques. Lacassagne et al. (1925) detected the radiation of polonium given to rabbits, using photographic plates applied to a smooth surface of paraffin-embedded organs, although the resolution was poor. In 1930, Lomholt demonstrated radioactive lead in rat bone by apposing frozen sections of rat tissues to a photographic emulsion. Gettler and Noms (1933) identified radium which had accumulated in human bones as a result of poisoning from drinking water. A few years later Pecher (1942) introduced whole-body autoradiography with radioactive calcium, strontium, and phosphorus in bone. An important advance in methodology was made when Belanger and Leblond (1946) obtained high resolution by painting a warm liquid photographic emulsion on radioactive sections, thus ensuring an intimate contact between section and emulsion with their “dipping” technique. Similar results were obtained with the “stripping” film method described by Pelc (1947). These techniques were widely used in light microscope autoradiography, especially in studies on calcified tissues. The organic and inorganic metabolism of bone, cartilage, dentin, cementum, and enamel was studied in developing and mature stages. Further progress was made when autoradiography was adapted for use with the electron microscope (Pelc et al., 1961; Granboulan, 1963; Salpeter and Bachmann, 1964). The sites of biosynthesis of organic and inorganic substances could thus be precisely localized in the various parts of a cell, as well as in a variety of extracellular matrices. It became possible to analyze molecular migration within cells and within their products, hence it can be said that autoradiography has introduced the time dimension in histology (Leblond, 1965). In fact, ultrastructural research conducted with the electron microscope by applying what nowadays can be considered conventional methods has provided, in the last two decades, an impressive number of new observations regarding cells and extracellular matrices. The state of knowledge is at a juncture where combined methodological approaches must be applied in order to make further advances. Thus electron microscopy combined with cytochemistry, immunocytochemistry,electron diffraction, and electron microprobe analysis offers great possibilities in biological research. However, if kinetic and dynamic information is needed in ultrastructural investigations, electron microscope autoradiography is the method of choice. Typical examples are given to ilkstrate this point: the presence of various types of granules and vesicles in the cellular processes of (1) the secretory odontoblasts and (2) the ameloblasts (Figs. 1 and 2, respectively). These electron micrographs are consistent with either exocytosis or pinocytosis. With the use of appropriate labeled precursors an unequivocal solution to this enigma can be derived. In the field of calcified tissues, this technique has been used mainly at the light microscope level; several excellent reviews and
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surveys can be consulted for information in this area (Leblond and Warren, 1965; Leblond and Weinstock, 1971, 1976; A. Weinstock, 1972a,b). In this article, a survey of electron microscope autoradiography in bone, dentin, and enamel is made from which cartilage is excluded.
11. Methodology Electron microscope autoradiography of calcified tissues requires a rigorous methodology. For quantitative observations, intravenous injections are necessary in order to deliver the radioactive material into the bloodstream at time zero. Rats, mice, and cats have been most commonly utilized. All the general procedural principles of electron microscope autoradiography are applicable to calcified tissues, and the reader interested in technical aspects is referred to a recent survey (Droz et al., 1976). In addition to general methodological difficulties, the specific problem of hardness complicates the fixation and sectioning of bone, dentin, and enamel. The importance of the nature of the fixative used and its buffer must be emphasized. In the case of protein synthesis studies, the newly synthesized labeled proteins must be preserved in the course of tissue processing, whereas the free radioactive amino acids must be washed out. For such investigations, a 4% formaldehyde solution seems to give the best results at the present time (Bergeron and Droz, 1968). Likewise, in the case of inorganic metabolic studies using 45Ca, phosphate buffers must be avoided. Two different procedures have been used in hard-tissue sectioning for autoradiography. Some workers have decalcified their specimens for several days. Ethylenediaminetetraacetic acid (EDTA) has been used frequently (Warshawsky and Moore, 1967). We personally prefer to avoid any decalcification procedure which lengthens noticeably the tissue-processing time and which can produce structural artifacts. Thus the risks of label extraction and displacement are significantly decreased. All developing and most adult calcified tissues can be easily prepared as ultrathin nondecalcified sections (with silver or pale-gold interference). Three conditions are necessary. It is important to use a well-polished 45" diamond knife, mounted on an ultramicrotome with a mechanical advance, and the embedding medium must approach, as closely as possible, the degree of hardness of the specimen to be cut. If, for example, Epon 812 is to be used, only Epon 812 resin and methyl nadic anhydride (MNA) should be. mixed with the catalyst; dodecenyl succinic anhydride (DDSA), which softens the epoxy resin, should be avoided. As for the autoradiographic techniques per se, we have applied the dipping method based on the work of Granboulan (1965), Salpeter and Bachmann (1963, and Larra and Droz (1970), using an Ilford L, nuclear research emulsion
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CALCIFIED TISSUE AUTORADIOGRAPHY
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diluted 1:s in distilled water. Two developers can be utilized. With Kodak Microdol X, the silver grains obtained have the shape of twisted filaments. Actually, phenidone development (Lettre and Paweletz, 1966) is preferred, because the dotlike shape of the grain geometrically occupies less surface area than any other shape. Since the mean size of a developed silver halide crystal is approximately 140 nm, structures measuring less than 100-120 nm cannot be considered the source of a silver grain without statistical analysis. If the metabolism of osteoblasts and osteocytes is to be investigated, there is no problem with section orientation, since these cells are oval or round. Such is not the case with odontoblasts and ameloblasts, for which oblique transverse sections may be indicated in order to cover, in one section, the various cell levels for quantitative evaluation. For this purpose, cell micrographs are enlarged to the same magnification on photographic paper of uniform origin. The nucleus, the organelles, and the cytoplasmic outlines, as well as the different parts of the extracellular calcified and noncalcified matrices, are cut out, and their relative section areas are determined by weighing. A silver grain lying completely or substantially over a cellular or extracellular structure is counted as a grain associated with that structure. If a grain is located between organelles or areas, half a grain is counted for each. By relating the number of silver grains to the surface area of the various structures studied, the radioactivity concentration in the various cellular and extracellular compartments can be obtained at consecutive time intervals following intravenous injection. Along these lines, statistical methods such as those developed by Parry (1976) will probably be very helpful in the future. Under certain circumstances the investigator can capitalize on the widespread uptake of the injected isotopes. In two series of newborn cats, we studied osteogenesis, dentinogenesis, and amelogenesis concurrently. This was done on the one hand after intravenous injections of p r ~ l i n e - ~(Frank H and Frank, 1969; Frank, 1970a,b), and on the other hand after intravenous injections of 45Ca (Frank et al., 1974; Nagai and Frank, 1974, 1975). Observations from these and other experiments are used to illustrate this article. F K 1 . Odontoblastic process (0)in the predentin of a newborn cat. Two types of vesicles are present, both of which show membrane fusion with the plasma membrane. Elongated rod-shaped vesicles (G) contain a dense filamentous material with dark granules. In the area marked by three mo ws , dark granules are observed in the extracellular space. Coated vesicles (c) are located in the odontoblastic process, as well as along the plasma membrane. Pd, Uncalcified collagen fibrils. x 16,000.
FIG.2. The Tomes’ process of a human secretory ameloblast, located in a thin zone of stippled material (preenamel) and enamel matrix (E). Round granules (C)limited by a membrane and containing stippled material are located in the cell process. Various stages of membrane fusion of these granules with the cell plasma membrane are visible. The membrane of one coated vesicle is continuous with that of a round granule (arrows). M, Mitochondria. X32,OOO. (From Frank, 1967.)
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ROBERT M. FRANK
111. Detection of Nucleic Acids in Calcified Tissues
Th~midine-~H, a specific precursor of DNA, has become a widely used marking tool for light microscope autoradiography concerning the proliferative rate and potential cellular components of bone and dentin, as well as of the enamel organ. The origin of osteoblasts and odontoblasts, their unique cellular life cycles, and their renewal have been studied by a large number of investigators. The sites of RNA transcription in osteogenic and odontogenic cells have been followed with ~ r i d i n e - ~(Droz H et al., 1976). Nucleic acids in calcified tissues have been less commonly detected by autoradiography at the electron microscope level. Scott (1967), after injecting thymidinesH into 18- to 21-day fetal rats, observed that the label was incorporated into two types of osteogenic cells derived from mesenchymal elements accompanying the blood capillaries. A spindle-shaped cell, type A, was considered an osteoblast precursor, since it had the characteristics generally associated with matrix production, including extensive development of the endoplasmic reticulum. A round-shaped cell, type B, was considered a preosteoclast and had the morphological features of a developing neutrophilic leukocyte. Scott (1967) thus confirmed the results of Young (1962) who used the same technique at the light microscope level. According to Young’s interpretation (1962), osteoblasts and osteoclasts developed directly from cells in the proliferating perivascular mesenchyme, for which he suggested the term “osteoprogenitor cells. ” On the basis of electron microscope cytochemistry of metaphyses from young guinea pigs, Thyberg et al. (1975) agreed with the presence of two types of perivascular osteogenic cells but, in contrast to Scott (1967), they considered the type-Bcell macrophage-like rather than granulocyte-like. Gothlin and Ericsson (1973) studied callus formation in parabiotic rats with electron microscope autoradiography. On the basis of labeling experiments with tritiated thymidine and thorium dioxide particles, they concluded that at least a proportion of osteoclasts may arise from the coalescence of monocytes. In view of these differing results further investigations are required to examine the long-debated problem of the origin of osteoclasts.
IV. Synthesis of Organic Matrices of Calcified Tissues Collagen, phosphoproteins, glycoproteins, and proteoglycans constitute the major components of the organic matrices of bone and dentin. It must be recognized that approximately 90% of these matrices is collagen. Eastoe (1 956) estimated that organic components constituted 30% of adult bone by weight. The situation is quite different in enamel where the newly secreted embryonic matrix makes up 20% of the tissue by weight (Deakins, 1942; Eastoe, 1960; Burgess and McLaren, 1965); after completion of mineralization, the tissue contains a
CALCIFIED TISSUE AUTORADIOGRAPHY
189
meager 0.5% organic material (Stack, 1954). Enamel matrix is composed essentially of proteins (the exact composition of which is still a matter of debate) and small amounts of glycoproteins and proteoglycans as well. Numerous light microscope autoradiographic studies have clearly shown that all these matrices are secretion products of specialized cells, namely, osteoblasts, odontoblasts, and ameloblasts (see reviews of Leblond and Weinstock, 1971, 1976; A. Weinstock, 1972a,b). Electron microscope autoradiography has in large part confirmed these findings and has in addition elucidated the precise intracellular and extracellular pathways of various participating organic molecules.
A. ELABORATION OF BONEMATRIX In the membranous type of ossification studied in the mandible of the newborn kitten (Frank and Lang, 1969), bone formation is characterized by the differentiation of a monolayer of osteoblasts lined up along a noncalcified osteoid matrix, or prebone, adjacent to calcified bone (Fig. 3). A round or oval-shaped nucleus with a dense nucleolus is often located in an eccentric position in the cell, farthest
FIG.3. Oval-shaped osteoblast with a well-developed endoplasmic reticulum (ER) and numerous mitochondria (Mi). The nucleus is located in the part of the cell opposite the osteoid matrix (OM). The collagen fibrils have a tendency to circumscribe the osteoblast (arrows). B, Bone. ~ 8 0 0 0 . (From Frank and Lang, 1969.)
FIG. 4. (A) Presence in the Golgi apparatus of a rat osteoblast of spherical portions containing an array of entangled threads. X 100,000. (From M. Weinstock, 1975.) (B) At a subsequent stage, the distended saccule becomes rectangular and the threads are aligned in parallel. X 100,ooO.(From M. Weinstock, 1975.) FIG.5 . (A) Later stages involve condensation of the contents into rodlike structures. X 100,OOO. (From M. Weinstock, 1975.) (B) Finally a Golgi secretory granule is formed with electron-opaque contents within which filaments are no longer evident. X 100,ooO. (From M. Weinstock, 1975.)
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191
from the bone matrix, as observed by Ascenzi and Benedetti (1959) and Cameron (1961). The Golgi apparatus of the osteoblast is prominent and occupies a juxtanuclear position. In thin sections, it appears to be composed of several stacks of flattened saccules. It is probable that, viewed three-dimensionally, these saccules are continuous, as shown for other cell types in relatively thick sections (0.5 -5 p m ) examined in the electron microscope. The Golgi stacks are polarized. The most outer, convex saccule is considered the forming face, whereas the inner or concave side is referred to as the maturing face. On the inner face of the Golgi apparatus, M. Weinstock (1972) and Leblond and Weinstock (1976) described spherical portions of saccules containing fine threads with no apparent organization (Fig. 4A). Beside these dilated units, rectangular portions containing parallel threads (Fig. 4B) were found, as were elongated denser structures considered secretory granules (Fig. 5A and 5B). These dense granules were morphologically very similar to elongated vesicles found in the Golgi zones of odontoblasts during dentinogenesis, except that the latter showed in addition periodically arranged electron-dense particles (Reith, 1968; Frank, 1970b; M. Weinstock and Leblond, 1974). Concentric arrays of rough-surfaced endoplasmic reticulum, closely associated with numerous mitochondria (Fig. 3), surround the nucleus as well as the Golgi zone. The
FIG.6. Overall view of the lining osteoblasts 5 minutes after intravenous injection of pr~line-~H. Prominent labeling of the endoplasmic reticulum (ER). N , Nucleus; OM, osteoid tissue; B, bone. x4550. (From Frank and Lang, 1969.)
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ROBERT M. FRANK
external rough-surfaced membrane of the outer nuclear envelope is continuous with the endoplasmic reticulum which appears as flattened cisternae or dilated sacs. The cytoplasm of the osteoblast contains in addition free ribosomes and some condensation of glycogen granules, as well as microtubules and microfilaments. The latter are also found in cell processes extending into the osteoid and bone matrix (Weinger and Holtrop, 1974). In the peripheral osteoblastic cytoplasm, adjacent to the osteoid, dense, elongated vesicles, considered secretory granules, have been observed to fuse with the cell plasmalemma. Invaginations in the cell membrane, in which small aggregates of material have been observed, were believed to correspond to secretory granules in the process of extruding their filamentous contents into the osteoid (A. Weinstock et al., 1975; M. Weinstock, 1975; Leblond and Weinstock, 1976). The bone organic matrix is mainly composed of type I collagen, in which the three-component a chains consist of a - 1(I) and a -2 in a 2: 1 ratio (Miller, 1973); some type I n collagen containing three a-l(II1) chains is also probably present. The other organic components are a phosphoprotein, rich in serine (Leaver and Shuttleworth, 1968), glycoproteins and proteoglycans (Herring, 1968), and
FIG.7. Numerous silver grains over the cistemae of the rough-surfaced endoplasmic reticulum in an osteoblast 5 minutes after intravenous injections of pr~line-~H. Mi, Mitochondria; N, nucleus. X13.000.
193
CALCIFIED TISSUE AUTORADIOGRAPHY
ENDOPLASMIC RETICULUM GOLGI APPARATUS MITOCHONDRIA NUCLEI PERIPHERAL CYTOPLASM OSTEOID
BONE
73 4
5'
30'
lh
24h
FIG.8. Distribution of radioactivity expressed as the number of silver grains per 1OOOpd in the various cell organelles of the osteoblast, the osteoid tissue, and the bone 5 minutes, 30 minutes, 1 hour, and 24 hours after intravenous injection of p r ~ l i n e - ~ H .
lipids. Except for a smaller amount of phosphoprotein, the composition of the osteoid is fairly similar to that of the organic dentin matrix (Leblond and Weinstock, 1976). With light microscope autoradiography, it was shown that collagen, glycoprotein, and proteoglycan precursors were initially elaborated in the osteoblast and then secreted into the extracellular osteoid (see review by Leblond and Weinstock, 1971). This sequence of events has been confirmed and refined by electron microscope autoradiography. Rohr (1965), with the aid of tritiated proline, studied collagen synthesis in rat osteoblasts and observed a prominent labeling of the endoplasmic reticulum, whereas the Golgi apparatus was slightly involved. He confirmed the observations of Ross (1965) and Ross and Benditt (1965) who, although recognizing that p r ~ l i n e - ~perhaps H passed through the Golgi apparatus of fibroblasts in healing wounds, described a direct transfer of the labeled proteins from the endoplasmic reticulum to the extracellular medium. Rohr (1965) thought that osteoblasts H behaved differently than chondroblasts, in which a transfer of p r ~ l i n e - ~from the endoplasmic reticulum to the Golgi apparatus had been shown (Revel and Hay, 1963; Rohr and Gebert, 1967). In these chondroblasts, silver grains were
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ROBERT M. FRANK
TABLE I CONCENTRATION OF RADIOACTIVITY IN THE OSTEOBLAST, OSTEOID TISSUE,
AND
BONEFOLLOWING
THE INJECTION OF PROLINE-3H"'*
Nucleus Mitochondria Endoplasmic reticulum Golgi apparatus Peripheral cytoplasm Osteoid tissue Bone
5 minutes
30 minutes
1 hour
24 hours
17.1 37.7 84 60.8 13.5 5.4 0
15.3 29.9 64.7
13.6 22.1 47.5 198.8 94 47.7 4.5
9.3 4.3 9.6 9.5 28.3 73.4 7.8
121.8
51.7 25.5 2.5
"From Frank and Frank, 1969. *Concentration is expressed as the number of silver grains per lOOpmZ at various time intervals following injection.
then observed over secretory granules which, by exocytosis, poured their contents into the extracellular matrix. Subsequent investigations demonstrated in fact that collagen biosynthesis in osteoblasts followed intracellular pathways similar to those observed in chondroblasts. After intravenous injection of p r ~ l i n e - ~into H newborn kittens, Frank and Frank (1969) observed a significant labeling of the endoplasmic reticulum within 5 minutes (Figs. 6, 7, and 8; Table 1). The number of silver grains increased progressively (Figs. 9, 10, 11A and 11B) over the Golgi apparatus and reached a maximum 1 hour after the injection (Fig. 8; Table I). Flattened and distended Golgi saccules were clearly labeled. It can be concluded that, from the sites of biosynthesis of the polypeptide chains in the endoplasmic reticulum, there is a transfer of synthesized protein to the Golgi apparatus. In the peripheral cytoplasm of the osteoblast (located between the most peripheral endoplasmic reticulum and the cell membrane), a progressive increase in silver grains was noted for up to 1 hour (Table I; Fig. 8), but Frank and Frank (1969) did not observe the labeling of secretory granules (Fig. 12). However, 24 hours after intravenous injection, a marked increase in silver grains was noted over the osteoid tissue, with a concomitant decrease over the cellular components and initial labeling of the calcified bone (Table I; Figs. 8, 12-14). M. Weinstock (1975) and M. Weinstock and Leblond (1974), after intravenous injections of p r ~ l i n eH - ~into young rats, confirmed the transfer of synthesized protein from the endoplasmic reticulum to the Golgi apparatus. The ergastoplasm was labeled 2 minutes after intravenous injection. In addition these investigators demonstrated that the collagen precursors were packaged in secretory granules in the Golgi apparatus. After 10 minutes, silver grains were seen over the distended spherical portions of the Golgi complex, and after 20 minutes the label was observed in the cylindrical portions, as well as in the secretory
CALCIFIED TISSUE AUTORADIOGRAPHY
195
granules in the Golgi region (Fig. 4A). Between 20 and 30 minutes, silver grains were located over secretory granules in the peripheral cytoplasm (Fig. 4B) and in the adjacent osteoid tissue, indicating that, after packaging of the collagen precursors into secretory granules within the Golgi apparatus, these granules migrate to the cell periphery where they pour their contents into the osteoid matrix by exocytosis. At 30 minutes and, to a greater extent at 90 minutes, prebone was labeled. M. Weinstock (1975) suggested that the spherical portions contained pro* chains, while the cylindrical portions contained triple helical procollagen molecules. In short-term organ cultures of chick embryo calvaria, A. Weinstock et al. (1975) confirmed these intra- and extracellular pathways with p r ~ l i n e - ~electron H microscope autoradiography. In addition, by biochemical analysis and subcellular fractionation, they showed that 85% of the intracellular radioactivity was related to collagenous protein. After polyacrylamide gel electrophoresis the pres-
FIG.9. Silver grains over saccules of the Golgi apparatus in an osteoblast 30 minutes after intravenous injection of pr~line-~H. Some grains are still located over the endoplasmic reticulum (Er). Mi, Mitochondria; OM, osteoid tissue. x 13,000.
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ROBERT M. FRANK
FIG. 10. Prominent labeling of the Golgi apparatus of an osteoblast 1 hour after intravenous Some silver grains are. located over dilated saccules (arrows) containing injection of pr~line-~H. entangled tine filaments. Er, Endoplasmic reticulum. X 18,500. (From Frank and Frank, 1969.)
ence of pro*-1 and pro*-2 polypeptide chains (2: 1) of procollagen was demonstrated. When the labeling patterns of the various organelles of the osteoblast and the osteoid tissue are compared for newborn cats (Frank and Frank, 1969) and young rats (M. Weinstock, 1975), it appears that there is faster collagen synthesis in the rat. With the present state of knowledge, questions now arise about the various mechanisms regulating the sequential and well-ordered series of such intracellular events. Along these lines, Scherft and Heersche (1975) demonstrated the important role played by microtubules in the transfer of secretory granules to the cell surface. Using the microtubule-disruptingagents, colchicine and vinblastine, in cultures of embryonic mouse radii, they observed the accumulation of numerous secretory granules in the osteoblast Golgi zone in the absence of exocytosis. Probably some effects of colchicine can also be attributed to a direct action on the cell membrane (Wunderlich et al., 1973). Intravenous administration of p r ~ l i n e - ~to H newborn cats allowed a better understanding of bone trabeculae formation (Frank and Frank, 1969). The transformation of a lining osteoblast (Fig. 3) into an osteocyte embedded in its lacuna is directly under cellular control. A striking “polarized” secretion of labeled proteins has been observed (Figs. 13 and 14), which gives rise initially to unilat-
CALCIFIED TISSUE AUTORADIOGRAPHY
197
eral development of the osteoid matrix. The latter undergoes progressive cdcification, so that some lining osieoblasts can be found directly adjacent to calcified bone (Fig. 15). Almost simultaneously, new osteoblasts differentiate on the medullary side of the first lining osteoblasts (Fig. 16). Autoradiography demonstrated dense p r ~ l i n e - ~labeling H over the newly differentiated osteoblasts compared to those adjacent to the bone matrix. Osteoid matrix secreted by the second line of osteoblasts (Fig. 16) completely surrounds the osteoblast on the side adjacent to the bone (Fig. 15). The width of this organic collagenous matrix progressively increases, with concomitant withdrawal of the second line of osteoblasts. As a result, the first osteoblast is surrounded on one side by calcified bone and on the other side by osteoid (Fig. 17) and thereby corresponds to an osteoid osteocyte, according to the terminology used by Dudley and Spiro
H Silver FIG. 11. (A) Portion of a rat osteoblast 20 minutes after intravenous ~ r o l i n e - ~injection. grains overlay secretory granules as well as cylindrical portions in the Golgi apparatus. Mi, Mitochondria; Er, endoplasmic reticulum. X25,OOO. (From M. Weinstock, 1975.) (B) Labeled secretoly granules at the cell membrane of a rat osteoblast at 30 minutes. X25,OOO. (From M. Weinstock, 1975.)
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ROBERT M.FRANK
FIG. 12. One hour after intravenous injection of p r ~ l i n e - ~silver H grains are seen over the osteoblast periphery (arrows) and in the osteoid tissue (OM). Mi, Mitochondria, N , nucleus; Er, endoplasmic reticulum. X 11,000. (From Frank and Frank, 1969.) FIG. 13. Numerous silver grains in the osteoid tissue (OM) along an osteoblast 24 hours after Cross-sectioned . osteoblastic processes are seen among the collaintravenous injection of p r ~ l i n e - ~ H gen fibrils. Go, Golgi apparatus; Er, endoplasmic reticulum. x 11,OOO.
CALCIFIED TISSUE AUTORADIOGRAPHY
199
FIG. 14. Numerous silver grains over the osteoid tissue 24 hours after intravenous injection of pr~line-~H. Only a few silver grains are located over the osteoblast. B, Bone; N, nucleus; Mi, mitochondria; Er, endoplasmic reticulum. X 13,000. (From Frank and Frank, 1969.)
(1961). Calcification now progresses in the osteoid all around the osteoid osteocytes (Fig. 18). Finally the osteocyte lacuna is completed with small canaliculi permeated by the cell processes (Figs. 18 and 19). The incremental repetition of these phenomena leads to growth in thickness of bone trabeculae. A similar sequence of events has been described by Schulz et al. (1974). The young osteocyte has a prominent Golgi apparatus, as well as a developed endoplasmic reticulum closely associated with mitochondria (Figs. 19 -21). Microfilaments and microtubules are observed in the cytoplasm. The cell does not completely fill the lacuna-an organic periosteocytic space containing an amorphous ground substance and sometimes noncalcified collagen fibrils can be seen between the calcified wall and the cell surface. With p r ~ l i n e - ~autoradiography H , silver grains have been seen over the osteocytic endoplasmic reticulum (Fig. 20) and the Golgi zone (Fig. 21), as well as over the organic periosteocytic space. However, the secretion cycle of the synthesized proteins seemed to be slower for the osteocyte, compared to that of the osteoblast. Indeed, 24 hours after intravenous p r ~ l i n e - ~injection, H several silver grains were still observed over the osteocyte cytoplasm.
FIG. 15. Lining osteoblast (Lo) which on one side is in direct contact with bone (right), whereas on the opposite side osteoid tissue (OM) has been elaborated. Differentiation of a new osteoblast is seen on the left. Mi, Mitochondria; Er, endoplasmic reticulum; N, nucleus. X 10,000. (From Frank and Lang, 1969.)
FIG. 16. Scarcity of silver grains in a lining osteoblast (0,)adjacent to osteoid matrix (OM) and bone (B) 5 minutes after intravenous injection of p r ~ l i n e - ~ H Conspicuous . labeling of the endoplasmic reticulum of an adjacent newly differentiated osteoblast (Q) engaged in active protein synthesis. X6OOO. (From Frank and Frank, 1969.)
CALCIFIED TISSUE AUTORADIOGRAPHY
20 1
FIG. 17. Beginning of calcification (arrows) in the osteoid tissue (OM) surrounding an osteoid osteoblast, according to the terminology used by Dudley and Spiro (1961). Cp, Cytoplasmic process; Mi, mitochondria; N, nucleus; B, bone. X9000. (From Frank and Lang, 1969.) FIG. 18. Further calcification around an osteoblast with delineation of the lacunar outline. OM, Osteoid tissue; B, bone; Cp, cytoplasmic process; Mi, mitochondria; N, nucleus. ~ 8 0 0 0 (From . Frank and Lang, 1969.)
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ROBERT M. FRANK
FIG. 19. Newly differentiated okteocyte in a bone lacuna. A well-developed Golgi apparatus (Go), in juxtanuclear position, is surrounded by endoplasmic reticulum (Er). Microfilaments and microtubuies are present in the upper part of the cell (Fi). S, Periosteocytic organic space. X5200. (From Frank and Lang, 1969.)
The osteoclasts present in the developing mandible of the newborn cat did not take up any label (Frank and Frank, 1969), confirming the light microscope autoradiographic studies of Tonna (1965). With the same technique BirkedalHansen (1974) incorporated p r ~ l i n e - ~into H bone; 6- 11 weeks later, osteoclastic bone resorption was experimentally induced. Practically no label was seen over active osteoclasts, suggesting that collagen destruction during bone resorption occurs extracellularly in proximity to the multinucleated cell. The biosynthesis of the other organic components of bone matrix, namely, phosphoproteins, glycoproteins, and proteoglycanshas not been extensively studied with electron microscope autoradiography (Leblond and Weinstock, 1976).
B . ELABORATION OF DENTINMATRIX Dentinogenesis is characterized by the differentiation of a layer of elongated cells, odontoblasts, possessing long, slender processes with lateral branches (Fig. 1). The odontoblast cell body is adjacent to a collagenous organic matrix, the predentin, and dentin arises through calcification of the latter. The odontoblast processes extending through the predentin reach dentinal tubules permeating calcified dentin.
FIG.20. Presence of silver grains over the endoplasmic reticulum of an osteocyte 1 hour after intravenous injection of pr~line-~H. S, Penosteocytic organic space. X 12,000. (From Frank and Frank, 1969.) FIG.21. Labeling of the Golgi apparatus (Go) of an osteocyte 1 hour after intravenous injection of pr~line-~H. N , Nucleus; S, periosteocyticorganic space. X 1 1 ,000. (From Frank and Frank, 1969.)
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ROBERT M.FRANK
FIG.22. Cross section of secreting odontoblasts at the nuclear level (N) 5 minutes after intravenous injection of p r ~ l i n eH. - ~ Numerous silver grains are located over the endoplasmic reticulum (Er) closely associated with several mitochondria (Mi). Ce, Pair of centrioles; Go, Golgi zone. X 11,000.
The differentiated odontoblast is a highly polarized secretory cell. Fairly good agreement has been reached regarding its ultrastructural organization, and there is a high degree of consistency when human odontoblasts are compared to cat and rodent cells (Nylen and Scott, 1958; Jessen, 1967; Takuma, 1967; Frank, 1968a; Reith, 1968; Garant et al., 1968; Matthiessen and von Bulow, 1970; Takuma and Nagai, 1971; Garant, 1972; Leblond and Weinstock, 1976). The nucleus is situated in the basal portion of the cell and is surrounded by an extensive roughsurfaced endoplasmic reticulum, closely associated with mitochondria (Fig. 22, 23). Both organelles are found in the basal and proximal cytoplasm but do not generally go beyond the terminal web marking the boundary between the odontoblast and its process. The Golgi apparatus is well-developed and located near the proximal side of the nucleus, where it is surrounded by cistemae of the
CALCIFTED TISSUE AUTORADIOGRAPHY
205
endoplasmic reticulum (Figs. 22, 23, 24). Appearing as a continuous network in thick sections, the Golgi apparatus has a general tubulocylindrical shape oriented parallel to the long axis of the odontoblast body. It is composed of several stacks of flattened saccules. Between the forming outer face of the Golgi apparatus and the surrounding endoplasmic reticulum smooth or “fuzz’ ’-coated vesicles have been observed (Leblond and Weinstock, 1976). On the maturing inner face of the Golgi apparatus, there are dense, elongated vesicles containing bundles of parallel filaments oriented lengthwise (Frank, 1968a; Reith, 1968; Frank, 1970b; Matthiesen and von Biilow, 1970; M. Weinstock, 1972; M. Weinstock and Leblond, 1974). These elongated rod-shaped vesicles (Figs 25 and 25) contain in addition dense granular particles arranged in transverse periodic bands or in more-or-less compact groupings without distinct periodicity. A variety of intermediate stages of formation between the distended Golgi saccules and the elongated rod-shaped vesicles have been described (Reith, 1968; M. Weinstock and Leblond, 1974; Leblond and Weinstock, 1976). These vesicles formed within the inner face of the Golgi apparatus are found in the proximal odontoblast cytoplasm, as well as in the odontoblast process, where they show membrane fusion with the plasmalemma (Figs. 1 and 26-28). In the inner maturing face of the Golgi apparatus, other elements were found. The most prominent were coated vesicles, often in contact with multivesicular and lysosome-like bodies. Confluence between coated vesicles and the dense, elongated granules has been described. These coated vesicles were also observed between the Golgi apparatus and the odontoblast process (Fig. 1). Microfilaments and microtubulels are disseminated throughout the odontoblast body cytoplasm with a general orientation parallel to the long axis of the cell. At the level of the terminal web, transverse microfilaments are oriented perpendicular to the long axis of the cell and delineate the actual boundary between the odontoblast cell body and cell process. The latter contains parallel groupings of microfilaments and microtubules as well as dense, elongated, rod-shaped vesicles and coated vesicles (Figs. 1 and 26 -28). The predentin matrix contains numerous collagen fibrils and an amorphous ground substance with fine, granular material (Fig. 1). From a biochemical point of view, collagen is by far the predominant component of the organic matrix of dentin. As in bone, it is of type I, in which the three-componenta chains consist ofa-l(1) anda-2 in a 2:l ratio (Miller, 1973). Type 111collagen containing three a - l ( I ) chains has also been found in dentin. Among the numerous amino acids found in dentin collagen, glycine constitutes about one-third of the residues, whereas proline and hydroxyproline account for slightly more than one-fifth. These are the most abundant amino acids found in dentin collagen. Another type of protein found in dentin is a phosphoprotein containing abundant serine and aspartic acid residues (Leaver and Shuttleworth, 1968). According to Butler er al. (1972), the dentin phosphoprotein extracted from rat incisor constitutes 10.8% of the proteinacous material, while collagen comprises 84%. Noncol-
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ROBERT M. FRANK
FIG. 23. Oblique section of an odontoblast 5 minutes after intravenous injection of pr~line-~H. The Golgi apparatus (Go) is devoid of silver grains. A distinct labeling of the endoplasmic reticulum (Er) is visible. N , nucleus; Mi, mitochondria. X12,OOO.
FIG. 24. Labeling of the juxtanuclear Golgi apparatus (Go) of newborn cat odontoblasts 1 hour after intravenous injections of pr~line-~H. N, Nucleus; Er, endoplasmic reticulum; Mi, mitochondria; Ce, centriole. x 12,000. FIG.25. Golgi apparatus of an odontoblast 1 hour after intravenous injection of proline-% Numerous dense, elongated, rod-shaped secretory vesicles (arrows) are covered by silver grains which are also located over Golgi saccules. X 18,000.
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ROBERT M.FRANK
FIG. 26. Longitudinal section of an odontoblastic process (0)in predentin (Pd) of a newborn cat 1 hour after intravenous injection of pr~line-~H. Silver grains are located over dense, elongated, rod-shaped secretory vesicles (arrows). The coated vesicles (c) are not labeled. D, Dentin. x 13,000. (From Frank, 1976.) FIG. 27. Longitudinal section of an odontoblastic process in a dentinal tubule near the
CALCIFIED TISSUE AUTORADIOGRAPHY
209
lagenous glycoproteins are also-preiient, as are gome proteoglycans, especially chondroitin sulfate A, citrate, and lactate, as well as some lipids. Although light microscope autoradiography showed that the precursors of the organic dentin matrix were elaborated in the odontoblast (see for reviews A. Weinstock, 1972a,b; Leblond and Weinstock, 1976), the intracellular mechanisms associated with various aspects of organic biosynthesis were far from clear. Concerning collagen biosynthesis, the majority of electron microscope observations indicated that the role of the odontoblast was major. Whereas a pinocytic function was attributed to the coated vesicles by Jessen (1967) and Reith (1968), Garant et al. (1968) thought that these organelles had a secretory function related to collagen and ground substance precursors. Using electron microscope autoradiography with prolinePH, Reith (1968) concluded that the collagen precursors were transferred through the cytoplasm without being packaged into secretory granules. This conclusion can probably be explained by the low p r ~ l i n e - ~dose H administered. By intravenous injection of tritiated proline and quantitative electron microscope autoradiography, Frank (1970b) studied collagen biosynthesis in the odontoblast of newborn cats and confirmed the optical level findings of Young and Greulich (1963) and Greulich and Slavkin (1965). The odontoblast plays a major role in the elaboration of dentin collagen precursors. No cells of the dental papilla showed such intense labeling as the odontoblast 5 minutes, 30 minutes, or 1 hour after intravenous injection. The sparse labeling of pulpal cells and lateral intercellular spaces of the odontoblasts (Table 11) does not give credibility to a pulpal origin for predentin collagen, although a few collagen fibrils are found in these locations (Frank, 1968a). Initial incorporation of p r ~ l i n e - ~into H the odontoblast occurs in the endoplasmic reticulum (Reith, 1968; Frank, 1970b; M. Weinstock and Leblond, 1974); 5 minutes after intravenous injection into newborn cats, a maximum of labeling is observed over the rough-surfaced cisternae (Table 11; Figs. 22, 23, 29). The silver grains are located on the free and attached ribosomes, as well as in the lumen of the cisternae (Figs. 22 and 23). The data suggest that this organelle is the site of the synthesis of the polypeptide chains of collagen precursors. The endoplasmic reticulum shows a progressive decrease in silver grains at 30 minutes, 1 hour, and 24 hours after the intravenous injections (Table 11; Fig. 29). In a measure proportional to the decrease in radioactivity in the ergastoplasm, a predentin-dentin junction, with some uncalcified collagen fibrils (F)in the intertubulardentin. In the odontoblastic process, there are dense, elongated, rod-shaped secretory vesicles covered by silver grains 1 hour after intravenous injection of pr~line-~H. Note also the presence of microfilaments and microtubules in the odontoblastic process. x 18,OOO. FIG. 28. Labeled, dense, elongated secretory vesicles in the odontoblast process in predentin Note the absence of silver grains over typical (Pd) 1 hour after intravenous injection of pr~line-~H. coated vesicles (c). ~21,000.
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ROBERT M. FRANK
TABLE I1 IN THE ODONTOBLAST, ODONTOBLAST PROCESS, PREDENTIN, CONCENTRATION OF RADIOACTIVITY AND DENTINFOLLOWING THE INJECTION OF 3H-PROLINEuD
Nucleus Mitochondria Endoplasmic reticulum Golgi apparatus Interodontoblast spaces Cytoplasm of odontoblast process Secretory granules in odontoblast process Coated vesicles in odontoblast process Predentin
5 minutes
30 minutes
1 hour
10.3 24 51.1 19.2 4.6 2.9 0 0 0.5
8.9 24.5 36.5 63.5 4.5 6.8 111 0 9.8
1.5 25 23.3 118.3 4.6 15.4 533 0 14
24 hours 1.5
42 6.2
1.7 5.3 34.3 245 0 69
"Frank, I970b. bConcentration is expressed as the number of silver grains per 100pd at various time intervals following injection.
progressive increase is observed in the Golgi apparatus, where the most dense labeling is reached 1 hour after the injection. Thus all conditions required for a transfer of the synthesized polypeptide chains from the endoplasmic reticulum to the Golgi zones seem to be fulfilled (Table 11; Fig. 29). In the Golgi apparatus, 1 hour after the intravenous injection, numerous silver grains are seen over flattened and distended saccules, as well as over the dense, elongated, rod-shaped secretory vesicles which contain the dark particles (Figs. 24 and 25) and which presumably arose by budding and condensation from the dictyosomes (Frank, 1970b). Between the Golgi apparatus and the odontoblastic process, that is, in the proximal cytoplasm, numerous labeled secretory vesicles are found. The collagen precursors packaged in the secretory vesicles are transferred to the odontoblastic process, where they are observed at the level of the predentin (Figs. 26 and 28) and the dentin (Fig. 27). The secretory granules, measuring about 0.4-0.5 p m in length and about 0.1-0.2 p m in width, have their long axes oriented approximately parallel to the odontoblast process (Figs 1, 26 and 27). They approach the lateral borders of the process, and their limiting membranes fuse with the plasmalemma (Fig. l), discharging their secretion products into the extracellular predentin matrix (Fig. 1 and 30). A prominent labeling of the predentin collagenous matrix is observed 24 hours after the intravenous injection (Table 11; Figs. 29, 30, and 31); at this time, dentin is only slightly labeled. These observations demonstrate that the collagen precursors synthesized in the endoplasmic reticulum are transferred to the Golgi apparatus, where they are packaged in the form of secretory granules. The latter migrate to the odontoblastic process, where they discharge their contents into the predentin by exocytosis. A transfer of the collagen precursors from the endoplasmic reticulum directly
21 1
CALCIFIED TISSUE AUTORADIOGRAPHY
to the extracellular space, such as that described by Ross (1965) and Ross and Benditt (1965) for the fibroblast, can be excluded in the case of the odontoblast. The endoplasmic reticulum terminates in the odontoblast body at the level of the terminal web, and we observed that the lateral interodontoblastic spaces were only very weakly labeled (Table 11; Fig. 29). Some silver grains, not associated with granules or vesicles, were seen in the cytoplasm of the odontoblast process (Table 11), especially 1 hour and 24 hours after injection, but their number was minimal when compared to those found over the secretory granules. No radioactivity has been observed over the coated vesicles present in the odontoblast process. It is highly probable that these vesicles are involved in pinocytosis, as suggested by Reith (1968) and Katchburian and Holt (1968). The above sequence of events in collagen biosynthesis was confirmed after intravenous injection of p r ~ l i n e - ~into H young rats (M. Weinstock and Leblond, 1974; Leblond and Weinstock, 1976). By 2-5 minutes after injection the label was restricted to the endoplasmic reticulum. Presumably the label corresponded to pro-a chains located within the cisternae. By 10 minutes, the silver grains appeared in the Golgi apparatus where spherical saccules with entangled threads were marked. At 20 minutes, label was detected within cylindrical portions of the Golgi saccules containing parallel threads. At the 30-minute interval a high
14
I
24 h
FIG. 29. Distribution of radioactivity, expressed as the number of silver grains per I O O p d , in the different organelles of the odontoblasts, the cytoplasm of the odontoblastic processes, their lateral interodontoblastic spaces, and predentin in the newborn cat after intravenous injection of proline-SH.
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ROBERT M. FRANK
CALCIHED TISSUE AUTORADIOGRAPHY
213
concentration of 3H was observed over prosecretory and secretory granules. Labeled secretory granules were seen between the Golgi apparatus and the odontoblast process, where M. Weinstock and Leblond (1974) observed membrane fusions between the secretory granules and the cell plasma membrane, followed by exocytosis. By 90 minutes and 4 hours, a significant labeling of the predentin was observed. No observation of phosphoprotein biosynthesis with electron microscope autoradiography has been made in dentin. However, at the optical level, using intravenous phosphateP3P and ~ e r i n e - ~ H as, well p r ~ l i n e - ~(for H purposes of comparison), in the rat, M. Weinstock and Leblond (1973) noted that within 30 minutes the labeled phosphorus, serine, and proline were taken up by odontoblasts and deposited into predentin. The proline label remained in the latter layer at least 4 hours after injection, whereas the labeled phosphorus and seine were displaced to the dentin side of the dentin-predentin junction, that is, the mineralization front, as early as 90 minutes after injection. The formation of glycoprotein in dentin was studied by electron microscope autoradiography by A. Weinstock et al. (1972) in young rats after intravenous injection of fucosePH, a sugar which is incorporated into glycoprotein without any significant conversion to other substances. By 5 -10 minutes after injection, the radioactivity was restricted to the Golgi apparatus of the odontoblasts. By 35 minutes, silver grains were observed over the dense, elongated, rod-shaped secretory granules in the Golgi zone, as well as in the odontoblast process, and there were a few silver grains in the adjacent predentin. By 4 hours, the radioactive reaction was present on the dentin side of the predentin-dentin junction. These results indicate that f u c o ~ e - ~isH added to forming glycoprotein in the Golgi apparatus and packaged into secretory vesicles in which the fucosecontaining glycoprotein is transported to the odontoblast process, with subsequent discharge into the predentin. The deposition of labeled glycoprotein and phosphoprotein at the dentin mineralization front may be related to the onset of mineralization (A. Weinstock et al., 1972). The presence of proteoglycan in the odontoblast and predentin of the rat incisor has been studied by ultrastructural cytochemistry, with the aid of ruthenium red (Nygren et al., 1976). Positive reactions were observed on the elongated, dense secretory vesicles and the cell coat of odontoblasts. Treatment with hyaluronidase prior to staining with ruthenium red abolished the staining of the vesicles but not that of the cell coat. Sulfated proteoglycan formation was FIG.30. Silver grains are present over the collagen fibrils of predentin adjacent to an odontoblastic process 24 hours after intravenous injection of pr~line-~H. x 13,000. (From Frank, 1976.) FIG 31 Predentin labeling 24 hours after intravenous injection of pr~line-~H. A few silver gmns are located over the odontoblast bodies (top). Er, Endoplasmic reticulum; Mi, mitochondria; F, transverse microfilamentsof the terminal web (W); 0, odontoblastic process and lateral branches. x11,ooo.
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ROBERT M.FRANK
traced with sulfateP5S (Leblond and Weinstock, 1976). The label was also observed over the elongated dense secretory granules 30-35 minutes after injection. The radioactive material was released into the predentin and subsequently reached the edge of the dentin by 4 hours, as in the case of the fucose label. It thus becomes evident that the secretory granules of the odontoblast are able to carry not only procollagen, but also glycoprotein, proteoglycan, and probably also phosphoprotein. However, it is unclear whether these organic components are transferred simultaneously in the same vesicle or if they are carried by different vesicles. Along these lines, M. Weinstock (1972) tried to determine the nature of the very dense particles found in the elongated secretory granules (Figs. 1, 24 and 25). They were not dissolved after EDTA or nitric acid treatment, indicating their organic nature. Leblond and Weinstock (1976) suggested that they might correspond to phosphoprotein. Numerous questions now arise about the essential mechanisms governing intracellular organelle polarization, as well as the elaboration, migration, and exocytosis of the secretory vesicles. Many investigators have emphasized the importance played by microfilaments and microtubules in morphogenesis and cytodifferentiation(for review, see Spooner, 1974). Since it has been shown that colchicine and related compounds are bound to microtubular proteins (Bonsy and Taylor, 1967) and inhibit polymerization of microtubules (Adelman et al., 1968), as well as dissociate the microtubular structure (Wisniewsky et al., 1968), it has been suggested that microtubules play an important role in secretory processes (Williams and Wolff, 1970; Diegelmann and Peterkofsky, 1972). The effects of various concentrations of colchicine and cytochalasin B on the polarization of mouse molar tooth germs cultivated in v i m were studied by Ruch et al. (1975). Colchicine produced inhibition of the polarization of the odontoblasts and ameloblasts, accumulation of secretory granules in the Golgi apparatus with inhibition of exocytosis, and dilatation of the odontoblastic processes. Cytochalasin B, besides its action on microfilaments, had important cytotoxic effects and delayed the polarization of the odontoblasts (Ruch et al., 1975). By light microscope autoradiography, using prolinesH in rats, Kudo (1975) studied the effect of colchicine on the secretion of matrices of enamel and dentin and found that the drug did not appreciably affect the incorporation of p r ~ l i n e - ~or H the synthesis of matrices but affected the secretion of matrices. There is no doubt that electron microscope autoradiography will in the future contribute significantly to a better understanding of these subtle differentiation and secretion mechanisms.
c. ELABORATION OF ENAMEL MATRIX Amelogenesis is under the control of the ameloblasts which differentiate from the inner epithelium of the enamel organ. The ultrastructure of the secretory
CALCIFIED TISSUE AUTORADIOGRAPHY
215
ameloblast is now well-documented in the human fetus (Ronnholm, 1962; Frank and Nalbandian, 1963, 1967; Frank, 1968a; Matthiesen and von Bulow, 1969; Egawa, 1970), in the newborn kitten (Frank and Nalbandian, 1963, 1967; Frank, 1968b; Kallenbach, 1977), and in rodents (Scott et al., 1959; Decker, 1963; Garant and Nalbandian, 1968; Jessen, 1968; Warshawsky, 1968; Reith, 1970; Moe, 1971; Katchburian and Holt, 1972; Kallenbach, 1973; Reith and Ross, 1973). The secretory ameloblast is composed of a cell body and a process, the so-called Tomes’ process. The actual boundary between both cell parts is marked by a terminal bar apparatus consisting of microfilaments interconnecting transversely the junctional complexes between adjacent ameloblasts (Fig. 40). According to Reith and Ross (1973), these filaments resemble myofilaments of smooth muscle and are contractile. The nucleus of the secretory ameloblast is located in the basal part of the cell. A prominent rough-surfaced endoplasmic reticulum surrounds the nucleus and the Golgi apparatus and occupies the body of the ameloblast. In both the human fetus and the kitten, the mitochondria are closely associated with the cisternae of the endoplasmic reticulum and are even found in Tomes’ process (Fig. 2), whereas in rodents they are concentrated in the basal part of the cell. A prominent Golgi apparatus is found in a juxtanuclear position, facing the developing enamel. Three-dimensionally the Golgi apparatus is a continuous network having the shape of a hollow cylinder with a central cytoplasmic core, the long axis of the cylinder being parallel to the longitudinal cell axis. In thin sections, it is resolved into stacks of several flattened sacs, presenting an outer or forming face in contact with endoplasmic reticulum and an inner or maturing face surrounding the central cytoplasmic core. In the latter, various types of vesicles and bodies are found (coated vesicles, multivesicular bodies, and so on). Derived from distended saccules, typical secretory granules (Figs. 32-35) are elaborated at this level. They are membrane-bound and contain a more-or-less dense, stippled material morphologically similar to the material which is found between the Tomes’ process and enamel matrix and which is referred to here as preenamel. Granules of this nature are observed throughout the whole proximal cytoplasm between the Golgi apparatus and the Tomes’ process, and fusion with the cell membrane is seen at the level of the Tomes’ process (Figs. 2 and 36). These secretory granules were referred to as “ameloblastic bodies” by Prenault (1924). Acid phosphatase activity was noted by Katchburian and Holt (1969) in Golgi saccules, adjacent vesicles, and in secretion granules located in the Tomes’ process. According to these workers, lysosomal enzymes may play a part in the development of enamel. The Tomes’ process, in addition to secretory granules and coated vesicles, contains mitochondria, endoplasmic reticulum cisternae, and ribosomes (Figs. 2 and 36). In the human fetus and the kitten, several hemidesmosomes are observed along the plasmalemma of the Tomes’ process, facing the developing enamel (Fig. 36). Numerous microfilaments and microtubules are found in the ameloblast body as well as in the Tomes’ process.
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ROBERT M. FRANK
FIG.32. Oblique section through the ameloblast layer 5 minutes after intravenous injection of p r ~ l i n e - ~ HThe . silver grains are located mainly over the Go@ apparatus. Er, Endoplasmic reticulum; N, nucleus. X 11,700. FIG.33. Several silver grains are located over dense secretory granules situated between the Golgi apparatus and the Tomes’ process 30 minutes after intravenous injection of p r ~ l i n e - ~ HEr, . Endoplasmic reticulum; Mi, mitochondria; Fi, microfilaments. X 20,000.
CALCIFIED TISSUE AUTORADIOGRAPHY
217
Whereas osteogenesis and dentinogenesis have much in common in human fetuses, kittens, and rodents, amelogenesis is quite different when comparing rodents and human fetal material. In agreement with Kallenbach (1977), we found that the ultrastructure of ameloblasts in kittens showed greater similarities to its human homologs. In addition, the enamel rod interrelationship and development are quite similar in the human fetus and kitten (Frank and Nalbandian, 1967; Kallenbach, 1977), whereas a complex ultrastructural rod architecture has been described in rodents (Frank and Sognnaes, 1970; Warshawsky, 1971). As soon as the first elements of the enamel matrix are deposited in extracellular positions around the Tomes’ processes, initiation of mineral deposition occurs, characterized by inorganic crystalline growth (Fig. 36). In contrast to bone and dentin, in which a noncalcified organic matrix of some dimension is first laid down (osteoid and predentin), enamel matrix begins to mineralize immediately after deposition. Biochemical investigations have shown that the embryonic organic matrix of enamel is composed essentially of protein (Eastoe, 1963; Glimcher et al., 1964). This enamel protein is rich in proline (25%), glutamic acid, leucine, and histidine and has small amounts of cysteine and tryptophan. The last-mentioned amino acid is not found in the dentin organic matrix. Eastoe (1965) suggested that the enamel protein be called “amelogenin” to distinguish it from collagen and keratin. Seyer (1972) found 26 proteins in bovine enamel matrix, which he separated into two major compartments, one of which had a molecular weight of 18,000 with a low content of phosphorus. The other, with a molecular weight of 6000, was phosphorylated. Carbohydrates constitute 1-2% of the organic enamel matrix, depending on the species and the stage of mineralization (Egyedi and Stack, 1956; Seyer and Glimcher, 1969). These carbohydrate components have been shown to exist in the form of glycoprotein and proteoglycans. Light microscope autoradiography has shown that organic enamel matrix precursors are initially located in an intracellular position over the ameloblasts before being secreted into the enamel matrix (for reviews see A. Weinstock, 1972a,b). With the same technique, a considerable diffusion of tritiated amino acids was noted within the calcifying enamel matrix, indicating great lability of the enamel proteins, in contrast to bone and dentin matrices where incorporation of the labeled amino acids appeared as stable bands of silver grains (Young and Greulich, 1963; Greulich and Slavkin, 1965; Cotton and Hefferen, 1966; A. A. Anderson, 1967). The steps in the secretion of enamel protein as seen by electron microscope autoradiography were first described by Warshawsky ( 1966) following tyrosine3H injections into young rats. He noted an initial conspicuous labeling of the endoplasmic reticulum cisternae, sites of protein synthesis. The newly synthesized proteins then migrated to the Golgi apparatus with subsequent labeling of secretory granules and enamel matrix. After intraperitoneal injections of
218
ROBERT M. FRANK
FIG. 34. Tomes’ processes of secretory ameloblasts and adjacent enamel matrix 1 hour after intravenous injection of pr~line-~H. Note the labeling of several secretory granules (G) containing
CALCIFIED TISSUE AUTORADIOGRAPHY
219
FIG. 37. Cells of the stellite reticulum 5 minutes after intravenous injection of p r ~ l i n e - ~ HNote . the scarcity of silver grains. N, nucleus; Ic, intercellular spaces. ~ 9 0 0 0 .
pr~line-~H and methi~nine-~H into rats, Johnson (1967) noted labeling of the endoplasmic reticulum and thought that the newly synthesized proteins could bypass the Golgi apparatus of the ameloblast. He felt that a greater number of silver grains was located over the cytoplasm of the Tomes’ process than over the secretory granules. A quantitative study of electron microscope autoradiography was accomH newborn plished by Frank (1970a) after intravenous injections of p r ~ l i n e - ~into kittens. At various time intervals following injection, a negligible number of silver grains was observed over the outer epithelium, the stellate reticulum (Fig. 37), and the stratum intermedium of the enamel organ. This contrasted with the
stippled material and the initial labeling of the enamel matrix (E). W, Terminal bar apparatus.
x11,Ooo. FIG.35. Labeled secretory granules (G) in the Tomes’ processes of ameloblasts 1 hour after intravenous injection of p r ~ l i n e - ~ HSome . silver grains are located over the adjacent enamel matrix (E). W, Terminal bar apparatus. x 11,000. FIG. 36. Exocytoses (through membrane fusion) of the contents of a secretory granule ( G ) in a Tomes’ process of an ameloblast 24 hours after intravenous injection of p r ~ l i n e - ~ H Note . the absence of label over the coated vesicles (c). Hemidesmosomes (Hd) are present along the cell membrane of the Tomes’ process. Note the labeling of the enamel matrix (E). The arrows indicate developing apatite crystals. S, Prism sheath; Pe, stippled material in preenamel; Mi, mitochondria. x24,000.
220
ROBERT M. FRANK
extensive labeling noted over the secretory ameloblast. By 5 minutes after the intravenous injection, labeling of the endoplasmic reticulum was observed (Table III; Fig. 38). Even more striking was the very large number of silver grains observed over the Golgi apparatus (Table 111; Figs. 32, 38, and 39). This conspicuous labeling cannot be considered an artifact, and it is interesting to note that in the electron microscope autoradiography study of Slavkin et al. (1976), 50% of the silver grains were over the Golgi apparatus and 40% over the endointo plasmic reticulum 5 minutes after intraperitoneal injection of trypt~phan-~H the rat. We observed a subsequent decrease in radioactivity in the Golgi zone at 30 minutes, 1 hour, and 24 hours (Table 111; Fig. 38). Two hypotheses can account for the rapid appearance of high radioactivity in the Golgi apparatus (Table 111). The first implies a very fast transfer of short polypeptide chains synthesized in the endoplasmic reticulum within, for example, the initial 2 or 3 minutes after intravenous injection. A less orthodox second possibility is that the Golgi apparatus of the ameloblast is the site of protein synthesis. It is interesting to recall that the Goigi apparatus of the odontoblast and the osteoblast of the same animal (Tables I and II) showed maximum labeling by 1 hour, whereas the ENDOPLASMIC RETICULUM NUCLEI YITOCWNDRIA PERIPHERAL CVTOPLASM GOLGl
APPARATUS
AMEL BODIES-PERIPHERAL C I T C:ITOPLASY -AMEL P R K E S S E S AMEL BODIES IN PROCESSES INTERAWELOBLAST SPACES PRE - E N I H E L
FIG. 38. Distribution of radioactivity, expressed as the number of silver grains per 100pm2, in the nucleus, the various organelles, the peripheral cytoplasm, the Tomes' process of the amebblasts, the preenamel, and the enamel 5 minutes, 30 minutes, 1 hour, and 24 hours after intravenous injection of p r ~ l i n e - ~in H the newborn cat.
221
CALCIFIED TISSUE AUTORADIOGRAPHY
TABLE 111 CONCENTRATION OF RADIOACTIVITY IN THE AMELOBLAST, ENAMEL AND PERIPHERAL DENTIN FOLLOWING THE INJECTION OF 3H-hOLINEa’* 5 minutes
Nucleus Mitochondria Endoplasmic reticulum Golgi apparatus Peripheral cytoplasm Secretory granules in peripheral cytoplasm Interameloblastic spaces Cytoplasm of the Tomes’ process Secretory granules in the Tomes’ process Coated vesicles in the Tomes’ process Preenamel Enamel Peripheral dentin ~~
~
10.5 39.2 39.8 215 14.7 33 7.7 0 33 0 17
6 0
30 minutes
1 hour
24 hours
13.3 36 32.8 I75 31 142 9.8 39 50 0 144 55 0
11 33 27 141 44 78 12 89 285 0 127 74 0
7.7 5.8 4.8 6.8 43.9 114 8.3 IS 111
~~
0 44
97 26
~
“Frank, 1970a. ‘Cpncentration is expressed as the number of silver grains per 100 pn? at various time intervals following injection.
radioactivity of their endoplasmic reticulum was highest by 5 minutes. However, there is no reason to assume that the same mechanism holds, since, as mentioned earlier, enamel protein is quite different from collagen. In fact, the first hypothesis seems to be the most likely when taking into account the experimental evidence of A. Weinstock (1970) who injected puromycin, an inhibitor of protein synthesis believed to act at the ribosome level. In rat ameloblasts, he noted distension of the Golgi saccules and the absence of secretory granules. In the kitten ameloblast, Frank (1970a) observed silver grains over flattened, distended Golgi saccules, as well as over numerous secretory granules (Figs. 32 and 39). Coated vesicles and lysosome-like bodies with a heterogeneous content (Fig. 32) did not take up the label. Secretory granules covered by silver grains were observed in the ameloblast body, between the Golgi zone and the Tomes’ process (Figs. 33 and 34). When the radioactivity of the peripheral cytoplasm of the ameloblast body, adjacent to the Tomes’ process, was studied (Table 111; Fig. 38), a large number of silver grains was noted over the secretory granules by 30 minutes when compared to the cytoplasm proper. The same observation can be made in the Tomes’ process (Figs. 34-36) where maximum labeling of the secretory granules was observed by 1 hour, followed by a decrease by 24 hours (Table 111; Fig. 38). The cytoplasm of the Tomes’ process showed the largest number of silver grains by 1 hour (Table III); however, this radioactivity was
222
ROBERT M.FRANK
FIG.39. Prominent labeling of the Golgi apparatus of secretory meloblasts 5 minutes after intravenous injection of p r ~ l i n e - ~ Has, seen in longitudinal section. Er, Endoplasmic reticulum. x 8000.
strikingly weaker than that of the secretory granules during the same time interval. The coated vesicles (Fig. 36) were not labeled at any time period, possibly indicating a pinocytotic function. The preenamel, consisting of stippled material located between the plasmalemma of the Tomes’ process and the enamel matrix proper (Figs. 34-36), was the site of significant labeling by 30 minutes and 1 hour. The mineralizing enamel matrix was diffusely labeled (Figs. 40-42), with maximum radioactivity at 24 hours. The rods, the rod sheath, and the interrod material had a similar concentration of granules. The labile character of the enamel protein, already noted by light microscope autoradiography, was confirmed by a uniform labeling of enamel matrix and also by the fact that a certain number of silver grains had diffused over peripheral dentin beyond the enamel-dentin junction (Table 111; Figs. 40-42). The radioactivity in the lateral intercellular spaces between adjacent ameloblasts is weak until one reaches the junctional complexes of the terminal bar apparatus, indicating that p r ~ l i n e - ~transfer H occurs mainly through the Tomes’ process to the preenamel and enamel matrix (Table III). Kallenbach (1977), who did not observe membrane fusion between the secretory granules and the Tomes’ process cell membrane in the kitten, questioned
CALCIFIED TISSUE AUTORADIOGRAPHY
223
FIG.40. Label is almost complete absent in the Tomes' processes of secretory ameloblasts 24 hours after intravenous injection of p r ~ l i n e - ~ HSilver . grains are present over the entire developing enamel matrix (E) and even in adjacent dentin (D). W, Terminal web apparatus. x6000. FIG.41. Junction between dentin (D) and enamel 24 hours after intravenous injection of proline3H.Diffuse labeling of enamel matrix (E). Note the presence of silver grains in adjacent dentin (arrows). X 13,000.
224
ROBERT M. FRANK
FIG.42. Silver grains are present throughout the developing enamel matrix 24 hours after intravenous injection of pr~line-~H. S, Prism sheath; D, dentin; TP, Tomes’ process devoid of silver grains. X 11,000. (From Frank, 1970a.)
how a distinction could be made (Frank, 1970a) between exportable and lysosomal proteins. When comparing morphological events, including membrane fusion phenomenon of secretory granules (Figs. 34-36) with the autoradiographical events, it becomes clear that the secretory granules elaborated in the Golgi apparatus migrate into the Tomes’ process and discharge their content toward the enamel matrix. A regular sequence of labeling indicates clearly the direction of the isotopic wave. The times of maximum radioactivity for the Golgi apparatus (5 minutes), for the secretory granules in the peripheral cytoplasm of the ameloblast cell body (30 minutes), and for the Tomes’ process (1 hour), followed by the enamel matrix (24 hours), indicated clearly that all the conditions were fulfilled to conclude a protein transfer from the Golgi apparatus to the cell periphery and the extracellular matrix via exocytosis involving the secretoxy granules. Such an interpretation is further substantiated by the puromycin experiment (A. Weinstock, 1970) in which inhibition of protein synthesis suppressed the formation of secretory granules in the ameloblast. By electron microscope autoradiographyperformed after intraperitoneal injection of trypt~phan-~H into newborn mice, Slavkin et al. (1976) confirmed the same sequence of labeling. By 5 minutes, 40% of the total silver grains were localized over the rough endoplasmic reticulum and 50% over the Golgi ap-
CALCIFIED TISSUE AUTORADIOGRAPHY
225
paratus. By 30 minutes, silver grains were predominantly observed over condensing vacuoles and secretory granules. The latter migrated to the Tomes' process, where they discharged their contents by exocytosis. The distribution of glycoproteins in secretory ameloblasts and enamel matrix was studied by electron microscope cytochemistry using phosphotungstic acid at low pH (Rambourg, 1967). Reaction products were observed within the stack of Golgi saccules (with increasing staining from the outer face to the inner face), as well as in the secretory granules (A. Weinstock and Leblond, 1971). The same workers followed the elaboration of the matrix glycoprotein of enamel in young rats by electron microscope autoradiography after intravenous galactose"H injections. An uptake of the label was observed 2.5 minutes after injection, indicating that galactosePH was incorporated into glycoprotein within this organelle. After 5-10 minutes, the label was found in the condensing vacuoles and in secretory granules of the Golgi region. By 20-30 minutes the label appeared in similar granules of the Tomes' process, as well as in the enamel matrix. Enamel matrix glycoproteins were therefore transferred to the developing enamel through a similar mechanism of exocytosis, that is, via secretory granules as observed for protein precursors. Similar pathways were described for proteoglycan using sulfate-35S label in the rat (A. Weinstock, 1972a) and kitten (Nagai and Nagai, 1977). Preliminary results obtained by A. Weinstock (1972a) indicated that radiosulfate-labeled material was transported to the growth regions of the enamel matrix via the secretory granules, and Nagai and Nagai (1977) confirmed these results. Based on these sophisticated methods of investigation, there are sufficient data for concluding that the organic enamel precursors, including protein, glycoprotein, and proteoglycan, are transferred from the Golgi apparatus to the secretory pole of the ameloblast via secretory granules correctly called ameloblastic bodies by Prenant (1924). Further work is needed in order to determine whether these secretory granules carry the various biochemical organic precursors simultaneously or separately. There is also some evidence for the presence of a lysosomal vacuole system in ameloblasts (Katchburian and Holt, 1969, 1972). Its precise relationship to the secretory granules also needs to be determined. Other fundamental questions concern the mechanism of ameloblast differentiation and polarization, as well as the factors governing both the intracellular transfer of organic precursors and the extracellular secretion of matrix. Ruch et al. (1972) demonstrated that polarization of postmitotic ameloblasts seemed to be conditioned by factors contained in the predentin which are bacterial collagenaselabile. Because of the relative impurity of collagenase, they could not include that collagen itself played a part in ameloblast differentiation. Therefore Ruch et al. (1974) used L-azetidine, an analog of proline, which suppresses the production of extracellular collagen. Their results suggest that collagen plays a part in ameloblast polarization, but L-azetidine could also affect predentin glyco-
226
ROBERT M. FRANK
protein and proteoglycan synthesis. According to Slavkin (1974), the odontoblasts, or extracellular molecules or matrix vesicles present in predentin, are capable of instructing nontooth, nonoral epithelium to differentiate into inner enamel epithelium and subsequently into ameloblasts. Several investigators have demonstrated the importance of microtubules and microfilaments in the intracellular events related to the secretory processes in ameloblasts, using colchicine (Kudo, 1975; Ruch et al., 1975) vinblastine (Moe and Mikkelsen, 1977a,b), and cytochalasin B (Ruch et al., 1975). In the future, the combined use of cytochemistry, immunocytochemistry, and electron microscope autoradiography will certainly contribute significantly to an understanding of the essential phases of differentiation and secretion related to the enamel organic matrix.
V. Transfer Routes of Inorganic Elements Bone, dentin, and enamel share an inorganic crystalline phase related to apatites, more precisely to hydroxyapatite, Ca,, (PO,),(OH),. Very few minerals have resulted in so many controversies and investigations among chemists, physicochemists, crystallographers, and biologists. Whereas the exclusive existence of crystalline calcium phosphate in calcified tissues has been long accepted, the presence of amorphous calcium phosphate (ACP) is also now acknowledged (Termine, 1972). Whether in bone, dentin, or enamel, the initiation of calcification and the subsequent growth of apatite crystals has always been observed in an organic matrix elaborated by cells, either mesenchymal as in the case of osteoblasts and odontoblasts, or epithelial as in the case of ameloblasts. For mesenchymal calcified tissues, the calcification phase is distinctly separate from the initial phase of organic matrix elaboration, whereas for enamel these two stages are concurrent. The exact sites of the initial mineral deposition in the extracellular organic matrices of calcified tissues have been the subject of heated controversy. However, it is now generally accepted that, in the early developmental stages of fetal woven bone (Bernard, 1969; Bernard and Pease, 1969; Ascenzi and Bonucci, 1970; Anderson and Reynolds, 1973), of calcifying cartilage (H. C. Anderson, 1967; Bonucci, 1967; Thyberg and Friberg, 1970; Balmain-Oligo and Juster, 1975), and of peripheral mantle dentin (Bernard, 1972; Eisenman and Glick, 1972; Sisca and Provenza, 1972; Katchburian, 1973; Larsson, 1973; Slavkin, 1975), the initial sites of calcification are in matrix vesicles originating from the cells. Needle-like apatite crystals appear in these vesicles, the triple-layered membrane of which is ruptured by subsequent mineral growth, giving rise to calcified nodules (Slavkin, 1975). The histochemical and biochemical analysis of
CALCIFIED TISSUE AUTORADIOGRAPHY
227
the vesicular contents has indicated that they contain all substances and enzymes capable of binding calcium and phosphates (Matthews and Martin, 1975; Slavkin, 1975), and it is implied that these inorganic elements transit through the cell. If the matrix vesicle theory can be accepted for the very early stages of calcification of fetal woven bone and peripheral mantle dentin, it must not be forgotten that the majorpart of bone (Fig. 43) and dentin matrices, as well as the entire ameloblast-secretedpart of enamel, undergo calcification in the absence of matrix vesicles (Frank, 1968a; Frank and Lang, 1969; Nygren et al., 1976; Landis ef al., 1977). It is highly probable that the main inorganic phenomenon during calcification consists of initial local deposition of an ACP followed by its transformation into minute apatite crystals which, through progressive growth, reach their mature size. ACP can be differentiated from the hydroxyapatite crystals by a calciudphosphate ratio lower than 1.67, by its irregular appearance under the electron microscope (often small spherical particles), and by the absence of fringes in x-ray and electron diffraction (Termine, 1972). The nucleation concept (Neuman and Neuman, 1958; Glimcher and Krane, 1968) still seems to be the most acceptable theory concerning calcification, but the biochemical and biophysical conditions required for the seeding of calcium phosphate in the organic matrices are still a matter of debate (Howell, 1971).
FIG.43. Beginning of calcification in the osteoid tissue (0).Some inorganic crystals are present over cross-sectioned collagen fibrils (arrows). Note the absence of matrix vesicles. Rounded mineralized islands arise from calcification on a few collagen fibrils and adjacent interfibrillar matrix (lower left). Cp, Cytoplasmic process of an osteoblast. ~35,000.
228
ROBERT M. FRANK
Calcification is characterized by the accumulation of calcium and phosphate ions in cell-elaborated organic matrices, but the transfer routes of these inorganic elements from the blood capillary lumens to the mineralization areas have not been studied to any extent by either light or electron microscope autoradiography (Leblond and Weinstock, 1976).
A. ELECTRON MICROSCOPE AUTORADIOGRAPHY OF 45CADURING OSTEOGENESIS The concept that the osteoblast plays a role in bone mineralization has become more and more important. In the view of Shapiro and Greenspan (1969), calcification is under cellular control, and mitochondria are responsible for the concentration of inorganic ions. These mitochondria liberate the calcium and phosphate ions under the influence of vitamin D,parathyroid hormone, and the intracytoplasmic calcium concentration. Calcium (Kashiwa, 1966; Rolle, 1969; Aaron, 1973) and phosphate (Kashiwa, 1968; Aaron, 1973) granules have been demonstrated histochemically in the osteoblast and osteocyte cytoplasm, and biochemical analysis has shown that these bone cells contain more calcium than the medullary cells and the surrounding extracellular fluid (Nichols et al., 1971;
FIG.44. Silver grains at the periphery of an osteocyte 30 minutes after intravenous injection of 45Ca. S, Periosteocytic organic space; B, bone; Mi, mitochondria with several dense granules. ~ 2 9 , 0 0 0 (From . Frank et nl., 1974.)
229
CALCIFIED TISSUE AUTORADIOGRAPHY INTERFIBROBL.
S-ES
FB#)BL*8tS
lNTEROSTEOBL. SPACES
d
30’
I:::::=:I 1-1 I:::::::I
lh
8h
FIG.45. Distribution of radioactivity expressed as the number of silver grains per 1OOOpm2 in the fibroblasts, osteoblasts, osteocytes, extracellular spaces, osteoid tissue, and bone 5 minutes, 30 minutes, 1 hour, and 6 hours after intravenous injection of 45Cainto the newborn cat. (From Frank et al.. 1974.)
Hirschmann and Nichols, 1972). As early as 1965, Baud and Dupont described in the osteocyte mitochondria the presence of dense granules 300-500 di in diameter. These granules (Fig. 44), containing calcium and phosphates, have since been described repeatedly. Martin and Matthews (1969) noted that they increased in epiphyseal chondroblasts toward the mineralizing zones and, in the view of Matthews et al. (1973), the mitochondria play a role in the intracellular regulation of calcium. The presence of calcium has been observed in osteoblasts and osteocytes with light microscope radioautography (Johnston, 1958), and the presence of 45Ca in several organelles of epiphyseal chondroblasts has been shown by electron microscope autoradiography (Matthews et al., 1968). Also, the matrix vesicle theory implies that calcium and phosphate transfer occurs via the cells through “osteoblast extrusions” (Bonucci, 1971). With electron microscope autoradiography, Frank et al. (1974) studied 45Ca transfer during intramembranousjaw ossification, using intravenous injections of 45CaC&,0.5 pCi/lO gm body weight, into newborn cats. By comparing the radioactivity in the various cell types (fibroblasts, osteoblasts, and osteocytes), certain observations could be made concerning the transfer of 45Ca from the capillary lumens to the bone tissue (Fig. 45). A maximum number of silver grains was observed over the medullary fibroblasts by 5 minutes, with a sub-
230
ROBERT M.FRANK
sequent progressive decrease up to 6 hours. 45Catransfer through the osteoblast front occurred principally through the cells since, at 5 minutes following injection, slightly less than half the number of silver grains was noted in the lateral interosteoblast spaces (Fig. 45). At this level, a progressive decrease in radioactivity was observed, with a zero value by 6 hours; whereas by this same time interval, the osteoblasts still showed some labeling. For 5 minutes, 30 minutes, 1 hour, and 6 hours after intravenous injection, the total radioactivity of the osteoblasts did not vary significantly (Fig. 45), but it is interesting to note that the silver grains in the osteocytes increased strikingly by 1 hour, disappearing totally by 6 hours (Fig. 46). This coincided with extensive labeling of bones by 6 hours (Fig. 4 3 , and morphologically a large number of silver grains was observed near the bone lacunae (Fig. 46). Hence all the conditions seemed to be fulfilled for possible 45Catransfer from the osteoblast to the osteocyte via their cell processes, where tight junctions have been described (Weinger and Holtrop, 1974). 45Catransfer from the osteoblast to the osteocyte and from there to the bone tissue through the organic periosteocytic spaces (Figs. 44 and 46) is in fact a reverse confirmation of the hypothesis of Talmage (1970) who proposed that calcium release from bone occurred through the cells, from the osteocyte to the osteoblast. If 45Ca can reach the bone through the bone
FIG.46. Numerous silver grains are located in the calcified bone matrix around an osteocyte lacuna (lower left) 6 hours after intravenous injection of 45Ca. The cytoplasm of the osteocyte is devoid of silver grains. Er, Endoplasmic reticulum; N, nucleus. X8000. (From Frank er al., 1974.)
23 1
CALCIFIED TISSUE AUTORADIOGRAPHY
m
80
N
t., (II
E
a 40
20 I
3
’
I
w n l
\ m
lacunae, a transfer from the osteoblasts to the osteoid and bone has also been observed (Figs. 45, 47, 48, 49 and 50). Analysis of the radioactivity variations in different parts of the osteoblasts (Figs. 51-53) showed, by 5 minutes, a maximum number of silver grains in the peripheral cytoplasm, situated between the most external cisternae of the endoplasmic reticulum and the cell membrane and indicating probable calcium penetration into the cell (Fig. 47; Table IV). Of all the cellular organelles, mitochondria showed the most intense labeling, with an increase up to 30 minutes followed by a significant decrease by 6 hours (Table IV; Figs. 47 and 52). This observation is in agreement with the concept in which the mitochondria are considered a reservoir and a regulating agent of calcium within the cell (Matthews et at., 1971). The endoplasmic reticulum showed an almost constant level of radioactivity (Table IV; Figs. 47, 52 and 53). In the Golgi apparatus an increase in silver grains was noted up to 30 minutes, reaching an almost steady state We did not observe any label within the secretory vesicles in the Golgi ap-
233
CALCIFIED TISSUE AUTORADIOGRAF’HY
TABLE IV CONCENTRATION OF RADIOACTIVITY IN THE OSTEOBLAST, OSTEOID TISSUE,AND BONEFOLLOWING THE INJECTION OF 45CAa’b 5 minutes
30 minutes
1 hour
6 hours
26.8 32.5 38.6 66.5 37.3 18.2 23.9
23.6 40.5 39.8 5.8 26.5 14.6
~
Nucleus Golgi apparatus Endoplasrnic reticulum Mitochondria Peripheral cytoplasm Osteoid tissue Bone
36.4 11.2 41.6 45.1 56.3 26.2 33.2
14.2 39.2 41.0 98.3 46.8 29.7 32.1
111.3
~~
=From Franker al., 1974. Concentration is expressed as the number of silver grains per 1000L./ d at various time intervals after injection.
paratus and transfer organic matrix precursors of the osteoid tissue. Likewise coated vesicles (Fig. 48) were not labeled. Silver grains were sometimes observed over osteoblast cell processes or matrix vesicles (Fig. 51). In this work it appeared that 45Catransfer from the capillary lumen to calcified bone occurred by two different routes. A direct transfer consisted of 45Camigration through the intercellular spaces of medullary fibroblasts and osteoblasts, reaching osteoid and bone. The second route (after passage through the extracellular space between fibroblasts) consisted of an intracellular transfer through the osteoblasts and the osteocytes to reach the bone tissue either directly or through the osteoid tissue as an intermediary. The second route is more important than the direct transfer.
B. ELECTRON MICROSCOPE AUTORADIOGRAPHY OF 45CADURING DENTINOGENESIS As is the case for bone, only a few investigators have studied the mechanisms of calcium transfer from the capillaries to the dentin. At the light microscope level, Kashiwa and Sigman (1966) demonstrated histochemically the presence of calcium granules in the odontoblast process. Using the potassium pyroantimonate technique, Ozawa et al. (1972) noted, with the electron microscope, reaction products in mitochondria, the Golgi apparatus, dense granules, and the interodontoblastic spaces. Using the same technique, Frazier and Nylen (1972) reported that electron-dense precipitates were localized in flattened sacs and vesicles of the Golgi region and in secretory granules. The reaction product was also found between cells, whereas the cytoplasm, mitochondria, and endoplasmic reticulum of the odontoblast were virtually free of reaction product. Similar
234
ROBERT M. FRANK
FIG. 51. Silver grains are present in an osteoblast 30 minutes after intravenous injection of 45Ca. N, Nucleus; Mi, mitochondria; Er, endoplasmic reticulum; OM, osteoid tissue. X9OOO. (From Frank er ai., 1974.) FIG. 52. Silver grains over a mitochondrion (Mi) and endoplasmic reticulum (Er) of an osteoblast 30 minutes after intravenous injection of 45Ca. Bundles of microfilaments (arrows)are disseminated in the cytoplasm. ~ 4 2 , 0 0 0 .(From Frank et al., 1974.)
CALCIFIED TISSUE AUTORADIOGRAPHY
235
FIG.53. Silver grains over the nucleus (N)and the endoplasmic reticulum (Er) of an osteoblast 1 hour after intravenous injection of 4Ta. NU, Nucleolus; Mi, mitochondria. X20,OOO. (From Frank et al.. 1974.)
investigations were made by Reith (1976) in odontoblasts of developing rat molar teeth. He found positive reactions in elongated dense secretory vesicles situated in the Golgi apparatus and at the distal pole of the cells. Lesser amounts of reaction product were found in the extracellular space, mitochondria, nucleus, and generally throughout the cell. Tissues pretreated to allow the escape of diffusible ions showed a positive calcium reaction only in the secretory vesicles. Reith (1976) considered that these results reflected the binding of calcium within the Golgi apparatus. By electron microscope autoradiography using 45Ca, Bauer (1968) compared the radioactivity of enamel and dentin in the mouse after intraperitoneal injection. With the same technique, Fromme et al., (1971) demonstrated the presence of calcium in the endoplasmic reticulum and in the odontoblast processes. Sayegh et al. (1976) injected 45Cainto young mice. By differential centrifugation, he recovered the mitochondrial fraction of the odontoblasts and found 45Ca activity associated with the microsomal supernatant as well as the membranous pellets containing mitochondria. The transport of 45Cato the odontoblasts was extremely rapid, occurring within 5 minutes after injection. That the transfer of 45Cadentin and enamel is extremely rapid has already been demonstrated by Munhoz and Leblond (1974) with light microscope autoradiog-
236
ROBERT M. FRANK P u l p a l spaces
I:::::::I
Pulpal fibroblasts
m
lnterodont .spaces
1:::::::I
j
Odon t o b I a s t s
1-
Pr e d e n t i n
D
Dentin
5’
30’
lh
6h
FIG.54. 45Caradioactivity concentration expressed as number of silver grains per 1OOOpm2 in the intracelluIar spaces of the dental papilla (pulpal spaces), in the pulpal fibroblasts, in the odontoblasts and their processes, in the lateral interodontoblastic spaces, and in the predentin and dentin at various time intervals following injection.
raphy. After intravenous injection of 45Ca into young rats, they sacrificed the animals which they perfused with a tracer-free CaCt, solution in an effort to remove unbound 45Cafrom the tissues. Within 30 seconds and 5 minutes after injection, they noted a maximum reaction at the dentin-predentin junction, with a gradual decrease toward the dentin-enamel border. However, in strong contrast to all previous investigators, they did not find any radioactivity over the odontoblasts. Following intravenous administration of 45Ca to newborn cats, Nagai and Frank (1974) studied the distribution of the label by electron microscope autoradiography without cold CaCh perfusion. A comparison was made between the various cellular and extracellular compartments of the pulpodentinal organ. The radioactivity of the odontoblast and its process was grouped under the collective term “odontoblasts” (Fig. 54). It was shown that, 5 minutes after intravenous injection, the extracellular spaces of the dental papilla were the most intensely labeled, followed by the pulpal fibroblasts (Fig. 54). At 30 minutes, the highest radioactivity was noted in the pulpal fibroblasts. At 1 hour, the odontoblast and the dentin (Fig. 54) were
231
CALCIFIED TISSUE AUTORADIOGRAPHY
the most densely labeled compartments, whereas at 6 hours the dentin had the maximum number of silver grains. Even when 45Ca showed an intracellular transfer through the odontoblast and its process, a significant part of the radioactive calcium diffused directly from the intercellular spaces of the dental papilla via the spaces located laterally between the odontoblasts, finally reaching the dentin (Fig. 54). It is interesting to note that after 5 minutes the radioactivity was slightly higher in the predentin than in the dentin; it was almost equal at 30 minutes, became strikingly higher in dentin at 1 hour, and even more so at 6 hours (Fig. 54). Two routes of almost equal importance for transfer through the layer of odontoblasts have therefore been demonstrated. The direct route from the capillary lumen follows the extracellular spaces of the dental papilla, the lateral interodontoblastic spaces, the predentin, and finally reaches the dentin. In the indirect route, after passage through the extracellular spaces of the dental papilla, the calcium followed a transcellular route through the odontoblast. This intracellular transfer of 45Ca through the odontoblast body consisted mainly of a progressive loading of the different cellular compartments over the first hour, followed by a decrease at 6 hours (Table V; Fig. 55). Among the cellular organelles, the mitochondria were richest in radioactive calcium (Fig.
I
5'
30'
Ih
I
6h
FIG.5 5 . Radioactivity concentration expressed as number of silver grains per 1 0 0 0 ~ m zin the odontoblast, its process, and the dentin at various time intervals following injection.
FIG. 56. Tangential section through the infranuclear region of two odontoblasts 1 hour after intravenous ‘ T a injection. Silver grains are seen in the endoplasmic reticulum (Er) and the Golgi apparatus (Go). Mi, Mitochondria. x 18,000. (From Magai and Frank, 1974.) FIG.57. Transverse section of an odontoblast 1 hour after intravenous45Cainjection. Labeling of the Golgi apparatus (Go) was noted. Mi, Mitochondria; Er, endoplasmic reticulum; g, crosssectioned secretory vesicles with dense granules. x24,OOO. (From Nagai and Frank, 1974.)
239
CALCIFIED TISSUE AUTORADIOGRAPHY
55). This observation confirms their importance in calcium storage and regulation. In addition to the mitochondria, the Golgi apparatus (Figs. 55, 56, 57), the endoplasmic reticulum (Fig. 56), and the nuclei (Fig. 55; Table V) accumulated calcium which was subsequently transferred to predentin and dentin through the odontoblast process (Table V; Figs. 55, 58-60). At 6 hours, a diffuse labeling was observed in the intertubular dentin (Fig. 60). However, a greater amount of radioactivity was present in the peripheral dentin all along the dentin-enamel junction (Fig. 62). The radioactivity concentration was clearly higher in whole dentin (177.8 graid1000 p d ) than in enamel (38.5 graindl000 p d ) . Under the experimental conditions used in this study, calcium transfer via elongated dense secretory vesicles was not observed in the odontoblast (Figs. 59 and 61). This contrasts with the ultrastructural cytochemical findings of Frazier and Nylen (1972) and Reith (1976). In our study, the calcium appeared to migrate through the cytoplasm of the odontoblast process, unassociated with secretory vesicles or coated vesicles (Figs. 58-61). However, it should be noted that we used relatively low doses of 45Ca. It might be that transfer via the secretory vesicles requires a certain minimal level of radioactivity to be detected. Indeed, using pr~line-~H, Kajikawa and Kakihara (1974) did not observe vesicular labeling after intraperitoneal injection of 10 p Ci/gm, whereas Frank (1970b) and Weinstock and Leblond (1974) did so but used larger intravenously injected doses. The absence of odontoblast labeling noted in light microscope autoradiography TABLE V CONCENTRATION OF RADIOACTIVITY I N THE ODONTOBLAST, ODONTOBLAST PROCESS, PREDENTIN, AND DENTIN FOLLOWING THE INJECTION OF 4sCA"*b
Nucleus Golgi apparatus Lysosomes Mitochondria Endoplasmic reticulum Cytoplasm Odontoblast process in predentin Secretory granules in process Odontoblast process in dentin Interdontoblast spaces Predentin Dentin
5 minutes
30 minutes
1 hour
6 hours
17.3 51.6 19.5
22.8 52.3 30.6 57.3 29.9 32.8 28.9
30.1 58.3 30.8 70.9 48.5
17.7 29.4 14.0 32.3 18.1 18.8 19.9 0 85.7 26.8 15.4
55.2
30.4 28.8 33.7 0 0 36.9 25.1 18.6
0 74.5
32.0 22.5 30.3
41 .O
52.0 0
164.3 35.5 28.2 56.7
177.8
"From Nagai and Frank, 1974. bConcentrationis expressed as the number of silver grains per 1OOOpmZ at various time intervals following injection.
FIG. 58. Section through the dentiwpredentin junction 30 minutes after intravenous 45Ca injection. Silver grains are present in the cytoplasm of an odontoblast process (Od). Pd, Predentin; D, dentin. ~32,000.(From Nagai and Frank, 1974.) FIG.59. Dentiwpredentin junction 1 hour after intravenous 45Cainjection. A silver grain is still present in the odontoblast process (Od), but most labeling is in the predentin (Pd). D, Dentin. ~ 2 9 , 0 0 0 (From . Nagai and Frank, 1974.)
240
FIG.60. Labeling of intertubular dentin (D) 1 hour after intravenous 45Ca injection. A silver grain is seen (at the left) over the odontoblast process (Od). Cp, Branch of an odontoblast process in a lateral branch of a dentinal tubule. X 14,300. (From Nagai and Frank, 1974.) FIG. 61. Dense, elongated, rod-shaped secretory vesicles (G)in the odontoblast process 1 hour after intravenous 45Cainjection. Silver grains appear in the cytoplasm of the process, but the vesicles are not labeled. Pd, Predentin. ~ 1 5 , 6 0 0 . FIG. 62. Junction between enamel (E) on the left and dentin (D) on the right. Numerous silver grains are present 6 hours after intravenous 4,Tainjection. x20,OOO. (From Nagai and Frank, 1974.) 24 1
242
ROBERT M.FRANK
by Munhoz and Leblond (1974), in contrast to the positive findings of ultrastructural cytochemistry, cell fractionation, and electron microscope autoradiography, is more difficult to explain and could perhaps be related to the use of cold calcium perfusion which removed the radioactive calcium in the cells and intercellular spaces.
c. ELECTRON MICROSCOPE AUTORADIOGRAPHY OF 45cA DURING AMELOGENESIS Calcium transpo,rt during amelogenesis was followed by light microscope histochemistry by Kashiwa and Sigman (1966) who noted stained granules in the cytoplasm of the ameloblast. With the electron microscope, Deporter and Ten Cate. (1976) studied the localization of alkaline phosphatase in relation to enamel formation in the mouse molar. Enzymic activity was localized in the stratum intermedium throughout amelogenesis and occurred in the ameloblasts only with the onset of enamel maturation. According to these investigators, the appearance of enzymic activity within the ameloblasts seemed to be related to calcium transport. Kuroda (1977), using the potassium pyroantimonate method, studied the electron microscope localization of ionic calcium in ameloblasts of rats. Secretory ameloblasts showed high concentrations of reaction product in nuclei Spaces-stratum intermedium Cells-stratum intermedium Interameloblast spaces
Amelobi a s t s
-
P r e enamel Enamel
5’
30’
-
Izizq /:::::::I
[ml
lh
Bh
FIG.63. Disttibution of radioactivity expressed as the number of silver grains per 1OOOpd in the stratum intermedium, the ameloblasts, the intercellular spaces, the preenamel, and the enamel 5 minutes, 30 minutes, 1 hour, and 6 hours after intravenous injection of 45Ca.
243
CALCIFIED TISSUE AUTORADIOGRAPHY
1 ' 5'
30'
I
I
lh
6h
FIG. 64. Radioactivity, expressed as,the number of silver grains per 1OOOpd in the ameloblast, its process, and the enamel at various time intervals following intravenous injection of Ca.
and mitochondria. The endoplasmic reticulum and Golgi region contained few deposits, whereas lysosomes, secretory granules, and nuclear membranes were largely negative. Diffuse deposits were observed throughout the cytoplasm of the cells and were particularly prominent in Tomes' processes. With light microscope autoradiography, Reith and Cotty (1962) demonstrated that, after intraperitoneal injection, 45Caentered the developing enamel via the enamel organ during matrix formation and also during later stages when matrix formation had ceased. Bawden and Wennberg (1977) studied in tissue culture the cellular influence on 45Cauptake in developing rat enamel. They hypothesized that calcium flux into newly formed enamel matrix is controlled in part by movement of the calcium which diffuses between the ameloblasts toward the enamel surface and away from the enamel through the ameloblasts. Bauer (1968) was the first to use electron microscope autoradiography after intraperitonealinjection of 45Cain mice, and he observed labeling of the developing enamel. With the same technique, Oka and Shimizu (1972) reported a clear labeling of the ameloblasts and concluded that 45Careaches enamel only through these cells; 15 minutes after intraperitoneal injection, they clearly noted labeling of the mitochondria, the endoplasmic reticulum, the Golgi apparatus, and the nuclei, as well as small vesicles. On the contrary, Munhoz and Leblond (1974) with light microscope autoradiography, reported, after the intravenous injection
244
ROBERT M. FRANK
of 45Ca, an intense labeling of developing enamel in rat incisor teeth at 30 seconds and 5 minutes, whereas the ameloblasts did not take up the label. Nagai and Frank (1975) followed 45Ca transfer during amelogenesis after intravenous injections into the newborn cat. Quantitative electron microscope autoradiography showed that 5 minutes after injection radioactivity was relatively high, on the one hand, in the intercellular spaces of the stratum intermedium and of the ameloblast layer and, on the other hand, in the cells of the stratum intermedium (Fig. 63). The extracellular spaces between the latter cells were labeled until 1 hour after injection, followed by a decrease at 6 hours. Two diffusion routes of 45Catransfer to enamel were observed (Fig. 63). The first (direct) route passing through the lateral intercellular spaces, between the ameloblasts, was relatively more important than the second transcellular route through the ameloblasts. The radioactivity in the lateral interameloblastic spaces increased slightly up to 30 minutes and decreased progressively at 1 hour and 6 hours (Fig. 63). A similar variation occurred for the intracellular radioactivity of the ameloblasts (Fig. 63). A detailed analysis of 45Catransfer through the ameloblast and its process is presented in Table VI and Fig. 64.At 5 minutes, the most intense labeling was observed in the basal peripheral cytoplasm adjacent to the stratum intermedium, specifically between the ameloblast plasmalemma and the basal endoplasmic reticulum as far as the basal terminal web. This indicates that 45Capenetrated the cell through its basal pole adjacent to the stratum intermedium. The mitochondria showed the most intense labeling of all the ameloblast organelles. The maximum TABLE VI CONCENTRATION OF RADIOACTIVITY IN THE AMELOBLAST, PREENAMEL (EXTRACELLULAR STIPPLED MATERIAL), AND ENAMEL FOLLOWING THE INJECTION OF 45CAa*b
Nucleus Golgi apparatus Mitochondria Endoplasmic reticulum Peripheral cytoplasm Lateral cytoplasm Cytoplasm of the Tomes' process Secretory granules in the Tomes' process Interameloblastic spaces Preenamel Enamel
5 minutes
30 minutes
1 hour
6 hours
24.5 19.8 37.8 23.1
25.6 27.1
29.7 30.2
46.4
42.3
31.9 30.4 21.3 36.4 0
30.5 19.9 29.5 22.9 0 39.0 16.3 34.5
19.3 13.1 28.9 20.5 18.3 21.6 7.6 0 32.7
41.5
21.8 25.2 0 45.7
5.4 23.4
53.8 11.1
30.4
43.5 38.5
"From Nagai and Frank, 1975. bConcentration is expressed as the number of silver grains per 1000pmz at various time intervals after injection.
CALCIFIED TISSUE AUTORADIOGRAPHY
245
FIG. 65. Transverse section through the infranuclear part of a secretov ameloblast. Labeling of the endoplasmic reticulum (Er) 30 minutes after intravenous 45Cainjection. Go, Golgi apparatus; Mi, mitochondria; E, lateral extracellular space between ameloblasts. X 22,000. FIG. 66. No silver grains are present in a secretory granule ( G ) of an ameloblast 1 hour after intravenous "Ca injection. Silver grains over the endoplasmic reticulum (Er). Mi, Mitochondria. X 16,000. (From Nagai and Frank, 1975.)
246
ROBERT M.FRANK
radioactivity for the endoplasmic reticulum (Fig. 65) and the Golgi apparatus was found at 30 minutes and 1 hour, respectively (Fig. 64;Table VI), raising the question of an eventual transfer from one organelle to the other. However, we did not observe any labeling within the secretory granules (Table VI; Fig. 66), and the 45Catransfer seemed to occur through the cytoplasm of the Tomes’ process and its cell membrane toward the preenamel and enamel, unassociated with membrane-bound formations. This observation is in agreement with the cytochemicalfinding of Kuroda (1977). However, as mentioned earlier in the case of dentinogenesis, we used relatively low doses of 45Ca, and heavier isotopic charges may be necessary to obtain labeling of secretory granules. In developing enamel, a progressive increase in 45Ca was noted. Indeed, as early as 5 minutes after intravenous injection, several silver grains were present in this tissue. The distribution of the silver grains was diffuse over rods, interrod substance, and rod sheathes. By 6 hours a higher number of silver grains was found in the peripheral enamel, along the enamel-dentin junction. Although our observations indicate a major, direct extracellular transfer route of 45Cato enamel, consistent with the findings of Munhoz and Leblond (1974), we observed, in contrast to these workers, labeling of the ameloblasts as well. This difference is probably related to variations in technical procedures, as mentioned earlier, perhaps to the omission of cold CaCI, perfusion in our study.
VI. Identification of Sensory Nerve Endings in Adult Dentin by Autoradiography The morphological basis of dental innervation is of major importance in understanding the sensory mechanisms in teeth, since the precise location of the receptors is paramount for an adequate interpretation of physiological data. Over a period of many years, the innervation of predentin and dentin was alternately confirmed and denied, based on silver impregnation methods combined with light microscopy (Fearnhead, 1967). Using the electron microscope, Frank (1966, 1968b,c) described unmyelinated nerve fibers in the inner third of fully formed human coronal dentin. Such an identification was based on the ultrastructural content of the intradentinal nerve fiber, typical of nerve endings observed in other parts of the body. This intradentinal receptor was not found in every coronal tubule. Complex infolding of the nerve ending in the odontoblast process has been observed (Frank, 1968b,c). At this level, tight junctions have been located (Frank, 1968~).With the use of lanthanum hydroxide labeling these were identified as gap junctions (Holland, 1976). The ultrastructural presence of nerve endings in the inner coronal dentin was further confirmed by Johansen (1967), Avery (1971), and Corpron et al. (1972). In addition to unmyelinated nerve fibrils, Corpron and Avery (1973)
CALCIHED TISSUE AUTORADIOGRAPHY
247
described Schwann cell-covered axons among coronal odontoblasts and predentin in mouse molar teeth. Convincing evidence for the sensory nature of the intradentinal nerve fibers was contributed by Arwill et al. (1973). In the teeth of cats subjected to sensory denervation by unilateral transection of the inferior alveolar nerve, these workers demonstrated after 2-4 weeks a total absence of impulse activity during electrophysiological recordings, regardless of whether or not the autonomic innervation was intact, and under the electron microscope nerve fibers were absent or showed degenerative changes. Control teeth responded to different pain stimuli, and ultrastructural evidence of nerve endings in the inner coronal dentin was found. After unilateral transection of the autonomic supply by removal of the cranial cervical sympathetic ganglion, no changes in the electrophysiological recordings of inner coronal dentin were observed from teeth subjected to sympathetic nerve supply resection, and ultrastructurally the intratubular nerves were present. Arwill et al. (1973) concluded that the structures designated “associated cells” (Arwill, 1967, 1968) were in fact intradentinal sensory nerve endings. Another demonstration of the sensory nature of the intradentinal nerve endings has been obtained by the application of autoradiographic methods. Droz and Leblond (1963) and Droz (1969) reported a migration of radioactive protein molecules from the neurons toward the nerve endings, and this axonal protein transport can be used to create a radioactivity map of nerve endings (Lasek, 1970; Lasek et al., 1968). Thus when prolinegH is injected into the trigeminal ganglion of rats (Byers and Kish, 1976) or cats (Weill et al., 1975), it is incorporated into radioactive proteins that move rapidly by axonal transport to the endings of these neurons. The sensory trigeminal origin of the intradentinal coronal nerve endings has thus been confirmed by light microscopy (Weill et al., 1975; Byers and Kish, 1976) and by electron microscope autoradiography (Byers and Kish, 1976).
VII. Conclusion It appears from this survey of electron microscope autoradiography that a significant amount of new information has been obtained relative to metabolic pathways and the dynamics of the various cells involved in the elaboration of calcified tissues. The cytodifferentiation of the osteoblasts, odontoblasts, and ameloblasts, as well as their function in the biosynthesis of organic molecules and extracellular matrix production, have been substantially clarified. The predominant role of these cells in the synthesis of collagen, enamel proteins, phosphoproteins, glycoproteins, and proteoglycans has been clearly demonstrated. Inorganic ion transfer during calcification has been studied primarily with 45Ca. During osteogenesis, dentinogenesis, and amelogenesis, it was shown that 45Ca
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ROBERT M. FRANK
followed two transfer routes from the capillaries to the mineralizing areas. The first route followed extracellular spaces exclusively, and the second route consisted of passage through the extracellular spaces followed by a transfer through the cells. Since in the osteoblast, odontoblast, and ameloblast mitochondria were the most heavily 45Ca-labeledorganelles, the absence of silver grains noted in the secretory granules of these cells should be verified. In the future many contributions can be expected from this technique, not only in the field of developmental biology but also in relation to the metabolism and the pathology of adult calcified tissues. Furthermore new knowledge will certainly be gained from the combined use of these techniques with cytochemistry, immunocytochemistry, electron diffraction, and electron microprobe analysis. Fundamental embryological issues, such as the epithelial-mesenchymal interactions in embryonic tooth formation, or the importance of the matrix theory in osteogenesis and dentinogenesis, should be examined with electron microscope autoradiography. But interesting results can also be expected from studies of physiological and pathological bone resorptions or experimentally produced diseases of the hard tissues in animal models or tissue culture. New perspectives made possible by electron microscope autoradiography, in the dynamic interpretation of physiological and pathological processes related to calcified tissues, are certainly far from being exhausted, and this technique will certainly continue to contribute meaningfully to the embryology, histology, cytology, physiology, and pathology of calcified tissues.
ACKNOWLEDGMENTS The author expresses his sincere thanks to Professor John Nalbandian for reviewing the manuscript. He also thanks Drs. P. Frank, N. Nagai, and 0. Leroy for their collaboration, as well as Mr. P. Steuer, Mrs. M. Paskov, and Mrs. C. Schaal for their skillful assistance.
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25 1
Larra, F., and Droz, B. (1970). J. Microsc. (Paris) 9 , 845-880. Larsson, A. (1973). 2. Anat. Entwicklungsgesch. 142, 103-1 15. Lasek, R. J. (1970). In?. Rev. Neurobiol. 13, 289-324. Lasek, R. J., Joseph, B. S., and Whitlock, D. G. (1968). Brain Res. 8, 319-336. Leaver, A. G., and Shuttleworth, A. (1968). Arch. Oral Biol. 13, 509-525. Leblond, C. P. (1965). Am. J. Anat. 116, 1-27. Leblond, C. P., and Warren, K. B., eds. (1965). “The Use of Radioautography in Investigating Protein Synthesis,” Symp. Int. SOC.Cell Biol., Vol. 4. Academic Press, New York. Leblond, C. P., and Weinstock, M. (1971). Biochem. Physiol. Bone, 2nd Ed. 3 , 181-200. Leblond, C. P., and Weinstock, M. (1976). Biochem. Physiol. Bone, 2nd Ed. 4 , 517-562. Lettre, H., and Paweletz, N. (1966). Naturwissenschafren 53, 268-271. Lomholt, S. (1930). J. Pharmacol. Exp. Ther. 40,235-245. Martin, J. H., and Matthews, J. L. (1969). Calcif. Tissue Res. 3 , 184-193. Matthews, I . L., and Martin, I. H. (1975). Colloq. Int. C.N.R.S. 230, 151-159. Matthews, J . L., Martin, J . H., Lynn, J. A., and Collins, E. J . (1968). Calcif. Tissue Res. 1 , 330-336. Matthews, J. L., Martin, J. H., Arsenis, C., Eisenstein, R., and Kuettner, K. (1971). In “Cellular Mechanisms for Calcium Transfer and Homeostasis” (G. Nichols, Jr. and R. H. Wasserman, eds.), pp. 239-252. Academic Press, New York. Matthews, J. L., Martin, J. H., Kennedy, J . N., 111, and Collins, E. J . (1973). Ciba Found. Symp. 11, 187-211. Manhiessen, M. E., and von Biilow, F. A. (1969). Z. Zelljorsch. Mikrosk. Anat. 101, 232-240. Matthiessen, M. E., and von Biilow, F. A. (1970). Z . Zellforsch. Mikrosk. Anat. 105, 569-578. Miller, E. J. (1973). Clin. Orthop. Relaa. Res. 92, 260-280. Moe, H. (1971). J. Anar. 108, 43-62. Moe, H., and Mikkelsen, H. (1977a). Acta Pathol. Microbiol. Scand. 8 5 , 73-88. Moe, H., and Mikkelsen, H. (1977b). Acta Pathol. Microbiol. Scand. 8 5 , 319-329. Munhoz, C. 0. G., and Leblond, C. P. (1974). Calcif. Tissue Res. 15, 221-235. Nagai, N., and Frank, R. M. (1974). Cell Tissue Res. 155, 513-523. Nagai, N., and Frank, R. M. (1975). Calcif. Tissue Res. 19, 211-221. Nagai, N., and Nagai, Y. (1977). Bull. Tokyo Dent. Coll. 18, 1-12. Neuman, W. F., and Neuman, M. D. (1958). “The Chemical Dynamics of Bone Mineral.” Chicago Univ. Press, Chicago, Illinois. Nichols, G., Jr., Hirschmann, P., and Rogers, P. (1971). In “Cellular Mechanisms for Calcium Transfer andHomeostasis”(G. Nichols, Jr. and R. H.Wasserman, eds.), pp. 211-235. Academic Press, New York. Nygren, H., Hansson, H. A., and Linde, A. (1976). Cell Tissue Res. 168, 277-287. Public Health S e w . Publ. 613. Nylen, M. U.,and Scott, D. B. (1958). U.S., Oka, N., and Shimizu, T. (1972). Jpn. J . Oral Biol. 14, 560-570. Ozawa, H., Yajima, T., and Kohayashi, S. (1972). J . Niigata Shigakai 2 , 29-42. Pany, D. M. (1976). J. Microsc. Biol. Cell. 27, 185-190. Pecher, C. (1942). Univ. Calif., Berkeley, Publ. Pharmacol. 2 , 117-139. Pelc, S. R. (1947). Nature (London) 160, 749. Pelc, S. R., Coombes, J. D., and Budd, J. C. (1961). Exp. CellRes. 24, 192-195. Prenant, A. (1924). Arch. Morphol. Gen. Exp. 19, i-95. Rambourg, A. (1967). C . R. Hebd. SLances Acad. Sci. 265, 1426-1428. Reith, E. J. (1968). In “Dentine and Pulp: Their Structure and Reactions” (N. B. B. Symons, ed.), pp. 19-41. Livingstone, London. Reith, E. J. (1970). J . Ulrrasrruct. Res. 30, 111-151. Reith, E. J. (1976). Am. J. Anat. 147, 267-272.
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Reith, E. J., and Cotty, V. F. (1962). Arch. Oral Biol. 7, 365-372. Reith, E. J., and Ross, M. H. (1973). Arch. Oral B i d . 18, 445-448. Revel, J . P., and Hay, E. D. (1963). Z . Zellforsch. Mikrosk. Anat. 61, 110-144. Rohr, H. (1965). Virchows Arch. Pathol. Anat. Physiol. 338, 342-354. Rohr, H., and Gebert, G. (1967). Beitr. Pathol. Anat. 91-116. Rolle, G. K. (1969). Calcif. Tissue Res. 3, 142-150. Ronnholm, E. (1962). J. Ulrrastruct. Res. 6, 229-248. Ross, R. (1965). Symp. Int. SOC. CellBiol. 4, 273-291. Ross, R., and Benditt, E. P. (1965). J. Cell Biol. 27, 83-106. Ruch, J . V., Karcher-Djuricic, V., and Gerber, R. (1972). Arch. Anat. Microsc. Morphol. Exp. 61, 127-138. Ruch, J. V., Fabre, M., Karcher-Djuricic, V.,and Staubli, A. (1974). Diflerentiation 2, 211-220. Ruch, J . V., Karcher-Djuricic, V., Sttiubli, A , , and Fabre, M. (1975). Arch. Anat. Microsc. Morphol. Exp. 64, 113-134. Saltpeter, M. M., and Bachmann, L. (1965). Symp. Int. SOC.Cell Biol. 4, 23-39. Sayegh, F. S., Porter, K., Sun, G., and Sellers, G. (1976). J . Dent. Res. 55, Spec. Issue B, B114 (abstr.). Scherft, J. P., and Heersche, J. N. M. (1975). Cell Tissue Res. 157, 353-365. Schulz, A., Donath, K., and Delling, G. (1974). Virchows Arch. Pathol. Anat. Histol. 364, 347356. Scott, B: L. (1967). J. Cell Biol. 35, 115-126. Scott, D. B., Nylen, M. U.,and Takuma, S. (1959). Rev. Belge Sci. Dent. 14, 329-342. Seyer, J. (1972). In “The Comparative Molecular Biology of Extracellular Matrices” (H.C. Slavkin, ed.), pp. 280-295. Academic Press, New York. Seyer, J., nd Glirncher, M. J. (1969). Biochim. Biophys. Acta 184, 509-522. Shapiro, I. M., and Greenspan, J. S. (1969). Calcif. Tissue Res. 3 , 100-102. Sisca, R. F., and Provenza, D. V. (1972). Calcif. Tissue Res. 9 , 1-16. Slavkin, H. C. (1974). “Embryonic Tooth Formation. A Tool for Developmental Biology,” Oral Sci. Rev., Vol. 4. Munksgaard, Copenhagen. Slavkin, H. C. (1975). Colloq. Int. C.N.R.S. 230, 161-177. Slavkin, H. C., Mino, W., and Bringas, P., Jr. (1976). Anat. Rec. 185, 289-312. Spooner, B. S. (1974). In “Concept of Development” (E. Lash and J. R. Whitaker, eds.), pp. 213-240. Sinauer Assoc., Stamford, Connecticut. Stack, M. V. (1954). J. Am. Dent. Assoc. 48, 297-306. Takuma, S. (1967). In “Structure and Chemical Organization of Teeth” (A. E. W. Miles, ed.), Vol. 1, pp. 325-370. Academic Press, New York. Takuma, S., and Nagai, N. (1971). Arch. Oral Biol. 16, 993-101 1. Talmage, R. V. (1970). Am. J. Anat. 129, 467-476. Tennine, J. D. (1972). CIin. Orthop. Relat. Res. 85, 207-241. Thyberg, J., and Friberg, U. (1970). J. Ulrrastruct. Res. 33, 554-573. Thyberg, J., Nilsson, S., and Friberg, U. (1975). Cell Tissue Res. 156, 273-299. Tonna, E. A. (1965). Symp. Int. SOC. Cell Biol. 4, 215-244. Warshawsky, H. (1966). Anat. Rec. (Proc.) 154, 438-439 (abstr.). Warshawsky, H. (1968). Anat. Rec. 161, 21 1-229. Warshawsky, H. (1971). Anat. Rec. 169, 559-584. Warshawsky, H., and Moore, G. (1967). J . Histochem. Cytochem. 15, 542-549. Weill, R., Bensadoun, R., and de Tourniel, F. (1975). C . R . Hebd. Siances Acad. Sci. 281, 647-650. Weinger, J. M., and Holtrop, M. E. (1974). Calcif. Tissue Res. 14, 15-29. Weinstock, A. (1970). J . Histochem. Cytochem. 18, 875-886.
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Weinstock, A. (1972a). In “Developmental Aspects of Oral Biology” (H. C. Slavkin and L. A. Bavetta, eds.), pp. 201-242. Academic Press, New York. Weinstock, A. (1972b). Biochem. Physiol. Bone, 2nd Ed. 2 , 121-154. Weinstock, A,, and Leblond, C. P. (1971). J . Cell Biol. 51, 26-51. Weinstock, A,, Weinstock, M., and Leblond, C. P. (1972). Calc(f Tissue Res. 8, 181-189. Weinstock, A., Bibb, C., Burgeson, R. E., Fessler, L. I., and Fessler, J. H. (1975). In “Extracellular Matrix Influences on Gene Expression” (H. C. Slavkin and R. C. Greulich, eds.), pp. 321330. Weinstock, M. (1972). Z . Zellforsch. Mikrosk. Anat. 129, 455-470. Weinstock, M. (1975). In “Extracellular Matrix Influences on Gene Expression” (H. C. Slavkin and R. C. Greulich, eds.), pp. 119-128. Weinstock, M., and Leblond, C. P. (1973). J . Cell Biol. 5 6 , 838-845. Weinstock, M., and Leblond, C. P. (1974). J . Cell Biol. 6 0 , 92-127. Wiesniewsky, H., Shelanski, M. L., and Terry, R. D. (1968). J. Cell Biol. 38, 224-229. Williams, J. A,, and Wolff, J. (1970). Proc. Natl. Acad. Sci. U.S.A. 67, 1901-1908. Wunderlich, F., Miiller, R., and Speth, V. (1973). Science 18, 1136-1138. Young, R. W. (1962). J . Cell Biol. 14, 357-370. Young, R. W., and Greulich, R. C. (1963). Arch. Oral Biol. 8, 509-521.
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INTERNATIONAL REVIEW OF CYTOLOGY VOL . 56
Some Aspects of Double-Stranded Hairpin Structures in Heterogeneous Nuclear RNA HIROTONAORA Molecular Biology Unit. Research School of Biological Sciences. The Australian National University. Canberra. Australia
I . Introduction . . . . . . . . . . . . . . . . . . . . I1. Occurrence of Double-Stranded RNA in “Uninfected” Eukaryotic Cells . . . . . . . . . . . . . . . . . . . . . . I11. Characteristics of Isolated dsRNA . . . . . . . . . . . . A . Resistance to RNases A and T , and Sensitivity to RNase I11 . B . Thermal and Chemical Denaturation . . . . . . . . . . C . Base Composition . . . . . . . . . . . . . . . . D . Buoyant Density in CsSO, . . . . . . . . . . . . . E . Reaction to dsRNA-Specific Antiserum . . . . . . . . . F . Interferon Induction . . . . . . . . . . . . . . . . G . Translational Inhibition . . . . . . . . . . . . . . H . Size . . . . . . . . . . . . . . . . . . . . . I . Sequence Complexity . . . . . . . . . . . . . . . J . Effect of Actinomycin D on dsRNA Synthesis . . . . . . IV . Some Comments on the HnRNA-mRNA Precursor-Product Relationship . . . . . . . . . . . . . . . . . . . . A . Size of the Putative mRNA Precursor Molecule . . . . . . B . HnRNA Sequences to Be Degraded in Nuclei . . . . . . V . Double-Stranded Hairpin Structures in HnRNA . . . . . . . A . HnRNA Origin of dsRNA . . . . . . . . . . . . . B . Double-Stranded Hairpin Structures . . . . . . . . . . C . In Vivo Existence of Structures . . . . . . . . . . . . D . Palindromic DNA Sequences from Which dsRNAs Are Transcribed . . . . . . . . . . . . . . . . . . . E . Location . . . . . . . . . . . . . . . . . . . . VI . Features of Eukaryotic mRNA . . . . . . . . . . . . . . . . . . . . . . . A . Untranslated Sequences of mRNA B . Double-Stranded Structures of mRNA . . . . . . . . . C . Presence of Sequences that Hybridize with Denatured dsRNA in the mRNA Molecule . . . . . . . . . . . . . . . D . Do mRNA Molecules Contain Sequences Transcribed from Repeated DNA Sequences? . . . . . . . . . . . . . E . Terminal Sequence Repetition in mRNA Molecules . . . . VII . A Model of Nuclear Processing of HnRNA . . . . . . . . A . A Model of DNA Sequences from Which HnRNA. dsRNA. and mRNA Are. Transcribed . . . . . . . . . . . . . . B . Support for the Model: The Double-Stranded Hairpin Structure Possesses a Processing Site for Generation of mRNA . . .
256 257 259 262 263 264 265 266 266 267 267 269 270 271 271 276 278 278 279 280 281 283 283 283 285 286 290 291 293 293 295
255 Copyright 0 1919 by Academic Press. lnc . All rights of reproduction in any form reserved. ISBN 0- 12-3643562
256
HIROTO NAORA C. Features of Double-Stranded Hairpin Structures of Bacteriophage High-Molecular-Weight RNA Revealed by RNase III . . . VIII. Occurrence of Eukaryotic RNases Specific for dsRNA . . . . IX. Summary and Concluding Remarks . . . . . . . . . . . Addendum (June 1978) . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .
297 299 303 305 306
I. Introduction A classic series of autoradiographic and biochemical experiments (Watts and Harris, 1959; Harris, 1959; Goldstein and Micou, 1959; Perry et al., 1961), together with results obtained from nuclear transplantation (Goldstein and Plaut , 1955) and enucleation experiments (Naora et al., 1960; Prescott, 1960), have revealed that some species of nuclear RNA are transported to the cytoplasm where they function in a variety of roles associated with protein synthesis. The nucleus is now known to contain the vast majority of the primary transcripts. In higher eukaryotes most of these are not identical to the functional form of cytoplasmic RNA but are subjected to a regulated breakdown or “processing” that includes phosphodiester bond cleavage, terminal addition of nucleotides, and modification of nucleosides before transport of the RNA to the cytoplasm (Darnell, 1976). Nuclear processing and/or nuclear-cytoplasmic transport of RNA in a given cell vary with changes in the physiological and pathological conditions of the cell (for example, Church and McCarthy, 1970) and in its environment (Schumm and Webb, 1975). Some FWA molecules restricted to the nucleus in normal cells are transported to the cytoplasm in regenerating liver (Church and McCarthy, 1967) and in tumor cells (Drews et al., 1968; Shearer and Smuckler, 1972; Garrett et al., 1973; Shearer and Mayer, 1974). Some tumor cells are unable to process rapidly at least part of the large heterogeneous nuclear RNA (HnRNA) molecules and retain unprocessed RNA molecules (Torelli et al., 1976). These observations clearly indicate that nuclear processing and/or resulting transport of RNA to the cytoplasm may be closely involved in regulatory mechanisms affecting cell function. At least a certain fraction of HnRNA is now thought to be a precursor of cytoplasmic mRNA, although uncertainty still exists. Most if not all HnRNA molecules have stable hairpin structures which include intramolecular doublestranded regions (Section V). In recent years, the question has been raised as to whether double-stranded hairpin structures are involved in the mechanism by which mRNA is processed in the nucleus and gene expression is subsequently regulated.
257
HAIRPIN STRUCTURES IN HnRNA
The purpose of this article is to review findings regarding the double-stranded hairpin structure in the HnRNA molecule and its possible involvement in nuclear processing into mRNA. This subject is an area of research that has been developed recently and is at present undergoing a period of intense investigation. Emphasis is given to the model proposed for the double-stranded hairpin structures relevant to a precursor-product relationship between HnRNA and mRNA. In this article, we present only a brief discussion of the problems arising from recent studies on the possible HnRNA-mRNA relationship and concentrate more on the published and unpublished results regarding double-stranded structures of HnRNA molecules. Several review articles have recently been published on the general problems of the HnRNA-mRNA relationship (Weinberg, 1973; Choi et al., 1974 Georgiev, 1974; Lewin, 1974, 1975a,b; Darnell, 1975; Molloy and Puckett, 1976; Naora, 1977) and the processing of RNA (Robertson and Dickson, 1975; Perry, 1976). Since these general matters are not the major subject of this article, the reader should refer to the above reviews and the published papers of the 29th Annual Symposium of the Biology Division of Oak Ridge National Laboratory (Progress in Nucleic Acid Research and Molecular Biology, Vol. 19, 1976) for a detailed survey.
11. Occurrence of Double-Stranded RNA in “Uninfected” Eukaryotic Cells Historically, studies on the occurrence of double-stranded RNA (dsRNA) relevant to this article originated with the discovery by Montagnier and Sanders (1963) of a double-stranded form of RNA in cells infected with encephalomyocarditisvirus, and subsequently by Montagnier (1968) of dsRNA in normal and cancerous cells. Since the work of Montagnier and Sanders in 1963, the double-stranded basepaired replicative form of RNA has been found in various groups of viruses, both in virus-infected cells and as an in vitro product of virus-induced enzymes (Bishop and Levintow, 1971). Research into the replication of virus-infected cells led to the unexpected finding of dsRNA in the uninfected control cells. Montagnier (1968) and Hare1 and Montagnier (1971) in fact reported that RNase-resistant dsRNAs were certainly present in various types of uninfected cells. The content of dsRNA in these cells was approximately 0.1-1.0% of the C content total RNA. It was noted that these dsRNA possessed a low G (41-43% G + C), and the dose of actinomycin D which inhibits rRNA synthesis did not suppress the synthesis of dsRNA, indicating a non-rRNA origin. Their preparations were not highly purified, but the double-strandedness of the RiqA was confirmed by the temperature-dependentRNase sensitivity and susceptibility at low ionic strength. Furthermore, the dsRNA fraction isolated from rat liver
+
258
HIROTO NAORA
cells was capable of more than 29% specific hybridization with rat liver DNA, suggesting it to be transcribed from the rat liver DNA (Harel and Montagnier, 1971).
There is now ample evidence c o d i n g the presence of dsRNA in a variety of uninfected normal animal cells, in rapidly growing cells as well as those not undergoing DNA synthesis and mitosis (Stem and Friedman, 1970,1971). These include sea urchin embryos (Kronenberg and Humphreys, 1972), Xenopus eggs (Chung, 1977), chick cells, rat liver, testis and kidney cells (Montagnier, 1968; Colby and Duesberg, 1969; Kimball and Duesberg, 1971; Monckton, 1974; Monckton and Naora, 1974), HeLa and other types of tissue-cultured cells (Montagnier, 1968; Stollar and Stollar, 1970; Jelinek and Darnell, 1972; Patnaik and Taylor, 1973; Jelinek et al., 1974; Calvet and Pederson, 1977), and various types of cancer cells (Montagnier, 1968; Patnaik and Taylor, 1973; Georgiev et al., 1973; Ryskov et al., 1976a; Mhsson et al., 1975; Naora, 1977). However, dsRNA has not been reported to occur in uninfected plant and lower eukaryotic cells. It should be mentioned here that the dsRNA preparations of the earlier experiments were mainly RNase-resistant fractions and were not highly purified. Therefore the preparations contained small fragments of single-stranded RNA (ssRNA), for example, poly-A segments (Monckton and Naora, 1973; Robertson and Hunter, 1975; Mhsson et al., 1975). Because of the general occurrence of dsRNA in normal uninfected cells of diverse genera, in cells of both normal and cancerous tissues under various physiological conditions, this form of RNA has evoked considerable interest. dsRNA must be involved in fundamental cellular events. The questions we immediately ask are concerned with the origin and functions of dsRNA of the normal cell. Here we refer to the possibilities in relation to these questions pointed out earlier by Montagnier and his colleague when they first found dsRNA in uninfected cells (Montagnier, 1968; Harel and Montagnier, 1971). These were: 1. All cells examined are latently infected with RNA viruses; therefore dsRNA is of viral origin. 2. Some cellular RNA species undergo self-replication. Thus dsRNA is a replicative form of cellular RNA. 3. Both strands of cellular DNA are transcribed at some sites, or complementary sequences are present on the same DNA strand.
Although the first possibility-latent viral origin4annot yet be completely ruled out, it seems to be unlikely in most cases for the following reason: As mentioned above, dsRNA can be isolated from varied and diverse sources. Furthermore, the fact that there are nucleotide sequences in the dsRNA molecule homologous to cell DNA, HnRNA, and mRNA (Sections V and VI) renders the
HAIRPIN STRUCTURES IN HnRNA
259
possibility unlikely. Recently, many of the nucleotide sequences in the dsRNA molecule have been found to represent transcripts of moderately repeated DNA sequences (Section 111, I). This observation further suggests that most dsRNA is not of viral origin. However, this does not imply that none of it is derived from the double-stranded forms of viral RNA. It is also possible that dsRNA does not originate from a single source but has a complex origin (Patnaik and Taylor, 1973). Although a possibility exists that some RNA might be self-replicated within the cell, there is as yet no compelling evidence either for or against this notion. There is no reason why some dsRNA should not be involved in the mechanism by which cellular RNA is self-replicated. In regard to the third possibility, there is evidence indicating that some dsRNA, if not all, is transcribed from “complementary sequences present on the same DNA strand. A particular form of complementary sequence on the same DNA strand is now known as a palindrome in chromosomes (Wilson and Thomas, 1974; Perlman et al., 1976; also Section V, D). The main topic of this article is concerned with the possibility that dsRNA is the transcript of palindromic DNA sequences from which the portion of mRNA precursor sequences is transcribed. It is remarkable that this possibility was pointed out in the first experiments carried out by Montagnier and his colleague nearly 10 years ago (Montagnier, 1968; Harel and Montagnier, 1971). There is a considerable body of evidence demonstrating that dsRNA is predominantly of nuclear origin in the wide taxonomic ranges of eukaryotic cells, that is, from evolutionarily less advanced eukaryotes (Kronenberg and Humphreys, 1972) to highly advanced organisms (Jelinek and Darnell, 1972; Patnaik and Taylor, 1973; Bases and Kaplan, 1973; Monckton and Naora, 1974; M h s son et al., 1975). Most if not all nuclear dsRNA is now known to be derived from HnRNA (Section V, A). However, some dsRNA is associated with mitochondria and hybridizes with mitochondrial DNA, indicating mitochondrial origin (Harel et al., 1975; Young and Attardi, 1975). This is not surprising since, as discussed in Section VII, A, dsRNA is probably involved in the processing mechanism of primary transcripts and mitochondria produce their own primary transcripts (Hendler et al., 1976; Kroon et al., 1976). Of course, this does not exclude the possibility that mitochondrial dsRNA has a special function other than the processing of primary transcripts. ”
111. Characteristics of Isolated dsRNA There are many reports on the characterization of dsRNA preparations isolated from uninfected animal cells. Most of them concern the double-strandedness of the preparations. The basic observations are summarized in Table I.
TABLE I.
References
Source
Resistance to RNases A and TI
+
Montagnier, 1968; De Maeyer et al. 1971; Hare1 and Montagnier, 1971
Rat liver, hamster cells
Stollar and Stollar, 1970
BHK-21 cells
Stem and Friedman, 1970, 1971
Burkitt's lymphoma, Human lymphocytes, chicken embryo fibroblasts
Kimball and Duesberg, 197 1
Rabbit kidney, chicken embryo fibroblasts, HeLa cells
Kronenberg and Humphreys, 1972
Sea urchin embryos
+
Jelinek and Damell, 1972 Robertson et al., 1977b
HeLa cell nuclei
+
Ryskovet al., 1973, 1976a,b; Kramerov et a[., 1977
Ehrlich carcinoma cells, rabbit bone marrow
Patnaik and Taylor, 1973
Ehrlich carcinoma cells, sarcoma-180 cells, L cells
+
Bases and Kaplan, 1973
HeLa cell nuclei
+
Monckton and Naora, 1974; Fry et al., 1978
Rat liver whole cells or nuclei Hen reticulocyte nuclei
+ +
PHYSICAL A N D CHEMICAL
Heat denaturation," T, 89°C (1 X SSC), 75°C (0.1 x SSC)
+ 67°C (1
+
86°C (0.01 M NaCI)
260
66) 29.5-43; NU = 2.11-4.64:l; G/C = 1.01-1.14:1
-
+ +
+
Base composition, G + C content
X SSC) -
90°C (1 X SSC), 76°C (0.1 X SSC)
-
Chicken embryo: 54.7; N U = 1.06:l; G/C = 1.28:l
29.5-41; N U = 2 . 1 1 4 . M l ; (includes poly A), G/C = 1.011.14:1 57.3; N U = 1.oO:I; G/C = 1.oO:l
Heat-denaturable, reassociation after denaturation
Short, 74; medium, 48-52; N U = 1.00-1.09:1; G/C 0.92-1.OO:l
49.5-76.5"C (0.0I x SSC) (gradual melting) -
74°C (0.1 X SSC), 94°C (2 X SSC) Reassociation after denaturation
43; NU = 0.98:1; G/C = 0.97:1
-
PROPERTIES OF ISOLATED dsRNA
Sizeb
Density in csso,
4-12s
-
12s
-
Chromatographic characteristicsc Cellulose
Special property Can induce interferon
-
Positive to dsRNAspecific antiserum
>4S
BD-cellulose
-
4 s and >4S
BD-cellulose
Can induce interferon
-
Can induce interferon
4s and
7s (rabbit kidney), 9s (chicken embryo), 11s (HeLa cells)
1.61-1.63 g d m l
-
4-6s
- 1.60 g d m l (from
-1OON
figures)
Short, 20-30N; medium, 100200N (5-6s); long, 300-SOON (7-1 1s);rabbit, SO- 150N
Medium and long: 1.61 g d m l
Cellulose
HAP, cellulose
Specific fingerprint pattern, sensitive to RNase Ill
HAP
4-13s
- 70N -
Rat liver, 60N (4-5s); Hen, 120N, WN, 65N, SON
Cellulose
Inhibitory to protein synthesis, dissociates at low salt
Cellulose
Positive to dsRNAspecific antiserum Contains nucleotide sequences corresponding to those of the ribosomebinding site of a globin mRNA
Cellulose
(coniinued)
26 1
262
HIROTO NAORA TABLE I
References
Source
Torelli er al., 1975
Human leukemic blast cells
Mbsson et al., 1975
Human lymphocytes, chronic lymphocytic leukemia
Pays, 1976
In vitro synthesis on rat liver chromatin and DNA
Resistance to RNases A and T I
+ +
Base composition, G C content
Tm
(%)
Heat-denaturable
-
+
-
+
+
Calvet and Pederson, HeLa cell, HnFUVP 1977; Kish and Pederson, 1977
Heat denaturation,"
93°C (0.3 M salt)
60.3-65.4
Reassociation after denaturation
Oligo(U) .poly(A) duplex
SSC,0.15 M Na chloride and 0.015 M Na citrate. bN, nucleotide.
A. RESISTANCE TO RNASES A
AND
TI
AND
SENSITIVITY TO RNASE 111
The resistance to RNases A and TI in 0.1 M or higher salt concentrations is one of the most important properties of dsRNA. This property has been used to characterize the double-stranded nature of RNA, but the RNase resistance of dsRNA is far from absolute (Loviny and Szekely, 1973; Edy et al., 1976). For example, dsRNA is resistant to RNase A when it is in solution at (1) a high salt concentration (higher than approximately 0.15 M),(2) a low enzyme concentration, and (3) an enzyme/RNA ratio of 1:lOO to 1:lO. However, at a high enzyme/RNA ratio (e.g., 1:l) or at a low salt concentration (lower than 0.1 M), dsRNA is susceptible to RNase-A digestion. Therefore a low enzyme/RNA ratio (lower than 1:lO) should be adopted for the treatment with RNase. Such low ratios have been used for the preparation of dsRNA from rat liver or mouse ascites tumor cells (Monckton and Naora, 1974; Ryskov et al., 1976a). In spite of the importance of enzyme/RNA ratios, these values have not been mentioned in most reports. Concentrations of RNase A for the preparation of dsRNA range from 2 p g/ml (Jelinek and Darnell, 1972) to 50pg/ml (Ryskov et al., 1973; Kramerov et al.,
263
HAIRPIN STRUCTURES IN HnRNA (continued)
Density in Sizeb
csso 4
Chromatographic characteristicsr
-
Cellulose
-
HAP
Special ProPe*Y Inhibitory to protein synthesis
-
20-30N some are IOON
1.62 g d m l
-
Sensitive to RNase I11
20-50N
1.610 g d m l
-
Sensitive to RNase
-
Sensitive to RNase I11
(reovirus RNA, 1.610 g d m l ) ;
oligo(U)*, pOlY(A)mn, 1.605 gm/d
rCellulose,BD-cellulose, or HAP means that the chromatographic pattern of the preparation is similar to that of synthetic or natural (viral or phage) dsRNA on cellulose, BD-celluIose, or hydroxyapatite, respectively.
1977) in high salt, but in general a concentration of 10-20 pg/ml is used. In contrast to RNase A, RNase TI is unable to destroy the double-stranded structures and can cleave only ssRNA under various conditions (Edy et al., 1976). Escherichia coli RNase 111, which digests dsRNA (Section VIII), is now widely used to investigate the double-strandedness of polyribonucleotides (Robertson and Hunter, 1975). RNase 111destroys most of the material resistant to RNases A and TI (Pays, 1976; Calvet and Pederson, 1977), supporting the idea that the RNase-resistant material from nuclear RNA is double-stranded.
B. THERMAL AND CHEMICAL DENATURATION Most of the dsRNA preparations isolated so far have been denaturable, indicating a base-paired double-stranded character. However, a significant portion of some dsRNA preparations remains resistant to RNases after thermal or chemical denaturation. For example, dsRNA from Burkitt lymphoma cells has a sharp transition at about 70°C, but only 60% of the material becomes sensitive to RNases following thermal denaturation (Stem and Friedman, 1971). A sharp,
264
HIROTO NAORA
E
+
90
R a t Liver (Monckton and Naora. corrected A for 01 conrenfi-
Seo Urchin (corrected for poly-A content) A Rot Liver(Montognier) 1 In vitro Products
4.
v
I
0.4
0.5
0.6
G + C Content
FIG. 1. The relationship between the heat denaturation temperature (T,) and the G + C content of various dsRNA. Values for T , and the G + C content of viral and phage dsRNAs are from Van Griensven et al. (1973). T , values were determined in 0.15 M NaCl by RNase resistance. A correction was made for the values obtained in different salt concentrations (Wetnur and Davidson, 1968; Casy and Davidson, 1977). Types of dsRNA are: TYMV, Turnip yellow mosaic virus; MS2, bacteriophage MS2; Newcastle, Newcastle disease virus; AMV, alfalfa mosaic virus; TMV, tobacco mosaic virus; rat liver: prepared by Montagnier (1968) and by Monckton and Naora (1974); sea urchin: prepared by Kronenberg and Humphreys (1972); in vitro products: prepared by Pays (1976).
almost complete transition is observed with other dsRNA preparations (Montagnier, 1968; Kimball and Duesberg, 197 1; Kronenberg and Humphreys, 1972; Monckton and Naora, 1974). Impurities in the preparation may in part account for the incomplete digestion by RNases after denaturation. The T , values for rat liver and sea urchin dsRNAs are 89"-9OoC in 1x SSC and 94" in 2~ SSC and are slightly lower than those expected from the G C content (Montagnier, 1968; Kronenberg and Humphreys, 1972; Monckton and Naora, 1974). dsRNA synthesized in vitro has significantly low T , values, compared with its G C content (Pays, 1976; see Fig. 1 and Van Griensven et al., 1973). This discrepancy may be partly accounted for by the shortness of the dsRNA and/or by the presence of impurities in the preparations.
+
+
C. BASECOMPOSITION
The base compositions of the dsRNA reported so far differ markedly (see Table I). In many cases, erroneous estimations may have been caused by insuffi-
265
HAIRPIN STRUCTURES IN HnRNA
cient purification. Most dsRNA is characterized by a low G + C (41-57%) content. These values are similar to those for HnRNA (4346% G C), cytoplasmic total mRNA (43-46% G C), and cellular DNA (4247% G C) for HeLa cells (see Naora, 1977). No modified nucleotide residue has been found in dsRNA, although this possibility has been suggested (Robertson et al., 1977b; Jelinek, 1977). It has been noted that some dsRNA is characterized by a high adenylic acid content; the N U ratio is much higher than 1. However, the G/C ratios of most dsRNA preparations are nearly equal to 1, showing the property of a base-paired structure. The high adenylic acid content is mainly due to the coexistence of poly-A segments in the dsRNA preparations, since poly-A segments of mRNA and HnRNA are resistant to RNases A and TI and are cofractionated on cellulose or hydroxyapatite columns with dsRNA (Kronenberg and Humphreys, 1972; Monckton and Naora, 1974; Robertson and Hunter, 1975; Mhsson et al., 1975). Poly-A sequences of HnRNA appear to be base-paired in the nucleus with oligo-U sequences (approximately 30 nucleotides long) (Molloy and Puckett, 1976; Edmonds et al., 1976) present in the HnRNA molecule and can be isolated as an oligo(U)*poly(A) duplex after RNase treatment. It is proposed that the base-paired oligo(U). poly(A) structure has an important role in the organization of HnRNA (Kish and Pederson, 1977; Dubroff, 1977). Some poly-A segments are probably single-stranded, but others occur as partially or fully paired oligo(U)* poly(A) duplexes. Approximately 13% of purified rat liver dsRNA is a poly-A segment (Monckton and Naora, 1974), while hen reticulocyte dsRNA preparations contain poly-A segments of only 2% (Fry et al., 1978).
+
+
+
D. BUOYANT DENSITY IN CsSO, A double-stranded form of viral and phage RNA has a characteristic buoyant density of 1.60-1.61 g d m l in a CsSO, gradient (Van Griensven et al., 1973). All the dsRNA preparations tested so far band at a density of 1.60-1.63 g d m l (Kimball and Duesberg, 1971; Jelinek and Damell, 1972; Jelinek et al., 1974; Pays, 1976; Kramerov et al., 1977; Calvet and Pederson, 1977). A short length of dsRNA causes the band in CsSO, to broaden (Jelinek et al., 1974; Pays, 1976). The above values are in good agreement with those for viral and phage dsRNA but are significantly different from those for ssRNA and DNA-RNA hybrids, for example, 1.68 g d m l for single-stranded tobacco mosaic virus RNA (Kimball and Duesberg, 1971), 1.65 g d m l for RNase-untreated bulk RNA (Pays, 1976), and 1.49-1.54 g d m l for DNA-RNA hybrids (Szybalski, 1968). This observation gives yet another indication that the RNAs are doublestranded.
266
HIROTO NAORA
E. REACTION TO DsRNA-SPECIFIC ANTISERUM A quantitative complement fixation assay, which specifically measures dsRNA, has been used to examine dsRNA from tissue-cultured (BHK 21) cells. Approximately 0.01% of the total RNA of these cells is double-stranded (Stollar and Stollar, 1970). However, the value obtained by this technique is markedly lower than those estimated by other methods (Section LI). dsRNAs from rat liver and testis give definite immunoprecipitationlines with an antiserum that reacts only with synthetic and natural dsRNA and not with dsDNA or ssRNA (Monckton and Naora, 1974). Since heat-denatured dsRNA does not react with the antiserum at all, the RNA is double-stranded.
F. INTERFERON INDUCTION There is a considerable body of evidence indicating that synthetic or natural dsRNA is an active component in the induction of interferon (Nagano, 1975). This raises the questions (1) whether dsRNA isolated from various sources of uninfected cells has antiviral activity, and (2) if so, whether homologous dsRNA can induce interferon in a given species of cells. Examination of dsRNA isolated from normal cells (rabbit kidney, mouse and rat liver cells, and human lymphocytes) and cancerous cells (Burkitt lymphoma and HeLa cells) has clearly demonstrated that these dsRNAs are quite capable of inducing interferon in cells of the same species of animal, as well as in those of other species of animals. For example, dsRNAs from rabbit kidneys and chicken embryos interfere with virus growth in rabbit kidney and chicken embryo cultures (Kimball and Duesberg, 1971). It has also been shown that antiviral activity by the dsRNA being tested is mediated by interferon (De Maeyer et al., 1971). A few lines of evidence indicate that the antiviral activity is indeed due to dsRNA used for the assay: (1) The ability to induce interference is completely lost when dsRNA is completely denatured (De Maeyer et al., 1971; Kimball and Duesberg, 1971). (2) The inducer and RNA band together in a CsSO, density gradient at a density of 1.61-1.63 g d m l (Kimball and Duesberg, 1971). (3) The RNA purified on a BD-cellulose column and further proved to be double-stranded has antiviral activity (Stem and Friedman, 1971). (4) The antiviral component and dsRNA have the same distinctive solubilities in salt solutions of different concentrations (Kimball and Duesberg , 1971). These dsRNAs are as potent or three times as potent as the synthetic polymer poly(1). poly(C) in the induction of interferon (De Maeyer et al., 1971; Stern and Friedman, 1971). The finding that the cells of a given animal possess dsRNA which induces interferon in other cells of the same species of animal raises the question of why its own dsRNA does not cause detectable interference in virus growth. In view of
HAIRPIN STRUCTURES IN HnRNA
267
the minimum level p g per chicken cell) of poly(I).poly(C) needed to cause interference (Colby and Chamberlin, 1969), the total amount of cellular dsRNA may be too low (Kimball and Duesberg, 1971). Most isolated dsRNA is derived from double-stranded hairpin structures of HnRNA (Section V) which are'complexed with specific proteins in the nucleus. It is also possible therefore that the activity that induces interferon is completely inhibited by complexing with proteins and/or by physical separation in the nucleus.
G. TRANSLATIONAL INHIBITION Synthetic [poly(I)-poly(C)], viral, and phage dsRNAs are extremely potent inhibitors of protein synthesis, mainly in reticulocyte lysates (Ehrenfeld and Hunt, 1971; Hunter et al., 1975; Robertson and Hunter, 1975; Emst et al., 1976) but not in plant cell-free systems (Grill et al., 1976; Naora et al., 1978). The addition of low levels (nanograms to micrograms per milliliter) of synthetic, viral, or phage dsRNA to reticulocyte lysates containing optimal concentrations of hemin results in an abrupt decline in protein synthesis after the synthesis has proceeded at an initial rate for several minutes. Inhibition occurs at the level of protein chain initiation, particularly in the formation of a 40s Met-tRN& complex (Beuzard and London, 1974; Clemens et al., 1975). This inhibition involves the phosphorylation by protein kinases of the Met-tRNA f binding factor (eIF-2) (Emst et al., 1976; Delaunay et al., 1977; Farrell et al., 1977). dsRNA isolated from HeLa cells and human leukemic blast cells inhibits globin synthesis in rabbit reticulocyte lysates under optimal conditions in a manner similar to that reported for viral or phage dsRNAs (Bases and Kaplan, 1973; Torelli et al., 1975). The inhibition is biphasic; a 4-minute lag period is observed before inhibition is detected. Inhibition can be induced by low levels (less than microgram amounts) of dsRNA samples (Bases and Kaplan, 1973; Torelli et al., 1975), and denaturation of dsRNA certainly abolishes the inhibitory activity (Bases and Kaplan, 1973). Once again, these results demonstrate that the material isolated from mammalian cells is as double-stranded as phage or viral dsRNA. The molecular mechanisms of inhibition by these mammalian dsRNAs have not yet been examined. However, since they have an inhibitory effect similar to that observed with viral and phage dsRNAs, this inhibition probably occurs through similar mechanisms.
dsRNA isolated from various sources of uninfected cells varies considerably in size. The variation may be partially due to the different sources and different isolation procedures.
268
HIROTO NAORA
Preparations of dsRNA may be roughly classified into three sizes: (1) short, smaller than 4s; (2) medium, 4-6s; and (3) long, 7-13s or longer. Mediumsized dsRNA is most commonly isolated and is derived from double-stranded hairpin structures of HnRNA (Section V). Even the dsRNA synthesized in vitro on DNA or chromatin includes some consisting of approximately 1 0 0 nucleotide pairs (Pays, 1976). Most dsRNA migrates on polyacrylamide gel at a rate similar to or slightly slower than that of 4 s RNA. It also sediments broadly at 4-6s upon centrifugation in sucrose gradient and is excluded in the void volume when cochromatographedwith tRNA on a Sephadex G-75 or G-100 column (Stem and Friedman, 1970; Ryskov et al., 1973; Monckton and Naora, 1974). The size of this type of dsRNA ranges from approximately 60 to 200 nucleotide pairs. For example, hen reticulocyte dsRNA shows at least six discrete peaks on polyacrylamide gel, four peaks of which migrate at a slower rate than 4s RNA (Fig. 2). The main peak of hen reticulocyte dsRNA (120 nucleotide pairs, estimated by electron microscopy) is 35-43% of the total dsRNA (Fry et al., 1978). Short dsRNA (20-30 nucleotide pairs) is also isolated from hen reticulocytes (Fry et al., 1978) and mouse carcinoma cells (Ryskov et al., 1973). Carcinoma short dsRNA is G + C-rich and seems to be different from longer dsRNA (Ryskov et al., 1973). The peaks of short dsRNA on polyacrylamide gel are broad, and the relative contents are variable in hen reticulocyte dsRNA (Fry et al., 1978). Whether some of the short dsRNA represents degradation products of longer dsRNA remains unclear at present. Short dsRNA also includes the (No. of nucleotide pairs)
120
2
90
6
4
Migration FIG. 2.
65 50
8
(cm)
Electrophoretic analysis of dsRNA from hen reticulocytes. RNA was analyzed on 10% polyacrylamide gels in a 30 mM phosphate buffer (pH 7.0). was monitored in a Joyce Loebl Scan 400. The lengths of fractionated dsRNA are expressed as the number of nucleotide pairs and are shown at the top of the figure. The length of dsRNA in the main peak was determined by electron microscopy (Fry and Naora, 1977), while the sizes of the other dsRNA fractions were calculated from their mobility relative to that of the main peak. (From Fry et al., 1978.)
HAIRPIN STRUCTURES IN HnRNA
269
oligo(U) * poly(A) duplex (approximately 20 to 50 nucleotide pairs) isolated from HnRNA (Section 111, C). In several cases, isolated dsRNA sediments at 7- 13s (and as high as 20s in some cases), which corresponds to about 500 (Jelinek and Darnell, 1972) to 2000 nucleotide pairs. There have been doubts regarding long dsRNA. For example, Kimball and Duesberg (1971) isolated long dsRNA, approximately 1l S , from HeLa cells, whereas Jelinek and Darnell (1972) isolated much smaller RNA from the same type of cells. Kramerov et al. (1977) isolated a significant amount of long dsRNA (300 to 800 nucleotide pairs) only by the hot phenol fractionation procedure (Georgiev et al., 1972) and not by other RNA extraction procedures (Kimball and Kuesberg, 1971; Scherrer and Darnell, 1962; Glisin et al., 1974). Apparently, this is neither an artifactual product which occurs during isolation nor a differential extraction of RNA (Kramerov et al., 1977). However, the fact that the intactness and purity of RNA extracted by these different procedures probably vary should be considered a reason for this discrepancy. Some long dsRNA may not be derived from double-stranded hairpin structures of HnRNA (Kramerov et al., 1977). It has been suggested that long dsRNA (approximately 20s) from mouse sarcoma-180 cells is probably of viral origin, since it has characteristics different from those of other dsRNA and is more similar to viral dsRNA (Patnaik and Taylor, 1973). This does not necessarily imply that all long dsRNA isolated from different sources is of viral origin. Some long dsRNA from Ehrlich carcinoma cells hybridizes with moderately repeated mouse DNA sequences (Kramerov er al., 1977). More information is required to determine the significance of long dsRNA.
I. SEQUENCE COMPLEXITY Evidence available to date indicates that approximately 20-60% of denatured dsRNA hybridizes rapidly with DNA under conditions where only RNA transcripts of repeated DNA sequences would be expected to hybridize (Harel and Montagnier, 1971; Jelinek and Darnell, 1972; Ryskov et at., 1973; Jelinek et al., 1974; Harel et al., 1975; Kramerov et al., 1977). For example, short (20 to 30 nucleotide pairs) and medium (100 to 200 nucleotide pairs) dsRNAs from Ehrlich carcinoma cells hybridize at the Cot, value of 1-2, and long (300 to 800 nucleotide pairs) dsRNA hybridizes at the Cot+ value of 12 (Kramerov et aE., 1977). This result clearly shows that many but not all dsRNA sequences are transcribed from moderately repeated DNA sequences. It should be pointed out here that in all cases the hybridization of denatured dsRNA does not exceed 60% at relatively low Cot values. Such poor hybridization at low Cot values may be due to technical difficulties, for example, self-hybridization, but probably is due to the presence of sequences transcribed from less repeated DNA sequences.
270
HIROTO NAORA
Indeed, hybridization certainly increases at high Cot values. However, the interpretation of the results at high Cot values involves a technically more complex situation (Harel et al., 1975) and must await further rigorous experiments. When denatured bulk dsRNA is annealed, a significant portion of material renatures rapidly at very low Cot values. For example, renaturation of medium dsRNA (100 to 200 nucleotide pairs) from Ehrlich carcinoma cells takes place at (Ryskov et al., 1976a; Kramerov et the Cott value of approximately 5 x al., 1977), corresponding in complexity to about 200 nucleotide pairs (Britten and Kohne, 1968). The complexity of HeLa cell dsRNA is also less than 500 nucleotide pairs (Robertson et al., 1977b). Therefore, since the medium-sized dsRNA is 100-200 nucleotide pairs long, it represents a more-or-less homogeneous population, and only one or perhaps a few kinds of sequence are present in this RNA. However, the complexity of long dsRNA (300 to 800 nucleotide pairs) is about 6000 to 30,000 nucleotides (Kramerov et al., 1977). Therefore there must be approximately 8 to 100 different kinds of these dsRNAs. Since there is no significant cross-reassociation between denatured long and medium dsRNA, each type must contain its own nucleotide sequences, which differ from each other (Kramerov et al., 1977). Once again, incomplete (60% or less) renaturation of denatured dsRNA takes place at low Cot values, and therefore some parts of dsRNA must renature at much higher Cot values (Ryskov et al., 1976a; Kramerov et al., 1977). Sequences involved in dsRNA are species-specific (Harel and Montagnier, 1971; Ryskov et al., 1973; Harel et al., 1975), but some of the dsRNAs isolated from a few tissues of a given species of animal, for example, mouse, are represented by similar nucleotide sequences (Ryskov et al., 1973). However, renaturation curves of denatured dsRNA differ markedly from one tissue to another (Ryskov et al., 1976a).
J. EFFECTOF ACTINOMYCIN D ON DsRNA SYNTHESIS
Low doses of actinomycin D, which halt rRNA synthesis without a significant effect on the synthesis of HnRNA and mRNA (Penman et al., 1968; Naora and Kodaira, 1969; Perry and Kelley, 1970; Kersten and Kersten, 1974), do not inhibit dsRNA synthesis, suggesting that dsRNA is not derived from rRNA or rRNA precursor molecules (Jelinek and Darnell, 1972; Patnaik and Taylor, 1973; Monckton and Naora, 1974; Harel et al., 1975). When total RNA, including both HnFWA and mRNA, synthesis is reduced by over 90% by high doses (5-10 g/ml for tissue-cultured cells) of actinomycin D, the synthesis of dsRNA is also inhibited (Kimball and Duesberg, 1971; Patnaik and Taylor, 1973). However, some dsRNA synthesis still continues, although the total amount of dsRNA synthesized in the presence of high doses of actinomycin D decreases
HAIRPIN STRUCTURES IN HnRNA
27 1
(Montagnier, 1968; Stem and Friedman, 1970, 1971; Kimball and Duesberg, 1971; Patnaik and Taylor, 1973). Up to 68% of RNA synthesized with high doses of actinomycin D is RNase-resistant (Stem and Friedman, 1970, 1971; Kimball and Duesberg, 1971). Therefore when the dsRNA content of cells exposed to high doses of the drug is measured, the relative percentage remains the same or is increased (Montagnier, 1968; Patnaik and Taylor, 1973; Hare1 ef al., 1975). The differential sensitivity of dsRNA synthesis to actinomycin D suggests that some dsRNA is synthesized on a DNA template in a manner similar to HnRNA. It is not known whether the RNase-resistant RNA synthesized in the presence of high doses of actinomycin D is identical to that found in the absence of the drug or whether it is a product of RNA-dependent RNA synthesis.
IV. Some Comments on the HnRNA-mRNA Precursor-Product Relationship In eukaryotes, where the transcription of genes and translation of mRNA are physically separated by the nuclear membrane, one of the central questions concerns how the primary gene products are processed in the nucleus and transported to the cytoplasmic translational machinery. This question leads us to ask whether HnRNA is a precursor of cytoplasmic mRNA. There is considerable evidence to suggest that some but not all HnRNAs are precursors of mRNA. It should be noted, however, that there is still no conclusive evidence for or against this possibility. In this article, we discuss only some of the problems arising from recent studies on this relationship.
A. SIZEOF
THE
PUTATIVE MRNA PRECURSOR MOLECULE
The results on the size of HnRNA or putative mRNA precursor molecules are most confusing and controversial (see Table 11). The tendency of RNA molecules to aggregate (Bramwell, 1972; Macnaughton et al., 1974; Getz et aE., 1975) makes it difficult to interpret many experiments with HnRNA. Applying mild denaturing conditions to HnRNA results in an incomplete disaggregation of RNA molecules, while strong denaturing conditions may introduce breaks in the HnRNA molecules or expose nicks (introduced in vivo or during isolation) in double-stranded hairpin structures, thereby causing fragmentation of the molecule. Thus it is not easy to fractionate intact large HnRNA and to determine its correct size. The actual size of HnRNA also depends upon how closely processing events follow transcription and the relative rates of both processes (Perry et al., 1976; Lizardi, 1976; McKnight et al., 1976). Small HnRNA that
TABLE I1 SIZESOF HnRNA CONTAINING SPECIFICMESSAGE SEQUENCES Cytoplasmic mRNA Organism or cell
Labeling period (minutes)
Fractionation"
Detection procedure"
Specific sequence
N
4
Size of HnRNA
Size
Reference
Hen oviducts
-
6 5 T , 10 minutes, SDSsucrose gradient
cDNA, RNA excess
Ovalbumin
16s
- 16s
Duck erythroblasts
-
Aqueous gel
cDNA, RNA excess
Globin
9s
<45S (- 14S, 28s)
Spohr et al., 1974
-
Foramide-sucrose gradient
cDNA, RNA excess
Globin
9s
14s
Mcnaughton et al., 1974
Hen immature red blood cells
-
Formamide gel
cDNA, RNA excess
Globin
9s
9s
Knochel and Grundmann, 1977
Rooster erythroblasts
-
DMSO-sucrose gradient
cDNA, RNA excess
Globin
9s
Not >28S (approx. 9- 15s)
Williamson and Tobin, 1977
Chicken 1 I-day embryonic erythroblasts
-
DMSO-sucrose gradient
cDNA, RNA excess
Globin
9s
Not >28S (approx. 9-15s)
Williamson and Tobin, 1977
McKnight and Schimke, 1974; Woo et al., 1975
Formamide-sucrose gradient
cDNA excess
Globin
10s
14s
Ross, 1976
Formaldehyde-treated RNA, 6 3 T , 15 minutes
cDNA excess
Globin
10s
15s
Kwan et al., 1977
5-60
100°C, 45 seconds, SDS-sucrose gradient
Globin DNA excess
a Globin p Globin
9.5s 10s
11s 15s
Curtis et al., 1977
5-15
Formamide-sucrose gradient
cDNA excess
Globin
10s
lOS, 15S, and 27s
Bastos and Aviv, 1977
Adenovirus type2-infected HeLa cells
1-2
Aqueous sucrose gradient
Adenovirus DNA excess
Adenovirus cytoplasmic mRNA
11-20s
>45s
Wall et al., 1972; Bachenheimer and Darnell, 1975
Adenovirus type. 2-infected KB cells
10-30
Formamide gel
Adenovirus DNA excess
Adenovirus cytoplasmic mRNA
11S, 19s 13S, 19s 20s IIS, 13s 13s
23S, 28s 25s 22s 22s 13s
Tal et a / . , 1974; Craig and Raskas, 1976; Raskas and Craig, 1976
Mouse fetal liver cells
15-20
Mouse spleen erythroid cells
10
DMSO-induced Friend cells
aRNA was fractionated by centrifugation on aqueous sucrose, SDS-sucrose, formamide-sucrose or DMSO-sucrose gradients, or by formamide gel electrophoresis. In some cases, RNA was treated with formaldehyde and/or heat before analysis. *Specific sequences were detected with cDNA or a specific DNA fraction in the presence of excess RNA, or with an excess of DNA.
274
HIROTO NAORA
contains a specific message sequence and that is either two to three times larger than or equal to mRNA in size has been obtained from globin and ovalbuminsynthesizing cells using denaturing conditions (see Table 11). However, a large, probably intact, HnRNA fraction has been successfully isolated from several sources under denaturing conditions (Holmes and Bonner, 1973; Derman and Darnell, 1974; Darnell, 1976; Bastos and Aviv, 1977). 27s HnRNA (4500 to 5000 nucleotides), which contains a globin message sequence but lacks a poly-A segment, was isolated by Bastos and Aviv (1977) after a short (5-minute) labeling period, using excess DNA complementary to mouse globin mRNA. This is good evidence showing that the putative mRNA precursor may be much larger than mRNA. The 27s HnRNA is then discretely processed to 15s RNA (- 1600 nucleotides), followed by polyadenylation. The intermediate precursor, 15s RNA, is then cleaved to 10s RNA (-750 nucleotides), which is subsequently transported to the cytoplasm as a cytoplasmic mRNA. All nuclear globin message sequences are conservatively transported to the cytoplasmic machinery (Bastos and Aviv, 1977), suggesting but not proving that the HnRNA that contains a globin message sequence is a precursor of cytoplasmic globin mRNA, if we assume that all cytoplasmic RNAs that contain globin message sequences are really globin mRNA. It is not known whether 27s RNA is the primary transcript of the globin genes or whether it is a previously processed molecule. In any case, HnRNAs that contain globin message sequences are at least seven times larger than cytoplasmic RNA. In some experiments (e.g., Macnaughton et al., 1974; McKnight and Schimke, 1974; Knochel and Tiedemann, 1975; Knochel and Grundmann, 1977), RNA has been detected by hybridization of labeled cDNA with excess amounts of RNA. This method can detect only the steady-state concentrations of the specific message sequences and not newly synthesized molecules. Since newly synthesized HnRNA is thought to be rapidly processed, the relative content of newly synthesized HnRNA as compared to processed RNA would be extremely low at the steady state. This may in part account for the failure of some groups to find large HnRNAs (see Knochel and Grundmann, 1977). Utilizing excess cDNA, Curtis et al., (1977) were also unable to detect large HnRNA containing the globin message sequence, but they observed that 11 and 15s HnRNA were the precursors of a-andp-globin mRNA, respectively. In mouse erythroid cells and hen reticulocytes, a similar size (15s) for newly synthesized (labeling for 10 minutes) HnRNA for globin has been obtained using excess cDNA (Kwan et al., 1977; Knochel and Grundmann, 1977). The reason for the discrepancy observed when similar types of cells and techniques are used is not clear. However, failure to detect large HnRNA may in part result from the use of long labeling times (10-60 minutes) (Table II). Furthermore, the breaks which occur in the HnRNA molecule under rigorous denaturing conditions may also account for the discrepancy.
HAIRPIN STRUCTURES IN HnRNA
275
In earlier experiments, HnRNA was thought to be huge (up to lOOS), up to 30 times larger than cytoplasmic mRNA (see Darnell, 1975). It seems apparent, however, that it is not as large as was expected. A recent experiment using the ultraviolet transcription mapping technique shows that the majority of HeLa cell mRNA molecules appear to be derived from HnRNA molecules which are only two to three times larger than the mRNA itself (Goldberg et al., 1977). Recent experiments on the organization of P-globin and ovalbumin genes show that the structural genes for these proteins do not reside in DNA as a single piece but are split into more than two fragments (Jeffreys and Flavell, 1977; Leder et al., 1977; Doe1 et al., 1977; Breathnach et al., 1977; see also Williamson, 1977). For example, the rabbit p-globin gene contains an extra DNA sequence-hereafter called an intervening sequence-as long as the structural gene forp-globin in the middle of this gene (Jeffreys and Flavell, 1977). It is certain from nucleotide-sequencing data that the intervening sequence never appears in the cytoplasmic mRNA molecule (Efstratiadis et al., 1977). It is not known whether the intervening sequence is transcribed together with the split coding sequences. Zfii is, the HnRNA that contains the globin message sequence must be at least twice as large as the cytoplasmic mRNA. Indeed, the size of the most readily detectable HnRNA that contains globin message sequences is 15S, that is, just twice as large as cytoplasmic globin mRNA (Kwan et al., 1977; Curtis et al., 1977; Bastos and Aviv, 1977; Knochel and Grundmann, 1977). HnRNA would be expected to possess in the molecule at least a few specific signals for removing portions of intervening and nonintervening sequence origin by nuclear processing enzyme(s). Indeed, some HnRNAs are known to contain several double-stranded hairpin structures which are thought to be specific signals for processing (Section V). The intervening DNA sequences, which may be repeated a few to 15 times, may be expected to contain small palindromes which do not self-associate (Williamson, 1977), implying that HnRNA also possesses double-stranded hairpin structures of intervening sequence origin. However, if the intervening sequences are not transcribed and only portions of the structural gene are transcribed separately, followed by splicing the small transcripts during processing, the primary products would not be large, and in some cases would be smaller than the final mRNA molecule. This is not the case for globin HnRNA, since discrete HnRNA molecules, which are larger than cytoplasmic mRNA, have been discovered (Bastos and Aviv, 1977). There may be another possibility, which is not discussed here, that the RNA polymerase skips over the intervening sequence in transcription. The experiment in which large HnRNA for ovalbumin failed to be detected (McKnight and Schimke, 1974) should be reattempted. The putative precursor of ovalbumin mRNA would be expected to be at least twice the size of that of cytoplasmic mRNA if the
276
HIROTO NAORA
intervening sequences are transcribed together with coding sequences (Breathnach et al., 1977; Doe1 et al., 1977). At present, it is important to know whether HnRNA contains the transcripts of intervening DNA sequences covalently linked to the coding sequences and, if so, whether the double-stranded structures are associated at specific sites for processing into each fragment. The ovalbumin gene is split into several fragments not only in oviduct DNA sequences but also in erythrocyte and 5-day embryo DNA sequences (Breathnach et al., 1977). It seems unlikely therefore that the split organization of the gene is peculiar to the nature of DNA of highly differentiated cells, such as globin or ovalbumin-synthesizing cells. A more precise analysis of HnRNA molecules transcribed from these genes would greatly contribute to our understanding of gene structure and the molecular mechanism whereby gene expression is posttranscriptionally controlled.
B. HNRNA SEQUENCES TO BE DEGRADED IN NUCLEI The majority of HnRNA molecules, isolated from a variety of eukaryotes, contain sequences transcribed from both nonrepeated and repeated DNA sequences (Davidson and Britten, 1973; Lewin, 1974, 1975a). In eukaryotes, most of the sequences involved in cytoplasmic mRNA are transcribed from nonrepeated DNA components (Section VI, D), indicating that most but not all of the transcripts from repeated DNA sequences do not enter the cytoplasm and must be degraded in the nucleus. Even large fractions of the transcripts from nonrepeated sequences are processed and not transferred to the cytoplasm (Galau et at., 1974; Birnie et al., 1974; Hough et al., 1975; Getz et al., 1975). For example, about 30% of the total nonrepeated DNA sequences is represented in HnRNA from sea urchin gastrula (Hough et al., 1975), while only 2.7% is represented in polysomal mFWA (Galau et al., 1974). However, the message sequence in HnRNA is not subject to degradation during processing, since practically no globin message sequences are degraded during the short period of nuclear processing and all message sequences are conservatively transported to the cytoplasm (Bastos and Aviv, 1977). A possibility that cannot be ruled out at present is that this may only be true of globin message sequences. The above result eliminates the possibility of wastage of HnRNA, at least for globin; that is, many extra copies of mRNA precursors might be transcribed, but only a limited number of mRNA sequences would enter the cytoplasm after processing, the remaining copies being totally degraded within the nucleus (Naora, 1977). There are other possibilities that may account for the extensive nuclear degradation of HnRNA sequences (see Perry et al., 1976; Naora, 1977). HnRNA might be a mixed population of at least two different classes, one
HAIRPIN STRUCTURES IN HnRNA
277
consisting of molecules that do not contain any translated sequences and do not migrate to the cytoplasm, and the other containing message sequences which are transferred to the cytoplasm after the removal of non-mRNA segments or without any prior processing of primary transcripts. HnRNA molecules of the former class are entirely unrelated to cytoplasmic mRNA and have unknown functions, but presumably play an important role in regulation. The recent model proposed by Davidson et al. (1977) is based on part of this possibility. The kinetic relationship between HnRNA and mRNA populations and between nuclear and cytoplasmic poly A is very complex (Lewin, 1975a,b; Perry et al., 1976) and appears inconsistent with the idea that all HnRNAs are obligatory precursors of cytoplasmic mRNA (see Naora, 1977). It seems certain at present that not all HnRNA molecules are precursors of cytoplasmic mRNA. This implies that some HnRNA molecules are totally degraded in the nucleus. Does the HnRNA that is totally degraded contain double-stranded hairpin structures similar to those of message-containing HnRNA? It seems likely that the role of HnRNA varies from one molecule to another. It is also possible that HnRNA consists only of molecules containing both untranslated and translated sequences, but that portions of only some types of HnRNA molecules are eventually converted to mRNA. After the removal of most but not all of the untranslated segments, processed mRNA moves to the cytoplasm. These untranslated segments may be present at or near the 5’-end of HnRNA and be transcripts of DNA sequences of regulatory loci which are recognized by RNA polymerase, repressors, derepressors, or other regulatory molecules (Georgiev, 1969). However, in some cases message sequences may be present at the 5’-end of HnRNA (Perry, 1976; Perry et al., 1976). It is premature at present to draw a definite picture of the nuclear degradation of HnRNA sequences, but a combination of the above possibilities seems likely. Extensive degradation of HnRNA sequences, which is observed in evolutionarily advanced eukaryotes, occurs to a lesser extent in some lower eukaryotes. In the water mold Achlya, for example, the complexities of nuclear and polysomal RNA are identical, and there is no detectable difference between the size distribution of poly-A-containingHnRNA and the mRNA populations (Timberlake et al., 1977). This suggests that HnRNA is transported to the cytoplasm as a cytoplasmic mRNA without detectable cleavage. It would be of interest to know whether the HnRNA of lower eukaryotes, such as Achlya, contains any double-stranded hairpin structures. With some exceptions, most mammalian and higher plant HnRNA is large enough to contain more than one mRNA sequence per molecule. So far, there has been no conclusive evidence as to whether an HnRNA molecule consists of one or more than one mRNA sequence. However, monocistronic HnRNA seems to be more likely (Darnell, 1976; Bastos and Aviv, 1977; see also Naora, 1977).
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V. Double-Stranded Hairpin Structures in HnRNA
A. HNRNA ORIGIN
OF
DsRNA
Since the synthesis of most dsRNA is not affected by a low dose of actinomycin D which completely suppresses rRNA synthesis (Section 111, J), nuclear rRNA and rRNA precursors are not likely sources of dsRNA. Indeed, no significant amount of dsRNA was obtained from rRNA precursors (Ryskov et al., 1973; Robertson et al., 1977b). There is also no resemblance between the fingerprint patterns of dsRNA and that of “RNase-resistant” material from 45s rRNA precursor (Robertson et al., 1977b). The double-stranded regions present in tRNA and bacterial tRNA precursors are about 6 to 12 nucleotide pairs long (Altman and Smith, 1971; Altman et al., 1975; Smith, 1975). Since eukaryotic tRNA precursors appear to contain short double-stranded regions as well and are more abundant in the cytoplasmic fraction (Burdon, 1975), dsRNA is unlikely to be derived from tRNA and tRNA precursors. Double-stranded structures have been proposed for some low-molecular-weightnuclear RNA (LnRNA) (Ro-Choi and Busch, 1974; see Naora, 1977). However, since these structures are also short, it appears unlikely that dsRNA is derived from LnRNA. It is now widely accepted that most if not all dsRNA is derived from HnRNA (Jelinek and Darnell, 1972; Kronenberg and Humphreys, 1972; Ryskov et al., 1973; Patnaik and Taylor, 1973; Monckton and Naora, 1974; Robertson et al., 1977b). Recent experiments involving fingerprint analyses of HnRNA and dsRNA show that HnRNA produces the patterns which include one identical to that of isolated dsRNA, suggesting the HnRNA origin of dsRNA (Robertson er al., 1977b). Isolated HnRNA has considerable secondary structure in solution; 57% (estimated from formaldehyde reactivity) to 68% (from hyperchromicity) of HnRNA nucleotides are actually in the form of base-paired structures (Holmes and Bonner, 1973). However, the dsRNA which can be isolated from HnRNA is a very small fraction of the HnRNA, and its content varies markedly: 0.2-0.5% and 2.5-3.1% for nuclear ribonucleoprotein (HnRNP) particles of mouse ascites tumor (Molnfir et al., 1975) and HeLa cells (Calvet and Pederson, 1977), respectively; 0.45% for rat liver HnRNA (R. P.Monckton and H. Naora, unpublished); 0.5-0.7 and 0.8-1.2% for mouse ascites cell 25-453 and >45S HnRNAs, respectively (Ryskov et al., 1973); and 3% for HeLa cell HnRNA (Jelinek and Damell, 1972). Variations in these values may result from the use of different materials andor preparation procedures, including column chromatography. Although in most cases isolated dsRNA is very heterogeneous, and the size of HnRNA appears ambiguous, the above values of the dsRNA content observed in various species of organisms roughly correspond on average to up to several
HAIRPIN STRUCTURES IN HnRNA
279
copies of double-stranded structures per HnRNA molecule. It should be pointed out that this is an average distribution for all HnRNA molecules and not a detailed picture of any particular one. In fact, preliminary electron microscope studies on rat liver HnRNA molecules reveal that some HnRNA molecules contain up to about 10 double-stranded structures on the long strands in an irregular manner, while other HnRNA molecules have none (H. Naora, unpublished).
B. DOUBLE-STRANDED HAIRPINSTRUCTURES Information suggesting that the double-stranded structures are part of singlestranded HnRNA comes from the following two lines of studies. First, it is known that fully double-stranded RNA is soluble in 2 M LiCl but partially double-stranded RNA is insoluble (Baltimore, 1966). Over 90% of the doublestranded structures present in HnRNA or whole-cell RNA preparations is recovered from the LiC1-insoluble fraction. However, dsRNA isolated from HnRNA or whole-cell RNA by RNase treatment is LiC1-soluble (Kimball and Duesberg, 1971; Jelinek and Darnell, 1972). Furthermore, when HnRNA is treated with E. coli RNase 111 or eukaryotic RNases specific to dsRNA, over 90% of the RNA undergoes limited cleavage and generates RNA fragments corresponding to mRNA in size (Section VII, B). These results suggest the presence of doublestranded regions interspersed among long regions of a single-stranded strand. Second, RNA synthesized in vitro and untreated with RNases has a density of 1.65 g d m l in CsSO,. After treatment with RNases, this changes to 1.62 g d m l (Pays, 1976). The density shift is also seen when HeLa cell HnRNA is digested with RNases under various conditions; the shift becomes more pronounced as digestion becomes more complete and finally reaches a value similar to that obtained for known dsRNA (Jelinek et a l . , 1974). There seem to be adequate evidence indicating that the double-stranded structures of HnRNA are in the form of intramolecular base pairing. The “snapback” treatment of RNA is a useful technique in examining the structure of HnRNA molecules. This treatment includes chemical or thermal denaturation of HnRNA, followed by rapid removal of denaturing agents or immediate chilling in high salt. If isolated dsRNA is derived from an intramolecular base-paired structure, the complementary strands should immediately reform the doublestranded structure after the treatment. Whereas, if dsRNA were in the form of intermolecular base pairing, the separated strands should remain single-stranded. Actually when high-molecular-weight HnRNA is subjected to the snap-back treatment, almost all double-stranded structures present in HnRNA before the treatment are recovered (Jelinek and Damell, 1972; Ryskov et a l . , 1973; Monckton and Naora, 1974; Jelinek et hl., 1974). This indicates that the struc-
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tures are indeed of the intramolecular hairpin type. Similar double-stranded hairpin structures are also found in low-molecular-weight HnRNA (R. P. Monckton and H. Naora, unpublished; Robertson et al., 1977b). However, recovery of dsRNA from low-molecular-weight HnRNA after the snap-back treatment is only 50% or less (R. P. Monckton and H. Naora, unpublished). It is not clear at present whether low-molecular-weight HnRNA contains the intermolecular base-paired structure as well or whether nicking of the intramolecular structures occurs in vivo or during isolation, resulting in such low yields of dsRNA. Once double-stranded structures are isolated from HnRNA, these RNAs are no longer in the form of intramolecular double-stranded structures (Jelinek and Darnell, 1972; Ryksov et al., 1973; Jelinek et al., 1974; Monckton and Naora, 1974). This indicates that the nucleotide linkages in the loop between the basepaired region are cleaved by RNases during the isolation of dsRNA. This loop region has been suggested to range from a few nucleotides to several thousand (Jelinek et al., 1974). However, since complete destruction of double-stranded hairpin structures does not occur until the HnRNA chain is reduced to less than several hundred nucleotides by alkali or RNase treatment (Jelinek et al., 1974), the loop should be short. It is conceivable that the polynucleotide chain is complexed consecutively with proteins immediately after transcription. If the two complementary sequences are separated from each other by a long distance on the newly transcribed HnRNA, these sequences would be complexed separately with proteins before base pairing and consequently would remain singlestranded.
c. In I / i V o EXISTENCE OF STRUCTURES There is a possibility that most if not all of the double-stranded structures observed in isolated HnRNA are generated by rapid intramolecular association during the isolation of HnRNA and are not the native form present in the nucleus. This possibility is particularly suggested by two different observations. First, HnRNA contains self-complementary single-stranded sequences which are capable of forming double-stranded hairpin structures under the appropriate annealing conditions and which are transcribed from repeated DNA sequences, as is dsRNA (Ryskov et al., 1974; Fedoroff et al., 1977). When mouse or rat HnRNA is annealed under conditions which include high Cot values, up to 20% of the HnRNA becomes RNase-resistant (Ryskov et al., 1974; Naora et al., 1975). Even by simply increasing the concentration of salt, more doublestranded hairpin structures become detectable (Pays, 1976). Second, HnRNA exists as a ribonucleoprotein complex (Georgiev, 1974) and is mainly associated with chromatin (Kimmel et al., 1976; Herman et al.,
HAIRPIN STRUCTURES IN HnRNA
28 1
1976a). Isolated HnRNA molecules usually possess more double-stranded structures than do HnRNP particles. During deproteinization, RNA-RNA association takes place easily (Weissmann et al., 1968). In spite of these observations, evidence available currently favors the view that double-stranded hairpin structures exist in nuclei. When prelabeled KB cells are incubated in the presence of an intercalating agent (ethidium bromide or riboflavin), which binds to double-stranded regions of RNA (Section VII, B), degradation of HnRNA is inhibited completely, without delay or after a short period (Brinker et al., 1973). Although no evidence has been presented for the in vivo intercalation of these agents between base pairs in the double-stranded regions of HnRNA, this result is consistent with the view that the double-stranded structures exist in HnRNA in vivo and are probably the recognition signals for nuclear processing enzyme(s). dsRNA can be isolated from the homogenates of sea urchin embryos by direct treatment with RNases prior to the isolation of bulk RNA (Kronenberg and Humphreys, 1972). Moreover, when HnRNP particles are incubated with RNases (TI and A) in the presence of 0.1 M salt, approximately 1% (HnRNP of Ehrlich carcinoma cells; Molnir et al., 1975) to 3% (HnRNP of HeLa cells; Calvet and Pederson, 1977) of the total RNA in these particles is RNaseresistant. This RNase-resistant RNA can be removed from particles by preincubation with E. coli RNase 111which digests dsRNA (Calvet and Pederson, 1977). These results indicate that double-stranded regions indeed exist in native ribonucleoprotein particles. Effective removal of double-stranded structures by RNase III further suggests that these structures are localized on the surface of or outside the particle. There seem to be two distinct classes of double-stranded structures in native HnRNP particles: stable and unstable. The first comprises approximately 1% of the total RNA of HeLa cell HnRNP particles and is resistant to RNase digestion at 0.13 M salt. Structures of the second type (2.5-3.1% of the total RNA of particles) are unstable at 0.13 M salt and become resistant to RNase digestion only at high ionic strength (Calvet and Pederson, 1977). Both types can be digested by RNase I11 under appropriate conditions. It is unknown whether the sequences involved in some of the unstable structures represent the selfcomplementary single-stranded sequences mentioned earlier.
DNA SEQUENCES FROM WHICHDSRNASARETRANSCRIBED D. PALINDROME The sequences involved in the double-stranded structures of HnRNA are probably transcribed from the palindromic portion of DNA. Eukaryotic chromosomes contain a considerable number of inverted-repeated sequences. For example, the rodent haploid genome possesses approximately 33 ,OOO to 42,000 inverted-
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repeated sequences (Cech and Hearst, 1975; Bell and Hardman, 1976; Szala et al., 1977), whereas Drosophila and human genomes contain 2000 to 4000 and 100,000 to 120,000 sequences, respectively (Schmid et al., 1975; Deininger and Schmid, 1976; Jelinek, 1977). Some of these sequences are palindromic, but others consist of two complementary regions separated by a long loop. These inverted-repeated sequences, which in human and mouse genomes are 150 to 6600 nucleotides long, are separated on DNA strands by a nonrepeated sequence. There are approximately 100,000 genes in the human genome (Lewin, 1974), suggesting that there is about one inverted-repeated sequence per gene. This is also found in Drosophila (Schmid et al., 1975; Wallace and Kass, 1976) and in rodents (Cech and Hearst, 1975; Bell and Hardman, 1976; Szala et al., 1977). There are dsRNA-derived sequences at both the 5'- and 3'-ends of mRNA molecules (Section VI,C), implying that there should be at least two invertedrepeated (probably palindromic) sequences in the gene. If it is assumed that the structural gene is interrupted with an intervening sequence (Section IV, A) and that the double-stranded hairpin structures are involved in the processing of the transcripts of the intervening DNA sequences, at least another one or two palindromic sequences would be expected. This estimate agrees with the number of double-stranded structures per HnRNA molecule obtained from the dsRNA content but is slightly higher than the number of inverted-repeated sequences in the gene. Church et al. (1974) isolated the fraction of mouse DNA which reassociated at the extremely low C o t value of 10-7-10-6 and contained palindromic sequences. At these C o t values, mouse satellite DNA does not reassociate. Mouse and human palindromic DNA sequences are about 100 and 300 nucleotide pairs long, respectively (Churchet al., 1974;Deininger and Schmid, 1976). They correspond to medium-sized dsRNAs from mouse ascites tumor cells and human cells (Section 111, H). The palindromic DNA fraction contains predominantly moderately repeated sequences from which many dsRNAs are transcribed but also possesses highly repeated and nonrepeated sequences in Drosophila (Schmid et al., 1975). Since the palindromic DNA fragments isolated hybridize extensively with dsRNA (Church et al., 1974), it is likely that sequences involved in the double-stranded hairpin structure of HnRNA are transcribed from at least some of the palindromic DNA sequences. Further support for this view has been obtained from fingerprint analyses, indicating that the in vitro transcript from inverted-repeated, probably palindromic, DNA sequences of HeLa cells are almost indistinguishable from those of dsRNA from HeLa cell HnRNA (Jelinek, 1977). Although there are several factors which influence the interpretation of the results (Robertson et al., 1977b; Jelinek, 1977), they suggest that dsRNA originates from palindromic DNA sequences.
HAIRPIN STRUCTURES IN HnRNA
283
E. LOCATION The location of double-stranded structures within an HnRNA molecule is of particular interest. RNA synthesized in vitro has double-stranded structures mainly at the 5’-end of the transcripts. In particular, RNA transcripts labeled in vitro with GTPyp2P showed a high resistance to RNase digestion, suggesting that at least a fraction of RNA chains is initiated in double-stranded structures (Pays, 1976). Electron microscope examination of rat liver HnRNA also revealed that the doublestranded structure existed at one end of some molecules (H. Naora, unpublished). However, this cannot be generalized, since the topography of rat liver HnRNA is quite irregular. In HeLa cell HnRNA, the repeated sequences are not detected in the 3‘terminal region (Molloy et al., 1974). Since many of the sequences involved in the double-stranded structures are transcribed from repeated DNA sequences (Section 111, I), the above result may suggest that there are no double-stranded structures near the 3’-end of the HnRNA molecule. However, if mRNA is mainly derived from sequences present near the 3’-end of the precursor molecule (Section IV, B), the double-stranded structures linked to both ends of the coding sequences (Sections VI, C and VII, A) should exist near the 3’-end. Of course, this does not necessarily exclude the possibility of a S’-terminal location. There are a few factors that influence the above view: (1) All dsRNAs are not transcripts of repeated DNA sequences (Section 111, I); (2) the degree of repetition may differ from one fraction of dsRNA to another; and (3) the location may depend on the type of HnRNA. Obviously, no clear picture of the location can be drawn at present.
VI. Features of Eukaryotic mRNA In this section, we briefly review findings on some features of the eukaryotic mRNA molecule which are relevant to the present topic, for example, untranslated sequences, secondary structures of the mRNA molecule, and terminal sequence repetition.
A. UNTRANSLATED SEQUENCES OF MRNA It is now well documented that eukaryotic mRNA is longer than would be expected from the known length of a specific protein. The proportion of the untranslated region which includes the poly-A segment, the cap structure, and
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HIROTO NAORA
part of the ribosome binding site to the entire length of mRNA varies significantly, depending upon the type of mRNA and methods of measurement. Untranslated regions of silkworm fibroin (Lizardi et al., 1975) and chicken myosin (heavy chain) (Mondal et al., 1974) mRNAs are only 6-17% of the total length. Since these mRNAs are 5370 and 16,000 nucleotides long, these values correspond to lengths of 290 to 2900 nucleotides, excluding poly-A segments. However, 64% of calf lensaA2-crystalline mRNA (1460 nucleotides long) correspondingto 740 nucleotides plus 200 adenylic acid residues is an untranslated sequence (Berns et al., 1974; Favre et al., 1974). The untranslated region is in general a few hundred to 1000 nucleotides in length (see Adams, 1977). In all cases where the precise lengths of the untranslated sequences have been determined, the mRNA molecule possesses these regions both at the 5’-end and preceding the poly-A segment at the 3‘-end (Proudfoot, 1976; Proudfoot and Longley, 1976;Proudfoot et al., 1976; Wilson et al., 1977; Cohen-Sold et al., 1977; Marotta et al., 1977; Baralle, 1977a,b; Efstratiadis et al., 1977; Chang et al., 1977). The length of the 3’-terminal untranslated region varies markedly. In rabbit a and p-globin mRNAs, 80 and 95 nucleotide residues are present in this region, respectively (Proudfoot, 1976; Proudfoot et al., 1976; Efstratiadis et al., 1977), whereas in human a- and p-globin mRNAs this region contains 112 and 135 nucleotides, respectively (Wilson et al., 1977; Marotta et al., 1977). A longer untranslated sequence (200 ? 50 nucleotides) at the 3’-end is reported to occur in mouse immunoglobulin light-chain mRNA (Milstein et at., 1974). When the 3’-terminal untranslated sequences of human and rabbit globin mRNAs for a and j3 chains are phylogenically compared, many (on average 83%) sequences are conserved; in particular, the nucleotide sequences proximal to the poly-A segment of human and rabbit j3 -globin mRNA are highly homologous (Proudfoot and Brownlee, 1976; Proudfoot and Longley, 1976; Proudfoot et al., 1976; Marotta et al., 1977; Efstratiadis et al., 1977). However, comparison of the nucleotide sequences of the 3’-terminal untranslated region of various types of eukaryotic and viral mRNAs, for example, rabbit and human a- and j3 -globin mRNAs, chicken ovalbumin mRNA, mouse immunoglobulin lightchain mRNA, SV40 early and late mRNA, shows no long common sequence but reveals the presence of the hexanucleotide sequence AAUAAA located about 20 residues from the poly-A segment (Proudfoot, 1976; Proudfoot and Longley, 1976; Proudfoot et al., 1976; Wilson et al., 1977; Efstratiadis et al., 1977). The sequence directly adjacent to the poly-A segment does not appear to be the same in most mRNAs, although the most common sequences are GC-poly(A) and GU-poly(A) (Nichols and Eiden, 1974). The 3’-terminal untranslated region possibly includes the signal to poly-A polymerase for posttranscriptional addition of the poly-A sequence and/or to RNA polymerase for the termination of transcription if mRNA shares its 3’-untranslated region with the end of the primary transcript.
HAIRPIN STRUCTURES IN HnRNA
285
The 5’-terminal untranslated regions of globin mRNAs are generally shorter than the 3’-terminal regions. Rabbit a - and @-globinmRNAs contain 37 and 53 nucleotides residues (excluding the capped nucleotide), respectively (Baralle, 1977a,b; Efstratiadis et al., 1977). These sequences show a substantial difference between a - and @-globin mRNA; only the short sequences, ACACUU and ACUCUU, located immediately adjacent to the cap structure are common to rabbit or human a-and@-globinmRNA, but not to other mRNAs, for example, reovirus mRNAs (Baralle, 1977a,b, Kozak, 1977; Chang et al., 1977). However, extensive homologies between human and rabbit are seen in this region of a - and @-globin mRNA (Baralle, 1977a,b; Efstratiadis et al., 1977; Chang et al., 1977). There seems to be evidence for the existence in eukaryotic (and viral) mRNA of a sequence in the 5’-terminal region which interacts with the 3’-terminal sequence of 18s rRNA (Dasgupta et al., 1975; Van de Voorde et al., 1976; Baralle, 1977a; Efstratiadis et al., 1977; Kozak, 1977) in a manner similar but not identical to that of bacterial mRNA (Shine and Dalgarno, 1974, 1975). The biological functions of the 5’-terminal untranslated sequence of mRNA are not well understood. A few experiments indicate that the cap structure plays an important role in stabilizing mRNA in the cell (Furuichi et al., 1977; Shimotohno et al., 1977). There is also evidence that the 5’-terminal m7G functions as a recognition signal for binding the ribosome to mRNA (Busch et al., 1976). The 5’-phosphorylated derivatives of m7G act as competitive inhibitors of “capped” mRNAs (Hickey et al., 1976, 1977) to an extent which probably depends on the length of the 5’-terminaI untranslated sequence (Suzuki, 1976, 1977; H. Naora, unpublished). Another possibility is that the 5’- and 3’-terminal untranslated sequences may be involved in mRNA turnover (Darnell, 1976) or in the processing of mRNA precursor into cytoplasmic mRNA. The latter possibility may be related to the following observations: (1) All the eukaryotic mRNAs so far examined contain long, untranslated sequences at or near the 5’- and 3’-ends. (2) There are sequences which are probably derived from those of dsRNA at or near both the 5’and 3’-ends of eukaryotic mRNAs (Section VI, C). (3) The 5‘- and 3’-terminal sequences of mFWA are similar within the molecule (Section VI, E).
B. DOUBLE-STRANDED STRUCTURES OF MRNA With the use of techniques of thermal denaturation and dye binding, it has been reported that 40-70% of nucleotides of isolated mRNA in solution are apparently in the form of double-stranded structures (Williamson et al., 1971; Houdebine et al., 1974; Bobst et al., 1974; Favre et al., 1974, 1975; Holder and Lingrel, 1975; Rhoads, 1975; Vournakis et al., 1976; Van et al., 1977). The
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thermal denaturation profile of rabbit globin mRNA is more or less different from that of a random polymer (A, U, G, and C) and remarkably different from that of double-stranded synthetic nucleic acids and natural DNA (Holder and Lingrel, 1975; Van et al., 1976). Although many intramolecular base pairings occur in a random polynucleotide sequence (Fresco et al., 1960; Gralla and De Lisi, 1974), the double-stranded structures which exist in rabbit globin mRNA are slightly longer than those which occur randomly (Van et al., 1976). Based on sequencing data, a small double-strandedhairpin structure has been proposed for the untranslated or translated sequences in immunoglobulin light-chain and globin mRNAs (Proudfoot and Brownlee, 1974; Proudfoot, 1976; Baralle, 1977b; Salser et al., 1976; Efstratiadis et al., 1977; Chang et al., 1977). However, it is unlikely that the mRNA contains an array of short, stable double-stranded structures in which 40-70% of the composite nucleotides of mRNA are involved (Naora and Fry, 1977). No secondary structures are electron microscopically visible in mRNA molecules from rat liver and rabbit and hen reticulocytes under various spreading conditions where short (20 to 30 nucleotide pairs) double-stranded structures of other types of RNA, that is, bacteriophage MS2 RNA, 28s rRNA, and HnRNA, are clearly demonstrated (Woo et al., 1975; Naora and Fry, 1977; H. Naora, unpublished). The thermal denaturation profile of bacteriophage MS2 RNA (total G + C content, 52%) suggests that its double-stranded structures do not have a C content (Van et al., 1976). It appears likely therefore that stable, high G long double-stranded structures (>20 to 30 nucleotide pairs), similar to those observed by electron microscopy in bacteriophage MS2 RNA, 28s rRNA, and HnRNA molecules, are entirely absent in mRNA molecules. However, this does not exclude the possibility that mRNA molecules contain unstable and/or short structures, that is, structures several nucleotide pairs long. The above view is further confirmed by the finding that no double-stranded structures are detectable in cytoplasmic mRNA by the techniques which detect double-stranded structures in HnRNA (Jelinek and Darnell, 1972; Ryskov et al., 1973; Naora e t a l . , 1975).
+
OF SEQUENCES THAT HYBRIDIZE WITH DENATURED DsRNA IN C. PRESENCE THE MRNAMOLECULE
In 1971, Hare1 and Montagnier noticed a small amount (1520%) of competitive inhibition by total cytoplasmic rat liver RNA, which included a small amount of mRNA, in a hybridization between rat liver dsRNA and homologous DNA. Later, Stampfer et al. (1972) found that approximately 5% of polysomal mRNA became RNase-resistant after annealing with nuclear RNA in excess. These were the first reports suggesting the possible involvement of dsRNA in an mRNA molecule, but their biological significance was not clear at that time. Work on sequences involved in both dsRNA and mRNA molecules was ini-
HAIRPIN STRUCTURES IN HnRNA
287
tiated independently in two laboratories almost at the same time, and the same conclusion was reached: There are sequences in mRNA which hybridize with denatured dsRNA and are probably derived from double-stranded hairpin structures of mRNA precursor molecules-hereafter called dsRNA-derived sequences (Georgiev et al., 1973; Naora et al., 1974). When labeled mRNA from rat liver, human HeLa and mouse carcinoma cells, or hen reticulocytes is annealed with denatured dsRNA from a homologous source, RNase-resistant complexes are formed (Naora et al., 1975; Darnell, 1976; Kramerov et al., 1977; Fry and Naora, 1977). Evidence has been presented that the complexes formed are not nonspecific aggregates of RNA molecules but specific hybrids between mRNA and dsRNA. For example, no significant RNase-resistant materials are formed in the presence of rRNA or tRNA (Naora et al., 1975). The fingerprint pattern of the hybrids is similar to that of dsRNA (Darnell, 1976). Most (98%) rat liver mRNA populations possess sequences which hybridize with dsRNA in mRNA (Naora and Whitelam, 1975). These sequences are also found in experiments involving molecular hybridization between labeled dsRNA and unlabeled mRNA. However, only part (about 25% above the background) of the dsRNA from mouse carcinoma, rabbit bone marrow, and hen reticulocytes hybridizes with the mRNA from homologous sources (Georgiev e f al., 1973; Ryskov et al., 1976a,b; Kramerov et al., 1977; Jelinek and Evans, cited in Robertson et al., 1977b; Fry et al., 1978). Hybrids are also formed to the same extent when dsRNA is annealed with HnRNA (Georgiev et al., 1973; Naora et al., 1975). Denatured dsRNA can hybridize with mRNA at the final “dsRNA-driven” C o t value of approximately lop3, indicating that sequences in mRNA hybridize with a fast-renaturing fraction of dsRNA. More slowly renaturing dsRNA also hybridizes with mRNA (Ryskov et al., 1976a). This suggests the presence of at least two types of sequences in mRNA. The heterogeneity of the sequences that hybridize with denatured dsRNA is also seen when medium-sized and long dsRNAs are annealed with mRNA (Kramerov et al., 1977). Hybrids formed between mRNA and dsRNA have a good fidelity of base pairing (Naora et al., 1975; Ryskov et al., 1976a). For example, those formed at a low input ratio (unlabeled rat liver dsRNNlabeled rat liver mRNA mass ratio = 1:1) exhibit a sharp melting profile with a T,,, difference of 6°C below that of the original rat liver ds RNA (Naora et al., 1975). It is not known to what extent the reduced T , is due to the difference in the base compositionsof dsRNA and mRNA and also to the shortness of the hybrid complexes (Hayes et al., 1970). However, if it is assumed that these factors are not significant, the T , of the hybrids indicates about 3% mismatching of the bases according to the calculations of Greenberg and Perry (1971). This suggests that the hybrid formation results from a specific interaction between mRNA and dsRNA. Sequences in mRNA that hybridize with denatured dsRNA are species-specific
288
HIROTO NAORA
but not tissue-specific (Naora et al., 1975; Ryskov et al., 1976a,b); rat liver mRNA hybridizes with denatured rat liver dsRNA, but mouse sarcoma mRNA does not (Naora et al., 1975). Rabbit liver mRNA forms hybrids with rabbit bone marrow dsRNA as efficiently as rabbit globin mRNA (Ryskov et al., 1976b). The sequences in mRNA are in general half (or less) the length of isolated dsRNA. This is shown by three different types of experiments. First, direct determination on hybrids formed between hen globin mRNA and denatured dsRNA following the removal of unpaired tails shows that the length is shorter than 60 nucleotide pairs. Since the major fraction of hen reticulocyte dsRNA is 120 nucleotide pairs in length, the size of the sequences that hybridize with dsRNA is one-half (or less) that of isolated dsRNA (Naora et al., 1978). As determined by gel electrophoresis, the sequences in mouse ascites and rabbit globin mRNAs are 10 to 60 (Ryskov et al., 1976a) and about 30 (Ryskov et al., 1976b) nucleotides in length, respectively. Since isolated dsRNAs from mouse carcinoma and rabbit bone marrow cells are 100 to 200 and 80 to 150 nucleotide pairs long, respectively (Table I), the hybridizable sequences also correspond to approximately one-half (or less) the length of isolated dsRNA. Second, when hybrids formed with mRNA and labeled dsRNA are isolated on a poly-U-Sepharose column, approximately 35% of the labeled dsRNA is bound to the mRNA. However, treatment of the hybrids with RNases results in a marked (30-75%) decrease in labeled materials (Ryskov et al., 1976a,b). This result suggests that the hybrids contain a duplex region as well as an unpaired tail of denatured dsRNA. Third, rat liver, HeLa cell, and mouse carcinoma dsRNAs hybridize with about 6-8% of the respective mRNA added (Naora and Whitelam, 1975; Darnell, 1976; Kramerov et al., 1977). If it is assumed that the length of an mRNA molecule is lo00 to 2000 nucleotides (Davidson and Britten, 1973; Perry et al., 1976; Darnell, 1976; Adams, 1977), the total length of the sequences involved is 60 to 160 nucleotides. Since, as described in Section VI, C, 1 and 2, there are sequences that hybridize with denatured dsRNA in two different regions of the mRNA molecule, the above values correspond to 30 to 80 nucleotides for each region, which represent one-half (or less) the length of isolated dsRNA. There is evidence indicating that the sequences are located both in the region preceding the poly-A segment at the 3'-end and in the region adjacent to the cap structure at the 5'-end. 1. The 3'-Terrninal Region of mRNA
Information on the presence of the sequences at the 3'-terminal region comes from three different sources. First, a polynucleotidephosphorylase which attacks only single-stranded polynucleotides from the 3'-end of the molecule under defined conditions (Grunberg-Manago, 1963) cannot efficiently digest the hy-
HAIRPIN STRUCTURES IN HnRNA
289
brids formed between labeled rat liver mRNA and denatured dsRNA after a short period of digestion. This indicates that there are sequences that hybridize with denatured dsRNA near the 3‘-end of the hybrids (Naora and Whitelam, 1975). Second, a more conclusive result has been obtained with short (approximately 80 or 200 nucleotides) copies of DNA complementary to the 3’-terminal sequence preceding the poly-A segment of hen globin mRNA. These cDNAs hybridize with denatured dsRNA from hen reticulocytes efficiently (Fry et al., 1978). Since the cDNAs used are exclusively or mainly the transcripts of the 3’-tenninal untranslated sequence, the above result clearly indicates the presence of sequences that hybridize with denatured dsRNA in the region preceding the poly-A segment. Third, poly-A-containing short fragments (one-half to one-third the total length) of rat liver mRNA and hen globin mRNA, obtained following brief exposure to alkali or CM-cellulose bound RNase A, hybridize with denatured dsRNA (H. Naora and J. M. Whitelam; H. Naora and K. E. Fry, unpublished).
2 . The 5’-Terminal Region of mRNA Non-poly-A-containing fragments of rat liver and hen globin po1y-Acontaining mRNA, obtained in the experiments mentioned above, also hybridize with denatured dsRNA to almost the same extent as poly-A-containing fragments, indicating that the 5’-terminal region also possesses the sequences (H. Naora and J. M. Whitelam; H. Naora and K. E. Fry, unpublished). Spleen phosphodiesterasehas been used to examine the location of the sequences (Naora and Whitelam, 1975). However, since the commercial preparation of this enzyme contains other enzyme activities, the results using this enzyme preparation seem to be complicated. More information comes from studies using the wheat germ cell-free system, which suggest that denatured hen reticulocyte dsRNA possesses nucleotide sequences corresponding to those of the ribosome-binding site, including codons for some amino acids adjacent to the initiation amino acid of a a-globin chain (Naora et al., 1978). Since dsRNA is not derived from mRNA (Section V), hen reticulocyte dsRNA seems to contain sequences probably identical to those in the 5’-terminal region of mRNA for a-globin. Georgiev and colleagues (Georgiev, 1969; Coutelle et al., 1970; Georgiev et al., 1972) considered mRNA to be localized near the 3‘-end of the mRNA precursor molecule (Herman et al., 1976a) and thus proposed a model suggesting localization of the double-stranded hairpin structure at the potential 5’-end of mRNA in the mRNA precursor molecule (Ryskov et al., 1976a,b). However, they did not comment on the possible existence of a similar structure at the potential 3’-end of the mRNA sequence. When all these facts are taken into account, it is suggested that, with some exceptions cytoplasmic mRNA contains sequences that hybridize with denatured dsRNA at or near both the 5’-end and the region preceding the poly-A segment at the 3‘-end. These sequences are probably derived from double-stranded hairpin
290
HIROTO NAORA
structures of HnRNA. However, it is possible that cytoplasmic mRNA coincidentally contains sequences that are similar to those of one strand of dsRNA but are not derived from double-stranded hairpin structures. Since, as mentioned in Section 111, I many but not all sequences involved in dsRNA are transcribed from repeated DNA sequences, this possibility cannot be completely ruled out at present, though it is unlikely.
FROM D. Do MRNA MOLECULES CONTAIN SEQUENCES TRANSCRIBED REPEATED DNA SEQUENCES?
Most mRNA hybridizes with DNA at high Cot values, implying that most mRNA in eukaryotes is transcribed from nonrepeated DNA sequences (Greenberg and Perry, 1971; Lewin, 1975b). An extreme case is the mRNA isolated from polysomes of sea urchin gastrulas (Goldberg et al., 1973); virtually all the mRNA sequences are derived from a nonrepeated DNA component. In other organisms, however, a certain fraction, approximately 20% in rat cells (Camp0 and Bishop, 1974), of mRNA sequences represents transcripts of the repeated DNA fractions. As mentioned earlier, many dsRNA sequences are transcripts of the repeated DNA component of the genome. The question is then raised whether a mRNA molecule contains repeated sequences covalently linked to the message sequence. There have been reports postulating that some of the repeated sequences found in the mRNA fraction are covalently linked to nonrepeated sequences. Crippa et al. (1973) and Dina et al. (1974) showed that repeated sequences (about 50 to 60 nucleotides long) were present as part of Xenopus mRNA at the 5'-end of the molecule. Recent experiments have further confirmed their earlier findings (Crippa, reference from Darnell, 1976). Firtel and Lodish (1973) and Lodish et al. (1973) demonstrated that about 10 and 25% of the cytoplasmic and nuclear poly-A-containing RNA of the slime mold Dictyostelium discoideum are transcribed from the repeated DNA fraction, respectively. The primary nuclear transcripts in Dictyosteliurn contain sequences with an average of 300 to 500 nucleotides at the 5'-end that are derived from the repeated DNA component and are covalently linked to the nonrepeated sequence. With transport of the nuclear RNA to the cytoplasm, the majority of the 5'-terminal repeated sequences are removed, leaving a short repeated sequence in the mRNA molecule. Based on these results, these workers have proposed a model in which cytoplasmic mRNA of Dictyostelium, mainly the transcript of nonrepeated DNA sequences, contains short transcripts of repeated DNA components at the 5'-end and also perhaps at the 3'-end of the same molecule. These results favor the idea that the 5'- and 3'-terminal sequences of mRNA are derived from a portion of the doublestranded hairpin structures. Recently, Jelinek et d . (reference from Darnell,
HAIRPIN STRUCTURES IN HnRNA
29 1
1976) showed that a large fraction of HeLa cell mRNAs might have some sort of repeated sequence at one or both ends, supporting the above-mentioned idea. However, different results have been obtained in an analysis of the sequences of mRNA isolated from Achlya (water mold), sea urchin embryos, and cultured human and rat celIs (Goldberg et al., 1973; Klein et al., 1974; Camp0 and Bishop, 1974; Timberlake et a l . , 1977). These mRNA fractions contain repeated sequence transcripts which are not covalently linked to nonrepeated sequence transcripts. Hence there exists a class of mRNA molecules composed entirely of sequences transcribed from repeated DNA components. The discrepancy may arise from the difference in materials, the degree of contamination of HnRNA in mRNA preparations, and the sensitivities and types of methods used for analyses. For example, only duplex fragments longer than approximately 40 nucleotide pairs can be quantitatively retained on hydroxyapatite under standard conditions, although a certain number of small fragments (17 nucleotide pairs) can be detected (Wilson and Thomas, 1973). It is possible that mRNA contains repeated sequence regions shorter than this.
E. TERMINAL SEQUENCE REPETITION I N MRNA MOLECULES If the double-stranded hairpin structures are linked to both ends of the message sequence in HnRNA as mentioned in Section VI, C and both structures are processed by the same enzyme, one would, though not necessarily, predict a similar feature in both structures that could determine the enzyme specificity. Furthermore, as pointed out in Section 111, I, many of the dsRNAs isolated from HnRNA are transcripts of moderately repeated DNA sequences. The question arises whether these structures within the HnRNA molecule are similar to or totally different from each other. This can now in part be examined, since (1) as described in Section VI, C, parts of sequences present at or near the 5 ' - and 3'-ends of mRNA are probably derived from double-stranded hairpin structures of HnRNA, and (2) the complete sequences of approximately 30 to 70 nucleotides adjacent to the cap structure and poly-A sequence are now available for human and rabbit a! - and P -globin mRNA (Proudfoot, 1976, Proudfoot and Longley, 1976; Proudfoot et al., 1976; Baralle, 1977a,b; Wilson et al., 1977; Cohen-Solal et al., 1977; Marotta et al., 1977; Efstratiadis et al., 1977; Chang et al., 1977). The results obtained in an assessment of sequence homology (Naora et al., 1978), using a simple matrix display procedure based on nucleotide-to-nucleotide matching (Gibbs and McIntyre, 1970), indicate that rabbit a-and p-globin mRNA show substantial homology in the terminal nucleotide sequences between the 5'- and 3'-ends, except for the cap nucleotide, m7G, and poly-A sequences which are posttranscriptionally added (Lewin, 1975a; Shatkin, 1976; Adams, 1977). A hypothetical RNA strand,
292
HIROTO NAORA
commonly complementary to both the 5‘- and 3’-tenninal nucleotide sequences, can be accommodated with the 5’- or 3’-terminal nucleotide sequence in the parts of thermodynamically stable double-stranded hairpin structures which are covalently linked to both ends of the message in the mRNA precursor molecule and are similar to each other (Fig. 3) (Naora et al., 1978). It is suggested from the sequencing data for some mRNA (Section VI, A) that the double-stranded structures that are similar within the mRNA precursor molecule seem to differ from one type of mRNA precursor to another but are perhaps “message-specific” (Naora et al., 1978). It should be noted here that, although there are small internal loops and bulges in the structures (see Fig. 3), these structures are approximately 16 to 40 nucleotide pairs long, which is in good agreement with the length
F‘, I I I
&ppp
FIG. 3.
-- 0 -8
--
?f
CACCAUG-
I 1 I
7-- A A A A A
Hypothetical primary and secondary structure of the presumptive precursor molecule (HnRNA) for rabbit a-globin mRNA. Nucleotide sequences in the region between two doublestranded structures are detached from the precursor molecule during nuclear processing and become mature mRNA after cap formation, methylation, and polyadenylation. There are a few undefined nucleotides in the RNA strand complementary to the 5‘- and 3’-terminal nucleotide sequences of the mature mRNA. The options for these nucleotides are shown, and the middle of the molecule is not shown. The arrows indicate the possible points of cleavage by processing enzyme(s). However, this does not necessarily imply that these are the first cleavage points in the process. Note the identical nucleotide sequence complementary to the terminal nucleotide sequences in the potential mRNA region and the similar structure of part of two double-stranded hairpin models. A similar model can be drawn for rabbit p-globin mRNA. (From Naora et al., 1978.)
HAIRPIN STRUCTURES IN HnRNA
293
determined experimentally for the dsRNA-derived sequences of rabbit and hen globin mRNA (Ryskov et al., 1976b; Naora et al., 1978). There is another body of evidence demonstrating the terminal repetition of nucleotide sequences. Avian sarcoma viral RNA possesses nucleotide sequences (21 nucleotides long) that are identical at both the 5’- and 3’-ends (Haseltine et al., 1977; Schwartz et al., 1977; Collett et al., 1977). Such repetition may lead to the completion of reverse transcription of the RNA genome and to the circularization of viral DNA (Haseltine et al., 1977; Schwartz et al., 1977). However, it could also be involved in the mechanism whereby the processing of the viral RNA transcribed from proviral DNA takes place within a nucleus in a manner similar to that for mRNA generation from the mRNA precursor molecule. It seems possible therefore that the 5 ’ - and 3’-terminal nucleotide sequences of avian sarcoma viral RNA are derived from an identical form of the double-stranded hairpin structures that are presumably present on the viral RNA precursor molecule.
VII. A Model of Nuclear Processing of HnRNA A, A MODELOF DNA SEQUENCES FROM WHICHHNRNA, DsRNA, AND MRNA ARETRANSCRIBED Figure 4 schematically illustrates the possible DNA sequences from which HnRNA, dsRNA, and mRNA are transcribed. A portion of the DNA sequences includes inverted-repeated sequences that presumably do not possess a long nucleotide sequence between them. HnRNA is a primary transcript from the DNA region which includes these sequences. As mentioned earlier (Section IV, A), the structural genes for /3 -globin and ovalbumin (and presumably some other proteins) are split into fragments and might be expected to contain small palindromes associated with them. If the whole region is transcribed, the transcript of intervening DNA sequence origin, including the double-stranded hairpin structures, should be totally removed during processing (see Fig. 4b). The doublestranded hairpin structure of non-intervening sequence origin would be subject to special cleavage: The middle of the sequences necessary for the double-stranded hairpin structures that are covalently linked to both potential 5‘- and 3’-ends of the message sequence is probably a cleavage point for processing enzyme(s). However, this does not necessarily imply that these are the first cleavage points in the process. The model that we propose here can accommodate the observation that the double-stranded hairpin structures of HnRNA are not preserved in toto during nuclear processing, but only half (or less) of one portion of the sequence necessary for double-stranded hairpin structures is retained during this process
294
HIROTO NAORA
(a)
DNA Non -reDeoted
5'
--- D'E'F'--
E D M'D'E'--
F ED
/ \ fIJ"IJ"uT ----- dxkkw-
.-BWF
m
U
Y
For drRNA
Y
U
FordrRNA
4
Intervening
sequence
Y
For HnRNA
FormRNA
(b)
5'--D
For mRNA
mRNA
Hn RNA
M
F'E:-
-
d,
!* F E D\T'-
N' F' - F B'- B
Double-stronded regions
u r lT ____-
-'
U
7 :
,
*;:I
D X'yX B W L w L F
-- 6D: - F'-(AAAA)
H
H
? Splice ?
p-
F
E D-
ICH
poly(A)
\
F
B
D-AAAA
3'
ICH
/
A portion of double-
poly(A)
rtronded region FormRNA
-
FIG.4. DNA sequences from which HnRNA, dsRNA, and mRNA (message sequences) are transcribed. Letters denote nucleotides which occur in sequence in a particular region and are complementary to nucleotides denoted by letters with primes. represents the regions corresponding to HnRNA, dsRNA, mRNA (message sequences), and intervening sequence (if present). Short, thick arrows show cleavage sites for the generation of mRNA during processing. The HnRNA and mRNA in (b) are transcribed from the region shown in (a).
and transported to the cytoplasm as part of cytoplasmic mRNA molecules at or near their 5'- and 3'-ends (Section VI, C). A typical example is shown in Fig. 3. The model requires that both the poly-A segment, if present, and the cap structure linked to the unprocessed HnRNA molecule be detached during processing and a new poly-A segment and cap structure be added to the 3'- and 5'-ends after and/or during the processing. Recent experiments have revealed that much of the poly-A segment attached to nuclear FWA decays in the nucleus and is not conserved (Perry et al., 1974, 1976; Lewin, 1975a). There is a considerable body of evidence demonstrating that polyadenylation occurs at various times before, during, and after processing in the nucleus and even on the
HAIRPIN STRUCTURES IN HnRNA
295
cytoplasmic polysomes (Derman and Darnell, 1974; Perry et al., 1974; Getz et al., 1975; Spohr et al., 1976; Bastos and Aviv, 1977; see also Perry, 1976). Large 27s HnRNA that contains the globin message sequence lacks a poly-A segment (Bastos and Aviv, 1977). If the poly-A segment is attached to the intermediate precursor RNA after removal of the sequence adjacent to the 3‘terminal region of the potential mRNA sequence, the nuclear poly-A segment will be conserved. Based on the observed similarity of the cap structures of HnRNA and mRNA and kinetic analyses of both cap structures, it has been postulated that some but not all of the cap structures of mRNAs are derived from those of HnRNAs, and that different species of mRNA might be located at different positions within their primary transcripts (Perry et al., 1975; Perry and Kelley, 1976; Perry et al., 1976). However, it is not yet clear to what extent the 5’-tenninal portions of HnRNA are conserved as compared with the cases in which the 3‘-terminal or internal region is excised and then capped (Perry and Kelley, 1976), or whether both the 5’- and 3’-terminal structures of HnRNA are excised and spliced together with the coding sequence during processing. Certainly, more information is required to clarify this point.
FOR THE MODEL:THE DOUBLE-STRANDED HAIRPINSTRUCTURE B. SUPPORT OF MRNA POSSESSES A PROCESSING SITE FOR GENERATION
The idea that the double-stranded hairpin structure possesses a processing site for the generation of mRNA is supported by the following observations: (1) the existence of dsRNA-derived sequences in mRNA; (2) special properties of “processing” enzymes, RNase I11 or similar types of eukaryotic RNases, which attack dsRNA or the processing sites in the double-stranded structures of precursor RNA; (3) in vitro formation of RNA fragments, similar to mRNA in size, from the HnRNA fraction by RNase I11 or similar types of eukaryotic RNases; (4) the finding of an analogous structure in prokaryotic precursor RNA molecules; (5) the inhibitory effect of intercalating agents on the in vitro or in vivo processing of HnRNA; and (6) discrete processing of large precursor molecuIes into small products. As mentioned earlier, the major part of the model is based on the finding of dsRNA-derived sequences in mRNA molecules. This has already been described in Section VI, C. Our knowledge of the sequence arrangements in the doublestranded hairpin structures of HnRNA is limited (Sections V and VI), although some progress has been made in the analysis of nucleotide sequences in the double-stranded region surrounding the processing site of bacteriophage T7 high-molecular-weight RNA molecule using RNase I11 (Section VII, C). An important observation which supports the model is that RNases, specific
296
HIROTO NAORA
for single endonucleolytic cleavage at particular sites involved in doublestranded RNA structures or specific for double-stranded RNA (Robertson et al., 1968; Robertson and Dunn, 1975; Ohtsuki et al., 1977), give limited cleavage of HnRNA in vitro and generate RNA fragments corresponding to mRNA in size (Gotoh et al., 1974; Robertson and Dickson, 1975; Nikolaev et al., 1975; Ohtsuki et al., 1977). For example, when HnRNA from duck reticulocyte nuclei is treated with calf thymus RNase, which preferentially cleaves double-stranded RNA, a population of RNA species varying in size between 8 and 20s is produced, but no acid-soluble material is concomitantly formed. Over 80% of HnRNA is cleaved by this enzyme, and fragments of 21, 16, and 10s accumulate (Ohtsuki et al., 1977). Since 16 and 10s RNA fragments roughly correspond in size to the in vivo cleavage products of HnRNA for globin, and also to cytoplasmic mRNA (Scherrer, 1973; Bastos and Aviv, 1977), the above result suggests that the in vitro fragments of HnRNA molecules generated by dsRNA-specific RNase may represent the intermediate and final forms of the RNA cleaved in nuclei. Similar cleavage of HnRNA from yeast or HeLa cell nuclei is also observed with the E. coli processing enzyme, RNase 111 (Gotoh et al., 1974; Nikolaev et al., 1975; Robertson and Dickson, 1975). It is of interest that specific cleavage of HnRNA is totally inhibited by ethidium bromide (Nikolaev et al., 1975). This observation is quite similar to that of the inhibition by ethidium bromide of specific cleavage of 45s rRNA precursors (Gotoh et al., 1974; Nikolaev et al., 1975; Ohtsuki et al., 1977). All these results further support the view that the double-stranded structures possess cleavage sites for the processing enzyme. In no case, however, is there any compelling evidence that the final in vitro products are actually intact mRNA. In this regard, an analysis of the terminal sequences of the in vitro cleavage products from globin HnRNA would be interesting. Agents, such as ethidium bromide and proflavin, which specifically intercalate between base pairs in helical regions of DNA and RNA (Lerman, 1964; Novogrodsky and Hurwitz, 1966; Waring, 1968; Kersten and Kersten, 1974) strongly prevent the nuclear cleavage of HnRNA in vivo (Brinker et al., 1973). Since an intercalating agent is a potent inhibitor of RNase I11 and similar RNases (Gotoh et al., 1974; Nikolaev et al., 1975; Ohtsuki et al., 1977), this result is in agreement with the view that the double-stranded hairpin structures present in HnRNA in vivo serve as recognition sites for the processing of HnRNA. The putative precursor (27s RNA) of globin mRNA is discretely processed to 15s and subsequently 10s in the nucleus, and the nuclear 10s RNA thus formed enters the cytoplasm (Bastos and Aviv, 1977). Such discrete processing suggests that the mRNA precursor cleaves endonucleolyticallyat specific sites in the large mRNA precursor molecule. A limited number of double-stranded structures in HnRNA (Section V, A) and the presence of endonucleasesin the nucleus specific for dsRNA (Section VIII) favor the above-mentioned view.
HAIRPIN STRUCTURES IN HnRNA
297
C. FEATURES OF DOUBLE-STRANDED HAIRPIN STRUCTURES OF BACTERIOPHAGE HIGH-MOLECULAR-WEIGHT RNA REVEALED BY RNASE111 The processing of mRNA is not peculiar to eukaryotic cells (Perry, 1976). Prokaryotic mRNA undergoes a minimal amount of processing, and the mechanism is probably much simpler than that in eukaryotic mRNA. When bacteriophage T7 infects E. coli, a long transcript (about 7500 nucleotides) which represents the entire early region of the phage genome is primarily formed (Dunn and Studier, 1973a,b; Studier, 1973; Durn et al., 1976). This high-molecular-weight RNA is a polycistronic mRNA precursor and is subsequently cleaved at specific sites by RNase 111, yielding five monocistronic mRNAs (Dunn and Studier, 1973a,b; Dunn etal., 1976; Rosenberg et al., 1975; Robertson and Dunn, 1975). RNase 111 (EC 3.1.4.24) is an E. coli endonuclease which gives a single endonucleolytic cleavage at a specific site involved in dsRNA structures (Robertson et al., 1968; Robertson and Dunn, 1975) and also functions as a processing enzyme in the maturation of primary transcripts of rRNAs (Nikolaev et al., 1973, 1975; Dunn and Studier, 1973b). Little is known about the primary and secondary structures surrounding the cleavage sites at the intercistronic regions of bacteriophage T7 high-molecularweight RNA molecules (Robertson et al., 1977a; Rosenberg and Kramer, 1977). Figure 5 shows the primary and possible secondary structure of the intercistronic region of genes 0.3 to 0.7 (Rosenberg and Kramer, 1977). In this region, cleavage occurs by RNase 111 only on one side of the internal loop located at the middle of a hairpin structure. The monocistronic mRNA molecules thus generated contain the nucleotide sequences UUUAU-OH at the 3'-end of one of the mRNA molecules and pGAU at the 5'-end of another molecule (Rosenberg and Kramer, 1977). The same nucleotide sequences have been identified in mRNA molecules cleaved from the intercistronic region of initiator RNA to gene 0.3, genes 0.7 to 1.O, and genes 1.O to 1.1 (Rosenberg et al., 1975; Robertson et al., 1977a). However, different nucleotide sequences are formed from regions of genes 1.1 to F5 RNA and F5 RNA to 1.3 (Robertson et al., 1977a), and from rRNA precursor molecules (Ginsburg and Steitz, 1975). Bacteriophage T4 species I RNA also generates different nucleotide sequences (Paddock et al., 1976), although these may be represented at secondary cleavage sites (Section Vm).Because of variations in nucleotide sequences surrounding the cleavage site of RNase 111, it seems likely that there is no single nucleotide sequence which uniquely specifies cleavage by the enzyme. Although this does not exclude the possibility that the enzyme requires any one of several different nucleotide sequences for recognition and cleavage, the absolute requirement of specific nucleotide sequences remains unclear at present. Under in vitro conditions, ssRNA can be cleaved at secondary sites, although these sites are not cleaved in vivo (Westphal and Crouch, 1975; Dunn, 1976;
298
HIROTO NAORA A
U
c
c
G
c
C-G U-A C-G G-U C-G U-G G-C G-C A-U A-U
u
U
A
C
d N o s e Ill
A
A
G- U A-U U-A A-U G-C 0.7 R N A
0.3RNA A-U
G-U .~-GCUUUAGAAUCUGCU
UACUUAUGAGGGAGUAAUUU-.~.
FIG.5 . The primary and possible secondary structure surrounding the cleavage site of RNase I11 at the gene 0.3 to 0.7 intercistronic region of bacteriophage T7 high-molecular-weight RNA (moditied from Rosenberg and Kramer, 1977). The cleavage site is shown by the arrow. The left and right sides of the structure are portions of transcripts of genes 0.3 and 0.7, respectively. Note that cleavage occurs at a point half way along the structure.
Dunn et al., 1976; Paddock et al., 1976). Nonspecific cleavage of RNA-RNA duplexes also occurs in vitro, yielding fragments with random end groups (Robertson et al., 1968; Schweitz and Ebel, 1971; Robertson and Dunn, 1975). It should be mentioned, however, that a specific form of double-stranded structure surroundingthe cleavage site may be important for correct recognition by the enzyme. Comparison of the possible secondary structures of different intercistronic regions of bacteriophage T7 high-molecular-weight RNA shows remarkable similarity in structure (Robertson et al., 1977a; Rosenberg and Kramer, 1977). This structure consists of a double-stranded hairpin form with five unpaired residues on the loop (Rosenberg and Kramer, 1977). The secondary structure of F5 FWA derived from the region between genes 1.1 and 1.3 by double cleavage in vitro resembles the structure of the “upper” stem of the hairpin present in the region of genes 0.3 to 0.7 (Fig. 5) (Robertson et al., 1977a). In vivo, most of the F5 RNA fragments remain attached to the 3’-end (Dunn, 1976). In the structures in these two regions, only 4 of the 24 nucleotides, excluding the regions of the hairpin loops, are different. Both double-stranded stems contain identical nucleotide sequences, and both cleavage sites are located 13 nucleotides below the top nucleotide pairs of the stem (Robertson et al., 1977a; Rosenberg and Kramer, 1977). However, significant differences are noted in nucleotide sequences of unpaired loops and cleavage sites.
HAIRPIN STRUCTURES IN HnRNA
299
Since no dsRNA fragment shorter than approximately 13 to 15 nucleotide pairs can be cleaved by RNase I11 (Crouch, 1974; Robertson and Dunn, 1975), a certain length of hairpin structure must be required. In the region of genes 0.3 to 0.7, cleavage occurs in the middle of a hairpin structure consisting of a total of 19 nucleotide pairs of two stems and 9 unpaired nucleotides of one internal loop. An interesting feature is the symmetric location of a special nucleotide sequence, GAGUG, in both the “upper” and “lower” stems of the hairpin structure. An attractive speculation would be that RNase 111, a dimeric protein (MW 50,000) of identical subunits (MW 25,000) (Dunn, 1976), may also require the symmetric feature of the double-stranded region for recognition and cleavage. We recall here that cleavage or processing occurs at the middle of the doublestranded hairpin structure of HnRNA (Sections VI, C and VII, A). Although the total length of the hairpin structure of HnRNA is much longer than that of bacteriophage T7 high-molecular-weightRNA (Section 111, H), the similarity of these structures suggests that the special arrangement of a certain length of double-stranded structures surrounding the cleavage site may be biologically important to processing events. There are several cases indicating that many double-stranded hairpin structures lack the features required for specific cleavage by RNase 111 (Simon and Studier, 1973; Dunn and Studier, 1975; Dunn, 1976; Branlant et al., 1976). It is conceivable therefore that double-stranded hairpin structures with a certain length of stem may be required for recognition but may not be sufficient for specific cleavage by RNase 111.
VIII. Occurrence of Eukaryotic RNases Specific for dsRNA The idea that the double-stranded hairpin structures of HnRNA molecules are recognized by nuclear processing enzyme@)as cleavage sites is supported by the discovery of nuclear enzymes similar to E. coli RNase III. A RNase which preferentially cleaves dsRNA-hereafter called dsRNasehas been isolated from several mammalian sources (Table 111). Purification of some dsRNases has been attempted by gel filtration, ion-exchange, or affinity chromatography andor glycerol gradient centrifugation. Among the enzymes so far isolated, the dsRNAses described by Ohtsuki et al. (1977) and Hall and Crouch (1977) have been most scrupulously characterized. Table 111 shows characteristicsof the dsRNases isolated from mammalian materials and compares them with those of E. coli RNase 111. The dsRNase purified from calf thymus by Ohtsuki et al. (1977) is free of detectable DNase and RNase H, but some of the enzymes described by others show activity capable of degrading RNA from RNA-DNA duplexes (Busen and Hausen, 1975; Rech et a l . , 1976; Hall and Crouch, 1977). ssRNA also serves as
TABLE III. CHARACTERISTICS OF EUKARYOTIC Product Length of PolY(C) 'PolY(1) 01
Enzyme source
Type of activity"
Molecular weight
Intncellular localization
HeLa cells
Endo
Nucleus
Human KB cells (RNase NU)
Endo
Cytoplasm (ribosomes)
Burkm's lymphoma, macrophage, lung, spleen
Requirement for ion Nai or Ki
MnPi or Mg2+
+
3'-P
Cytoplasm (membranebound endoplasmic reticulum)
Human and whale pancrease
30,000
Calf thymus (enzyme fraction Ila)
30.000
Calf thymu\
Terminus
PolY(C) .poly(G) cleaved (no. of nucleotides)
Endo
60.000
Nucleus
Krebs I1 ascites cells
Cytoplasm
Krebs I1 ascites cells (RNase D)
Cytoplasm (cytosol)
5'-P
10
+
+
-
t
+
+
Chicken embryo Nuclease DI
Endo
60,00062.500
Cytoplasm
5'-P
11-12
Nuclease D11
Endo
38,00040,000
Nucleus
5'-P
3
+
+
Escheri
Endo
45,00055,000; 50,000 (dimer)
Ribosomes
5'-P
13-15
+
+
"Endo" means endonucleolytic activity.
?M
I
dsRN ases COMPARED WITH E. coli RNase I11
Substrate
ssRNA
dsRNA
RNADNA duplex
Others
Specific cleavageb E . coli 30s pre-rRNA.
HeLa cell
Reference B u g and Schlssinger,
1974; Nikolaev er a / . . 1975
pre-rRNA (Probably attack singlestranded region bounded by double-smnded structure)
+
8s bacteriophage
( E . co/i tRNA" ' precursor)
A transcript.
80 M3 RNA. bacteriophage T7 RNA
Altman er a / . . 1975; Bothwell and Altman,
1975
Stern and Wilcrek. 1973
Bardon e r a /. . 1976
Busen and Hausen. 1973
i
+
i
45s pre-rRNA,
Ohtsuki
el
al.. 1977
reticulocyte HnRNA
+
+
Almost (trace)
-I
+
Rech
+
i
Hall and Crouch. 1977
Hall and Crouch, 1977
Robertson and Mathews,
I973
+
el
a / . . 1976
(but PI)
+
i
+
+
t
+
45s pre-rRNA. HnRNA, bacteriophage Tl RNA
(specific)
Robenson er a/..1% Crouch, 1974; Gotoh era/.. 1974; Nikolacv er d.,1975; Robertson and D ~ M .
1975; Darlir, 1975; Robertson and Dickson.
1975; Dunn, 1976; Robenson c r d . . 19771; Rosenberg and Kramer,
I977
*Enzymes cleave at the specific sites of natural RNAs, yielding fragments similar or identical in size or in other criteria to the in vivo products. pre-RNA, rRNA precursor.
30 I
302
HIROTO NAORA
a substrate for most purified dsRNases, even after intensive purification by column chromatography on hydroxyapatite, phosphocellulose, DEAESephadex, CM-cellulose, poly (U)-Sepharose and poly(C) - poly(1)-Sepharose and/or by repeated glycerol gradient centrifugation (Biisen and Hausen, 1975; Ohtsuki et al., 1977; Hall and Crouch, 1977). The enzyme described by Rech et al. (1976) may be exceptional, as it shows a very low level of activity against ssRNA. In some cases, two enzyme activities can be differentiated. For example, enzyme activity against dsRNA is more sensitive to N-ethylmaleimide, an inhibitor of sulfhydryl enzymes, and also to high temperature (SOOC), than that against ssRNA (Ohtsuki et al., 1977), At present it is unclear whether the two activities are due-to separate enzyme proteins or to a single enzyme molecule possessing both types of RNase activity. RNase I11 is known to exhibit a general specificity for dsRNA (Robertson et al., 1968; Robertson and Dunn, 1975; Dunn, 1976; Dunn ef al., 1976). At low enzyme and moderate salt concentration, the enzyme recognizes particular nucleotide sequences in the double-stranded structures of bacteriophage T7 highmolecular-weight RNA (Section VII, C)-hereafter called primary sites-and cleaves them to fragments similar to those of in vivo products. ssRNA can also be cleaved by this enzyme at the secondary cleavage sites under in vitro conditions that include a high enzyme/substrate ratio and low ionic strength (Dunn, 1976). Since dsRNA inhibits cleavage in ssRNA by RNase III, it appears likely that the specific cleavage sites in ssRNA may have a double-stranded character (Dunn and Studier, 1973b; Robertson and Dunn, 1975). It seems possible that the RNA structure may determine the primary and secondary cuts by RNase I11 or that the salt concentration affects the enzyme directly, causing a change in its conformation or in its monomer-dimer equilibrium with resulting changes in its specificity (Dunn, 1976). RNase I11 is characterized by an absolute requirement for divalent cations, including Mg2+ and Mn2+, and for monovalent cations, including NH4+, K+, and Na+ (Robertson et al., 1968). Furthermore, this enzyme cleaves RNA chains to yield 5 '-phosphate and 3'-hydroxyl termini (Robertson and Dunn, 1975). Since HnRNA processing takes place discretely in the nucleus (Bastos and Aviv, 1977), the processing enzymes(s) have to reside in the nucleus and be mainly endonuclease(s). Obviously, dsRNases from semen (Libonati and Floridi, 1969; Libonati et al., 1975) and serum (Stem, 1970; Norlund et al., 1970; Torrence et al., 1973; Bardon et al., 1976) are not included in the processing of HnRNA. The dsRNases isolated from calf thymus (Ohtsuki et al., 1977), chicken embryos (particularly nuclease DII) (Hall and Crouch, 1977), and HeLa cells (Birge and Schlessinger, 1974; Nikolaev et al., 1975) are of nuclear origin and may be possible candidates for processing activity. RNase NU (endonuclease attacking unstable RNA) isolated from human KB cells exists in ribosomes (Altman et al., 1975; Bothwell and Altman, 1975), and RNase D is prepared
HAIRPIN STRUCTURES IN HnRNA
303
from cytosol of Krebs I1 ascites cells (Rech et al., 1976). In the latter case, however, it is highly possible that the enzyme resides in the nucleus but readily leaks out of the nucleus when nuclei are exposed to the low salt of the medium (Rech et al., 1976). Thus RNase D may also be a strong candidate for processing activity. One type (nuclease DI) of dsRNase from chicken embryos seems to be of cytoplasmic origin and shows a low level of ability to hydrolyze dsRNA, suggesting that it may not be a processing enzyme (Hall and Crouch, 1977). It is not known whether some RNases exhibiting enhanced activity against dsRNA, as compared to ssRNA (Bardon et al., 1976), are processing enzymes. Many mammalian dsRNases show a strong resemblance to E. coli RNase 111. This includes a preference for dsRNA, a requirement for monovalent and divalent cations, and an endonucleolytic mode of action which generates 5‘phosphate termini (Table 111). The dsRNase purified from calf thymus (Ohtsuki et al., 1977) cleaves both native and synthetic dsRNAs endonucleolyticallyin a manner quite similar to that of E. coli RNase I11 with regard to its ionic requirement. RNase NU from human KB cells also cleaves at specific sites on natural RNAs, for example, 8s bacteriophage X transcript, @ 80 M3 RNA, and bacteriophage T7 RNA, but the divalent cation requirement and mode of action are different from those of E. coli RNase 111 and other mammalian dsRNases (Altman et al., 1975; Bothwell and Altman, 1975). Some dsRNases isolated from mammalian cells attack natural RNAs, such as 45s rRNA precursor and HnRNA, yielding RNA fragments corresponding to the respective rRNAs and “mRNA” in size in a manner similar to that of E. coli RNase 111. Much of this activity is inhibited by ethidium bromide (Section VII, B). These results certainly favor the view that some of the mammalian dsRNases listed in the table are involved in the processing of precursor RNAs. Of course, their relationship to the in vivo mechanism remains to be shown. It is of biological interest that specific cleavage, at least as far as size is concerned, of mammalian precursor RNAs occurs both by E. coli RNase I11 and mammalian dsRNases. This probably implies that the biochemical properties of the specific cleavage sites of precursor RNAs are preserved throughout evolution and are more or less common in various types of RNA molecules. There are other classes of processing enzymes, including “trimming, ” “scavenger,” and tRNA maturation enzymes. Since these types of enzymes are not within the scope of this article, the reader should refer to other publications (Winicov and Perry, 1975; Sierakowska and Shugar, 1977).
IX. Summary and Concluding Remarks
As this article shows, the double-stranded hairpin structures of HnRNA have provided valuable information on the possible precursor-product relationship
304
HIROTO NAORA
between HnRNA and mRNA. Much of the work described here has been done over the past several years, and intensive investigation continues. However, we have realized the existence of gaps in our factual knowledge of the functions of dsRNA in normal, uninfected eukaryotic cells. These gaps are replaced here with a hypothesis. It has been necessary to consider much unpublished, and therefore unconfirmed, data to construct this hypothesis. There is considerable evidence to suggest a possible precursor-product relationship of HnRNA and mRNA, although further conclusive experiments are required. Because of rapid technological progress, it has been possible to isolate successfully an HnRNA that contains a specific message sequence and is larger than cytoplasmic mRNA. This molecule is a putative mRNA precursor which must be cleaved during processing in the nucleus for the generation of mRNA. However, this does not imply that all HnRNAs present in the nucleus are precursors of cytoplasmic mRNA. In this article, emphasis is placed on the possible role of double-stranded structures in the nuclear cleavage of HnRNA. There is now ample evidence to demonstrate that most if not all HnRNA molecules have double-stranded hairpin structures from which dsRNAs are derived. The role of these structures is hypothesized as follows: The middle of the sequences necessary for the doublestranded hairpin structures covalently linked to the potential 5’- and 3’-ends of the message sequences of the mRNA precursor molecule is a cleavage point for the nuclear processing enzyme(s). Hence approximately half of one portion of the sequence necessary for double-stranded hairpin structures is retained during processing. Several properties of dsRNA, mRNA, and HnRNA support this model. However, there may be double-stranded hairpin structures different from those shown in the model. It would be of biological interest to determine whether the DNA sequences intervened in the structure gene are actually transcribed to the mRNA precursor molecule, and whether the primary transcript contains the doublestranded hairpin structures that are of intervening DNA sequence origin and may differ in nature. The role of double-stranded hairpin structures may not be confined to nuclear cleavage of the HnRNA molecule. For example, HnRNA is associated with a nuclear structure through the double-stranded structures (Herman et al., 1976b). Alternatively, the double-stranded hairpin structures or products of these structures may become active after nuclear cleavage and play a regulatory role in gene expression and/or replication. It has been postulated that some regulatory RNAs are present in the nucleus (Frenster, 1976; Dickson and Robertson, 1976; Maclean and Hilder, 1977). Another possibility is that the sequences involved in the structures are transcripts of DNA sequences of regulatory loci which are recognized by chromosome or nuclear components for structural organization or by RNA polymerases or regulatory molecules. A detailed model is not yet available for this function. In this article, these possibilities were not discussed.
HAIRPIN STRUCTURES IN HnRNA
305
Several of the problems arising from the model of possible signals for nuclear processing await resolution. These problems and other questions are considered in the relevant sections of this article. Extrapolation into the future will become easier when the preparations of single species of double-stranded structures and purified nuclear processing enzyme(s) are available. The model given in this article is intended to be a guide to further experimentation, and it is our hope that it will provide motivation for future investigations.
ACKNOWLEDGMENTS
The author wishes to thank his colleagues, Dr. N. J . Deacon and Mrs. K . E. Fry, for their valuable comments on the manuscript. The excellent and untiring assistance of MIS. E. McGahey and Miss G. M. Philip in preparing the manuscript is gratefully acknowledged.
Addendum (June 1978) Since this article was completed, several reports have been brought to my attention. It was found that dsRNA is highly resistant to digestion by RNase A in high salt, but sensitive in low salt in which the secondary structure of dsRNA is still maintained (Libonati and Palmieri, 1978). Therefore, the secondary structure is probably not the only important factor in determining the susceptibility of dsRNA to RNase A, but also the effects of salt on the enzyme molecule and/or the binding of enzyme to RNA may be involved in the degree of dsRNA digestion. The size distribution on gels and differential melting profiles of the dsRNA preparations isolated from various tissues of different organisms show speciesspecific, but not tissue-specific, patterns (Naora and Fry, 1978), correlating well with the results obtained in hybridization experiments (Section 111, I). It is now known that many genes, such as genes for immunoglobulin, globin, ovalbumin, SV40, polyoma virus, adenovirus, Rous sarcoma virus, murine leukemia virus, tRNA, and rRNA, possess intervening sequences and consist of discontinuous segments (Gilbert, 1978; Marx, 1978; Doolittle, 1978). For example, mouse p-globin gene contains at least one (550 nucleotide pairs long) and possibly two other intervening sequences (Tilghman et al., 1978a). It is of interest that these intervening sequences are transcribed into mRNA precursor and excised during nuclear processing (Tilghman et al., 1978b; see Marx,1978). This finding raises an interesting question Concerning a specific signal for excision and ligation on the HnRNA molecule (Tonegawa et al., 1978). However, more information is required before any conclusion is made concerning excision and ligation. The intervening DNA sequences of the ovalbumin gene have been further characterized (Weinstock etal., 1978; Lai etal., 1978). As menticned in
306
HIROTO NAORA
Section IV, A, it is now expected that a putative mRNA precursor for ovalbumin mRNA is in fact larger than cytoplasmic mRNA. More observations have been made concerning the size of HnRNA that contains specific message sequences (Section IV, A). Large HnRNAs, i.e., 28s and 16.5s RNA that contain globin mRNA sequence and 40s RNA that possesses immunoglobulin mRNA sequence, are subject to the stepwise cleavage and processing in the nucleus (Strair et al., 1977; Gilmore-Hebert and Wall, 1978). In another experiment, however, larger than 16s HnRNA that contains globin mRNA sequence was not detected (Ross and Knecht, 1978; Ross, 1978). The notion that all eukaryote mRNA is monocistronic no longer seems to be correct. Evidence has been presented that some cytoplasmic mRNA contains more than one message sequence (Chen and Spector, 1977; Roberts and Herbert, 1977). Comparison of nucleotide sequences between the intercistronic region of polycistronic mRNA and the portions that are subject to cleavage during nuclear processing would be very interesting.
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INTERNATIONALREVIEW OF CYTOLOGY, VOL. 56
Microchemistry of Microdissected Hypothalamic Nuclear Areas M. PALKOVITS First Department of Anatomy, Semmelweis University Medical School, Budapest, Hungary
I. Introduction . . . . . . . . . . . . . . . . .
. . .
II. Microdissection of the Hypothalamus . . . . . . . . . . . A. Topography of Major Hypothalamic Regions . . . . . .
B. Topography of Individual Hypothalamic Nuclei . . . . . . UI. Microchemistry . . . . . . . . . . . . . . . . . . . A. Putative Neurotransmitters and Their Related Enzymes . . . B. Neuronal Peptides (Neurohonnones) . . . . . . . . . . IV. General Considerations . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .
315 315 315 318 320 321 329 335 336
I. Introduction The hypothalamus has been considered to be the source of neuronal peptides, hormones, and neurotransmitters that influence the pituitary gland and other target tissues outside the brain. Unfortunately, neither a generally acceptable nomenclature nor a detailed topographical description of the hypothalamic structures has been developed as yet. Several important technical advances have enabled us to visualize the cells producing the hormones and transmitter amines. It has also become possible to measure concentrations of neuronal peptides and neurotransmitters, as well as the activity of several enzymes, in small pieces of tissue no larger than an individual hypothalamic cell group. This development of biochemical and microdissecting techniques calls for further topographical knowledge of the hypothalamus. Therefore, this article includes a detailed description of the hypothalamic areas and the topographical distribution of various substances measured in microdissected hypothalamic nuclei.
11. Microdissection of the Hypothalamus A. TOFQGRAPHY OF MAJORHYFQTHALAMIC REGIONS The hypothalamus can be divided into three parts: anterior, middle, and posterior (Fig. 1). Although the preoptic region and the mammillary body actually 315 Copyright @ 1979 by Academic Press, Inc. Ail rights of feeproduction in any form reserved. ISBN 0-12-364356-2
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FIG. 1. Schematic drawings of the rat brain and hypothalamus. (A) Sagittal section of the brain. The preoptic area and hypothalamic regions are demarcated. (B-D) Frontal sections of the hypothalamus at various distances (A, rostral; P, caudal) from the bregma (B) level (0 pm). A, Anterior hypothalamus: medial part ( E ) and lateral parts ( M ); M, middle part of the hypothalamus: medial-basal hypothalamus ( ), medial-dorsal hypothalamus ( %), and lateral parts ( 3 ); P, posterior hypothalamus: medial part ( I l l 1 1 ) and lateral parts ( ). PO, preoptic area; MB, mammillary body; OB, olfactory bulb, OT, olfactory tubercle; CC, corpus callosum; S, septum; F, fornix;AC, anterior commissure; Th, thalamus; ME, mesencephalon; MO, medulla oblongata; CP, posterior commissure; CS, superior collicle; CI, inferior collicle; IC, internal capsule; MFB, medial forebrain bundle; OC, optic chiasm; TO, optic tract; ME, median eminence; FM, fasciculus mammillothalamicus; S, pituitary stalk.
are not part of the hypothalamus, they are included in several descriptions oft. organ. Microdissection of the major hypothalamic regions can be performed on both fresh and- frozen brain. Coronal sections can be cut either by free hand on the surface of a hard but nonrigid (rubber) black sheet or with the aid of a cryostat at a temperature of -10°C. The rostral (preoptic) border of the hypothalamus is approximately 0.5 mm
317
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behind the bregma level in 150- to 200-gm rats. [The bregma level using as 0 p m corresponds to the coronal level, passing through the bregma, perpendicularly on the skull, bisecting the crossing fibers of the anterior commissure (Fig. lA)]. The anterior hypothalamus extends caudally as far as the caudal edge of the optic chiasm (Fig. 1A). This region can be divided, by a vertical section passing through the fornix, into a medial and two lateral parts (Fig. 1B). The medial portion contains the anterior hypothalamic, suprachiasmic, and magnocellular periventricular nuclei, since the cell and fibers of the medial forebrain bundle and the supraoptic nuclei occupy the lateral part of the anterior hypothalamus. The paraventricular nucleus is located partly in the anterior and partly in the middle portion of the hypothalamus. The middle portion of the hypothalamus starts from the caudal edge of the optic chiasm, rostrally, and ends at the level of the separation of the pituitary stalk (3.5 mm behind the bregma), caudally (Fig. 1A). A vertical cut through the fornix and the mammillothalamic tract divides into the middle hypothalamus medial and lateral parts (Fig. 1C). The medial portion can be further divided, by a horizontal cut bisecting the third ventricle, into a basal and a dorsal portion (Fig. 1C). The basal part, called the medial-basal hypothalamus, contains the median eminence, the retrochiasmatic area, and the arcuate and ventromedial nuclei as well. The medial-dorsal part is occupied by the paraventricular and dorsomedial nuclei, while the medial forebrain bundle and the perifornical nucleus can be localized in the lateral part of the middle hypothalamus. The posterior hypothalamus is relatively small and is bordered by the mammillary body caudally (Fig. 1A). Its medial part contains the caudal portions of the arcuate nucleus, as well as the ventral and dorsal premammillary and posterior TABLE I
WEIGHTSOF THE HYPOTHALAMIC REGIONSOF 200 f 10 gm R A T S ~
Region
Total hypothalamus Anterior hypothalamus
Medial Lateral Middle hypothalamus Medial-basal Medial-dorsal Lateral Posterior hypothalamus Medial Lateral
Mean ? SEM (mg) 18.77 7.50 3.26 4.24 7.35 1.60 1.67 4.08 39.2 1.91 2.01
rf-
2
2 2 rf-
%
2 %
? ?
rf-
0.58 0.45 0.34 0.32 0.38 0.14 0.21 0.35 0.36 0.29 0.29
Percentage of total hypothalamus
Percentage of major regions
40.0
-
7.4 22.6
43.5 56.5
39.2
-
8.5 8.9 21.7 20.8 10.2 10.6
21.8 22.1 55.5
"Length of the sagittal suture (bregma-lambda distance) = 0.72
?
-
48.8 51.2 0.05 mm; n = 10.
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M. PALKOVITS
hypothalamic nuclei, while the lateral part is practically equal with the territory of the medial forebrain bundle (Fig. 1D). The wet weights of the total hypothalamus of 200 k 10 gm rats are approximately 18-19 mg. The weights of various hypothalamic regions are summarized in Table I.
B. TOFQGRAPHY OF INDIVIDUALHYPOTHALAMIC NUCLEI Recent developments of microdissecting techniques (like the so-called punch technique) have allowed individual hypothalamic nuclei to be removed for per-
FIG.2. Topographical distributions of hypothalamic nuclei in a sagittal view (looking from the third ventricle). Coordinates in millimeters (200 2 10 gm live rats). Vertical axis = bregma level; horizontal axis = horizontal plane through the interventricular line with a 5" nose-down position of the head. OC, Optic chiasm; AC, anterior commissure; F, fornix; FM, fasciculus mammillothalamicus; F'G, pituitary gland; MB, mammillary body; NIST, nucleus interstitialis striae terminalis; NPOm, medial preoptic nucleus; NPE, penventricular nucleus; NPM, magnocellular periventricular nucleus; NSC, suprachiasmatic nucleus; NSO, supraoptic nucleus; NHA, anterior hypothalamic nucleus; NPV, paraventricular nucleus; NVM, ventromedial nucleus; NPF, perifornical nucleus; NDM, dorsomedialnucleus; NA, arcuate nucleus; NHP, posterior hypothalamic nucleus; NPMD, dorsal premammillary nucleus; NPMV, ventral premammillary nucleus; NSM, supramammillary nucleus.
319
MICRODISSECTED HYPOTHALAMIC NUCLEAR AREAS
I
I
I
I
I
a
9 a
FIG.3. Rostrocaudal extensions of the hypothalamic nuclei, of their subdivisions, and of the major hypothalamic tracts. Bregma level = 0 pm. A, Rostral; P, caudal to the bregma. For abbreviations see legend for Fig. 2.
forming microassays of various substances (Palkovits, 1973, 1975). The brains are cut in a cryostat at - 10°C, or with a modified tissue chopper (Zigmond and Ben-An, 1976) or with a vibratome (Jacobowitz, 1974). The nuclei are removed from the serial frontal sections using stainless-steel cannula (Palkovits, 1973; Brownstein et al., 1976~).The inside diameters of the cannula depend on the size of nuclei to be removed. Cannula of 0.2, 0.3, 0.5, and 1.0 mm inside diameter are generally used. Tissue pellets punched out from 300-pm thick sections weigh 10, 20, 60, and 230 pg, respectively (Palkovits, 1973). In regard to the brain the term “nucleus” refers to an area where the density of cells is higher than in the adjacent regions. Based on this definition, 16 hypothalamic nuclei can be distinguished (Palkovits, 1975). Their topographical distributions in the rat are shown in Fig. 2 to give a three-dimensional impression of their size, shape, and localization. According to the different types of cells within hypothalamic nuclei, they can be subdivided into groups which have been detailed elsewhere (Palkovits, 1975). The rostrocaudal extensions of hypothalamic nuclei and their subdivisions are summarized in Fig. 3. (The coexistence of section profiles of hypothalamic nuclei in certain coronal sections of the hypothalamus can be easily determined in the figure.) The sectioning of the brain and the necessary steps in the precise dissection of the nuclei have been detailed previously (Palkovits et al., 1974b; Palkovits, 1975).
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111. Microchemistry
During the last few years, several sensitive, specific microassays for measuring neuronal peptides, neurotransmitters, and related enzymes have become available. Biochemical microtechniques sensitive enough to detect substances in less than 1 mg of brain tissue are suitable for studies on hypothalamic nuclei. The recently developed enzymic isotopic techniques, radioimmunoassays, and mass fragmentography have been proved to be the most suitable for this purpose. There is an increasing number of sensitive, specific microassays for the determination of various substances in the central nervous system. In Table I1 miTABLE I1 MICROASSAYS FOR INDIVIDUAL HYPOTHALAMIC NUCLEI
Substance NE DBH
E PNMT DA TH Serotonin Tryptophan hydroxylase Histamine Histamine N-methyltransferase COMT MA0
Studies on the hypothalamic distribution of various substances Palkovits et al. (1974b) Versteeg et al. (1976) Saavedra et al. (1974a) van der Gugten et at. (1976) Saavedra et al. (1974b) Palkovits et al. (1974b) Versteeg et al. (1976) Saavedra et al. (1974a) Saavedra et al. (1974~) Brownstein et al. (1975d) Brownstein et al. (1974b) Saavedra et al. (1976) Saavedra et al. (1976) Saavedra et al. (1976) Hirano ef at. (1975)
ACh CAT GABA GAD LH-RH Somatostatin CRF Vasopressin Oxytocin TRH Substance P Neurotensin Enkephalins
Brownstein et al. (1975b) Tappaz et al. (1977) Tappaz et al. (1976) Palkovits et al. (1974a) Brownstein et al. (1975a) Palkovits et al. (1976a) Lang et al. (1976) George and Jacobowitz (1972) George et al. (1976) Brownstein et al. (1974a) Brownstein et al. (1976b) Kobayashi et al. (1977) Kobayashi et al. (1975)
Microassays Henry et al. (1975) Cuello et al. (1973) Molinoff et al. (1971) van der Gugten et al. (1976) Axelrod (1962) Henry et al. (1975) Cuello et al. (1973) Coyle (1972) Saavedra et al. (1973) Kizer et al. (1975) Taylor and Snyder (1972) Taylor and Synder (1972) Axelrod and Cohn (1971) Wurtman and Axelrod (1963) Goldberg and McCaman (1973) Hanin and Schuberth (1974) Bull and Oderfeld-Novak (197 1) Tappaz et al. (1977) Albers and Brady (1959) Arimura er al. (1973) Arimura e l al. (1975) Lang et al. (1974) George et al. (1972) George et al. (1976) Bassiri and Utiger (1972) Powell et al. (1973) Brown et at. (1977) Miller et al. (1975)
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32 1
croassavs first used for determining substances in individual hvpothalamic nuclei are summarized. (The table also includes a list of the original descriptions.) Of course, since that time several other new microtechniques have been published in the literature. The wet weight of the tissue samples from hypothalamic nuclei vary between 60 and lo00 p g in that rat. Such a small amount of brain tissue can be measured directly only with difficulty. Therefore determination of the protein content of tissue samples seems to be more feasible (Lowry et al., 1951). Concentrations or activities of substances measured can be expressed as a function of the protein content.
A. PUTATIVE NEUROTRANSMITTERS AND THEIRRELATED ENZYMES Neurotransmitters measured by radioenzymic microassays are widely distributed throughout the hypothalamus. 1. Norepinephrine (NE) NE concentrations in hypothalamic nuclei were measured by using modifications (Palkovits et al., 1974b; van der Gugten et al., 1976) of the enzymic isotopic assays described by Cuello et al. (1973) and Henry et al. (1975). Compared to other brain regions the hypothalamus is rich in NE (20-90 pg/mg protein). It is unevenly distributed throughout the hypothalamic nuclei (Fig. 4), the paraventricular, periventricula, and dorsomedial nuclei having higher con-
FIG. 4. NE concentrations in hypothalamic nuclei (Palkovits et al.. 1974b; Versteeg et al., 1976). Sagittal drawing of the hypothalamus, I, Very high concentrations; m ,High concentrations; %, Moderate concentrations; +:.: , low concentrations. OC, Optic chiasm; AC, anterior commissure; TH, thalamus; MB, mammillary body; A, pars distalis; I, intermediate lobe; N, pars nervosa. 1, Median eminence; 2, arcuate nucleus; 3, ventromedial nucleus; 4, dorsomedial nucleus; 5 , medial preoptic nucleus; 6, nucleus interstitialis striae terminalis; 7, suprachiasmatic nucleus; 8, anterior hypothalamic nucleus; 9, supraoptic nucleus; 10, periventricular nucleus; 1 1, paraventricular nucleus; 12, retrochiasmatic area; 13, perifornical nucleus; 14, posterior hypothalamic nucleus; 15, dorsal premammillary nucleus; 16, ventral premammillary nucleus; 17, medial forebrain bundle. Abbreviations, numbers, and concentration symbols used here apply to Figs. 5-22 also.
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centrations (Palkovits et al., 1974b; Versteeg et al., 1976). The hypothalamus itself does not contain NE-synthesizing cell bodies, only noradrenergic nerve fibers and terminals originating from the NE cells in the lower brainstem (Dahlstrom and Fuxe, 1964). After total surgical isolation of the hypothalamus, most of the NE disappears from the deafferentated hypothalamic nuclei (Brownstein et al., 1976d; Palkovits et al., 1976b, 1977).
2 . Dopamine-P-hydroxylase (DBH) This enzyme is characteristic of the biosynthetic pathway of NE.DBH activity as measured by the assay of Molinoff et al. (1971) is high in hypothalamic nuclei where NE is present in high concentrations (Fig. 5). A slight correlation between DBH activity and NE concentration can also be observed in other hypothalamic nuclei (Saavedra et al., 1974a). 3 . Epinephrine Concentrations of epinephrine in hypothalamic nuclei are more than 10 times less than NE concentrations, although hypothalamic nuclei contain epinephrine in the highest concentrations in the central nervous system (van der Gugten et al., 1976). The distribution of epinephrine is uneven throughout the hypothalamus (Fig. 6). The highest concentrations are found in the periventricular and dorsomedial nuclei, while in others (suprachiasmatic, anterior and posterior hypothalamic, premammillary, ventromedial, perifornical nuclei) the possible concentrations of the epinephrine are less than the sensitivity of the assay used (0.2 pg/pg protein).
4. Phenylethanolamine N-Methyltransferase (PNMT) The conversion of NE to epinephrine is catalyzed by PNMT which shows high activity in the brain regions (the C1 and C2 regions in the medulla) where epinephrine-containing cell bodies have been demonstrated by immunohistochemical technique (Hokfelt et al., 1974). Epinephrine and PNMT are located
TH
FIG. 5 .
,+'
DBH activity in hypothalamic nuclei (Saavedra ef al., 1974a).
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FIG. 6 . Epinephrine concentrations in the hypothalamic nuclei (van der Gugten et al., 1976).
in nerve fibers and endings within the hypothalamus where PNMT activity can be measured by the technique of Axelrod (1962). The uneven distribution of PNMT activity in hypothalamic nuclei is demonstrated in Fig. 7 (Saavedra et al., 1974b).
5 . Doparnine (DA) DA levels are particularly high in the median eminence and arcuate nucleus of the hypothalamus (Palkovits etal. , 1974b; Versteeg et al., 1976) as measured by enzymic isotopic assays. Relatively high DA levels are found in the dorsomedial nucleus and in the retrochiasmatic area (Fig. 8). Part of the DA found in the hypothalamus is present in cell bodies in the arcuate and periventricular nuclei (the A 12 catecholaminergic cell group) (Dahlstrom and Fuxe, 1964; Fuxe, 1965), as well as within and in the vicinity of the dorsomedial and posterior hypothalamic nucIei (the A1 1 and A13 cell groups) (Bjorklund and Nobin, 1973; Jacobowitz and Palkovits, 1974). Axons arising from dopaminergic cell bodies in the A13 cell group (incertohypothalamic DA system) probably terminate in the dorsal regions (paraventricular and dorsomedial nuclei) of the hypothalamus (Bjorklund et al., 1975; Palkovits et al., 1977a), while the A12 DA cells innervate the median eminence and other hypothalamic cell groups.
FIG. 7 . PNMT activity in the hypothalamic nuclei (Saavedra et al., 1974b).
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FIG. 8. DA concentrations in the hypothalamic nuclei (Palkovits et al., 1974b; Versteeg et al., 1976).
6 . Tyrosine Hydroxylase (TH) TH is a rate-limiting enzyme involved in the synthesis of NE and DA. The enzyme, as measured by the technique of Coyle (1972), is present in hypothalamic nuclei containing DA cell bodies and in nuclei where only catecholaminergic nerve terminals are found (Fig. 9). This finding is in a good agreement with studies obtained by electron microscope autoradiography (Pickel et al., 1975) in which TH was localized on the membranes of the endoplasmic reticulum and the Golgi apparatus in the cell bodies and in neurotubules of nerve fibers and terminals. The highest TH activity in the hypothalamus is found in the median eminence (Saavedra et al., 1974a). It has also been reported that the enzyme activity in the intermediate lobe of the pituitary gland (Fig. 9) is also relatively high (Saavedra et al., 1975). 7. Serotonin Like catecholamines, serotonin is found in all hypothalamic nuclei, but in different concentrations (Saavedra et al., 1974~).It is present in high concentrations in the arcuate, suprachiasmatic, and perifornical nuclei (Fig. 10) and also in the medial forebrain bundle, which contains serotonin fibers traveling through the lateral hypothalamus to more rostral brain areas. Serotonin-containing cells
,.;/
FIG.9.
TH activity in the hypothalamic nuclei (Saavedra et al., 1974a).
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FIG. 10. Serotonin concentrations in the hypothalamic nuclei (Saavedra et al., 1974).
of the rostral raphe nuclei appear to provide the major part (about two-thirds) of the serotonin in the rat hypothalamus (Palkovits et al., 1977b). Complete surgical deafferentation of the medial-basal hypothalamus results in a 40-70% decrease in serotonin in the arcuate and ventromedial nuclei and in the median eminence (Brownstein et al., 1976d). 8. Tryptophan Hydroxylase
This compound is a rate-limiting enzyme involved in the synthesis of serotonin. It is present in high concentrations in brain areas containing serotonin perikarya (raphe nuclei) and in lower concentrations in other brain regions where serotonin is located in nerve terminals (Brownstein e f al., 1975d). In the hypothalamus the enzyme is found in the highest concentrations in the perifornical nucleus and in the medial forebrain bundle (Fig. 11). Its presence in the median eminence, in the pituitary gland, and in all hypothalamic nuclei suggests the local, hypothalamic synthesis of serotonin.
9. Histamine The highest concentration of histamine in the rat hypothalamus is found in the median eminence (Brownstein et al., 1974b). A relatively high histamine level is present in the basal (arcuate and suprachiasmatic nuclei) and in the posterior
FIG. 1 1 .
Tryptophan hydroxylase activity in the hypothalamic nuclei (Brownstein et al., 1975d).
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hypothalamic nuclei (premammillary and posterior hypothalamic nuclei), while the anterior hypothalamus is poor in histamine (Fig. 12). In the absence of histochemical techniques for the visualization of histamine, the extra- or intraneuronal localization of histamine is still unknown. An unchanged level of histamine in the medial-basal hypothalamus following its total surgical deafferentation suggests a local origin of the intrahypothalamic histamine (Brownstein et al., 1976d). 10. Histamine N-Methyltransferase This compound is the main enzyme in the catabolysis of histamine. Its activity has been determined by the method of Taylor and Snyder (1972). Enzyme activity in the hypothalamus, compared to that in other brain regions, is moderate (Saavedra et al., 1976). Within the hypothalamus enzyme activity varies. In contrast to the concentration of histamine, the concentration of histamine N-methyltransferase in the anterior hypothalamus is relatively high, while it is only moderate in the median eminence and quite low in the ventromedial and arcuate nuclei (Fig. 13). 11. Catechol 0-Methyltransferuse (COMT) COMT is the main catabolytic enzyme for the biogenic amines. Activity in individual brain nuclei has been measured by the method of Axelrod and Cohn (1971). The enzyme is present in all regions of the central nervous system (Saavedra et al., 1976). The relatively high COMT activity in the median eminence and in the periventricular nucleus suggests a high degree of catabolic O-methylation of catecholamines in these areas. Relatively low activity of COMT is found in the arcuate, suprachiasmatic, ventromedial, and anterior hypothalamic nuclei (Fig. 14). 12. Morwamine Oxidase (MAO) This enzyme has been quantitated according to the method of Wurtman and Axelrod (1963), using serotonin as a substrate (Saavedra et al., 1976). Similar TH
FIG.12.
Histamine concentrations in the hypothalamic nuclei (Brownstein et al., 1974b).
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327
FIG. 13. Histidine N-methyltransferase activity in the hypothalamic nuclei (Saavedra et al., 1976).
measurements have been made on individual hypothalamic nuclei by Hirano et al. (1975). MA0 is found to be unevenly distributed in the rat hypothalamus (Fig. 15). The activities are relatively high but do not exceed half of that present in the locus coeruleus. In general, MA0 activity is about two orders of magnitude higher than that of COMT or histamine N-methyltransferase (Saavedra et al., 1976). Within the hypothalamus, the nuclei of the middle hypothalamus and the penventricular nucleus are rich in MA0 (Fig. 15). 13. Acetylcholine (ACh) Determinations of ACh in individual brain areas can be made by enzymic isotopic assay (Goldberg and McCaman, 1973), by mass fragmentographictechniques (Hanin and Schuberth, 1974), or by bioassay (Paton and Vizi, 1969). Since no systematic studies have been made on the rat hypothalamus for the determination of ACh levels in individual nuclei, Fig. 16 shows data available in the literature (Cheney et al., 1975; Jacobowitz and Goldberg, 1977; Vizi and Palkovits, 1978). In general, the hypothalamus and the median eminence contain ACh in moderate or low concentrations.
FIG. 14. COMT activity in the hypothalamic nuclei (Saavedra et al., 1976).
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FIG. 15. M A 0 5HT activity in the hypothalamic nuclei (Saavedra et al., 1976).
14. Choline Acetyltransferase (CAT)
CAT has been used as a marker to localize cholinergic neurons in the central nervous system. Although its distribution correlates well with the amount of ACh in discrete brain regions (Vizi and Palkovits, 1978), CAT is not a rate-limiting enzyme for ACh. It is synthetized in cholinergic cell bodies and transported through the axons to the nerve terminals. Only parallel measurements on the concentration of ACh and the activity of CAT give information about the possible cholinergic character of nerve elements in certain brain nuclei. In the hypothalamus (Fig. 17), compared to other brain regions, CAT activity is low and unevenly distributed in the individual cell groups (Brownstein et al., 1975b; Uchimura et nl.. 1975). Only small changes in CAT activity were found in the arcu& and ventromedial nuclei after surgical deafferentation of the medial-basal hypothalamus, while the activity did not change at all in the median eminence (Brownstein el al., 1976d). This observation suggests the existence of a cholinergic tuberoinfundibular pathway similar to the dopaminergic pathway. Cell bodies in the arcuate nucleus can be visualized by a stain specific for acetylcholinesterase (Mkszhros et al., 1969).
15. y-Aminobutyric Acid (GABA) The distributions of GABA in certain hypothalamic nuclei has been determined recently (Tappaz et al., 1977). A relatively high level of GABA is found
FIG. 16. ACh concentrations in the hypothalamic nuclei.
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FIG. 17. CAT activity in the hypothalamic nuclei (Brownstein et al., 1975b).
in the dorsomedial nucleus, while the magnocellular nuclei (supraoptic and paraventricular nuclei), ventromedial nucleus, and medial forebrain bundle contain moderate amounts (Fig. 18). The concentration of GABA in the dorsomedial nucleus and median eminence is found to be quite low. 16. Glutamic Acid Decarboqdase (GAD) This enzyme converts glutamic acid into GABA. GAD activity has been assayed in a large number of individual brain nuclei, including those of the hypothalamus (Tappaz et al., 1976). The anterior hypothalamic, paraventricular, and dorsomedial nuclei, and the medial forebrain bundle are relatively rich in GAD (Fig. 19). As a consequence of total surgical isolation of the medial-basal hypothalamus, the enzyme activity falls markedly in the arcuate and ventromedial nuclei but not in the median eminence (Brownstein et al., 1976d). This observations suggests the presence of GABA-ergic cell bodies in the hypothalamus, probably innervating the median eminence. B . NEURONAL PEPTIDES (NEUROHORMONES) Several important technical advances have permitted characterization and assaying of biologically active peptides in small, well-defined brain areas. One
FIG. 18. GABA concentrations in the hypothalamic nuclei (Tappaz et al., 1977).
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FIG. 19. GAD activity in the hypothalamic nuclei (Tappaz et af., 1976)
group of peptides has been characterized as neurohormones, while others have been described as neurotransmittersor “modulators of cell activities. ” However, the so-called neurohormones are widely distributed not only in the neuroendrocrine hypothalamus but throughout the entire central nervous system. They are present also in nerve terminals, suggesting a local, probable neurotransmitter function. However, peptides present in the median eminence, such as substance P, may participate in endocrine regulation, being neurohormones rather than neurotransmitters. The presence of biologically active peptides both in the endocrine hypothalamus and in the extrahypothalamic brain suggests that, depending on the site of action, neuronal peptides in the central nervous system might have both neurohormonal and neurotransmitter character. Several neuronal peptides in the hypothalamus can be determined by radioimmunoassay. 1. Luteinizing Hormone-Releasing Hormone (LH-RH) This hormone has been isolated and characterized as a decapeptide which promotes the release of both LH and follicle-stimulating hormone (Baba et a l . , 1971; Matsuo et al., 1971). LH-RH has been measured in the hypothalamic nuclei by radioimmunoassay as described by Arimura et al. (1973). A very high concentration of LH-RH (22 pg/ng protein = 0.5 ng) is present in the rat median eminence, and much lower concentrations in the arcuate nucleus (Palkovits et aZ., 1974a). Except for one subdivision of the ventromedial nucleus, LH-RH in the hypothalamic nuclei may be present in undetectable amounts (Fig. 20). The preoptic regions also contain LH-RH, mostly in the organon vasculosum laminae terminalis (OVLT) and the neighboring area, including the medial preoptic nucleus (Brownstein et al., 1975c; Wheaton et al., 1975; Kizer et al., 1976). Depending on the species and the technique used, LH-RH cell bodies can be visualized by immunocytochemistry in both extra- and intrahypothalamicregions (the preoptic region and arcuate-premammillary nuclei, respectively). Surgical deafferentation of the hypothalamus causes LH-RH to fall by 70-90% in the medial-basal hypothalamus (Weiner et al., 1975; Brownstein et a l . , 1976a) but not in the OVLT.
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33 1
FIG.20. LH-RH concentrations in the hypothalamic nuclei (Pakovits et at., 1974a).
2. Growth Hormone Release-Inhibiting Hormone (Somatostatin) This hormone has been identified as a tetradecapeptide (Brazeau et al., 1973). The highest concentration of somatostatin in the brain is in the median eminence (Brownstein et al., 1975a), as measured by radioimmunoassay (Arimura et al., 1975). Levels of somatostatin are high in the hypothalamus, but it is present in measurable amounts in all parts of the central as well as the peripheral nervous system. Within the hypothalamus (Fig. 21), the arcuate, periventricular, ventral premammillary , and ventromedial nuclei have more somatostatin than the other hypothalamic nuclei (Brownstein et al., 1975a). All the subdivisions of the ventromedial nucleus contain about the same concentrations of somatostatin as the rostrocaudal subdivisions of the arcuate nucleus (Palkovits et al., 1976a). Somatostatin-like activity has been estimated by bioassay in extracts of hypothalamic nuclei in the rat (Vale et al., 1974), and the distribution is similar to that obtained by radioimmunoassay. Somatostatin-synthesizing cell bodies have been demonstrated in the periventricular nuclei of the anterior hypothalamus and preoptic regions by immunocytochemical techniques (Alpert ef al., 1976). Somatostatin is present in nerve terminals in the median eminence, as well as in the nuclei of the medialbasal hypothalamus. As a consequence of the total or anterior isolation of the medial-basal hypothalamus from the remainder of brain, 70-80% of the somatostatin disappears from the deafferentated hypothalamus (Brownstein et al., 1977).
FIG.21.
Somatostatin concentrations in the hypothalamic nuclei (Brownstein et al., 1975a).
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3. Corticotropin-Releasing Factor (CRF) Although several substances inducing ACTH release are known, CRF itself has not yet been identified. Because of the lack of synthetic CRF, antibodies against this factor as well as immunocytochemical assays and radioimmunoassays for localizing and measuring it in the central nervous system are not available. The CRF activity of individual hypothalamic nuclei has been determined by bioassay (Lang et al., 1974). The highest CRF activity was measured in the median eminence, but all the hypothalamic nuclei contain CRF-like substance(s) in detectable concentrations (Lang et al., 1976). In the arcuate, suprachiasmatic, supraoptic, and paraventricular nuclei significantly more activity is detected than in other hypothalamic nuclei (Fig. 22). 4. Vasopressin
The octapeptide vasopressin is produced by magnocellular neurosecretory cells in the supraoptic nuclei and in smaller amounts in the paraventricular nuclei. This peptide is bound to a carrier molecule (neurophysin 11) and transported within neurosecretory granules along the axons of the supraopticohypophyseal tract to the posterior pituitary for storage. By using radioimmunoassay (George et a f . , 1972), levels of arginine vasopressin have been measured in the hypothalamic nuclei of the rat (George and Jacobowitz, 1975). As expected, the median eminence contains vasopressin at a very high level. It can be found in fairly high concentrations in the retrochiasmatic area (probably in the fibers of the supraopticohypophysealtract passing through this area) and also in the supraoptic, paraventricular, and suprachiasmatic nuclei (Fig. 23). The presence of vasopressin in the suprachiasmatic nucleus has also been corroborated by immunocytological examinations (Vandesande et al., 1974; Zimmerman et al., 1975).
5 . Oxytocin This nonapeptide is synthesized in the paraventricular and supraoptic nuclei and is bound to a carrier protein (neurophysin I). Within the supraopticohypophyseal tract, similarly to those of vasopressin content, reach the posterior pitui-
FIG.22. CRF activity in the hypothalamic nuclei (Lang et al., 1976).
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FIG. 23. Vasopressin concentrationsin the hypothalamic nuclei (George and Jacobowitz, 1975).
tary after having transversed the retrochiasmatic area and median eminence. With the use of radioimmunoassay (George et al., 1976) a rather high oxytocin concentration was found in the median eminence, about 30 times less than in the pituitary gland (George et al., 1976). Oxytocin is present in relatively large amounts in the supraoptic and paraventricular nuclei and in lower concentrations in the arcuate, anterior hypothalamic, and medial preoptic nuclei, as well as in the retrochiasmatic area (Fig. 24). Oxytocin in the arcuate nucleus and retrochiasmatic area is probably located in axons passing through these areas to reach the median eminence, while oxytocin in the anterior hypothalamic and medial preoptic nuclei is probably present in the accessory magnocellular cells forming cell groups within the anterior hypothalamus (Palkovits et al., 1974~). 6. Thyrotropin-Releasing Hormone (TRH) This hormone has been identified as a tripeptide by Burgus et al. (1969) and Bpler et al. (1969). TRH, measured by radioimmunoassay (Bassiri and Utiger, 1972), is not restricted to the hypothalamus, since about 70-75% of it is found in the extrahypothalamic brain (Winokur and Utiger, 1974; Jackson and Reichlin, 1974; Oliver et al., 1974). The highest concentration of TRH is found in the median eminence, and is one to three orders of magnitude higher than that in other brain regions (Brownstein et al., 1974a). Within the hypothalamus (Fig. 25) high levels of TRH are encountered in the medial part of the ventromedial
FIG.24. Oxytocin concentrations in the hypothalamic nuclei (George et al., 1976).
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FIG. 25. TRH concentrations in the hypothalamic nuclei (Brownstein et nl., 1974a).
nucleus, in the periventricular, arcuate, and dorsomedial nuclei. These data are in accordance with the results obtained from a bioassay for TRH (Krulich et al., 1974). The localization of TRH-synthesizing cells in the central nervous system is still unknown. It is suggested that they might be located in extrahypothalamic regions, because 75% of the TRH found normally in the medial basal hypothalamus disappears following surgical isolation of this region, while the level of TRH does not change elsewhere in the brain (Brownstein et al., 1975e). 7. Substance P This substance was detected in 1931 (von Euler and Gaddum, 1931), purified (Chang and Leeman, 1970) and characterized only 40 years later (Chang et al., 1971). This undecapeptide has been synthesized (Tregear et al., 1971), and a sensitive, specific radioimmunoassay for it has been developed by Powell et al. ( 1973). The level of substance P is higher in the mesencephalon, hypothalamus, and preoptic area than in other regions of the brain (Brownstein et al., 1976b). Within the hypothalamus (Fig. 26), substance P is unevenly distributed. The periventricular, anterior hypothalamic, dorsomedial, paraventricular, and ventral premammillary nuclei have higher levels of substance P than other hypothalamic nuclei. The median eminence is relatively poor in substance P. The medial forebrain bundle appears progressively to be depleted of its substance P in a
FIG. 26. Substance-P concentrations in the hypothalamc nuclei (Brownstem et nl., 1976b).
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FIG.27. Neurotensin concentrations in the hypothalamic nuclei (Kohayashi et al., 1977).
rostrocaudal direction from the preoptic region through the hypothalamus into the mesencephalon (Brownstein et al., 1976b). 8. Neurotensin This compound is a vasoactive peptide detected in hypothalamic extracts during the course of purification of substance P (Carraway and Leeman, 1973). About 30% of the neurotensin content of the brain is found in the hypothalamus, the bulk of the remainder being located in the midbrain and brainstem. The distribution of neurotensin in individual brain nuclei has been determined by radioimmunoassay (Brown et al., 1977) recently (Kobayashi et al., 1977). Neurotensin is found in the highest concentration in the median eminence, followed by the preoptic and other hypothalamic nuclei (ventromedial, periventricular, suprachiasmatic, arcuate nuclei) and the retrochiasmatic area (Fig. 27). 9. Enkephalins Two opiate-likepentapeptides, methionine-enkephalinand leucine-enkephalin, have been demonstrated in the brain, and the structure of methionine-enkephalin has been shown to be identical to that of thep-lipotorpin 61-65 (Hughes et at., 1975; Simantov and Snyder, 1976). The distribution of enkephalin in the central nervous system has been investigaed by immunohistochemical techniques (Simantov et al., 1977) and also by radioimmunoassay (Miller et al., 1975; Kobayashi et al., 1978). The concentration of enkephalins in the medial hypothalamus is more than twice as great as that in the lateral hypothalamus, but seven times less than that in the globus pallidus (Kobayashi et al., 1978). The concentrations of enkephalins, as well as of other p -1ipotropin fragments (endorphins, a-MSH, ACTH), in individual hypothalamic nuclei have yet to be measured.
IV. General Considerations Neurochemical measurements give very little information about the cellular distribution of various substances in the central nervous system. These studies
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M. PALKOVITS
must be completed with histofluorescence and immunocytochemical observations. Because of technical problems neither histofluorescence nor immunocytochemical studies have revealed a similar distribution of substances observed using enzymic isotopic assays or radioimmunoassay. By measuring concentrations of neuronal peptides it may be possible that different forms of these substances can be detected by radioimmunoassay and immunocytochemistry. Radioimmunoassay seems to be more effective in detecting free neuronal peptides, while immunocytochemistry detects mainly a bound form. Neurotransmitters and their related enzymes, as well as the neuronal peptides measured, are present in different concentrations in all the nuclei of the hypothalamus. There are nuclei which contain certain substances in higher concentrations than other cell groups, but particular centers for the localization of synthesis or action or these substances cannot be distinguished. There are substances which may act, depending on the site of action, both as neurohormones and as neurotransmitters. Therefore the terms ‘‘neurohormone and “neurotransmitter” concern the type of action rather than the chemical nature of a substance. High concentrations of neuronal peptides in hypothalamic nuclei may represent their presence in nerve terminals where they may act as neurotransmitters at the synaptic level. Neurotransmitters and neuronal peptides in the hypothalamus are located mainly in nerve terminals rather than in cell bodies. Most of these substances are of extrahypothalamic origin, which means that nerve fibers or pathways of different chemical nature exist between the hypothalamus and extrahypothalamic brain regions. Like many other substances, neurohormones are found in the highest concentrations within the median eminence. Only the minority of these neurohormones originate from the so-called hypophysiotrophic area of the hypothalamus. This observation contradicts a special role for the hypophysiotrophic area in neurohormonal regulation of the pituitary gland. The explanation of a possible role of this area seems to be supervised. The chemical nature of the majority of hypothalamic cell bodies is still unknown. Characterization of these cells, that is, determination of their synthetic activity is yet to be made. ”
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Subject Index
A
c
Actin, amoeboid cells and, 118-123 Actinomycin D, double-stranded RNA synthesis and, 270-271 Aequorin, intracellular calcium and, 153-159 Amelogenesis, electron microscope autoradiography of 4SCa during, 242-246 Amoeboid cells, 117-118 actin and, 118-123 categories of, 59-61 contractile and/or cytoskeletalproteins of, 132 cytoplasmic structure and contractility in virro, 96-97 early model systems of contractility, 97-100 models of, 100-1 17 cytoplasmic structure and contractility in vivo, 61, 65-67 environmental effects, 91-96 historical sketch, 62-63 light and electron microscopy and, 74-91 physical evidence for structure, 67-74 terminology and, 63-65 movement of, 58-59 myosin and, 123-127 regulation of, 128-132 summary and future challenges, 132-134 Antiserum, double-stranded RNA and, 266 h n a m III, intracellularcalcium and, 152-153 Autoradiography, intracellular calcium and, 168- 172
Calcified tissues electron microscope autoradiography detection of nucleic acids in, 188 methodology, 185-187 synthesis of organic matrices of, 188-189 bone, 189-202 dentin, 202-214 enamel, 214-226 Calcium fluorescent chelate probe and, 159-165 intracellular autoradiography and, 168-172 electron probe microanalysis and, 173-178 luminescent proteins and, 153-159 metallochrome dyes and, 151-153 total cellular, assay of, 147-150 45Calcium electron microscope autoradiography melogenesis and, 242-246 dentinogenesis and, 233-242 osteogenesis and, 228-233 Cephalopods anatomical outline of nervous system effectors, 3 ganglia, 2-3 receptors, 2 squid giant fiber system, 3-5 cytological features of synapses in ganglia distribution of presynaptic elements in Octopus brain, 10 examples of Octopus brain synapses, 5-8 presumed neurotransmitters in ganglia, 10-13 synapses of squid giant fiber system, 8-10 nerve endings in effectors biochemical and cytological data, 16-17 ultrastructural observations, 14-16
B
Bacteriophage, high-molecular weight RNA, double-stranded hairpin structures and, 297-299 Bone, matrix, elaboration of, 189-202 Brain cells, morphology in culture, 48-51
341
342
SUBJECT INDEX
special features in peripheral afferent pathways, 13-14 Cerebral cortex, development in vivo, 28-38 Chelate, fluorescent, intracellular calcium and, 159- 165 D
Dentin adult, identification of sensory nerve endings in, 246-247 matrix, elaboration of, 202-214 Dentinogenesis, electron microscope autoradiography of 45Ca during, 233-242 Deoxyribonucleic acid model of sequences from which RNAs are transcribed, 293-295 support for, 295-296 palindromic structures, ds RNAs and, 281282 repeated sequences, mRNA and, 290-291 Double-stranded RNA addendum, 305-306 characteristics, 259-262 base composition, 264-265 buoyant density in CsSO,, 265 actinomycin D effect on synthesis, 270-271 interferon induction, 266-267 reaction to specific antiserum, 266 ribonucleases and, 262-263 sequence complexity, 269-270 size, 267-269 thermal and chemical denaturation, 263265 translational inhibition, 267 denatured, hybridization with mRNA, 286290 eukaryotic ribonucleases and, 299-303 hairpin structure, 279-280 processing site for generation of rnRNA, 295-296 bacteriophage high-molcular weight RNA, 297-299 hn RNA origin of, 278-279 hn RNA sequences to be degraded in nuclei, 276-277 in vivo evidence of structure, 280-281 Occurrence in uninfected eukaryotic cells, 257-259 palindromic DNA and, 281-282
size of putative mRNA precursor molecule, 27 1-276
E Effectors, cephalopod, 3 nerve endings in, 14-17 Electron probe, intracellular calcium and, 173178 Enamel, matrix, elaboration of, 214-226 Eukaryotic cells, double-stranded RNA in, 257-259
G Ganglia, cephalopod, 2-3 cytological features of synapses in ganglia, 543 presumed neurotransmitters in, 8-10
H Hairpin structures, double-stranded, 279-280 location, 283 Hypothalamus microchemistry , 320-32 1 neuronal peptides, 329-335 putative neurotransmitters and related enzymes, 321-329 microdissection topography of individual nuclei, 318-319 topography of major regions, 315-318 I
Inorganic elements, transfer routes of, 226-228 amelogenesis and, 242-246 dentinogenesis and, 233-242 osteogenesis and, 228-233 Interferon, double-stranded RNA and, 266-267
M Metallochrome dyes, intracellular calcium and, 151-153 Murexide, intracellular calcium and, 151-152 Myosin, amoeboid cells and, 123-127
N Nerve endings, sensory, identification in adult dentin, 246-247 Nervous system anatomical outline in cephalopods effectors, 3 ganglia, 2-3
343
SUBJECT INDEX receptors, 2 squid giant fiber system, 3-5 columnar organization, 51-53 development in vitro, 46 morphology of brain and retinal cell cultures, 48-51 development in vivo cerebral cortex, 28-38 retina, 38-46 Neurotransmitters, putative hypothalamic, 321-329 Nucleic acids, detection in calcified tissue, 188
0 Octopus brain, distribution of presynaptic elements in, 10 examples of synapses, 5-8 Osteogenesis, electron microscope autoradiography of 45Ca during, 228-233
P Peptides, neuronal, 329-335 Protein(s), luminescent, intracellular calcium and, 153-159 R
Receptors, cephalopod, 2 Retina, development in vivo, 38-46 Retinal cells, morphology in culture, 48-51 Ribonuclease( s) double-stranded RNA and, 262-263
eukaryotic, ds RNA and, 299-303 Ribonucleic acid, see also Double-stranded RNA Ribonucleic acid eukaryotic messenger double-stranded structures, 285-286 hybridization with denatured ds RNA, 286-290 repeated DNA sequences and, 290-291 terminal sequence repetition, 291-293 untranslated sequences, 283-285 heterogeneous nuclear, double-stranded RNA and, 278-283 model of nuclear processing, 293-299 to be degraded in nuclei, 276-277 Ribonucleic acid messenger, double-stranded RNA and, 271276 repeated DNA sequences and, 290-291 terminal sequence repetition in, 291-293 untranslated, 283-285
5 Squid, giant fiber system, 3-5 synapses of, 8-10
T Tissue(s), fixation for cytology chemical, 165- 167 freeze-drying, 167-168
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Contents of Previous Volumes Volume 1 Some Historical Features in Cell BiologyARTHURHUGHES Nuclear Reproduction-€. LEONARD HUSKINS Enzymic Capacities and Their Relation to Cell Nutrition in AnimalS-GEORGE 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 Development-G. FRANK-HAWSER Structural Agents in Mitosis-M. M. SWANN Factors Which Control the Staining of Tissue Sections with Acid and Basic Dyes-MARCUS SINGER The Behavior of Spermatozoa in the Neighborhood of E~~s-LoRDROTHSCHILD The Cytology of Mammalian Epidermis and Sebaceous Glands-WILLIAM MONTACNA The Electron-Microscopic Investigation of Tissue Sections-L. H. BRETSCHNEIDER The Histochemistry of Esterases-G. GOMORI
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. GAILLARD 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. CONWAY A Critical Survey of Current Approaches in Quantitative Histo- and CytochemistryDAVIDGLICK NucIeo-cytoplasmic Relationships in the Development of Acerabularia-J. HAMMERLING Report of Conference of Tissue Culture Workers Held at Cooperstown, New York-D. J. HETHERINGTON AUTHOR INDEX-SUBJECT
INDEX
Volume 3
The Nutrition of Animal Cells-CtiAmTY 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 with Special Reference to Plants-J. CHAYEN Study of Tissue Enzymes-cHR. DE DUVE 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 HistochemistryMultienzyme Sequences in Soluble ExtractsA. G. EVERSON PEARSE HENRYR. MAHLER Microscopic Studies in Living Mammals with The Nature and Specificity of the Feulgen Transparent Chamber Methods-Roy G. WILNucleal Reaction-M. A. LESSLER LIAMS 345
346
CONTENTS OF PREVIOUS VOLUMES
The Mast Cell-G. ASBOE-HANSEN Elastic Tissue-EDWARDS w . DEMPSEYA N D ALBERTI. LANSING The Composition of the Nerve Cell Studied with AND New Methods-SVEN-OLoE BRATTGKRD HOLGERHYDEN AUTHOR INDEX-SUBJECT
INDEX
Volume 4 Cytochemical Micrurgy-M. J . KOPAC Amoebocytes-L. E. WAGE Problems of Fixation in Cytology, Histology, and Histochemistry-M. WOLMAN Bacterial Cytology-ALFRED MARSHAK Histochemistry of Bacteria-R. VENDRELY Recent Studies on Plant Mitochondria-DAVID P. HACKETT The Structure of Chloroplasts-K. MUHLE-
on Cytokinesis and Amoeboid MovementDOUGLAS MARSLAND Intracellular pH-PETER C. CALDWELL The Activity of Enzymes in Metabolism and Transport in the Red Cell-T. A. J. PRANKERD Uptake and Transfer of Macromolecules by Cells with Special Reference to Growth and Development-A. M. SCHECHTMAN Cell Secretion: A Study of Pancreas and Salivary Glands-L. C. J. JUNQUEIRA A N D G . C. HIRSCH The Acrosome Reaction-JEAN C. DAN Cytology of Spermatogenesis-VIsHwA NATH The Ultrastructure of Cells, as Revealed by the Electron Microscope-FRITIOF s. SJOSTRAND AUTHOR INDEX-SUBJECT
INDEX
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 Mammalian 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 MusCle-JOHN W. HARMON The Mitochondria of the NeUTOn-wARREN ANDREW The Results of Cytophotometry in the Study of the Deoxyribonucleic Acid (DNA) Content of A N D C. VENthe Nucleus-R. VENDRELY DRELY
Protoplasmic Contractility i n Relation to Gel Structure: Temperature-Pressure Experiments
The Antigen System ofParameciuma u r e l i a 4 . H. BEALE The Chromosome Cytology of the Ascites Tumors of Rats, with Special Reference to the Concept of the Stemline C~H-SAJIRO MAKIN0
The Structure of the Golgi Apparatus-ARTHUR W. POLLISTER A N D PRISCHIA F. 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-ArtTHUR J. HALE The Dynamic Cytology of the Thyroid Gland-J. GROSS Recent Histochemical Results of Studies on Embryos of Some Birds and Mammals-ELI0 BORGHESE Carbohydrate Metabolism and Embryonic Determination-R. J . O’CONNOR Enzymatic and Metabolic Studies on Isolated Nuclei-G. SIEBERTA N D R. M. S. SMELLIE 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
347
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 S3'-Sulfate-D. D. DZIEWIATKOWSKI The Structure of the Mammalian Spermatozoon-DON W. FAWCETT The Lymphocyte-0. A. TROWELL The Structure and Innervation of Lamellibranch Muscle-J. BOWDEN Hypothalamo-neurohypophysial NeurosecretionJ. C. SLOPER Cell Contact-PAUL WEISS The Ergastoplasm: Its History, Ultrastructure, and Biochemistry-FRANGOISE HAGUENAU Anatomy of Kidney Tubules-JOHANNES RHODIN
Structure and Innervation of the Inner Ear Sensory Epithelia-HANS ENGSTROMAND JAN WERSALL The Isolation of Living Cells from Animal Tissues-L. M. RINALDINI AUTHOR INDEX-SUBJECT
INDEX
Volume 8 The Structure of CytOplaSm-cHARLES
AUTHOR INDEX-SUBJECT
INDEX
Volume 9 The Influence of Cultural Conditions on Bacterial Cytology-J. F. WILKINSON A N D J. P. DUGUlD
Organizational Patterns within ChromosomesBERWIND P. KAUFMANN, HELENGAY,A N D MARGARET R. MCDONALD Enzymic Processes in Cells-JAY BOYDBEST The Adhesion of Cells-LEONARD WEISS Physiological and Pathological Changes in Mitochondrial Morphology-CH. ROUILLER The Study of Drug Effects at the Cytological Level-G. B. WILSON Histochemistry of Lipids in Oogenesis-VlsHWA NATH Cyto-Embryology of Echinoderms and Amphibia-KUTsuMA DAN The Cytochemistry of Nonenzyme ProteinsRONALDR. COWDEN AUTHOR INDEX-SUBJECT
INDEX
OBER-
LING
Wall Organization in Plant Cells-R.
A Survey of Metabolic Studies on Isolated Mammalian Nuclei-D. B. ROODYN Trace Elements in Cellular Function-BERT L. VALLEE AND FREDERICL. 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
D. PRES-
TON
Submicroscopic Morphology of the SynapseEDUARDO DE ROBERTIS The Cell Surface of Paramecium-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
348
CONTENTS OF PREVIOUS VOLUMES
Epidermal Cells in Culture-A.
GEDEONMA-
TOLTSY AUTHOR INDEX-SUBJECT INDEX CUMULATIVE SUBJECT INDEX (VOLUMES
Sequential Gene Action, Protein Synthesis, and Cellular Differentiation-REED A. FLICKINGER
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 MechanismsdHARLES B. METZ The Growth-Duplication Cycle of the Cell-D. M. PRESCOTT Histochemistry of Ossification-RoMuLo L. CABRINI Cinematography, Indispensable Tool for Cytology*. M. POMERAT
The Composition of the Mitochondrial Membrane in Relation to Its Structure and Function- ERICG. BALLAND 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 NATHAND G. P. DUTTA A N D CHOCell Renewal-FELIX BERTALANFFY SEN LAU AUTHOR INDEX-SUBJECT
INDEX
Volume 14
Inhibition of Cell Division: A Critical and Experimentat Andysis-SEYMOUR GELFANT EIectron 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 Hearing-J. A. VINNIKOV AND L. K. TITOVA JOHNL. HAMERTON Connective Tissue and Serum Proteins-R. E. Chromosomal Evolution i n Cell Populations-T. MANCINI C. Hsu The Biology and Chemistry of the Cell Walls of Chromosome Structure with Special Reference to Higher Plants, Algae, and Fungi-D. H. the Role of Metal Ions-DALE M. STEFFENNORTHCOTE SEN Development of Drug Resistance by StaphyloElectron Microscopy of Human White Blood Cells and Their Stem CdlS-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 Smoke on the Cell and Its Nucleic AcidThe Nature and Stability of Nerve Myelin-J. B. AND RUDOLF CECILIE LEUCHTENBERGER FINEAN LEUCHTENBERGER Fertilization of Mammalian Eggsin Vitro-C. R. The Tissue Mast Wall-DOUGLAS E. SMITH AUSTIN AUTHOR INDEX-SUBJECT INDEX Physiology of Fertilization in Fish Eggs-TOKI-0 YAMAMOTO AUTHOR INDEX-SUBJECT
INDEX
AUTHOR INDEX-SUBJECT
INDEX
Volume 13 The Coding Hypothesis-MARTYNAS YEAS Chromosome Reproduction-J. HERBERTTAYLOR
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-
349
CONTENTS OF PREVIOUS VOLUMES LEONARD MACHLISAND ERIKARAWITSCHERKUNKEL The Cellular Basis of Morphogenesis and Sea Urchin Development-T. GUSTAFSON AND L. WOLPERT Plant Tissue Culture in Relation to Development CytOlOgy
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. ROODY N AUTHOR INDEX-SUBJECT INDEX Histochemical Localization of Enzyme Activities by Substrate Film Methods: Ribonucleases, Volume 16 Deoxyribonucleases, Proteases, Amylase, and Hyaluronidase-R. DAOUST Ribosomal Functions Related to Protein SyntheCytoplasmic Deoxyribonucleic Acid-P. B. GAsis-Tow 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,A N D J. ERMAN J. KATZ Microbeam and Partial Cell 1rradiation-C. L. The Use of Heavy Metal Salts as Electron SMITH Stains-C. RICHARD ZOBELAND MICHAEL Nuclear-Cytoplasmic Interaction with Ionizing BEER Radiation-M. A. LESSLER AUTHOR INDEX-SUBJECT INDEX I n Vivo Studies of Myelinated Nerve FibersCARLCASKEY SPEIDEL Respiratory Tissue: Structure, Histophysiology , Volume 19 Cytodynamics. Part I: Review and Basic Cytomorphology-FELIX D. BERTALANFFY “Metabolic” DNA: A Cytochemical Study-H. AUTHOR INDEX-SUBJECT INDEX ROELS The Significance of the Sex Chromatin-MuRRAY L. BARR Volume 17 Some Functions of the Nucleus-J. M. MITCHIThe Growth of Plant Cell Walls-K. WILSON SON Reproduction and Heredity in Trypanosomes: A Synaptic Morphology on the Normal and DegenCritical Review Dealing Mainly with the Aferating Nervous System-E. G. GRAYA N D R. rican Species in the Mammalian Host-P. J. W. GUILLERY WALKER Neurosecretion-W. BARGMANN The Blood Platelet: Electron Microscopic StudSome Aspects of Muscle Regeneration-E. H. ies-J. F. DAVID-FERREIRA BETZ, H. FIRKET,AND M. REZNIK The Histochemistry of MucopolysaccharidesThe Gibberellins as Hormones-P. W. BRIAN Phototaxis in PlaIItS-WOLFGANG HAUFT ROBERTC. CURRAN Respiratory Tissue Structure, Histophysiology, Phosphorus Metabolism in Plants-K. S. Cytodynamics. Part 11. New Approaches and ROWAN AUTHOR INDEX-SUBJECT INDEX Interpretations-FELIX D. BERTALANFFY
3 50
CONTENTS OF PREVIOUS VOLUMES
Volume 20 The Chemical Organization of the Plasma Membrane of Animal Cells-A. H. MADDY Subunits of Chloroplast Structure and Quantum Conversion in Photosynthesis-RODERIC B. PARK Control of Chloroplast Structure by Light-LEsTER PACKER AND PAUL-AND& SIEGENTHALER
The Dynamism of Cell Division during Early Cleavage Stages of the Egg-N. FAUTREZFIRLEFYN AND J. FAUTREZ Lymphopoiesis in the Thymus and Other Tissues: AND Functional Implications-N. B. EVERETT RUTH W. TYLER(CAFFREY) Structure and Organization of the Myoneural Junction<. COERS The Ecdysial Glands of Arthropods-WILLIAM S. HERMAN Cytokinins in Plants-B. I. SAHAISRIVASTAVA
The Role of Potassium and Sodium Ions as 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,GITTA ASCH,AND JAQUELINE Roos Cytology and Cytophysiology of Non-MelanoVolume 23 phore Pigment Cek.-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 NORREVANG Physiological Clocks-R. L. BRAHMACHARY Dynamic Aspects of Phospholipids during ProCiliary Movement andcoordination in Ciliatestein Secretion-LOWELL E. HOKIN BELAPARDUCA The Golgi Apparatus: Structure and FunctionElectromyography: Its Structural and Neural H. W. BEAMSA N D R. G. KESSEL Basis-JOHN V. BASMAJIAN The Chromosomal Basis of Sex DeterminationCytochemical Studies with Acridine Orange and KENNETH R. LEWISA N D BERNARD JOHN the Influence of Dye Contaminants in the AUTHOR INDEX-SUBJECT INDEX Staining of Nucleic Acids-FREDERICK H.
KASTEN Experimental Cytology of the Shoot Apical Cells Volume 24 during Vegetative Growth and Flowering-A. Synchronous Cell Differentiation-GEORGE M. NOUGAR~DE PADILLA A N D IVANL. CAMERON Nature and Origin of Perisynaptic Cells of the Mast Cells in the Nervous SyStem-YNGVE Motor End Plate-T. R. SHANTHAVEERAPPA OLSSON A N D G . H. BOURNE Development Phases in Intermitosis and the AUTHOR INDEX-SUBJECT INDEX Preparation for Mitosis of Mammalian Cells in VI'ITO-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 WlSCHNlTZER LEY A N D 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 tiatiOn-GAJANAN v . SHERBET A N D M. s. A. PEPE LAKSHMI
351
CONTENTS OF PREVIOUS VOLUMES Cytology of Intestinal Epithelial CdS-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 Beat 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 CARIUERE Strandedness of Chromosomes-SHELDON WOLFF Isozymes: Classification, Frequency, and Significance--CHARLEs R. SHAW The Enzymes of the Embryonic NephronLUCIEARVV 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 SUPPO~-GILBERT N. LING The Cell Periphery-LEONARD WEISS Mitochondria1 DNA: Physicochemical Properties, Replication, and Genetic Function-P. BORSTA N D A. M. KROON Metabolism and Enucleated CellS-KONRAD KECK Stereological Principles for Morphometry in Electron Microscopic Cytology-EwALD R. WEIBEL Some Possible Roles for Isozymic Substitutions during Cold Hardening i n Plants-D. W. A. ROBERTS AUTHOR INDEX-SUBJECT
INDEX
Volume 27 Wound-Healing in Higher Plants-JACQUES LIPETZ
Chloroplasts as Symbiotic Organelles-DENNIS L. TAYLOR The Annulate Lamellae-SAUL WISCHNITZER Gametogenesis and Egg Fertilization in Planarians-4. BENAZZILENTATI Ultrastructure of the Mammalian Adrenal CorteX-sIMON IDELMAN The Fine Structure of the Mammalian Lymphoreticular SyStem-IAN CARR Immunoenzyme 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 LymnUeU EggzHRISTIAAN P. RAVEN The Environment and Function of Invertebrate Nerve Cells-J. E. TREHERNE AND R. B. 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-REN6 SlMARD The Origin of Bone CellS-MAUREEN OWEN Regeneration and Differentiation of Sieve Tube EhIentS-wILLIAM P. JACOBS Cells, Solutes, and Growth: Salt Accumulation in Plants Reexamined-F. C. STEWARD A N D 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-HIDEKO KAJI
352
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 A N D BETTY1. ROOTS Functional Electron Microscopy of the Hypothalamic Median Eminence-HIDESHI KOBAYASHI, TOKUZO MATSUI,AND SUSUMl ISHII Early Development in Callus Cultures- MICHAEL M. YEOMAN AUTHOR INDEX-SUBJECT
INDEX
Volume 30
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 Hernoflagellates-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 . ADIYODl
High-pressure Studies in Cell Biology-ARTHUR M. ZIMMERMAN Micrurgical Studies with Large Free-Living Amebas-K. W. JEONAND 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 TisSUCS-SHINJIRO KOBAYASHI AUTHOR INDEX-SUBJECT
Volume 32
The Use of Ferritin-Conjugated Antibodies in Electron Microscopy~OUNClLMAN MORGAN
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. HAMKALO Studies on Freeze-Etching of Cell MembranesCell Disjunction (“Mitosis”) in Somatic Cell KURTMUHLETHALER Reproduction-ELAINE G. DIACUMAKOS, SCOTTHOLLAND, AND PAULINE PECORA Recent Developments in Light and Electron Microscope Radioautography-C. C. BUDD Neuronal Microtubules, Neurofilaments, and Microfilaments-RAYMOND B. WUERKER Morphological and Histochemical Aspects of AND JOEL B. KIRKPATRICK Glycoproteins at the Surface of Animal Cells- A. RAMBOURC Lymphocyte Interactions in Antibody Responses-J. F. A. P. MILLER DNA Biosynthesis-H. S. JANSZ,D. VAN DER MEI, AND G. M. ZANDVLIET Laser Microbeams for Partial Cell IrradiationW. BERNSA N D CHRISTIAN SALET MICHAEL Cytokinesis in Animal Cells-R. RAPPAPORT Mechanisms of Virus-Induced Cell FusionThe Control of Cell Division in Ocular L e n s - C . V. HARDING, J. R. REDDAN,N. J. UNAKAR, GEORGEPOSTE Freeze-Etching of BacteriarHARLEs c . REMAND M. BAGCHI The Cytokinins-HANS KENDE SEN AND STANLEY W . WATSON Cytophysiology of the Teleost Pituitary- MAR- The Cytophysiology of Mammalian Adipose TIN SAGE AND HOWARD A. BERN Cells-BERNARD G . SLAVIN
Volume 31
AUTHOR INDEX-SUBJECT
INDEX
AUTHOR INDEX-SUBJECT
INDEX
353
CONTENTS OF PREVIOUS VOLUMES
Volume 34 The Submicroscopic Morphology of the Interphase Nucleus-SAUL WISCHNITZER The Energy State and Structure of Isolated Chloroplasts: The Oxidative Reactions Involving the Water-Splitting Step of Photosynthesis- ROBERTL. HEATH Transport in NeUrOSpOrU-GENE A. SCARBOROUGH
Mechanisms of Ion Transport through Plant Cell Membranes-EMANUEL ERSTEIN Cell Motility: Mechanisms in Protoplasmic Streaming and Ameboid Movement-H. W. STOCKEM, A N D K. E. WOHLEKOMNICK, FARTH-BOTTERMANN The Gliointerstitial System of MoIIuscs- GHISLAIN NICAISE Colchicine-Sensitive Microtubules-LYNN MARGULIS AUTHOR INDEX-SUBJECT
INDEX
Volume 35 The Structure of Mammalian ChromosomesELTONSTUBBLEFIELD Synthetic Activity of Polytene ChromosomesHANSD. BERENDES Mechanisms of Chromosome Synapsis at Meiotic Prophase-PETER B. MOENS Structural Aspects of Ribosomes-N. NANNINGA
Comparative Ultrastructure of the Cerebrospinal Fluid-Contacting Neurons-B. VlCH AND I. VIGH-TEICHMANN Maturation-Inducing Substance in StarfishesHARUOKANATANI The Limonium Salt Gland: A Biophysical and Structural Study-A. E. HILLAND B. s . HILL Toxic Oxygen Effects-HAROLD M. SWART2 AUTHOR INDEX-SUBJECT
INDEX
Volume 36 Molecular Hybridization of DNA and RNA in SiIU-WOLFGANG HENNIG The Relationship between the Plasmalemma and Plant Cell W~~-JEAN-CLAUDE ROLAND Recent Advances in the Cytochemistry and Ultrastructure of Cytoplasmic Inclusions in Mas-
tigophora and Opalinata (Protozoa)-G. P. DUTTA Chloroplasts and Algae as Symbionts in MolIUSCS-LEONARDMUSCATINE AND RICHARD W. GREENE The Macrophage-SAIMoN GORDON A N D ZANVIL A. COHN Degeneration and Regeneration of Neurosecretory Systems-HORST-DIETER DELLMANN AUTHOR INDEX-SUBJECT
INDEX
Volume 37 Units of DNA Replication in Chromosomes of Eukaroytes-J. HERBERTTAYLOR Viruses and Evolution-D. C. REANNEY Electron Microscope Studies on Spermiogenesis in Various Animal SpeCieS-GONPACHIRO YASUZUMI Morphology, Histochemistry, and Biochemistry of Human Oogenesis and OVUhtiOn-SARDUL S. GURAYA Functional Morphology of the Distal LungKAYEH. KILBURN Comparative Studies of the Juxtaglomerular Apparatus-HIRoFuMI SOKABE A N D MIZUHO OGAWA The Ultrastructure of the Local Cellular Reaction to Neoplasia-IAN CARRA N D J. C. E. UNDERWOOD
Scanning Electron Microscopy in the Ultrastructural Analysis of the Mammalian Cerebral Ventricular System-D. E. SCOTT, G. P. KOZLOWSKI, A N D M. N. SHERIDAN AUTHOR INDEX-SUBJECT
INDEX
Volume 38 Genetic Engineering and Life Synthesis: An Introduction to the Review by R. Widdus and c. Auk-JAMES F. DANIELLI Progress in Research Related to Genetic Engineering and Life Synthesis-Rov WIDDUS AND CHARLES R. AULT The Genetics of C-Type RNA Tumor VirusesJ. A. WYKE Three-Dimensional Reconstruction from Projections: A Review Of AlgOnthmS-RICHARD GORDONAND GABORT. HERMAN
3 54
CONTENTS OF PREVIOUS VOLUMES
The Cytophysiology of Thyroid Ceh-VLADIMIR R. PANTIC The Mechanisms of Neural Tube FormationPERRY KARFUNKEL The Behavior of the XY Pair in MammakALBERTO J. SOLARI Fine-Structural Aspects of Morphogenesis in Acetabuiaria-G. WERZ Cell Separation by Gradient Centrifugation-R. HARWOOD
Volume 41
Fine Structure of the Thyroid Gland-HisAo FUJITA Postnatal Gliogenesis in the Mammalian BrainA. PRIVAT Three-Dimensional Reconstruction from Serial Sections-RANDLE w. WARE A N D VINCENT LOPRESTI
SUBJECT INDEX
The Attachment of the Bacterial Chromosome to the Cell Membrane-PAUL J. LEIBOWITZ AND MOSELIOSCHAECHTER Regulation of the Lactose Operon in Escherichia coli by c A M P - G . CARPENTER A N D B. H. SELLS Regulation of Microtubules in TetrahymenaNORMAN E. WILLIAMS Cellular Receptors and Mechanisms of Action of SUBJECT INDEX Steroid Hormones-SHUTSUNG LIAO A Cell Culture Approach to the Study of Anterior Volume 39 Pituitary Cells-A. TIXIER-VIDAL, D. GOURDJI,AND C. TOUCARD Androgen Receptors in the Nonhistone Protein Immunohistochemical Demonstration of NeuFractions of Prostatic Chromatin-TuNc YUE rophysin in the HypothalamoneurohypoWANGA N D LEROYM. NYBERG physial System-W. B. WATKINS Nucleocytoplasmic Interactions in Development of Amphibian Hybrids-STEPHEN SUBTELNY The Visual System of the Horseshoe Crab Limulus polyphemus-WOLF H. FAHRENBACH The Interactions of Lectins with Animal Cell SUBJECT INDEX SUrfaCeS--GARTHL. NICOLSON Structure and Function of Intercellular Junctions-L. ANDREW STAEHELIN Volume 42 Recent Advances in Cytochemistry and UltraRegulators of Cell Division: Endogenous Mitotic structure of Cytoplasmic Inclusions in CilioInhibitors of Mammalian CellS-BISMARCK B. phora (Protozoa)-G. P. DUTTA LOZZIO,CARMEN B. LOZZIO,ELENAG. BAMStructure and Development of the Renal GlomBERGER, A N D STEPHEN V. LAIR erulus as Revealed by Scanning Electron MiUltrastructure of Mammalian Chromosome croscopy-FRANC0 SPINELLI Aberrations-B. R. BRINKLEY A N D WALTER Recent Progress with Laser Microbeams- MIN. HITTELMAN CHAEL w. BERNS Computer Processing of Electron Micrographs: A The Problem of Germ Cell Determinants-H. W. Nonmathematical Account-P. W. HAWKES BEAMSA N D R. G. KESSEL Cyclic Changes in the Fine Structure of the SUBJECT INDEX Epithelial Cells of Human EndometriumMILDRED GORDON Volume 40 The Ultrastructure of the Organ of CortROBERTS. KIMURA B-Chromosome Systems i n Flowering Plants and Endocrine Cells of the Gastric Mucosa-ENmco Animal Species-R. N. JONES SOLCIA,CARLOCAPELLA,GABRIELE VASThe Intracellular Neutral SH-Dependent Protease SALLO, A N D ROBERTOBUFFA Associated with Inflammatory ReactionsMembrane Transport of Purine and Pyrimidine HIDEOHAYASHI Bases and Nucleosides in Animal CellsThe Specificity of Pituitary Cells and Regulation RICHARD D. BERLIN A N D JANET M. OLIVER of Their ACtlVltieS-VLADIMIR R. P A N T ~ C
SUBJECT I N D E X
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-RoDY P. COX A N D JAMESc. KING Morphology and Cytology of the Accessory Sex AND Glands in Invertebrates-K. G. ADIYODI R. G. ADIYODI SUBJECT INDEX
355
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. KNIGCE, S. A. JOSEPH,J. R. SLADEK,M. F. NOTTER,M. MORRIS, D. K. SUNDBERG, M. A. HOLZWARTH,G. E. HOFFMAN,A N D L. O’BRIEN SUBJECT INDEX
Volume 44 The Nucleolar StruCtUre-sIBDAS GHOSH The Function of the Nucleolus in the Expression of Genetic Information: Studies with Hybrid Animal Cells-E. SIDEBOTTOM A N D I. 1. DEAK Phylogenetic Diversity of the Proteins Regulating Muscular Contraction-WILLIAM LEHMAN Cell Size and Nuclear DNA Content in Vertebrates-HENRYK SZARSKI Ultrastructural Localization of DNA in Ultrathin Tissue Sections-ALAIN 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, JORG REINICKE WECKE,A N D BERNHARD Cyclic AMP and Cell Behavior i n Cultured CellS-MARK c . WILLINCHAM Recent Advances in the Morphology, Histochemistry, and Biochemistry of Steroid-Synthesizing Cellular Sites in the Nonmammalian Vertebrate OVary-sARDUL s. GURAYA SUBJECT INDEX
Volume 45 Approaches to the Analysis of Fidelity of DNA Repair in Mammalian Cells-MicmEL W. LIEBERMAN The Variable Condition of Euchrornatin and Heterochromatin-FRIEDRICH BACK Effects of 5-Bromodeoxyuridine on Tumorigenicity, Immunogenicity, Virus Production, Plasminogen Activator, and Melanogenesis of Mouse Melanoma CellS-SELMA S~LACI 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 CeIl Differentiation Mechanisms-VICTOR NIGONA N D JACQUELINE GODET Cytophysiology of Corpuscles of Stannius-V. G. KRISHNAMURTHY Ultrastructure of Human Bone Marrow Cell Maturation-J. BRETON-GORIUSA N D F. REYES Evolution and Function of Calcium-BindingProteins-R. H. KRETSINCER 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 Tract-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 PIXCUrSOr Cells Of MeChanOCyteS-ALEXANDER J. FRJEDENSTEIN SUBJECT INDEX
356
CONTENTS OF PREVIOUS VOLUMES
Volume 48 Mechanisms of Chromatin Activation and RePreSSiOn-NORMAN MACLEAN A N D VAUGHAN A. HILDER Origin and Ultrastructure of Cells in Virro-L. M. FRANKSAND PATRICIAD. WILSON Electrophysiology of the Neurosecretory CellKINJIYAGl AND SHIZUKO IWASAKI Reparative Processes in Mammalian Wound Healing: The Role of Contractile Phenomena- GIULIOGABBIANI AND DENYSMON-
New Aspects of the Ultrastructure of Frog Rod Outer SegInc2ntS-JuRGEN ROSENKRANZ Mechanisms of Morphogenesis in Cell CulA N D I. M. GELFAND tures-J. M. VASILIEV Cell Polyploidy: Its Relation to Tissue Growth and Functions-W. Y A . BRODSKY AND I. v. URYVAEVA Action of Testosterone on the Differentiation and Secretory Activity of a Target Organ: The Submaxillary Gland of the Mouse-MoNIQuE CHR~TIEN 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 CeU Organelles of Anoxic Energy ProdUCtiOn-IMRE ZS.-NAGY SUBJECT INDEX
Volume 50
Volume 51 Circulating Nucleic Acids in Higher Organisms-MAURICE STROUN, PHlLlPPE ANKER, PIERRE MAURICE,AND PETER B. GAHAN Recent Advances in the Morphology, Histochemistry, and Biochemistry of the Developing Mammalian O V ~ - S A R D U L s. GURAYA Morphological Modulations in Helical Muscles (Aschelminthes and Annelida)-GluLro LANZAVECCHIA Interrelations of the Proliferation and Differentiation Processes during Cardiac Myogenesis and Regeneration-PAVEL P. RUMYANTSEV The Kurloff Cdl-PETER A. REVELL Circadian Rhythms in Unicellular Organisms: An Endeavor to Explain the Molecular Mechanism- HANS-GEORG SCHWEIGER A N D MANFRED SCHWEIGER SUBJECT INDEX
Volume 52 Cytophysiology Celk-ELADlo
of Thyroid Parafollicular A. NUNEZAND MICHAELD.
GERSHON Cytophysiology of the Amphibian Thyroid Gland through Larval Development and Metamorphosis-ELIANE REGARD The Macrophage as a Secretory Cell-Roy C. PAGE, PHILIP DAVIES,AND A. c. ALLISON Biogenesis of the Photochemical ApparatusTIMOTHY TREFFRY Extrusive Organelles in Protists-KLAUS HAUSMANN
Cell Surface Enzymes: Effects on Mitotic ActivLectins-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 GIUNNELL Chemosensory Responses of Swimming Algae and Protozoa-M. LEVANDOWSKY AND D. C. R. HAWSER Morphology, Biochemistry, and Genetics of Plastid Development in Euglena grucilis-V. NIGONAND P. HEIZMANN Plant Embryological Investigations and Fluorescence Microscopy: An Assessment of Integration-R. N. KAPILA N D S. C. TIWAIU The Cytochemical Approach to Hormone Assay-J. CHAYEN
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Binding and Patterns of Axonal Projections-DoNALD W. PFAFFAND LILYC. A. CONRAD Ancient Locomotion: Prokaryotic Motility Systems-LELENG P. TO AND LYNNMARGULIS An Enzyme Profile of the Nuclear EnvelopeI. B. ZBARSKY SUBJECTINDEX Volume 55
Chromatin Structure and Gene Transcription: Nucleosomes Permit a New S y n t h e s i s THORU PEDERSON The Isolated Mitotic Apparatus and Chromosome Motion-H. SAKAI Contact Inhibition of Locomotion: A Reappraisal-JOAN E. M. HEAYSMAN Morphological Correlates of Electrical and Other SUBJECT INDEX Interactions through Low-Resistance Pathways between Neurons of the Vertebrate CenVolume 54 tral Nervous System-C. SOTELOAND H. KORN Microtubule Assembly and Nucleation-MARC Biological and Biochemical Effects of W. KIRSCHNER The Mammalian Sperm Surface: Studies with Phenylalanine Analogs-D. N. WHEATLEY specific Labeling Techniques-JAMES K . Recent Advances in the Morphology, Histochemistry, Biochemistry, and Physiology of KOEHLER Interstitial Gland Cells of Mammalian The Glutathione Status of Ceh--NECHAMA s. O V ~ ~ ~ - S A R DS. U LGURAYA KOSOWERAND EDWARD M. KOSOWER Correlation of Morphometry and Stereology Cells and SeneSCenCe-ROBERT ROSEN with Biochemical Analysis of Cell FractionsImmunocytology of Pituitary Cells from Teleost Fishes-E. FOLLBNIUS,J. DOERR-SCHOTT, R. P. BOLENDER Cytophysiology of the Adrenal Zona FasAND M. P. DUBOIS CiCUlamsASTONE G. NUSSDORFER, GIUSEPFollicular Atresia in the Ovaries of NonmammaPINA MAZZOCCHI, AND VIRGILIO MENEGHELLI lian Vertebrate&RINIvAs K. SAIDAPUR Hypothalamic Neuroanatomy: Steroid Hormone SUBJECTINDEX
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