INTERNATIONAL REVIEW OF CYTOLOGY V73, Volume 73 (v. 73)

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME73

ADVISORY EDITORS

H. W. BEAMS

ROBERT G. E. MURRAY

HOWARD A . BERN

RICHARD NOVICK

GARY G. BORISY

ANDREAS OKSCHE

PIET BORST

MURIEL J . ORD VLADIMIR R. PANTIC

STANLEY COHEN RENECOUTEAUX MARIE A. DIBERARDINO CHARLES J. FLICKINGER

W. J. PEACOCK DARRYL C. REANNEY LIONEL I. REBHUN

OLUF GAMBORG M. NELLY GOLARZ DE BOURNE

JEAN-PAUL REVEL

YUKINORI HIROTA

WILFRED STEIN

K. KUROSUMI GIUSEPPE MJLLONIG ARNOLD MITTELMAN

HEWSON SWIFT

JOAN SMITH-SONNEBORN

DENNIS L. TAYLOR

AUDREY MUGGLETON-HARRIS

TADASHI UTAKOJl ROY WIDDUS

DONALD G . MURPHY

ALEXANDER L. 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 lndies

Worcester Polytechnic Institute Worcester, Massachusetts

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

VOLUME 73

1981

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

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9 8 7 6 5 4 3 2 1

Contents .

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protoplast Methodology . . . . . . . . . . . . . . . . . . . . . . . . Experimental Work with Protoplasts . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 13 17 18 19

LISTOF CONTRIBUTORS. . . . . . . . . . . . . . . .

Protoplasts of Eukaryotic Algae MARTHAD . BERLINER I. I1. 111. IV .

Polytene Chromosomes of Plants WALTERNAGL

I. I1. I11. IV . V. VI . VII .

Introduction . . . . . . . . . . . Historical Background . . . . . . . Development through Endocycles . . Occurrence and Induction . . . . . Structure of Plant Giant Chromosomes Functional and Evolutionary Aspects . Conclusions . . . . . . . . . . . References . . . . . . . . . . .

Endosperm-Its

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Morphology. Ultrastructure. and Histochemistry S. P . BHATNAGAR AND VEENASAWHNEY

Introduction . . . . . . . . . . . . . . . . The Central Cell . . . . . . . . . . . . . . The Primary Endosperm Nucleus . . . . . . . The Endosperm Cell . . . . . . . . . . . . v . Types of Endosperm Formation . . . . . . . . VI . Ruminate Endosperm . . . . . . . . . . . . VII . Cell Wall Formation . . . . . . . . . . . . VIII . The Endosperm Haustoria . . . . . . . . . . IX . Aleurone Tissue . . . . . . . . . . . . . . X . Functions of Endosperm and Endosperm Haustoria I. I1. 111. IV .

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CONTENTS

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XI . Conclusions and Prospects . . . . . . . . . . . . . . . . . . . . . . . References

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The Role of Phosphorylated Doiichols in Membrane Glycoprotein Biosynthesis: Relation to Cholesterol Biosynthesis JOAN TUGENDHAFT MILLSA N D ANTHONY M. ADAMANV

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction I1 . Structure and Biosynthesis of Carbohydrate Units of Animal Glycoproteins . . I11. Lipid Intermediates in Bacterial Systems . . . . . . . . . . . . . . . . IV . Lipid Intermediates in Eukaryotic Systems . . . . . . . . . . . . . . . V . Relationship of Dolichylsaccharides to Asparagine-Linked Carbohydrate Units of Glycoproteins: Processing and Maturation of Protein-Bound Saccharide Units . VI . Cellular Location of Dolichol-Dependent Glycosylation Reactions . . . . . . VII . Regulation of Giycoprotein Biosynthesis: interdependence of Glycoprotein and Cholesterol Biosynthetic Pathways . . . . . . . . . . . . . . . . . . . . VIII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Mechanisms of Intralysosomal Degradation with Special Reference to Autophagocytosis and Heterophagocytosisof Cell Organelles HANSGLAUMANN. J A N L . E . ERICSSJN. A N D

I . Introduction . I1 . Autophagy . 111. Heterophagy . References .

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LOUIS

MARZELLA

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Membrane Ultrastructure in Urinary Tubules

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LELIOORCI FABIENNE HUMBERT. DENNIS BROWN.A N D ALAINPERRELET 1. Introduction . . . . . . . . . . . . . . . . . . . . . I1 . The Freeze-Fracture Technique . . . . . . . . . . . . . 111. General Morphology of the Freeze-Fractured Plasma Membrane IV . Glomerulus . . . . . . . . . . . . . . . . . . . . . V . Proximal Tubule . . . . . . . . . . . . . . . . . . . VI . Loop of Henle (Thin Descending and Ascending Limb) . . . VI1 . Thick Ascending Limb of Henle and Distal Convoluted Tubule VIIl . The Collecting Tubule . . . . . . . . . . . . . . . . . IX . Discussion . . . . . . . . . . . . . . . . . . . . . . X . Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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CONTENTS

Tight Junctions in Arthropod Tissues NANCY J . LANE

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Fine Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Functional Significance . . . . . . . . . . . . . . . . . . . . . . . . IV . Arthropod Groups in Which Tight Junctional Structures Have Been Reported . . V . Junctions Exhibiting Certain Features in Common with Tight Junctions . . . . . VI . Comparison of Arthropod Zonulae Occludentes with Those of Vertebrates and Lower Chordates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Stages in the Assembly and Breakdown of Arthropod Tight Junctions . . . . . VIII . Models Constructed to Clarify the Features of Arthropod Tight Junctions . . . . IX . Possible Explanations for the Existence of Both Simple and Complex Tight Junctions in Arthropods-Evolutionary Implications . . . . . . . . . . . . . . . . . X . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

244 247 261 273 291

295 300 305 309 312 313

Genetics and Aging in Protozoa JOANSMITH-SONNEBORN

I . Introduction . . . . . . . . . . . . . . I1. Clonal Aging in Ciliates . . . . . . . . . 111. Ciliate Fertilization: Marvel or Menace . . . IV . V. VI . VII .

Environmental Modulation of Longevity . . Aging in Muiticells and Unicells . . . . . Mechanisms of Cellular Aging . . . . . . Summary . . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

INDEX. . . . . . . . . . . . . CONTENTS OF RECENTVOLUMES .

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

ANTHONY M. ADAMANY (103), Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461

MARTHAD. BERLINER (1), Department of Biology, Simmons College, Boston, Massachusetts 02 I I5 S. P. BHATNAGAR ( 5 3 , Department of Botany, University of Delhi, Delhi, India DENNISBROWN(183), Institute of Histology and Embryology, University of Geneva Medical School, 121 I Geneva 4, Switzerland

JAN L . E. ERICSON(149), Department of Pathology, University Hospital, Uppsala University, 5-751 22 Uppsala, Sweden HANS GLAUMANN (149), Department of Pathology, Huddinge Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden FABIENNE HUMBERT (1 83), Institute of Histology and Embryology, University of Geneva Medical School, 121I Geneva 4, Switzerland NANCYJ . LANE(243), A.R.C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, England LOUIS MARZELLA(149), Department of Pathology, Huddinge Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden JOAN TUGENDHAFT MILLS*(103), Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 WALTERNAGL(Zl), Division of Cell Biology, Department of Biology, The University of Kaiserslautern, 0-6750 Kaiserslautern, Federal Republic of Germany

LELIOORCI(183), Institute of Histology and Embryology, University of Geneva Medical School, 121 I Geneva 4 , Switzerland *Present address: Department of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021.

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LIST OF CONTRIBUTORS A I N PERRELET ( 1 83), Institute of Histology and Einhryology, Universiv of Genevu Medical School, 1211 Geneva 4 , Switzerland

VEENASAWHNEY ( 5 9 , Department of Botany, University of Delhi, Delhi, Indiu JOAN SMITH-SONNEBORN ( 3 19). Zoology and Physiology Department, University of Wyoming, Laramie, Wyoming 82071

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME73

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INTERNATlONAL REVIEW OF CYTOLOGY, VOL. 73

Protoplasts of Eukaryotic Algae MARTHAD. BERLINER Department of Biology, Simmons College, Boston, Massachusetts I. Introduction . . . . . . . . . A. Objectives . . . . . . . . B. Definitionof Terms . . . . . c . scope . . . . . . . . . . 11. Protoplast Methodology . . . . . A. Organisms . . . . . . . . B. Culture Conditions . . . . . C. Enzymes and Other Methods . D. Osmotica . . . . . . . . E. Separation . . . . . . . . F. Viability . . . . . . . . . G. Regeneration . . . . . . . 111. Experimental Work with Protoplasts A. Cell Walls . . . . . . . . B. Organelles . . . . . . . . C. Morphogenesis . . . . . . D. Physiology . . . . . . . . IV. Conclusions . . . . . . . . . A. Results through 1980 . . . . B. Suggestions for Future Work . References . . . . . . . . . Note Added in Proof . . . . . .

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I. Introduction A. OBJECTIVES Protoplasts of higher plants have become important experimental tools in the past decade (Cocking, 1980), whereas protoplasts of eukaryotic algae, which were first reported in 1970 by both Chardard and Gabriel, have not received the attention they deserve as potential models for many current aspects of cellular research. Stewart’s book (1974) on algal physiology and biochemistry does not mention the word protoplast, and neither does Pickett-Heaps’ (1975) compendium on the green algae. The literature on algal protoplasts through 1977 has been reviewed by Adamich and Hemmingsen (1980) but is limited to protoplast production methods without discussion of experimental work and of their future in research in areas such as somatic hybridization, morphogenesis, and organelle structure and function. 1 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-364473-9

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This article proposes to bring information on protoplast induction up to date and to discuss the advantages and problems of working with protoplasts of eukaryotic algae. B. DEFINITION OF TERMS

Within the framework of this review, the term protoplast is defined as a viable cell whose wall and other materials external to the plasmalemma have been removed, but which retains all internal components (Adamich and Hemmingsen, 1980). Additional criteria are ( a ) spherical shape, usually of greater volume than the vegetative cell, ( b )osmotic fragility, ( c ) physioiogical activity, ( d ) internal structural integrity, and ( e ) the potential for cell wall regeneration and cell division. Physiological activity and internal structure need not necessarily be similar to that of intact cells as required by Adamich and Hemmingsen (1980), and regeneration and division are frequently difficult to ascertain.

c. SCOPE This article is limited to eukaryotic algae, since they most closely parallel the extensive work on vascular plant protoplasts and have little relationship to protoplast studies with prokaryotic blue green algae (Adamich and Hemmingsen, 1980). In addition, this article will not consider what have been described as “wall-less’’ algae such as Dunaliella (Marano, 1976), Asteromonas (Floyd, 1978), and Ofmansiella (Hargraves and Steele, 1980), because glycoprotein wall materials have recently been demonstrated for these types of organisms by improved cytochemical methods (Oliveira et al., 1980), and also because these cells are neither affected by hydrolytic enzymes nor are they osmotically fragile (Berliner, 1981). Wall-less cells occurring as transient stages as in Chlamydomonas gametogenesis are also not included (Matsuda, 1980).

11. Protoplast Methodology

A. ORGANISMS The review by Adamich and Hemmingsen (1980) lists the following genera of eukaryotic algae (Stewart, 1974) for which protoplast induction has been reChlorella, Cosmarium, Klebsorported: Chlorophyceae-Chlamydomonas, midium, Micrasterias, Mougeotia, Spirogyra, Stigeoclonium, Ulothrix, Zygnema. Bacillariophyceae-Nitzschia. PyrrophyceaeGonyaulax. Rhodophyceae-Porphyridium. Subsequent reports of other true protoplast obtention from additional genera

PROTOPLASTS OF EUKARYOTIC ALGAE

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are: Chlorophyceae-Boergesenia (Kosuge and Tazawa, 1979), Chlorosarcinopsis (Berliner, 1981), Cladophora (Berliner, 1981), Closterium (Berliner, 1978), Cylindrocystis (Berliner, 1981) , Derbesia (Wheeler and Page, 1980), Enteromorpha (Millner et al., 1979), Fritschiella, Mesotaenium (Berliner, 1981), Netrium (Berliner, 1978), Nitella (Abe et al., 1980; Kuroda, 1980), Oedogonium (Berliner, 198l), Polyphysa (Acetabularia) (Zimmer and Werz, 1980), and Staurastrum (Berliner, 1978). Haptophyceae-Hymenomonas (Safa-Esfahani, 1980). All are fresh-water organisms except for Boergesenia (Kosuge and Tazawa, 1979), Chlorosarcinopsis (Berliner, 1981), Enteromorpha (Millner et al., 1979), Gonyaulux (Adamich and Sweeney, 1976), Hymenomonas (SafaEsfahani, 1980), Polyphysa (Acetabularia) (Zimmer and Wen, 1980), and Porphyridium (Clement-Metral, 1976). All these organisms are either unicellular or uniseriate filaments except for Enteromorpha (Millner et al., 1979) which forms a macroscopic tubular thallus. Usually little has been known about the structure and chemical composition of the cell walls and mucilagenous matrices that envelop the genera and species of algal cells used for protoplast induction. One of the exceptions was the deliberate selection by Braun and Aach (1975) of Chlorella species whose walls did not contain sporopollenin. All reports of successful protoplast obtention were from mature vegetative cells except for Derbesia maturing gametophytes (Wheeler and Page, 1980) and for gametes in Chlamydomonas (Schlosser et al., 1976). Attempts to obtain protoplasts from zygotes of Closterium and Cosmarium were not successful (Berliner, 1981). ,

CONDITIONS B. CULTURE Algae from which protoplasts will be induced have always been grown as liquid cultures, but there is no uniformity among the media and all are chemically undefined, containing either seawater or soil extract, and sterilized by filtration or autoclaving. The pH is often not adjusted nor monitored during protoplast formation. All commonly used enzymes and most osmotica such as mannitol substantially lower the pH in the range of 4.0-5.5. Whether or not the algae were axenic is often not mentioned, and both bacteria-free and contaminated cultures have been used in obtaining protoplasts. Rendering algal cultures axenic is often difficult because the mucilagenous slime that naturally surrounds many algal species is a normal habitat for bacteria. The method of Bradley and Pesano (1980) holds the most promise for bacterial removal without toxicity to the algae. Protoplasts induced from nonaxenic cultures rarely survive because the bacteria thrive on the enzyme and the osmoticum .

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The optimal physiological state of the alga prior to protoplast induction has often not been ascertained. Where this has been studied, it has generally been found that cultures in log phase (Berliner, 1977), just prior to division (Marchant and Fowke, 1977) or just after division (Berliner and Wenc, 1976a,b), yield the largest number of protoplasts in the shortest time. In Chlorella it has been observed that the cultures must be monitored microscopically for autospores since these do not form protoplasts (Aach et al., 1978; Berliner, 1977). Light has been one of the other factors often mentioned just in passing rather than extensively studied for its effect on protoplast induction. The temperature at which protoplast induction has been most successful has generally been within the normal culture range for that species, although Millner et al. (1979) found that a low temperature of 10-12°C was optimal for protoplast formation in Enferomorpha. Temperature-sensitive mutants, which form a cell wall at 25°C but not at 35”C, have been used to study cell wall development in Chlarnydomonas (Loppes and Deltour, 1978). Hemmingsen (1971) found that protoplasts were formed by the marine diatom, Nitzschia, only in calciumand/or magnesium-deficient media while Safa-Esfahani (1980) determined that Ca2+ was necessary for protoplast formation.

C. ENZYMES AND OTHERMETHODS Although exogenous hydrolytic enzymes are the main inducers of protoplast formation, other methods have been successful. In algae that form naked zoospores, mechanical disruption of the sporangia have released these “natural” protoplasts (Kosuge and Tazawa, 1979; Robinson and Schlosser, 1978). By the same token the natural weak point at the isthmus of constricted desmids has been used to induce protoplast release by osmotic means alone (Berliner and Wenc, 1976a,b; Chardard, 1970). Detergents and high osmotica induced protoplast emergence between the theca of Gonyaulax (Adamich ‘and Sweeney, 1976). The exogenous enzymes that have been used by most investigators are unspecific mixtures of unknown exact composition that vary from lot to lot. This is true for the most common of these: Driselase, Onozuka Cellulase, Cellulysin, and Macerozyme. Snailgut juice or Helicase, which is commonly used for fungal protoplast induction, has been used only once on algae (Gabriel, 1970). All of these unpurified enzymes contain large amounts of proteases and peroxidases and desalting them, which is often done in higher plant protoplast procedures, only serves to concentrate these lytic fractions (Berliner, 1981). Commercially available enzymes are also contaminated with fungal and/or actinomycete hyphal fragments and conidia and should be sterilized by ultrafiltration. The only specific enzymes that have been used are the autolysines of Chlamydomonas reinhardii (Schlosser et al., 1976; Robinson and Schlosser, 1978). These enzymes are isolated from synchronized cultures of the tissue when gametes fuse and their walls are shed. This instance of the large-scale production

FIGS. 1-10, Fig. 1. Cludophora sp. Protoplast emerging from tip of ultimate cell of filament. Walls remain intact. Figs. 2-5. Zygnemu sp. protoplast formation. Fig. 2. Normal vegetative filament. Fig. 3. Early plasmolysis. Fig. 4. Lateral release of one protoplast per cell. Fig. 5. End of protoplast process. Side walls about to disintegrate leaving unaffected septa. Figs. 1-5. Phase contrast at magnification in Fig. 3. Figs. 6-9. Mesotaenium caldariorum protoplast regeneration. Phase-contrast, all at magnifications in Fig. 8. Fig. 6. Early budding with contents equally divided between the protoplast and the bud. Similar to Fig. 17. Fig. 7. Protoplast with some remaining cell content attached to regenerated filament. Fig. 8. Both original protoplast (left) and its bud have elongated and regenerated a cell wall as seen in Fig. 15. Fig. 9. Protoplast (center) forming walled bud (right) and extruding a vacuole (left). Fig. 10. Nerrium digitus. Formation and release of two protoplasts per cell at dissolved area in side wall (arrow) with eventual disappearance of all wall material. Normarski interference phase contrast.

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of protoplasts from vegetative cells by a “nonforeign” enzyme appears to be useful only for species of Chlamydomonas since their cell walls contain no cellulose, only glycoproteins with galactose and arabinose as the major sugars (Robinson and Schlosser, 1978). Exogenous hydrolytic enzymes act in different ways on algal cell walls during and after protoplast release. The entire wall may be dissolved as with Chlorosarcinopsis (Figs. 11 and 18) and Chlorella (Aach et al., 1978;Berliner, 1977); or a terminal pore is formed in a filamentous species such as Cludophora (Berliner, 1981) (Fig. 1). In other filamentous species such as Zygnema, Mougeotia, and Spirogyra (Marchant and Fowke, 1977; Ohiwa, 1977), the protoplasts are released through a lateral pore and only the tubular walls dissolve, leaving intact septa in the induction medium (Fig. 5 ) . In those algae that have a natural cleavage area in their cell walls, of modified or different chemical composition, this is invariably the locus through which the protoplasts emerge, as in Gonyuulax (Adamich and Sweeney, 1976), Nitzschia (Hemmingsen, 197l), Hymenomonas (Safa-Esfahani, 1980) and in desmids (Berliner and Wenc, 1976b). In the constricted desmids, such as Cosmarium and Micrasterias, the empty walls remain intact in the enzyme solution (Berliner and Wenc, 1976c) while in the unconstricted desmids such as Netrium (Berliner and Wenc, 1976b) and Mesotaenium (Fig. 10) the wall dissolves following protoplast release. Lastly, in Gonyaulax (Adamich and Sweeney, 1976)there remained a carbohydrate-rich pellicle, and in Enteromorpha (Millner et al., 1979) there remained patches of undissolved cell wall on the surface of the spherical and osmotically fragile protoplasts. Whether these are spheroplasts is a question of semantics (Adamich and Hemmingsen, 1980).

D. OSMOTICA Since induced protoplasts of fresh-water algae have lost the mechical barrier that maintains their internal osmotic pressure, they can only retain their integrity and viability in an osmotically protective medium of equal or greater tonicity than that of the normal internal cellular osmolarity. The protoplasts of marine algae such as Chlorosarcinopsis and Acetabularia (Zimmer and Werz, 1981) are osmotically fragile but do not require external osmotic protection beyond that of sea water. In only one instance (Ohiwa, 1977) has the internal osmotic pressure been measured and the information used to determine the minFIGS.1 1 and 12. Chlorosarcinopsis halophila: transmission electron microscopy of protoplast formation. (1 la) Protoplast with well-preserved organelles. C, chloroplast; L, lipid bodies; M, mitochondria; N , nucleus with nucleolus and perforated double membrane; V, vacuole with intravacuolar membranes; s, starch. ( 1 lb) Higher magnification of plasmalemma showing total absence of wall material, ER, endoplasmic reticulum. Fig. 12. Vegetative cell showing thick fibrillar inner (IW) and outer (OW) walls, which interfere with successful fixation of organelles.

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PROTOPLASTS O F EUKARYOTIC ALGAE

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imal hypertonicity needed to protect protoplasts. The internal osmotic pressure of Spirogyra was 0.38 M sucrose-equivalent and that of Zygnema was 0.27 M sucrose-equivalent. The surface tension on the giant protoplasts of Nitella in nonenzyme medium was 0.2 dynekm-' or equivalent to that of mature sea urchin eggs (Kuroda, 1980). The substances most commonly used as osmotica in all plant protoplast work, including algae, are mannitol, sorbitol, MgSO, and KCI, either alone or in combination. These substances are rarely metabolized and often do not support the growth of contaminants. Several studies on the effective molar range of these substances have been done (Berliner and Wenc, 1976a; Chardard, 1972; and Marchant and Fowke, 1977) and have generally concluded that mannitol and sorbitol are the least toxic and generally most effective in a range of 0.3 to 0.5 M mannitol or equivalent. Only for the large thallus of Enteromorpha (Millner et al., 1979) was a 1.2 M concentration of sorbitol required to induce and maintain protoplasts. The cells of fresh-water algae are invariably plasmolyzed in the effective osmotica, and this plasmolysis is often the best indicator of the preprotoplast stage (Berliner and Wenc, 1976a). On the other hand, marine algae such as Gonyaulax (Adamich and Sweeney, 1976), Chlorosarcinopsis (Berliner, 1981), Enteromorpha (Millner et al., 1979), and Nitella (Abe et al., 1980) usually are not plasmolyzed at any hypertonicities prior to protoplast release. Some researchers have preplasmolyzed cells in osmoticum prior to adding the enzyme (Berliner and Wenc, 1976a, b; Ohiwa, 1978) to try to shorten the time that cells need to be exposed to the enzyme, but with little success. Ohiwa (1978) did establish that only protoplasts of Spirogyra and Zygnema that had been preincubated in the osmoticum had the potential for completing protoplast fusion.

E. SEPARATION There are few details available on the separation of protoplasts from the inducing medium. Removal of the enzyme and maintenance of the osmoticum are imperative in preserving morphologically and physiologically intact protoFIGS. 13-17. Cell wall regeneration in protoplasts of Mesoraenium caldariomm. Magnification in Fig. 15 applies to Figs. 13, 14,and 17 as well. Figs. 13-16,ultraviolet microscopy of Calcofluorstained cells. Fig. 13. Elongated protoplast with multilayered accumulation of amorphous wall material not visible in phase-contrast microscopy as in Fig. 8. Protoplast still osmotically fragile. Fig. 14.Early septum formation and amorphous wall deposition. Fig. 15. Protoplast bud elongation into filament of diameter similar to that of control cell on the left. Both the protoplast and the filament have Calcofluor-positive walls. Fig. 16.Short and stubby regenerated protoplast as in Fig. 14,which has divided and is shedding Calcofluor-positive primary wall (arrow). Fig. 17. Dark-filled light microscopy of early budding protoplast and normal cell. Cell contents are outlined but cell wall deposition is not visible. FIG. 18. Scanning electron @croscopy of Chlorosarcinopsis halophila protoplast with conspicuous lipid globules just beneath the tom plasma membrane (Fig. l l a ) (arrow).

PROTOPLASTS OF EUKARYOTIC ALGAE

11

plasts that will have the potential to regenerate. Both Millner et al. (1979) and Adamich and Sweeney (1979) used a Ficoll gradient, while the latter and Berliner (1981) (Fig. 20) have provided detailed flow diagrams of separation techniques.

F. VIABILITY Protoplast viability has most commonly been assessed by the exclusion of vital dyes by living cells (Berliner et al., 1978). The dyes most commonly used are Trypan Blue (Berliner et al., 1978), Crystal Violet (Clement-Metral, 1976), Neutral Red (Millner et ul., 1979), and Fluorescein Diacetate (Marchant and Fowke, 1977; Safa-Esfahani, 1980). The latter author also correlated viability to counts of cells that regenerated flagellae and became motile. Colony counts of regenerated cells in soft agar were done by Braun and Aach (1975). The last two methods measure regeneration potential rather than protoplast viability.

G. REGENERATION Regeneration of an outer wall followed by cell division is the desired outcome of most protoplast experimental work. Its successful achievement has eluded many investigators and the various methods that have been described in any detail are all derived from experience with higher plant protoplasts. The modifications that have succeeded have little rational basis and are most often the results of chance or guesswork. The flow diagram for regeneration of protoplasts of Mesotuenium (Fig. 20) incorporates most of the factors that must be considered and modified for each organism. These are: (1) the pH which universally needs to be raised as soon as enzyme is removed; (2) the osmoticum which must be decreased as soon as a cell wall matrix is laid down-this to be checked by using Calcofluor fluorescence in ultraviolet light of polysaccharide materials; (3) Ca2+ ions to stabilize the naked plasmalemma; (4) diffuse or no light; ( 5 ) temperature usually slightly higher than for normal growth; (6) shaking and aeration which although often tried seem to make little difference. All detailed reports of regeneration and cytokinesis have considered these criteria (Berliner, 1981; Marchant and Fowke, 1977; Ohiwa, 1978, 1980; Safa-Esfahani, 1980). ~~

~~

FIG.19. Chlorosnrcinopsis halophila protoplast regeneration after 72 hours. The outer wall (OW) has the typical wavy appearance of the control vegetative cells (Fig. 12). Oblique sections through the inner wall (IW) show both granular and fibrillar regions. The wall patches (WP) are temporary and disappear after the first division. The single chloroplast is well-developed with many osmiophilic droplets and some starch. The double arrows in the center point to spindle-organizing microtubules. Triple arrow in lower left point to the early cytoplasmic invagination preceding cytokinesis and cross-wall formation. C, Chloroplast;0, osmiophilic granules; Ve, vesicles; S, starch.

step 1

step 2 18-20h

step 3

J

60-90

mirm.

3 days1

FIG.20. Flow diagram for protoplast formation and regeneration in Mesotaenium caldariorum, Step 1, Axenic strain growing on agar slant inoculated into liquid medium at 2loC, 15 hours light/9 hours dark cycle. Growth for 1 month on rotary shaker. Step 2: Culture transferred to Petri dish to which are added final concentration of 3.3% Cellulysin, 0.6 M mannitol, and 5 mM CaCI2, pH 4.5, for protoplast induction. Step 3: After 24-48 hours, protoplasts in enzyme-induction medium are

13

PROTOPLASTS OF EUKARYOTIC ALGAE

The experimental applications of cell wall regeneration are discussed in the next Section (111,A). In Chlorosarcinopsis (Berliner, 1981) and in Derbesia (Wheeler and Page, 1980) it was noted that cell wall formation begins with a very noticeable increase in rough endoplasmic reticulum and vesicles, whereas this was not found in Chlamydomonas (Robinson and Schlosser, 1978).

111. Experimental Work with Protoplasts A. CELLWALLS Protoplast formation and regeneration are the theoretically ideal tools for the understanding of cell wall structure and formation, and several studies have used algal models. Most authors do not distinguish between primary and secondary cell walls, yet some algae such as Cosmarium have conspicuous primary walls that disintegrate in the induction medium whereas the secondary walls remain unaffected. Often certain portions of the secondary walls are more resistant to enzyme digestion than others. An example is Zygnema (Fig. 5 ) whose septa remain as H configuration after protoplast release. When the composition of cell walls has been known, the information has been used to advantage by Braun and Aach (1975) who used a Chlorella strain known not to contain the resistant polysaccharide sporopollenin. Safa-Esfahani (1980) used similar information about coccolith composition to devise regeneration media for protoplasts of Hymenomonas. The time required for de novo regeneration of coccoliths was 24 hours which is identical to the time required to build a new wall for a daughter cell under the same conditions. Zimmer and Werz (1981) have reported that microtubules or microfilaments are not involved in Acetabularia protoplast regeneration. Most algae have complex, multilayered, microfibrillar walls with interspersed dispensed into glass centrifuge tubes and allowed to settle for 60-90 minutes, and then gently spun for 2 minutes (3a); supernatant is decanted and replaced with culture medium 0.4 M mannitol + 5 mM CaCl,, pH 6.8-7.2 (3b); step 3b is repeated, except that the supernatant is replaced with medium without CaCl,. Step 3 is carried out at room temperature in the light. Step 4:Protoplast suspension (3c) dispensed into corresponding wells of unsealed Well-Plates (Linbro) and placed on one sheet of bond paper at 23°C for 16 hours in the light and at 19°C for 8 hours in the dark. A single sheet of paper towel was placed on top of the plate. The bond paper and paper towel are used to diffuse the light. Step 5: After 3 days, 1.5 ml from each well, 2 ml per well was pipetted into each well from a second plate. Equal amounts of medium + 0.2 M mannitol added to all wells to obtain a final concentration of 0.3 M mannitol. This step repeated at 3-day intervals to obtain final concentrations of 0.2 M, and 0.5 M mannitol. Wall deposition starts in 0.4M mannitol without enzyme (step 4)and is completed in 0.2 M mannitol.

+

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MARTHA D. BERLINER

amorphous areas (Millner et al., 1979) that should lend themselves to understanding the sequential nature of wall formation and degradation (Fig. 19). Robinson and Schlosser (1978) studied the effects of different types of inhibitors on cell wall regeneration by protoplasts of Chlamydomonas smithii. These were ion chelators such as EDTA; sugar metabolism inhibitors such as 2deoxy-d-glucose; modifiers of cell surface properties such as concanavalin A; inhibitors of translation such as cycloheximide, and inhibitors of transcription such as actinomycin D. Cell wall regeneration was reversibly inhibited by cycloheximide and reversibly disturbed by concanavalin A. Actinomycin D irreversibly inhibited cell wall regeneration, resulting in unusual membrane configurations. Chelators and 2-deoxy-d-glucose had minimal effects. Similar studies with Polyphysa cell wall regeneration (Zimmer and Werz, 1980) showed that the concanavalin A inhibition was completely reversible by the application of the hapten sugar a-methyl-D-mannoside. In addition they found that Con A dissociated the synchrony between cell wall formation and nuclear division. Cell wall deposition was suppressed but mitosis was unaffected. Coumarin has been found to be a reversible inhibitor of cell wall and septum formation in regenerating Mougeoria and Hymenomonas protoplasts (Marchant, 1979; Safa-Esfahani, 1980), but not of cortical microtubules normally associated with active wall deposition. In further work with regenerating Mougeotia protoplasts, freeze-etch studies revealed that microfibrils were deposited within 60 minutes and that in 2 hours a dense mat of wall material was deposited, followed by projections of the plasma membrane (Marchant and Hines, 1980). In regenerating protoplasts of Chlorosarcinopsis, Calcofluor-positive temporary wall patches (Fig. 19) were deposited first, followed by the normal fibrillar material (Berliner, 1981); whereas in Mesotaeniurn amorphous wall was present on the budding protoplasts (Figs. 13 and 17). A temperature-conditioned mutant of Chlamydomonas (Loppes and Deltour, 1978) that does not form cell wall at 35°C showed a correlation between cell wall formation and phosphatase activity as the temperature was lowered to 25°C. This “conditional protoplast” was due to a single nuclear gene. In giant cells of Boergesenia (Kosuge and Tazawa, 1979) the cell wall is laid down on the protoplast at the time of abrupt increase of the surface tension on the plasmalemma. The direction of regenerating protoplast elongation did not always coincide with the axis of the chloroplast helix in Spirogyra and Zygnema (Ohiwa, 1977). B. ORGANELLES For most algae, the ultrastructure of the protoplast is better defined than in vegetative cells, whose walls are often impervious to fixative penetration (Figs. lla,12, and 19) (Berliner, 1981; Robinson and Schlosser, 1978). The organelles

PROTOPLASTS OF EUKARYOTIC ALGAE

15

of protoplasts have been studied by phase-contrast microscopy, fluorescence microscopy, scanning and transmission electron microscopy, and freezeetching, yielding a variety of unrelated observations about intracellular changes during protoplast formation and regeneration that extend the in-depth survey of Pickett-Heaps (1975) on the ultrastructure of the green algae-the taxonomic group where the majority of protoplasts have been obtained. From all the transmission electron microscopy studies of protoplasts, it appears that the enzymatic cell wall removal does not seem to substantially alter the normal internal structure, nor that the osmoticum has more than a transient effect. Lipid was accumulated during protoplast formation in Chlorosarcinopsis halophila (Fig. 18), but disappeared as soon as new cell wall was laid down (Fig. 19). Robinson and Schlosser (1978) found unusual membrane configurations only in Chlamydomonas reinhardii protoplasts whose cell wall regeneration was inhibited by actinomycin D. As soon as the inhibitor was removed, normal appearance of the membranes and cell wall deposition was reestablished. Membranes of Mougeotia protoplasts (Marchant, 1978a,b) studied by scanning electron microscopy allowed visualization of the cytoplasmic face of the plasmalemma with its associated microtubules, which have also been demonstrated by negative staining (Marchant and Hines, 1980). These microtubules have been postulated to be involved in the stabilization of the plasmalemma and in the control of orientation of the cell wall microfibril deposition (Marchant and Hines, 1979). The vacuole membrane was much harder to break than the plasma membrane in Cosmariurn protoplasts (Berliner et al., 1978). Broken protoplasts resulting from lowering the osmoticum yielded fractions that can be separated into portions predominantly made up of nuclei, chloroplasts, vacuoles, and cytoplasmic materials. The critical temperature for membrane lysis in protoplasts of Chlamydomonas reinhardii was 40°C. Polycations such as polyL-ornithine and lysine, spermine, and spermidine, within a narrow concentration range, provided thermal protection to the plasmalemma at 45°C (Hasnain et a / ., 1980). This was not the case for streptomycin sulfate and was probably due to this substance’s mode of action as an inhibitor of membrane-protein synthesis. Chlamydomonas cells need not be flagellated to form protoplasts (Schlosser et al., 1976), and regenerating Hymenomonas protoplasts re-form basal bodies and flagella as soon as the enzymes are removed (Safa-Esfahani, 1980). When these latter protoplasts are placed in coumarin, not only is cell wall formation inhibited, but the number of basal bodies, mitochondria, nuclei, and chloroplasts is increased (Safa-Esfahani, 1980). Protoplast fusion has been used to test the survival of organelles from different genetic pools. Ohiwa (1978, 1980) fused Spirogyra and Zygnema protoplasts by using polyethylene glycol, and identified the resulting somatic hybrids on the basis of chloroplast shape, number of pyrenoids and their size, and the location of the nucleolus. He found that regeneration of fusion products depended on the

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MARTHA D. BERLINER

ratio of Zygnema to Spirogyra protoplasts. Normal regeneration occurred when the fusion was between several Zygnema and one Spirogyra protoplast but not in the reverse proportions. One: one fusions did not survive to regenerate. Intraspecific fusions between Spirogyra protoplasts were common, and rare in Zygnema. In fusion products, division took place only in Zygnema nuclei. There was no nuclear fusion or coexistence, and the nuclei of the genus with the smallest number degenerated. Ohiwa thus postulated (1978) that the amount of cytoplasm in a somatic hybrid determined which nucleus would survive. If the nucleoli were lost, then the chloroplast of that genus degenerated, whereas if the nucleoli were maintained, so was the cytoplasmic integrity. Nuclei in intergeneric fusions that would degenerate, did so in 12-20 hours by the rupture of the nuclear envelope, extrusion of the nucleolus, and condensation of the chromatin (Ohiwa, 1980). Fowke et al. (1979) introduced the organelles of Chlamydomonas reinhardii into carrot cells by the fusion of a wall-less Chlumydomonas mutant to a carrot protoplast. Most of the algal organelles degenerated rapidly with the chloroplasts lasting the longest time. C, MORPHOGENESIS

During regeneration of Mougeotia protoplasts (Marchant and Hines, 1980), the cortical microtubules, which are originally free, associate and cross-bridge with the plasmalemma during cell wall regeneration. When microtubule polymerization is inhibited, the protoplasts remain spherical with disoriented microfibrils. Nevertheless, the microtubules are not solely responsible for morphogenesis because protoplasts in coumarin did not regenerate a wall but had the same number and distribution of microtubules bridged to plasmalemma (Marchant, 1979). Flagellated cells were found in regenerating Stigeoclonium protoplasts 12- 16 hours after protoplast isolation. Zoospores in this organism normally differentiate from vegetative cells, but thus seem to be able to arise directly from protoplasts. Flagella are normally shed prior to protoplast formation (Hemmingsen, 197 I ; Safa-Esfahani, 1980) but reappear early in regeneration in Hymenornunas (Safa-Esfahani, 1980). Autospores have been reported to develop within protoplasts of Chlorella (Aach et al., 1978). D. PHYSIOLOGY Photosynthesis and its associated metabolic processes is the most commonly used measure of physiological activity in protoplasts. In Chlorella vulgaris photosynthesis as measured by O2 evolution was 337 for vegetative cells, 70 for protoplasts, and 234 in cells in mannitol but without enzyme (Webb et al., 1980). In Enteromorpha (Millner et al., 1979) O2 evolution by protoplasts was

PROTOPLASTS OF EUKARYOTIC ALGAE

17

only slightly higher than in the vegetative thallus, but O 2 consumption by protoplasts was more than five times higher. This was probably due to cell injury since 90% of the protoplast were dead in 24 hours and there was no regeneration. In Porphyridium (ClCment-Metral, 1976) the protoplasts evolved oxygen without a Hill oxidant. Autofluorescence of chlorophyll in ultraviolet light was used to trace the fate of Chlamydomonas ‘‘protoplasts” of a cell-wall-less mutant into chloroplast-less carrot cell protoplasts (Fowke er al., 1979). Photosynthesis and starch accumulation continued in cultured fusion products from Spirogyru and Zygnema protoplasts (Ohiwa, 1978). Gonyaulax protoplasts became motile before new cell wall maturation, resumed chemical and mechanically stimulated bioluminescence in approximately 1 hour after removal of the enzyme, and quickly reestablished the normal rhythmic light-emission pattern (Adamich and Sweeney, 1976). Labeled [14C]mannitolentered protoplasts faster than in whole and plasmolyzed cells and located in all areas of the cytoplasm except the nucleus and the pyrenoids (Berliner and Martindale, 1981).

IV. Conclusions A. RESULTSTHROUGH 1980 Since the review of the literature through 1977 (Adamich and Hemmingsen, 1980), as many papers have been published on protoplasts of eukaryotic algae as in the 10 previous years, and double the number of genera are now available for protoplast studies. Nevertheless, of the 28 genera of algae, only 3 are not in the Chlorophyceae and only 1 is an alga with a macroscopic thallus. Axenic culture methods for other groups of algae are being developed and these should soon be added to the list of protoplast sources. Algal protoplasts have begun to be used in well-designed experiments aimed at understanding all aspects of the sequences and kinetics of cell wall assembly. Interspecific and intergeneric protoplast fusions have been regenerated and the resulting somatic ‘‘hybrids ” cultured. Elucidation of membrane physiology using protoplasts of giant cells has now been reported, Yet as of now there is insufficient information on which to base broad correlations of morphogenetic phenomena among such a diverse group of algal species, nor is it possible to make such generalizations for universal plant cell mechanisms. Perhaps some of the suggestions in the next section will answer such broad questions. FOR FUTURE WORK B. SUGGESTIONS

Protoplast fusion to create intergeneric hybrids is perhaps the most tantalizing goal of investigators in this field. Large marine algae such as Laminaria and

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MARTHA D. BERLINER

Ulva could be sources of much-needed protein if they could be modified to grow faster, with thinner walls and less mucilage, and with more complete proteins. Fresh-water algae could in turn be modified to become halotolerant and grow in brackish waters as sources of single-cell protein. Nitrogen-fixing genes could be introduced into protoplasts of nontoxic algae that form pond scums or blooms. On a smaller laboratory scale, questions such as the following could be answered by using algal protoplasts. In genera with both homo- and heterothallic species, are there differences in their ability to form protoplasts and have them fuse? In algae with gametophyte and sporophyte generations, are there again differences in their haploid vs diploid protoplasts? So far, no protoplasts have been obtained from zygotes. In another aspect, studies can elucidate the role of the plasmalemma to the passage of large molecules normally barred by the cell wall. The possibilities are truly endless, and the choices among algal cells is rapidly expanding. One or more should fulfill many experimental needs.

ACKNOWLEDGMENTS The author’s research, which forms part of this article, has been supported by grants from the Simmons College Fund for Research, The Research Corporation, and the National Science Foundation. I acknowledge with thanks the work of my students Katherine Wenc, Josephine Damico, and Nancy Wood; of my assistants Irene Carlson and Paul Martindale; and of my colleague Elizabeth Guth. The literature search for this paper was concluded in March 1981.

REFERENCES Aach, H. G., Bartsch, S., and Feyen V. (1978). Planfa 139, 257-260. Abe, S., Takeda, J., and Senda, M. (1980). Plant Cell Physiol. 21, 537-546. Adamich, M., and Hemmingsen, B. B. (1980). In “Handbook of Phycological Methods: Developmental and Cytological Methods” (E. Gantt, ed.), pp. 153-157. Cambridge Univ. Press, London and New York. Adamich, M., and Sweeney, B. M. (1976). Planta 130, 1-5. Berliner, M. D. (1977). Plant Sci. Lett. 9, 201-204. Berliner, M. D. (1978). 2. Pflunzenphysiol. 88, 341-348. Berliner, M. D. (198 1). Unpublished observations. Berliner, M. D., and Guth, E. A. (1980). Bot. SOC. Am. Meet., Vancouver, B . C . p. 12 (Abstr.). Berliner, M. D., and Martindale, P. T. (1981). Planr Sci. Lett. 20, 371-377. Berliner, M. D., and Wenc, K. A. (1976a). Microbios Lett. 2, 39-45. Berliner, M. D., and Wenc, K. A. (1976b). Protoplasma 89, 389-393. Berliner, M. D., and Wenc, K. A. (1967~).Appl. Environ. Microbiol. 32, 436-437. Berliner, M. D., Wood, N. L., and Damico, J. (1978). Protoplasma 96, 39-46. Bradley, P. M., and Pesano, R. L. (1980). J . Cell B i d . 87, 228a. Braun, E., and Aach, H. G. (1975). Planta 126, 181-185. Chardard, R. (1970). 95‘ Cong. Natl. SOC. Suv. Reims 3, 315-320.

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Chardard, R. (1972). C.R. Acad. Sci. Paris. 274, 1015-1018. Clement-Metral, J. D. (1976). J. Microsc. Bioi. Cell. 26, 167-172. Cocking, E. C. (1980). In “Advances in Protoplast Research” (L. Ferenczy and G. L. Farkes, eds.), pp. 3-18. Academic Press, New York. Floyd, G. L. (1978). J. Phycol. 14, 440-445. Fowke, L. C., Gresshoff, P. M., and Marchant, H. J. (1979). Planta 144, 341-347. Gabriel, M. (1970). Protoplasma 70, 135-138. Hargraves, P. E., and Steele, R. L. (1980). Phycologia 19, 96-102. Hasnain, S. E., Khan, M. A., and Upadhyaya, K. C. (1980). Indian J . Biol. Sci. 18, 1037-1039. Hemmingsen, B. B. (1971). Ph.D. Thesis, Univ. California, San Diego, California. Kosuge, Y., and Tazawa, M. (1979). Bot. Mag. Tokyo 92, 315-323. Kuroda, K . (1980). Cell Biol. Inr. Rep. 4, 195-199. Loppes, R., and Deltour, R. (1978). Enp. Cell Res: 117, 439-441. Marano, F. (1976). J. Microsc. B i d Celf. 25, 279-282. Marchant, H. J . (1978a). Exp. Cell Res. 115, 25-30. Marchant, H. J . (1978b). In “Scanning Electron Microscopy 11” (R. P. Becker and 0. Johari, eds.), pp. 1071-1076. AMF, O’Hare, Illinois. Marchant, H. J. (1979). Nature (London) 278, 167-168. Marchant, H. J . , and Fowke, L. C. (1977). Can. J . Bot. 55, 3080-3086. Marchant, H. J . , and Hines, E. R. (1980). Planta 146, 41-48. Matsuda, Y. (1980). Plant Cell. Physiol. 21, 1339-1342. Millner, P. A., Callow, M. E., and Evans, L. V. (1979). Planta 147, 174-177. Ohiwa, T. (1977). Cell Strucr. Funcr. 2, 249-255. Ohiwa, T. (1978). Protoplasma 97, 185-200. Ohiwa, T. (1980). Protoplwna 102, 77-95. Oliveira, L., Bisalputra, T., and Antia, N.J.(1980). New Phyrof. 85, 385-392. Pickett-Heaps, J. P. (1975). “Green Algae. ” Sinauer, Sunderland, Massachusetts. Robinson, D. G., and Schlosser, U. (1978). Planr 141, 83-92. Safa-Esfahani, A. R. (1980). Ph. Thesis, Iowa State University, Ames. Schlosser, U., Sachs, H., and Robinson, D. C. (1976). Protoplasma 88, 51-64. Stewart, W. D. P., ed. (1974). “Algal Physiology and Biochemistry.” Univ. of California Press, Berkeley. Webb, D. T., Berliner, M. D., and Carlson, I. (1980). Pflanzenphysiol. 96, 325-329. Wheeler, A. E., and Page, J. 2 . (1980). Bot. SOC.Am. Meet., Vancouver, B.C. p. 125. (Abstr.). Zimmer, B., and Wen, G. (1980). Exp. Cell Res. 126, 299-310. Zimmer, B., and W e n , G. (1981). Exp. Cell Res. 131, 105-111. NOTE ADDEDIN PROOF. Two significant additional reports have recently been published. (1) Hermesse, M.-P., and Matagne, R. F. (1980). Arch. Inf. Physiol. Biochem. 88, B282, B291. The authors present the conditions for PEG-induced somatic fusions between nutritionally cornplementing protoplasts of Chlumydomonas rheinhardii and an analysis of the resultant products. (2) Klein, K., Wagner, G., and Blatt, M. R. (1980). Plantu 150, 354-356. These authors demonstrated the presence of actin in burst protoplasts of Mougeoriu and hypothesized that these filaments mediate chloroplast movement.

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INTERNATIONAL REVIEW OF CYTOLOGY,VOL. 73

Polytene Chromosomes of Plants' WALTERNAGL Division of Cell Biology, Deparment of Biology, The University of Kaiserslautern, Kaiserslautern, Federal Republic of Germany Introduction . . . . . . . . . . . . . . . . . . . . Historical Background . . . . . . . . . . . . . . . . Development through Endocycles . . . . . . . . . . . . Occurrence and Induction . . . . . . . . . . . . . . . Structure of Plant Giant Chromosomes . . . . . . . . . . A. General Aspects . . . . . . . . . . . . . . . . . B. The Phaseolus Polytene Chromosomes . . . . . . . . . C . The Tropaeolum Polytene Chromosomes . . . . . . . . D. The Rhinanthus Polytene Chromosomes . . . . . . . . VI. Functional and Evolutionary Aspects . . . . . . . . . . . A. Functional Aspects . . . . . . . . . . . . . . . . B. Evolutionary Aspects . . . . . . . . . . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . I. 11. 111. IV. V.

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I. Introduction Polytene, or giant, chromosomes have been attracting the attention of cell biologists since their discovery. Much of the substance of genetics, cytogenetics, and cell biology is based on the study of polytene chromosomes in Diptera. Actually, one can see them as the connecting link between genetics and gene physiology on the one hand, and molecular biology of DNA and chromatin on the other. Giant chromosomes allow the direct microscopic observation of chromosome organization, gene location (e.g., by in situ hybridization), differential gene activity (puffing), heterochromatin (satellite DNA), underreplication, and many other events. Therefore, many models of chromosome organization and function have been developed from investigations on polytene nuclei. Polytene chromosomes are structures occurring in highly endopolyploid interphase nuclei; to be precise, they are cable-like bundles of sister chromosomes that are the result of endocycles, that is, chromosome-replication cycles without nuclear division. Although the salivary gland chromosomes of dipteran larvae are the best-studied ones, polytene chromosomes are neither unique to salivary 'Dedicated to Professor Dr. Elisabeth Tschermak-Woess, the pioneer of polyteny in plants

21 Copyright @ 1981 by Academic Press, Inc. All rights of reproduction i n any form reserved. ISBN 0-12-364473-9

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glands nor to Diptera. They have been found in various tissues of insects and a few highly specialized cell types of different animals and plants (for reviews see Beermann, 1962; Geitler, 1965; Nagl, 1978a). The intention of this article is to review findings of polytene chromosomes in plants and their development and structure in comparison with giant chromosomes in Diptera, and to discuss some functional and evolutionary aspects of polyteny . The recently developing ideas about the spatial arrangement of chromatin as a link between DNA thermodynamics and regulation of gene expression are discussed. 11. Historical Background

The occurrence of polytene nuclei in plants was detected as early as 1898 by Osterwalder, who described giant chromosomes in the antipodal cells of Aconitum. Woycicki (1907) observed giant nuclei in the suspensor of Tropaeolum that “appeared to be undergoing mitosis although no division figures were observed. ” Evidently, the author recognized polytene chromosomes. But as the structures were not understood by these authors, and because Aconitum displays polytene chromosomes only occasionally, the descriptions were forgotten by the scientific community. As late as 1956, Tschermak-Woess and other cytologists at the Botany School of the University of Vienna reinvestigated Aconitum and other species in a systematic study on endopolyploidy, and polytene chromosomes were indeed found again (Table I). Because plant polytene chromosomes are not normally banded, or not banded throughout, they were first called “giant chromosome-like structures” or “plant giant chromosomes. ” Today it is known that the structure of polytene chromosomes can vary in both plants and animals, from strongly bundled, banded organization to a more loosely bundled, unbanded organization. Due to the fundamental similarities of all polytene structures, no terminological distinction is made now. Only several minor differences between the typical dipteran polytene chromosomes (e.g., in Drosophila or Chironomus) and most of the plant chromosomes have been recorded. Giant chromosomes in plants are not somatically paired, except occasionally in Bryonia (Barlow, 1975). In the embryo-suspensor of Phaseolus, the homologues are sometimes found in the vicinity (Nagl, 1974a). As a consequence of the unpaired situation, the haploid number of polytene chromosomes is counted in antipodal cells, but a diploid number in suspensor cells, and a triploid number in endosperm and endosperm haustoria. (It should be noted that unpaired giant chromosomes can also be found among insects.) 111. Development through Endocycles

The term “endocycle” was introduced to designate a DNA-replication cycle within the nuclear envelope and without spindle formation, that is, the endqmito-

POLYTENE CHROMOSOMES OF PLANTS

23

tic cycle and the endoreduplication cycle (Nagl, 1978a). Both cycles irreversibly lead to endopolyploidy and are evidently under strict genetic control. This strict control may be seen as the main distinction from other cell cycles leading to somatic polyploidy, such as the restitution cycle and mitosis in binucleate cells with spindle fusion. Endomitosis (Geitler, 1939) and endoreduplication (Levan and Hauschka, 1953) differ from each other in that structural changes comparable with those seen in mitosis occur in the former event, while no mitosis-like process can be seen during an endoreduplication cycle. As pointed out by D ’Amato ( 1954), the crucial difference between endomitosis and endoreduplication is polytenization in the latter event, the most common process of endonuclear chromosome multiplication to be found in animals and plants. (Regarding the question of a dispersion stage as expression of plant endomitosis, refer to Section V.) Polytene nuclei of low order (exhibiting from one to a few endoreduplication cycles) do not show polytene chromosomes during interphase, but their condition can be visualized by induction of polyploid mitoses (e.g., by phytohormones). If the nuclei are in the polytenic state, then the mitotic figure exhibits diploid “pairs, ” diploid “groups of four, ” and diploid “groups of eight” chromosomes, respectively, now commonly called diplochromosomes, quadruplochromosomes, and octuplochromosomes. Figures 5b and c show parts of such “endoreduplication mitoses. This type of mitosis sometimes occurs spontaneously, particularly in cell cultures, but also can be induced by phytohormones and other agents in plants (Grafl, 1939; Levan, 1939; D’Amato and Avanzi, 1948; D’Amato, 1952; Nagl, 197Oe) and animals including humans (Schwarzacher and Schnedl, 1965, 1966; Herreros and Giarelli, 1967; Nasjleti and Spencer, 1968; reviewed by D’Amato, 1977). The prerequisite for the origin of polytene chromosomes is, therefore, an endocycle during which the chromosomes stick togehter at least at their centromeres (centromeric heterochromatin?). Moreover, no chromatin condensation must take place throughout the endocycle. Therefore, the highly endopolyploid nuclei of Heteroptera and other genera, in which endomitotic chromosome condensation occurs, never exhibit giant chromosomes, Very rarely a polytenic nucleus undergoes endomitosis or even mitosis; as a consequence the polytene chromosomes condense and fall apart into the single sister chromosomes (endochromosomes) in both insects (Bier, 1969) and plants (Nagl, 1970b; Marks and Davies, 1979). DNA replication in endocycles can follow different pathways: either the total genome is replicated (as in the mitotic cycle); or a small portion of the nuclear DNA is not replicated or is not as often replicated as the main portion of the genome (i.e., DNA underreplication). In a few cases, only a small portion of the genome (e.g., a single gene) is extrareplicated on the diploid or polyploid level (i.e., DNA amplification). Figure 1 shows the genomic result of different cell cycles. Some examples may elucidate that scheme. Gage (1974) studied the DNA from highly polyploid silk gland nuclei with the DNA isolated from other somatic tissues by means of buoyant density centrifugation, DNA-RNA hybridi”

24

WALTER NAGL

FIG.1. The genetic result of the mitotic cell cycle and of some endo cycles: (a) mitotic cycle, (b) endoreduplication cycle, (c) underreplication cycle, (d) amplification cycle (R, DNA replication; DIV, cell division; UR, underreplication; A, amplification;DEG, degradation of extra DNA copies). (Modified from Nagl, 1976a.)

zation, and DNA-reassociation kinetics. As the DNAs were qualitatively similar, he concluded that the total genome was replicated during endopolyploidization. The giant nuclei, which exhibit levels of endopolyploidy between 500,000 C and 2 million C, display numerous cable-like structures each about 0.5 p m in diameter (Gage, 1974; Rasch, 1974; Nakanishi et al., 1969). In the giant nuclei of mouse trophoblast the constitutive heterochromatin and the satellite DNA therein are replicated in step with the euchromatin and main band DNA, respectively (Barlow and Sherman, 1974; Sherman et al., 1972). Also in the rat trophoblast, there is no evidence for underreplication. While the facultative heterochromatin (sex chromatin) always forms ‘‘polytenic clumps” (Zybina and Mosjan, 1967; Nagl, 1972a), the euchromatin only sometimes joins to giant chromosomes (Zybina, 1977; Nagl, 1978a; Nagl and Fuhrmann, 1981). DNA underreplication has been observed only during somatic polyploidization so far. Most frequently the proximal (centromeric) heterochromatin, and the satellite DNA located therein, as well as the nucleolus organizers (ribosomal DNA sequences), are underreplicated (reviewed by Laird, 1973; Nagl, 1978a). DNA underreplication has also been recorded from polytene nuclei in several cells of the embryo-suspensor of Tropaeolum and Phaseolus (see Section V for

POLYTENE CHROMOSOMES OF PLANTS

25

details). Recently, Laird (1980) has discussed the structural paradox occumng in polytene chromosomes with varying levels of polyteny. DNA amplification is well known from the salivary gland chromosomes of sciarid flies (“DNA puffs”), but it evidently also takes place in plant giant nuclei (Nagl, 1972b; Nagl and Rucker, 1976; Forino et al., 1979). Some findings will be discussed in Section V. In some cases of insects and most examples of plants, polytene chromosomes can be seen only in some nuclei of a tissue, or only during some years, while otherwise the nuclei display different structures (see, for instance, Fig. 3). Neither the functional significance nor the environmental stimuli leading to such differences are understood. Nutritive factors, effects of temperature, and other possibilities have been discussed (reviewed by Nagl, 1978a). In plants there is a general rule: the development of polytene chromosomes is strongly supported by the presence of large blocks of heterochromatin in the chromosome complement, probably due to the general “stickiness” of heterochromatin. For instance, the polytene chromosomes of Tropaeolum and Rhinanthus, which regularly can be observed, are nearly entirely composed of constitutive heterochromatin, while the euchromatin only rarely joins up. If heterochromatin underreplication takes place, no polytene chromosomes originate (Nagl, 1976b; Nag1 et al., 1976). In Phaseolus, where also giant chromosomes develop regularly in the giant cells of the suspensor, polytenization clearly starts at the heterochromatic regions and proceeds during each endocycle also within euchromatic regions (Nagl, 1974a).

IV. Occurrence and Induction As noted in Section I, polytene chromosomes can be found within various animal and plant taxa, although only in a limited number of species. Banded polytene chromosomes have been reported to occur in the macronucleus anlage of several hypotrichous ciliates (e.g., Ammermann, 1971; Prescott et al., 1973). In these protists they are a transitory structure; during a certain stage they are dissected by membranes, and most of the DNA is degraded. The remaining gene-sized DNA sequences are amplified to form the amitotically dividing macronucleus. From the large group of invertebrates, polytene chromosomes are so far known only among the insects (Collembola, Diprera; some Orthoptera exhibit polytene structures similar to those in plants). In vertebrates, weakly bundled polytene structures have been recorded only in the giant cells of trophoblast in some rodents (for reviews see Beermann, 1962; Hennig, 1974; Nagl, 1978a). All known examples of polyteny in plants are summarized in Table I. It must be emphasized that these examples certainly represent only a small number of cases of polyteny among plants, because only a few species have been studied in

26

WALTER NAGL TABLE I RECORDS OF POLYTENE CHROMOSOMES IN PLANTS Species

Aconitum neomontanum A . ranunculifolium A . variegatum A lisma plantago-aquatica Allium amophilum Allium ursinum

Tissue

Antipodal cells Suspensor Antipodal cells Synergids Endosperm haustoria

Bryonia dioica

Anther hairs

Clivia miniata Cymbidium (hybrid) Dicentra spectabilis Eruca sativa Gagea lutea Papaver rhoeas Phaseolus caffer Phaseolus coccineus

Antipodal cells Protocorm culture Antipodal cells Suspensor Suspensor Antipodal cells Suspensor Suspensor

Phnseolus Phaseolus Phaseolus Phaseolus

Endosperm Suspensor Suspensor Suspensor Suspensor

hysterinus multijlorus tuberosus vulgaris

Endosperm Roots (?)" Cotyledon culture

Pisum sativum Rhinanthus alectorolophus Rh. aristatus Rh. minor Salvia horminum Scilla bifolia Thesium alpinum Triticum aestivum

Endosperm haustorium Glandular hairs Antipodal cells Endosperm haustorium Antipodal cells

Triticum durum Tropaeolum majus Zea mays

Antipodal cells Suspensor Endosperm

(?) Doubtful report.

References

Tschermak-Woess (1956) Bohdanowicz (1973) Hasitschka-Jenschke (1957) Hasitschka-Jenschke (1957) Geitler (1955), Hasitschka-Jenschke (1957), Turala (1966) Tschermak-Woessand Hasitschka (1954), Barlow (1975) Tschermak-Woess (1957b) Nagl and Rucker (1972) Hasitschka-Jenschke (1959) Corsi (1972) Hasitschka-Jenschke(1962) Hasitschka (1956) Nagl (1974a) Nagl (1962, 1974a), Avanzi et al. (1970), Brady (1973a,b), Brady and Clutter (1972, 1974), Clutter et al. (1974), Schweizer (1976), Schweizer and Ambros (1979), Forino et al. (1 979) Nagl (1970a) Nagl (1974a) Nagl (1974a) Nagl (1974a) Nagl (1969a-c), Schweizer (1976), Bhattacharya (1 978a) Nagl (1971) Bhattacharya (1978b) Marks and Davies (1979)

Tschermak-Woess (1957a, 1967) Gostev and Asker (1978) Nagl (1976~) Erbrich (1965) Bennett et al. (1973), Bennett and Smith (1975) Ivanovskaya (1968, 1973) Nagl (1976b), Nagl et al. (1976) Tschermak-Woess and Enzenberg-Kunz (1965), Stephen (1973)

POLYTENE CHROMOSOMES OF PLANTS

27

this regard. As most of the angiosperms undergo somatic polyploidization (about 90%; Partanen, 1963) and most of the mature cells exhibit various levels of polyploidy (about 70 to 80%; Butterfass, 1966; Frisch and Nagl, 1979), one must expect to find more species that also develop polyteny. However, as can be seen from Table I, most examples are from cells within the ovule (antipodal cells, synergids, endosperm and endosperm haustoria, cells of the suspensor-i.e., the nutritive organ of the young embryo in many species). It is evident that polytene chromosomes originate, with some exceptions, only in cells of high endopolyploidy levels, and that these levels are only.reached in highly active cells (see Section VI for details). As many of the listed cells are only short-lived, some experience is required in order to detect the polytene nuclei in plant ovules. Figure 2 shows how to find the giant nuclei in the Phaseolus bean and the Rhinanthus endosperm (for Phaseolus see also the instruction for preparation of suspensors and polytene chromosomes as given by Nagl, 1974a). In Rhinanthus, the giant chalazal haustoria can be seen already as hills on the surface of the young seed (Tschermak-Woess, 1957a). Under certain experimental conditions it was possible to induce the formation of polytene chromosomes in species where normally none are found. In sterile organ cultures of protocorms of the orchid Cymbidium, whose cells alsovexhibit heterochromatin amplification, a few cells developed cable-like polytene chromosomes (Nagl and Riicker, 1972). The induction required high levels of 2,4-dichlorophenoxyaceticacid, a synthetic auxin, in the culture medium, but this was certainly not the only factor. Besides phytohormones, several other

/

\

MH

FIG.2. Where to find plant polytene chromosomes. (a) Median section through a bean of Phaseofuscoccineus (Em, embryo; En, endosperm; S,suspensor;M = rnicropyle). (b) Endospenn of a young ovule of Rhinnnrhus alecrorolophus (CH, chalazal haustorium; MH, micropylar haustorium). The arrows indicate the cells whose nuclei exhibit polytene chromosomes. X150. (a, redrawn from Nagl, 1974a; b, redrawn from Tschermak-Woess, 1957a.)

28

WALTER NAGL

factors are known to control the mitotic as well as the endomitotic cycle (reviewed by Nagl, 1978a). If polytenization is seen as the normal consequence of endocycles leading to sufficiently high ploidy levels, then the induction of polyteny requires a more intense study of the regulation of endocycles. This should be possible in vitro, as polyteny has not only been found to occur in cultured protocorm pieces, but also was obtained in cultured cells of Drosophila that were originally obtained from diploid embryos (Shields et al. (1975). Marks and Davies (1979) found polytene nuclei in cultured cotyledons of Pisum sativum, but the inductive factors have not been studied. Another question refers to the side-by-side packaging of the individual endochromosomes and the exactness of banding patterns. As discussed in Section 111, one factor of polytenization is absence of chromosome coiling. As this is an event determined by the kind of endocycle that is passed through, again the question may be more related to the control of cell cycle curtailment (before or during mitosis) than to the regulation of nuclear structure itself. However, in insects and plants nutritive factors and environmental factors have been found to influence the degree of bundling of the endochromosomes (reviewed by Nagl, 1978a). Mayfield and Ellison (1975) have put forward the hypothesis that sequence-specific nonhistone proteins account for the binding together of the chromomeres of each band. Actually, several nonhistone proteins are not found in polytene nuclei of Drosophila, but in “reticulate” nuclei (Elgin and Boyd, 1975). A specific nonhistone protein or modified histone H1 may be responsible for the condensation and stickiness of heterochromatin (Holmgren et al., 1976; Blumenfeld et a f . , 1977). Gruzdev and Belaya (1968) suggested a role of disulfide bridges in the formation of polytene chromosomes and (1973) also the involvement of bivalent ions (see also the model of Zainiev and Gruzdev, 1973; and the discussion by Nagl, 1976a). The arrangement of chromatin within the nucleus must not be seen, however, only as the consequence of endoreduplication cycles. As will be discussed in Section VI, spatial chromatin organization is now envisaged to play a key role in gene expression, as it determines the portion of the genome that can be transcribed.

V. Structure of Plant Giant Chromosomes A . GENERAL ASPECTS

As mentioned in the preceding sections, the occurrence of polyteny and the appearance of the polytene chromosomes may vary within a given tissue, and from year to year. In the antipodal cells of Pupaver rhoeas the sister chromosomes range from an obviously polytenic organization to an isolated arrangement

FQLYTENE CHROMOSOMES OF PLANTS

29

FIG. 3. Semidiagrammatic drawings of some possible structures of endopolyploid antipodal nuclei in Papaver rhoeas: (a) endochromocenters with radiating euchromatic portions of the chromosomes, (b) decondensed, isolated chromatin fibers, (c) coiled chromatin fibers, (d) polytene chromosomes. (Modified from Hasitschka, 1956.)

within the nucleus in either a condensed or decondensed state (Hasitschka, 1956; Fig. 3). In Phaseolus suspensor cells, the polytene nuclei may appear banded, or granular, or looped, and their ends split more or less; during degeneration, they often disintegrate along their whole length into single chromatids (Nagl, 1970c; see also the following paragraph). In general, plant polytene chromosomes are composed of loose cable-like structures at low ploidy levels, and they display a granular appearance with at least some heterochromatic bands at higher ploidy levels. The frequent absence of distinct bands in the euchromatin can be attributed to the weak bundling tendency as a consequence of the dispersion stage of heterochromatin during endomitosis or late S period (Nagl, 1972c, 1976a, 1977a, 1978a). During the dispersion stage the heterochromatin decondenses to the level of euchromatin (Heitz, 1929; Geitler, 1941; Tschermak-Woess and Hasitschka, 1953; Nagl, 1968, 1972c; Tschermak-Woess, 1971; Barlow, 1976). These changes could prevent the formation of a clear banding pattern (Fig. 4). Evidently the corresponding chromomeres of the endochromosomes do not match up exactly to form bands. However, in many cases a few bands can be observed (cf. Figs. 5c, 6, and llc). In Phaseolus, bands are more frequently seen; their distinctiveness can be greatly enhanced by inactivating transcription through chilling the plants, or by exposing them to other disadvantageous condi-

31)

UALTER NGGL

b

Q

c c

d

FIG. 4 , Theoretical organization of polytene chromosomes and their microscopic appearance: ( a h ) Lliptcra, ( c and (11 Anpiuspemata, (hkxiified from Nsgl, 1Y76a I

ilntl

tions (,cf. Fig, 7, inset). Similar effects of temperature c1r drugs have been reported in animal systems (reviewed by Nagl. 19783). Thc hetemchrc~maficportions appear either in the form of cottipact and sometimes vacuolatcd blocks, o r in the forms of groups of large granules, the so-called hcterochrcimonieres. Smaller portions of intercalary heterochromatin may be observed as heterochromatic bands. A widely established feature of the heteroT chromatin in plant giant chromosorncs is its ability to produce nucleolar material. It is not yet discerned what kind of RNA (rRNA, mRNA?) is located in such additional micronucleoli. The endosperm nuclei of Phaseolus (see the following section) and of Zeu mays (up to 384 n) exhibit polytene chromatin fibers (Tschermak-Woess and Enzenberg-Kunz, 1965: Fig. Sa). They producc many micronuclcoli, particularly at the nuclcolus-associated heterochromatin. The pattern and amount of tnicronucleoli-producingsites is highly variable among the studied varieties (cultivars, inbred lines). Recent analysis of the DNA isolated from fruits and lcavcs by melting and renaturation kinetics did not give evidence for ribosomal DNA amplification (Monika Jeanjour, unpublished; Fig. lob). Figures S to 9 and 1 1 give examples of the morphology of plant polytene chromosomes. Some of them are composed of euchromatic and heterochromatic sections, others of almost exclusively heterochromatin. In the following paragraphs the best-studied systems will be put forward i n detail.

B . T H EPhcrseolus POLYTENE CHROMOSOMES The polytene chromosomes in the embryo-suspensor of Phaseolus coccineus were detected some 20 years ago (Nagl, 1962). The cells of the suspensor show a gradual increase in their degree of endopolyploidy from the embryo to the basal cells (Table II), and only the latter giant cells display polyteny (Fig. 2 4 . The 1 1

POLYTENE CHROMOSOMES OF PLANTS

31

FIG. 5. Examples of polyteny in plants. (a) Highly endopolyploid nucleus from the endospenn of Zeo mays; the chromatin is organized into threads. (Redrawn from Tschermak-Woess and Enzenberg-Kunz, 1965.) (b) Part of a 16-ploid prophase from the stem pith of Kniphofio natalensis exhibiting octuplochromosomes; this indicates that the interphase nucleus had low-level polytene chromosomes, each composed of eight endochromosomes. (Redrawn from Fenzl and TschermakWoess, 1954.) (c) Quadruplochromosome from the root of Allium cepa. (Redrawn from Levan, 1939.) (d) Polytene chromosome in an antipodal nucleus of Aconitum variegotum, displaying some bands. (Redrawn from Tschennak-Woess, 1956.)

32

WALTER NAGL TABLE 11 ENDOWLYPLO~DY LEVELS IN T H E SVSPENSOR CELLSOF Phaseofus coccineus"

Location of cells

Polyploidy level

Embryo Suspensor (close to embryo)

2n 4n 8n I6 n 32 n

64n Suspsnsor (median region)

Suspensor (basal giant cells)

128 n 256 n 512 n 1.024 n 2.048 n 4,096 n 8,192 n

Mean nuclear volume

Mean nuclear DNA content

Wn3)

(Pg)

277 529 1,412 2,230 4,761 12,488 26,261

52,857 116,227

240,250 479,453 826,550 1,700.000

2.7 5.4 10.8 22.0 44.0 W.0 170.0 340.0 650.0 1,400.0 2,800.0 5,750.0 12,000.0

Modified from Nag1 (1974a).

pairs of polytene chromosomes can be distinguished by means of their heterochromatic bands and have an average length from 39 p m (chromosome pair 1 1) to 62 p m (chromosome pair I ) at 4096-ploidy (Nagl. 1967). As metaphase chromosomes are 1.3 to 2.7 p m long, the polytene chromosomes are about 30 times longer than the metaphase chromosomes. The suspensor giant chromosomes do not usually exhibit a characteristic longitudinal differentiation in bands and interbands such as is known from polytene chromosomes in Diptera (Fig. 6). Using silver-staining and Giemsa C-banding techniques, Schweizer and Ambros (1979) were able to demonstrate that the chromosome maps drawn by Nagl (1967, 1974a) should be revised with respect to the nucleolus-organizing regions. An actinomycin D binding map was proposed by Cionini and Avanzi (1972) for the nucleolus-organizing chromosomes 1 and 5 , and a scanning densitometric map by Forino et al. (1979). The latter authors undertook also to find a precise nomenclature for the chromosome regions similar to what exists for human chromosomes. The Phaseolus polytene chromosomes exhibit high structural variability and functional structures along their total length and in distinct regions. When plants developing seedpods are grown at lower temperatures (12°C during day, 8°C during night), the polytene nuclei are inactivated, and the giant chromosomes condense to form bands and interbands (Nagl, 1969a, 1970c; Linnert 1977; Fig. 7). A similar condensation of the normally granular polytene chromosomes takes place after inactivation of RNA synthesis by actinomycin D and after in vitro

’. I .

d

FIG. 6. Several phenotypes of the suspensor polytene chromosomes of Phaseolus coccineus in comparison to prophase chromosomes: (a) prophase; (b) polytene chromosome 5 with decondensed nucleolus-organizing region and additional micronucleolus formed from the centromeric heterochromatin; (c) polytene chromosome 4 with splitting short arm; (d) polytene chromosome 5 forming several loops and micronucleoli; (e) polytene chromosome (not identified) in the banded state. (Modified from Nagl, 1967, 197Od.)

FIG.7. Microphotos of Phaseolus metaphase and polytene chromosomes. The polytene nucleus from a basal cell of the suspensor is stained by the Giemsa C-banding technique (X400). the metaphase (round insert. x 1600) by the silver method for nucleolus organizers, the banded polytene chromosome (rectangular inset. x 1600) is unstained, taken in the phase-contrast microscope. (Moditied from Schweizer and Ambros, 1979. and Nagl, 1969a.)

POLYTENE CHROMOSOMES OF PLANTS

35

treatment of isolated suspensor cells with Ca2+ ions (Nagl, 1969b; reviewed 1974a). In Phaseolus coccineus a conspicuous structural change of the polytene chromosomes occurs after a temperature elevation from 12/8 to 22°C (and also other temperature levels) in many seeds. Within 20 minutes all the polytene chromosomes of a nucleus develop loops like lampbrush chromosomes, first in distinct regions, later along the whole euchromatic portions. The loops, which are radiating from the outer chromatids of the polytene chromosomes leaving a more condensed central core, have a total length between 4 and 50 p m and a diameter of 0.25 to 0.40 p m . The terminal parts of some loops form spherical networks, which become increasingly loaded with droplets, evidently material synthesized by the chromatin fibrils. Finally, the droplets fuse to form nucleolus-like bodies, which are released from the chromosomes (for details see Nagl , 1970~). The validity of the general rule that decondensed (puffed) chromatin is more active in RNA synthesis than condensed chromatin has been shown by [3H]uridine autoradiography (Nagl, 1969b). The activity of the suspensor polytene chromosomes is apparently also under the control of light (photoperiodic effects: Nagl, 1973a). Structural modifications in distinct regions of the polytene chromosomes have been recorded in the euchromatic, heterochromatic, and nucleolus-organizing regions. Two puffs, somewhat different in their structure, are found nearly throughout the lifetime of the giant cells in chromosome pair 2 (Nagl, 1969c, 1974a). The same polytene chromosomes exhibit, in addition, an additional nucleolus (micronucleolus), which is regularly formed on one of the heterochromatic telomeric bands (Fig. 8). In very old suspensor cells, or after actinomycin D treatment, the puffs disappear. Other polytene chromosomes show puffs of various structures (granular expansion, fibrillar networks, etc.) at unidentified regions (Nagl, 197Od). The heterochromatic regions have been found to be also “puffed” in certain chromosomes of some giant cells (Nagl, 1967). Later, these regions were interpreted as DNA puffs similar to those occumng in sciarid flies by Avanzi et al. (1970) and Forino el al. (1976, 1979). Although these investigations by means of autoradiography and scanning densitometry of Feulgen-stained preparations cannot be considered as proof of DNA amplification, evidence for differential DNA replication has also been obtained by CsCl density centrifugation of DNA and filter hybridization of DNA and ribosomal RNA from different Phaseolus organs (see later). The nucleolus organizers show the most important structural modifications in connection with high or low synthetic activity (Nagl, 1970~).In active cells they split up into oligotene bundles, single chromatids, and probably submicroscopic loops; in inactivated cells the nucleolus organizers condense to heterochromatic knobs, and the intranucleolar fibrils fuse to a banded thread. In active suspensor cells the nucleolus is highly lobed and vacuolated, and many micronucleoli are

36

WALTER NAGL

FIG, 8 . Diagrammatic representation of a pdytcne chromosome (probably no. 2) with two puffed regions (P,and P,). heterochromatic hands (HI. and a micronucleolus fonned at the telonirric heterochromatin. The kinetochore region (K)is also clearly visible. (According to Nagl, 1974a.)

released from its surface. These micronucleoli and those that originated on heterochromatic bands and euchromatic loops migrate through the nucleoplasm to the nuclear envelope and are released as free RNP aggregates into the cytoplasm. Sometimes long nuclear evaginations can be seen, which translocate a micronucleolus deep into the cytoplasm and release it by breakdown of the membranes (Nagl, 1973b). In the electron microscope, clusters of RNP granules can be seen in the cytoplasm that gradually disperse throughout the cell (Nagl and Fuhrmann, 1981). While the micronucleoli protruding from the main nucleolus evidently contain ribosomal material, it has not yet been discerned whether the other micronucleoli contain ribosomal or informosomal (messenger) RNP. In situ hybridization studies did not yield unequivocal results (Avanzi et ul., 197I , 1972; Brady and Clutter, 1972). The development of polytene chromosomes during the endomitotic growth of the suspensor cell nuclei occurs gradually. In smaller nuclei only the heterochromatin of the sister chromatids (or endochromosomes) sticks together, while the euchromatic regions are radiating in all directions. In larger nuclei the polytene chromosomes become more and more distinct with every round of replication. DNA synthesis in the suspensor polytene chromosomes was first studied by Avanzi e t a / . ( 1 970). The authors suggested the occurrence of extra replication in certain regions, which was believed to occur in step with the general replication (see also the foregoing on DNA puffs). The DNA core of some of the micronucleoli formed by such regions hybridized with [ 3H]rRNA, but that of others did not. Therefore, it was concluded that genes different from those for rRNA might also be amplified (Avanzi et ul.. 1972). The behavior and structure of the Phaseolus polytene chromosomes and micronucleoli strikingly resemble observations made in salivary gland chromosomes of Sciarida (Brito-da-Cunha et a ] . , 1969), in the macronucleus anlage of Sglonychia (Gil et al., 1972), in the oocytes of Acheta (Jaworska and Lima-de-Faria, 1973), and in many other systems. The finding that the Feulgen-DNA values of different-aged suspensor nuclei follow a geometric series does not confirm the suggestion of DNA amplification (Brady, 1973a). Lima-de-Faria rt ( I / . (1979, however, em-

POLYTENE CHROMOSOMES OF PLANTS

37

phasized according to their biochemical results that some regions of the polytene chromosomes may be amplified, as they detected a suspensor-specific satellite DNA, while others might be underreplicated: thus the sum equals, in statistical terms, the complete replication value. Brady (1973b) and Brady and Clutter (1974) studied DNA replication in the endomitotic cell cycle of the suspensor cell nuclei. They found autoradiographic evidence of late replication in the heterochromatin, and suggested that this might possibly by misinterpreted as DNA amplification. The total replication of the polytene chromosome complement requires 4 to 6 hours. The labeling pattern after exposure to [3H]thymidineis changing during the S period, and the chromosomes themselves were said to undergo structural changes during DNA replication (Brady and Clutter, 1974), but this was not confirmed by other authors (reviewed by Nagl, 1974a). Avmzi et al. (1970) observed lateral differences of DNA replication, in that only some of the endochromosomes incorporated [3H]thymidine at a given time. In a few cells, the polytene chromosomes have been found in a dispersion stage, that is, all the chromatin is equally decondensed to the level of chromomeres, including the heterochromatin (Nagl, 1965). This dispersion stage was thought to be the structural expression of endomitosis in plants (Geitler, 1953; Tschermak-Woess, 1971), but this was later doubted (Barlow, 1976; see review and discussion by Nagl, 1978a). The DNAihistone ratio has been measured by means of the FeulgedFast green absorption values for different regions of the Ph. coccineus polytene chromosomes (Diez and Cionini, 1971; Cionini, 1971). Different ratios were found between different chromosomes and between different regions of individual chromosomes. In addition, the ratio in active regions was discovered to be lower than that in inactive, condensed regions. Although the data obtained may be affected by methodological errors in the cytochemical procedures and do not take into accoiint possible modifications of the histones, they show that the study of the histones may reveal interesting aspects of polytene chromosome formation and function (see also the notes given by Hagl, 1974a). Polytene chromosomes of smaller size are found in the chalaza1 endosperm nuclei of Phaseolus; they display a chromomeric (banded) structure. If gibberellic acid is injected into ovules, the polytene chromosomes develop loops and numerous additional nucleoli, which can be envisaged as expression of increased gene activities (Nagl, 1971). Moreover, [3H]uridine incorporation confirms stimulated RNA synthesis, and [3H]thymidine incorporation indicates that the phytohormone also stimulates new rounds of DNA replication. C. THE Tropueolum POLYTENE CHROMOSOMES

The suspensor of the nasturtium. Tropueolum mujus, is a large tripartite structure that evidently serves as haustorium for the nutrition of the embryo. Most of

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its cells become highly endopolyploid, up to 2048 n. While some of the endopolyploid nuclei exhibit the same structure as the diploid nuclei (i.e., a very diffuse euchromatin and several dotlike chromocenters), and others lack chromocenters due to heterochromatin underreplication, several nuclei show polytene chromosomes (Nagl, 1976b). The polytene chromosomes are composed mainly of heterochromatin displaying some bands, while the euchromatin forms more diffuse tails and loops (Fig. 9a). Heterochromatin underreplication in many of the suspensor cell nuclei has been demonstrated by DNA measurements in single cells (scanning cytophotometry ), structural analysis. and melting profiles of isolated DNA (Nagl, 1976b, 1978b; Nagl et al.. 1976; Fig. IOa). The satellite DNA of Tropueolum is evidently not underreplicated as was shown by in situ hybridization (Deumling and Nagl, 1978). The localization of the satellite DNA in nucleolus-organizing regions indicates that it is of ribosomal nature. The underreplicated DNA within the heterochromatin, therefore, is composed of heterogeneous repetitive DNA sequences, which do not form a satellite on CsCl density centrifugation.

D. THERhinunthus POLYTENECHROMOSOMES Tschermak-Woess ( 1957a, 1967) described the occurrence of polytene chromosomes in the endosperm haustoria (particularly the chalaza1 haustoria) of ovules of Rhinanrhus alecrorolophus, Rh. minor, Rh. serotinus, and Rh. arisratus. The haustorial cells contain two giant nuclei each, which reach levels of 384-ploidy [endosperm nuclei start with 3 n (33) due to double fertilization]. The two sister nuclei exhibit in about 80% of all haustoria studied the same level of polyploidy, in 20%, successive levels. The polytene chromosomes (normally 2 1) display a granular, chromomeric organization. The subterminal centromere cannot be identified in the polytene chromosomes, but the region, where the centromere is to be expected, is composed of particularly dense heterochromatin, which is in connection to the nucleolus in most chromosomes. The larger part of the giant chromosomes is built up of granular heterochromatin, and diffuse, weakly bundled euchromatin is only found for a short distance at one end (Fig. 1 1b). While in the triploid endosperm nuclei 9 chromosomes are nucleolusorganizing ones (i.e., 3 in the haploid complement), in the polytene nuclei up to 21 chromosomes are connected to large, often ramified nucleoli with their short arms (heterochromatic ends). Others form a small additional nucleolus at their heterochromatic regions. It has not yet been determined whether these micronucleoli are not visible in the diploid nuclei due to their tiny size, or whether they are produced only in the polyploid state in order to increase the amount of ribosomal RNA or a certain messenger RNA. The application of the silver-staining tech-

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FIG.9. Polytene nuclei in two plant species. (a) Tropaeolum majus. suspensor; the arrow indicates some bands: Feulgen-stained, phase-contrast, X 860. (Modified from Nagl, 1976b.) (b) Scilla bifolia, antipodal cell; note the diploid and low-polyploid nuclei in the surrounding area; the inset shows a polytene chromosome at maximal development; Feulgen-stained, X 700. (Modified from Nagl, 1976c.)

WALTER NAGL

m

90

80

loo

*C

a

z 2

75

I0

90

a5

95

100

'C

b FIG. 10. DNA melting profiles used as a test for differential DNA replication in polytene nuclei. (a) Melting profiles of Tropoeolum DNA isolated from leaf buds (A),globular embryos (m), and buspensors (a); the melting temperature of the suspnsor DNA is shifted to higher values, indicating that the AT-rich heterochromatin is underreplicated in many nuclei. (From Nagl. 1978h.) (b) Derivative melting profiles of maize DNA isolated from leaf buds (e).young leaves (0).and unripe fruits (mainly endosperm, A):the melting fine structures do not differ, indicating that there is no gene amplification in the endosperm nuclei. (Courtesy of Monika Jeanjour.)

nique for active ribosomal genes and of in situ hybridization could help to elucidate the function of these micronucleoli. The growth and development of the polytene chromosomes was studied by Tschermak-Woes ( 1957a). At low levels of endopolyploidy, the individual sister chromosomes can be seen as thick chromatin fibers wound around each other. Later, at 12 n to 24 n, the chromosomes elongate and decondense so that only chromomeres remain visible. The chromomeres still resemble the location of the former fibers and allow the counting of endochromosornes. These cable-like

POLYTENE CHROMOSOMES OF PLANTS

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structures are more or less distinguishable until degeneration. However, during further polytenization the endochromosomes and their chromomeres, respectively, fuse at some sites. During senescence of the endosperm haustoria, first the euchromatin and granular heterochromatin spreads and is degraded; later the heterochromatin becomes pycnotic and degenerates. Tschermak-Woess and Hasitschka-Jenschke (1963) studied the behavior of the

FIG. 11. Camera-lucida drawings of polytene chromosomes in two plant species. (a) Dodecaploid nucleus in the chalazal haustorium of Rhinanrhus; the single endochromosomes can be followed up. (b) One L polytene chromosome and several K chromosomes. The L chromosome shows the tiny euchromatic end (at left), the main body composed of granular heterochromatin, and the condensed heterochromatic end producing micronucleoli; some K chromosomes lie isolated, while some others form a starlike endochromocenter. (a and b, modified from Tschermak-Woess, 1957a, 1967.) (c) Polytene nucleus in an antipodal cell (16 n) of Dicentra spectabilis. (Modified from HasitschkaJenschke, 1959.)

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additional small chromosomes in polytene nuclei of the species Rhrnnnthus minor. This and the other Rhinanthirs species have, besides seven large chromosomes. four \mall chromosomes per haploid complement. The regular number of four of these small chromosomes has thrown doubt on their interpretation as accessory B chromosomes, Therefore, the chromosome complement was designed to be composed of 7 L (large) and 4 K (small) chromosomes (TschermakWoess, 1967). The small K chromosomes exhibit a totally different behavior during polyploidization than the large L chromosomes. While the latter form the deccribed polytene chromosomes, the former remain tiny individual chromatin rods, or fuse to small. sometimes starlike endochromocenters (Fig, 1 I ) . It was suggested that the reason for this different behavior is the euchromatic nature of the K chromosomes (Tschermak-Woess, 1967).

VI. Functional and Evolutionary Aspects A. FUNCTIONAL ASPECTS The functional significance of endopolyploidy and polyteny has been subjects of discussion since their discovery (Geitler, 1953; D’Amato, 1964; Torrey, 1965: Nagl. 1976a. 1978a). As the synthesis of DNA, the largest natural polymeric macromolecule, is very expensive in te.ms of energetics, if is very unlikely that the wide occurrence of endopolyploidy and polyteny is without any selective advantage. Actually a number of advances can be seen in endocycles and the endopolyploid state. But let us first consider some objections to a functional role of somatic polyploidy. Evans and Van’t Hof (1975) argued that endopolypioidy cannot have a function in differentiation and development, because species seem to exist that differentiate in the diploid state. This argument, however, does not bear in mind that evolution has developed different strategies for most events that occur in living matter. For instance, the C4 pathway of photosynthesis is not without function and advantage, because it can be found in a limited number of plants only. Many textbooks of cell biology and developmental biology either do not deal with endopolyploidy and polyteny, or misrepresent these characters of most higher organisms as exceptions or even as pathological slips of a cell. Therefore, many biologists do not take into consideration that polyteny at each level can play some role in the complex differentiation process of multicellular organisms. What advantages can be proposed for growth by endocycles? First, RNA synthesis is not interrupted during an endocycle, but it is during the mitotic cycle. So the cell is liberated for continuous and increasing RNA synthesis and therefore an increased capacity of protein synthesis (Bennett, 1973; Nagl, 1973c; Clutter et al., 1974). This enables rapid growth and differentiation, which is

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apparently of outstanding importance for nutritive cells of the embryo and other highly active but short-lived cells. Actually, such cells exhibit the highest degree of polyploidy and the largest polytene chromosomes. The inverse relationship between the rate of RNA synthesis and the rate of cell division has been demonstrated in animal systems (Flickinger et al., 1970) and plant systems (Clutter et al., 1972; Walbot et al., 1972). As in insect larvae, so also in plant embryology, growth by endocycles allows a rapid and early function of cells in comparison to mitotically growing tissues. For instance, the chalaza1 haustorium of Rhinanthus exhibits full function in supporting the embryo with nutritive material during polytenization (Tschermak-Woess, 1957a). The suspensor in Phaseolus ovules is fully differentiated and active, while the embryo is still in a very early proliferative stage; very similar situations can be found in mammalian trophoblasts and ovarian nurse cells of insect oocytes (reviewed by Nagl, 1978a; Nagl and Fuhrmann, 1981). Cell differentiation and function are initiated in many organisms only after cell division stops (reviewed by Cameron and Jeter, 1971). The large-scale synthesis of fibroin in the silk gland of Bombyx mori depends on the completion of polyploidization (Nigon et al., 1978). Mammalian bone marrow megakaryocytes do not undergo thrombopoiesis before they have reached the octoploid state (Paulus, 1968). The nurse cells (trophocytes) of meroistic insect ovaries, which support the oocytes with ribosomal and informosomal ribonucleoprotein, are always polyploid (Bier, 1969). Embryos of plant species that have evolved a suspensor depend on this polytenic organ in order to undergo normal growth and differentiation (Corsi, 1972; Haq et al., 1973; Nesling and Morris, 1979; Yeung and Susses, 1979). Bennett et al. (1973) found that the number of polytenic antipodal cells formed in Triticum aestivum embryo sacs determines the number of endosperm cells and thus seed size. Also the secretory activities of plant nectaries apparently depend on polyploidization (Mikhailova, 1969; Kartashova et al., 1970), as well as those of honeybees (Merriam and Ris, 1954) and glands in insects (Swift, 1962) and starfishes (Vorobiev and Leibson, 1974). Summing up, it must be emphasized that there is overwhelming evidence for a particularly active state of polyploid and polytenic cells, which is apparently the direct consequence of the multiplication of chromosomes. A further advantage of endocycles can be seen in their immunity from disturbances that easiIy affect mitosis with its more complex events (TschermakWoess, 1971). This advantage was shown by experiments employing actinornycin D and histones, which inhibit mitosis in root tips of Allium carinatum much more than endomitosis (Nagl, 19700. Polyploidy has also been found to protect cells from damage by irradiation under certain conditions (von Wangenheim, 1976). Polyploidy also decreases the sensitivity of cells to alkylating mutagens (Zutshi and Kaul, 1975). Endopolyploidy leads to a number of quantitative changes in the cell. Butter-

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fass (1973) reviewed the effects of the nuclear DNA content on the number of chloroplasts and found a strict positivc correlation. A we!!-known effect of polyploidy and polyteny is the influence on cell size. Cell eiongation in roots is frequently a consequence of polyploidy (Torrey, 1965; Capesius and Bopp, 1970; Capesius et NI., 1972). The different size of individual scales of a butterfly wing is merely the consequence of different levels of polyploidy found in the scale cells (Kuhn, 1965; see further examples in the reviews by Nagl, 1976a, 1978a). The highly tissue-specific pattern of various degrees of polyploidy and plyteny has been summarized under the term ’ ’karyological anatomy ’ ’ (Geitler, 1952; D’Amato, 1977). In this sense, polyteny is not understood so much as a mechanism increasing the synthetic capacity of a cell, as it i s as a mechanism of “organ modeling” (Nagl, 1981). Polytcne chromosomes may also help to elucidate the biological significance of non-protein-coding DNA. Within the cell nucleus, the huge amount of never expressed DNA (approx. 97-99%; Galau rt al., 1976; Kamalay and Goldberg, 1980) exhibits evidently different conformations due to its sequence diversity, which is reflected by confornational differences of the chromatin. In this way different chromatin domains, or polytene chromosome bands, can react to dif= ferent electrodynamic environments, and hence release specific patterns of domains into the “active conformation.” The latter can be seen as the prerequisite for transcription. Evidence for the control of puffing patterns by a changing electrodynamic environment was given by Kroeger and Miiller ( 1973) and Lezzi {1967), long before the basic structure of chromatin was elucidated. These authors reported that certain concentrations and combinations of ions selectively stimulated one or another puffing pattern in isolated polytene chromosomes. The huge amount of non-protein-coding DNA in eukaryotes may, therefore, not be seen as “selfish” or “junk” (Doolittle and Sapienza. 1980; Orgel and Crick, 19801, but as necessary for the control of differential transcription: according to the conformation of a DNA and chromatin region, it will react to a changing ~ l ~ ~ ~ r ~environment y ~ a ~ iini ac t ~ e ~ o d y n ~ m ifavored ~ ~ l l yway. By this, a certain pattern of acceptor sites for RNA polymerase is brought into a accessible position.

B . EVOLUTIONARY ASPECTS That polyteny is found in certain species only, and that the degree of polyteny is highly species-specific, raises the question of the evolution of polytene chromosomes. Understanding the evolution may help to elucidate the function of polyteny, as “the study of genomic regulation and the study of evolution must be considered the two sides of the same coin” (Galau el al., 1976). Nagl (I978a, 1979) pointed out that diversification of biomatter during phylogenesis (speciation, splitting events) and ontogenesis (cell differentiation) seems to depend

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mainly on the same mechanisms of DNA increase (or decrease) and sequence reorganization, rather than on gene mutations. The genetic distance in terms of the number of nucleotide substitutions seems to be nothing but a consequence of time after splitting; but polyploidization, saltatory replications, and karyotype repatterning are apparently the driving factors of speciation (see also Wilson et al., 1977; Avise, 1976). The initial steps of speciation seem to be the results of accident rather than of adaptation (Dover, 1978), and gene mutations account only for the fine tuning of metabolism and behavior to the selection pressure exerted by the changing environment. This idea is confirmed by the fact that closely related species with nearly identical genes differ extremely in their DNA contents (the so-called 2C value paradox; see reviews by Nagl, 1976a, 1978a). It is also consistent with the fact that the protein-coding genes differ only slightly between organismically diverged organisms, but that the amount of noncoding (mainly repetitive DNA) varies extremely (references ifn Hinegardner, 1976; Nagl, 1978a, 1979). The evidence for a role of polyploidy and related events of genome variation in evolution will not be discussed here in more detail. In the context of this article it is more interesting to search for the relationship between phylogenetic and ontogenetic changes in the nuclear state, as well as for their causality. The evolution of cell cycle types is a fairly neglected point. As with other evolutionary aspects of diversification in organisms, the evolution of the common mitotic cycle-with its G,, S, Gz periods, karyokinesis, and cytokinesis from an archtype similar to that found in prekaryotes (Prescott, 1981)-has to be considered a consequence of regulatory changes. The same is valid for the evolution of the “curtailed” cell cycles, such as the restitution, endomitotic, and endoreduplication cycles (Nagl, 1981; Fig. 12a). Uncoupling of cytokinesis and karyokinesis has let to the multinucleate state, a quasi-polyploid state. Uncoupling of the chromosome-reduplication cycle from karyokinesis has led to nuclear restitution, the mechanism of polyploidization that is most closely related to mitosis. Uncoupling of chromosome condensation, as well as both spindle formation and breakdown of the nuclear envelope, has led to the endomitotic cycle. And shortcut of the cell cycle after S phase, omitting any event that reverts to mitosis, created the endoreduplication or polytenization cycle. Finally, the “economic strategy” of nature led to cell cycles, which are curtailed already during the S period. resulting in underreplication of the late-replicating heterochromatin. DNA amplification may be seen as the ultimate reduction of such an underreplication cycle (Nagl, 1974b). Why do not all organisms develop polytenization cycles? The answer is rather speculative, although founded on exact data. Nagl (1976d) found a negative relationship between the basic genome size (2C DNA content) and the tendency to undergo endocycles. Moreover, the level of polyteny is higher is species with very low 2C values than in those with some higher ones. (The Diptera belong to

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the eukaryotes with the smallest genomes; also, the plant species with large polytene chromosomes-such a5 Phasanlirs, Rhinanttius, and Tropaiwluitirange among the species with the smallest 2C values.) This led to the conclusion that “ontogenetic lateral ” DNA increase (by polytenization) is an evolutionary

-

v)

u)

0 C

0

m 0

c

C

0

I

--E v o Iu t i o n

b

FIG. 12. The evolution of endocycles. (a) The circle indicates the mitotic cell cycle of a typical plant ( G , , presynthetic interphase; S, period of DNA synthesis; G,, premitotic interphase; D, dispersion stage; P, prophase: M. metaphase: A, anaphase: T. telophase). The numbered symbols indicate rhe progressive curtailment of the mitotic cycle, probably by silencing the corresponding genes ( I , omission of cytokinesis leading to a multinucleate state: 2, omission of karyokinesis leading to a polyploid state: 3, omission of mitosis except the dispersion stage. leading to the plant type of endomitosis, which is often not discernible from endoreduplication; 4. return to the G, period immediately alter DNA replication leading to the polytenic state: 5 , curtailment of the cell cycle during the S period leading to differential DNA replication. (b) Polyteny understood as evolutionary strategy: Species with a small genome (represented by a small chromosome) multiply their genome “laterally” in certain highly active cells, that is, they show polytene or polyploid nuclei: if the genome had been enlarged “tandemly” during evolution (largc chromosome), this would not be necessary. (According to Nagl. 1976d, 1981.)

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strategy to compensate missing “phylogenetic tandem” DNA increase (e.g., by saltatory replications) (Nagl, 1976d, 1977b; Fig. 12b). The various interrelationships between the ontogenetic and phylogenetic programs of DNA variation have been summarized in the “DNA optimization model” (Nagl, 1978a). The functional significance of both phylogenetic and ontogenetic variations in the quantity of nuclear DNA may be seen, as discussed above, in adaptation to optimal synthetic activities and in the ability to control cell sizes (see also Cavalier-Smith, 1978). In addition, the phylogenetic state of nuclear DNA apparently has an adaptive value with respect to its “nucleotypic effects” (Bennett, 1973) on cell cycle duration, cell size, organ size, and minimal generation time. In reference to this phylogenetic state, the ontogenetic changes are seen as predefined processes in order to adapt the mature cell and organ to its function (Nagl, 1978a). The key question as to how both phylogenetic and ontogenetic changes in DNA content are controlled touches on the general question about the regulation of differentiation, or more generally, on diversification of biomatter. Science has not made any progress in this direction, as best shown by the cancer problem. The main reason for this lack of success’might perhaps be seen in our thinking in terms of linear causality, an adaptation to everyday life (Riedl, 1977) that is however inappropriate in biology (Wright, 1979). Both phylogenesis and ontogenesis control themselves through feedback loops, which do not have one cause for one effect; nor can they be seen independently from all other feedback loops to which they are connected. It is very likely that phylogenesis is the result of the self-organizing and self-instructing ability of biomatter, leading to increase of molecular and organismic complexity. Adaptation and selection are secondary factors, which never can “cause” the origin of evolutionary novelty, and which never can “cause” the apparent evolution to increasing complexity. One example may be allowed: the evolution of the human (and mammalian) placenta cannot be the result of adaptation and selection, in spite of its high adaptive value. Such a complex organ requires a very long evolution to allow time for many genetic and regulatory changes to take place so that the organ can take over nutrition, excretion, respiration, and hormonal and immunological control for the helpless and undeveloped embryo. Actually, placenta-like structures can be found to have occurred long before the evolution of Placentalia, as today visible in Reptilia and even Tunicata (see review by Nagl and Fuhrmann, 1981). The driving force for this evolution is probably again the same as for differentiation: destabilization of the genome by chance (mutation, temperature change, fertilization, radiation, etc.) can induce a reaction (“fluctuation”) of the nucleus in order to adapt to the new thermodynamic constraints. Although the stimulus was accidental and can be variable, the reaction is unique and determined: it follows the laws of thermodynamics far from equilibrium (Prigogine, 1976) and is determined by what I will call “thermodynamic code” of the DNA. This code includes DNA mass (mainly noncoding sequences), DNA conformation, DNA-

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protein interaction (nearly exclusively electrostatic), and chromatin conformation. Evidence for such a basic physical “control” of cell cycles, cell differentiation, and neoplasia is accumulating (e.g., Prigogine, 1978, 1979; Lamprecht and Zotin, 1978; Popp, 1979; Ryan, 1980; reviewed by Nag1 and Fuhrmann, 1981). Nicolini (1979, 1980. 1981) has demonstrated particularly well that the thermodynamics of chromatin organization is the key to the understanding of cell differentiation and cell proliferation. In this sense. differential gene activity is one of the consequences of differentiation, but never its “cause. ” Polyteny is, according to this hypothesis, a switch in the complex contro! system of differentiation, induced (but not caused) by a fluctuation that is brought about by a hormone gradient or anything else. According to the thermodynamic code (i.e., the evolutionary state of the DNA), and controlled by numerous feedback loops between chromatin organization, cell surface and cell position, and all the accepted regulatory machines. the cell reacts necessarily with polytenization. Another cell will not react in this way, because it may exhibit a different chromatin conformation or a different position within a hormone gradient, or some other variation. The main exception to the conservative view is that, in my opinion, regulatory RNAs or proteins, hormones, and glycoproteins-which all are gene products themselves-cannot be envisaged as the basic control mechanism. Before they can activate a set of genes involved in DNA replication, their own genes have to been activated, and this process again needs gene products as activators, and so on. Therefore, the only realistic view of organisms (i.e., dissipative structures) would be based on nonequilibnum thermodynamics as the fundamental control factor of differentiation and morphogenesis. The question as to the significance of endocycles and polyteny now has a new dimension. Avoiding the illegitimate question as to the cause of polyteny, polyteny is understood as the most striking reaction of the genome according to the thermodynamic code of the species, and in accordance with its microenvironment. It is the switch to a new type of chromatin organization that enables the cell to work with maximal efficiency in situations that require rapid, undisturbed, and high function. Experiments on cell and tissue cultures should allow verification or refutation of this hypothesis.

VII. Conclusions Polytene chromosomes are more or less compact bundles of laterally rnultiplied interphase chromosomes that occur in certain highly active and specialized cell types among various unrelated animals and plants. Prerequisites for their development are endoreduplication cycles without chromatin condensation. The limitation of polyteny to certain species with rather small genome size is understood as an evolutionary strategy meant to substitute for lacking phylogenetic

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DNA increase. It is apparently necessary that these cells reach functional size and activity in a short time, as for instance during embryogeny as haustoria. The DNA optimization model (Nagl, 1978a) was used to describe the somatic patterns of endopolyploidy as a predefined process depending on the phylogenetic state of the genome. Recent progress in nonlinear thermodynamics of dissipative structures allowed proposal of the hypothesis that the “coupling factor” that relates the ontogenetic events to the phylogenetic state of the DNA may be seen in terms of some “thermodynamic code. This code is assumed to be present in such entities as the sequence arrangement, structural conformation, or electrostatic protein binding, of the non-protein-coding, mainly repetitive DNA sequences, which vary widely among species. In accordance (by numerous feedback loops) with its position and microenvironment, the cell either reacts or does not by polytenization. This represents then one of the switches in genome and chromatin organization within the nucleus, governing the direction of differentiation and function. Polyteny may well be utilized as a model system for understanding the role of chromatin patterns in determining the direction of differentiation, due to the good microscopic visibility of all changes. Plant polytene nuclei with their high structural variability may not be of as much help in describing puffing patterns, but they might rather be very useful in the study of the interrelationships among environment, DNA content, cell function, and the like, in relation to the key role of chromatin organization (as switches of differentiation). The search for additional species exhibiting polytene chromosomes is an urgent responsibility of plant cell biology in order to find more suitable systems than are known today. Moreover, the induction of endocycles and polyteny in cell and tissue culture may provide much information about the controlling factors leading to this type of chromatin organization. ”

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INTERNATlONAL REVIEW OF CYTOUKiY, VOL. 73

Endosperm-Its Morphology, Ultrastructure, and Histochemistry S. P. BHATNAGAR AND VEENASAWHNEY Department of Botany, University of Delhi, Delhi, India I. Introduction . . . . . . . . . . The Central Cell . . . . . . . . The Primary Endosperm Nucleus . . The Endosperm Cell . . . . . . . Types of Endosperm Formation . . . A. Nuclear Endosperm . . . . . . B. Cellular Endosperm . . . . . . C. Helobial Endosperm . . . . . VI. Ruminate Endosperm . . . . . . VII. Cell Wall Formation . . . . . . . VIII. The Endosperm Haustoria . . . . . IX. Aleurone Tissue . . . . . . . . Function of Aleurone Tissue . . . . X. Functions of Endosperm and Endosperm XI. Conclusions and Prospects . . . . References . . . . . . . . . . 11. 111. IV. V.

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I. Introduction Our awareness of sexuality in plants dates back to the time of Herodotus in the fifth century B.C. Amici in 1824 for the first time observed the growth of the pollen tube into the pistil. Subsequently Strasburger (1884) gave an account of the discharge of male nuclei and fusion of one of them with the egg nucleus. Later, in 1898, Nawaschin showed that both the sperms released by a pollen tube are involved in fertilization. The second male nucleus unites with the secondary nucleus, which is the fusion product of the two polar nuclei of the central cell of the female gametophyte. Normally all the three nuclei are haploid and therefore the fusion product is triploid. The endosperm is formed by the repeated divisions of the primary endosperm nucleus. The endosperm is a very important tissue because it is the main. source of food for the embryo in angiosperms. In gymnosperms, the endosperm is haploid and is already present at the time of fertilization; this is unlike angiosperms, where endosperm formation is postponed until after fertilization. In almost all angiosperms, except a few apomicts and the families Orchidaceae, Podostemaceae, and Trapaceae, triple fusion is a must for the 55 Copyright @ 1981 by Academic F’ress. Inc. All rights of reproduction in any form reserved. ISBN 0- 12-364473-Y

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normal growth and development of the endosperm. Its failure leads to seed abortion. The endosperm may be completely consumed by the developing embryo as in pea, Arachis and Cucurbita, or if may persist to serve the function of storage tissue to support the embryo growth during germination as in cereals, coconut. Phorni.r. and Ricinus. Light-microscopic studies of the endosperm have been known for quite some time. It is only ~n the last decade that our understanding of the development of the endosperm, its haustoria. and their functions has increased to a considerable extent because of the use of electron microscopy and other sophisticated techniques such as autoradiography and histochemistry . Results of recent ultrastructural work, coupled with histochemical studies, reveal that endosperm is a very interesting tissue. This article attempts to discuss these recent investigations.

11. The Central Cell The female gametophyte is primarily a seven-celled structure consisting of two synergids and an egg at the micropylar end, three antipodal cells at the chalazal end, and a central cell with two polar nuclei, one from each pole. Most of the earlier studies on the central cell were confined to the number of polar nuclei involved in secondary fusion, the time and place of their fusion, and development of the secondary nucleus and its fertilization with one o f the male gametes to form the primary endosperm nucleus. Recently electron-microscopic and histochemical investigations have been carried out on the cell wall, cytoplasm. polar nuclei, and secondary nucleus of the central cell. These studies reveal that the central cell is a highly active cell that performs several important functions in the embryo sac. The central cell is the largest cell of the embryo sac with two unusually large haploid polar nuclei, but in Liliurn regale, one of the nuclei is haploid and the other is triploid (Mikulska and Rodkiewicz. 1967). The polar nuclei are large. elongated, and flattened on their facing surface (Fig. { A ) . The two polar nuclei are generally similar in size and shape, and once they come together it becomes very difficult to distinguish the micropylar one from the chalazal. When there is a difference in size, it i s usually the micropylar one that is larger. In the Fritillaria type of embryo sac, it is the reverse (Maheshwari, 1950). In Epidendrum scutella the nuclei have lobed outlines and exhibit a number of finger-like processes at the opposite faces (Cocucci and Jensen, 1969a). In contrast with the egg nucleus, the chromatin clump in the polar nuclei is uniformly distributed. The polar nuclei lie very close to each other and are separated by a thin layer of cytoplasm in which organelles and vacuoles may be present (Jensen, 1965; Cocucci and Jensen, 1959a: Van Went, 1970a; Schulz and Jensen, 1973).

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The nuclear membrane surrounding the polar nucleus contains many pores. In Gossypium hirsutum the nuclear membrane shows two types of projections entering into the surrounding cytoplasm. These are ( a ) connections with the ER ( b ) numerous small projections of the inner nuclear membrane into swellings of the outer membrane. These projections show considerable complexity and have membrane-bound structures within them. Their formation and function are not understood (Jensen, 1965). Each polar nucleus contains a large dense nucleolus in the granular nucleoplasm. A micronucleolus occurs in the polar nucleus of barley (Cass and Jensen, 1970) and Cupsellu (Fig. 1A; Schulz and Jenson, 1973). In cotton, there is no evidence of micronucleoli or nucleolar budding (Jensen, 1965). The fusion time of polar nuclei differs from species to species. In Capsella the nuclei are usually completely fused at the time of fertilization (Schulz and Jensen, 1973). In cotton (Jensen, 1965) and E . scutella (Cocucci and Jensen, 1969b) the fusion begins before fertilization but is not completed. In cotton, it is accomplished only after the arrival of the male nucleus which triggers the final fusion (Jensen, 1965, 1972). Studies carried out on seven European species of Melampyrum (Greilhuber, 1973) reveal that the polar nuclei do not fuse and only the upper polar nucleus is fertilized thereby giving rise to a diploid primary endosperm nucleus. The fate of the lower polar nucleus is not known. The fusion of the polar nuclei begins when they come closer. The mechanism has been described in Gossypium (Jensen, 1964; Jensen and Fisher, 1967) and Capsella (Schulz and Jensen, 1973). The fusion process involves the evagination of the nuclear envelopes of both the nuclei to make contact at several points (Fig. 1A). The outer membranes fuse at these points, followed by the fusion of the inner nuclear membranes to form nuclear bridges (Fig. 1B). These bridges enlarge and join to complete the process of fusion, which is also accompanied by the complete fusion of nucleoli. The secondary nucleus usually lies below the egg and is separated from the antipodal cells by a large central vacuole. It is connected with the egg apparatus and the antipodals by cytoplasmic strands. In addition to the large vacuole, many small vacuoles also occur in the cytoplasm of the central cell. Both large and small vacuoles are devoid of structural contents in Capsella (Schulz and Jensen, 1973). According to Maze and Lin (1975), granular components of unknown nature occur in the vacuoles of Stipa elmeri. Ryczkowski (1964) and List and Steward (1965) found that the central cell vacuole is a reservoir of sugars, amino acids, and inorganic salts. Secondary nucleus has a chalaza1 position in plants that possess the helobial type of endospenn (Maheshwari, 1950). The newly formed polar nuclei give a strong positive Feulgen reaction indicating high DNA content (Hu, 1964b; Hu and Chu, 1964). The mature nuclei, however, do not have a demonstrable amount of DNA or show a very faint reaction with Feulgen staining (see Kapil and Bhatnagar, 1975). The decrease in

FK. 1. (A. E) Cupsella. ( A ) Polar nuclei. One ofthe nuclei shows a small micronucleolus. The membranes of the two evaginate to make contact. X6420. (B) Fusing polar nuclei showing cytoplasmic pockets between nuclear bridges (arrows). The outer and the inner membranes of the two nuclci are continuous. 'x 13.380. Mn, micronucleolus; NI,nucleolus; Pn,polar nucleus; V , vacuole. (From Schulz and Jcnscn. 1973.)

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density at maturity is ascribed to increase in nuclear size and the resultant dilution of DNA (Pritchard, 1964; Hu, 1964b). Nucleoli of polar nuclei are rich in RNA and protein (Alvarez and Sagawa, 1965; Jensen, 1965). The high rate of protein synthesis is attributed to the large nucleolar volume of the secondary nucleus (Wallace, 1963). In addition, the large size of the nucleolus might also be indicative of a high rate of ribosome production since the nucleolus has been shown to be the site of ribosomal RNA synthesis (Brady and Clutter, 1972; Mac Gregor, 1972). The central cell cytoplasm is rich in endoplasmic reticulum, plastids, mitochondria, Golgi bodies, ribosomes, microbodies, and spherosomes. It has large amounts of RNA and protein in Stellaria media (Pritchard, 1964), Gossypium (Jensen, 1965; Jensen and Fisher, 1967), and Vanda (Alvarez and Sagawa, 1965). The ash content of the cytoplasm is high (Jensen, 1965). Shortly before fertilization, concentration of protein drops sharply (Alvarez and Sagawa, 1965). In Trudescantia during fertilization there is an increase of cytoplasmic RNA of the central cell (Vassileva-Dryanovska, 1966). The starch is abundant in Triticum (Hu, 1964a), cotton (Jensen, 1965), Zea (Diboll, 1968), and Hordeum (Cass and Jensen, 1970), but poor in Stellaria (Pritchard, 1965). It forms a kind of halo around the polar nuclei. The starch content in wheat is very high before pollination and remains so until triple fusion, but later when primary endosperm nucleus is formed, no starch is visible (Hu, 1964b). According to Luxova (1968.), the gradual hydrolysis of the reserve starch in the egg and the central cell provides the energy needed for fertilization. Both smooth and rough ER are present in the central cell cytoplasm. E . scutellu shows a large amount of smooth tubular ER (Cocucci and Jensen, 1969a). In cotton (Jensen, 1965), Zea (Diboll and Larson, 1966; Diboll, 1968), Cupsella (Schulz and Jensen, 1973), and Helianthus (Newcomb, 1973a), rough ER is present. ER membranes in Petunia may be partly covered with ribosomes (Van Went, 1970a). In Helianthus some of the rough ER is organized into circular patterns that encircle small vacuoles in the cytoplasm (Newcomb, 1973a). In cotton, rough ER cisternae frequently terminate in whorls that suggest the possibility that ER is being synthesized (Jensen, 1965; Schulz and Jensen, 1977). Most of the ER is found parallel to the plasma membrane and the nuclear envelope (Van Went, 1970a; Schulz and Jensen, 1973). The ER connections with the nuclear envelope are infrequent and their number increases greatly during the early development of the endosperm (Schulz and Jensen, 1973). In Petunia the outer membranes of the two nuclear envelopes are sometimes connected by ER sheets that are generally found in the periphery of the cells (Van Went, 1970a). In Capsella ER contains long needle-shaped crystals whose chemical nature is not yet known (Jensen, 1972; Schulz and Jensen, 1973). The central cell cytoplasm is fiIled with ribosomes near the secondary nucleus and in the chalaza1region of the egg apparatus. Before fertilization, the ribosomes

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are usually unattached and unaggrcgated. In cotton (Jensen, 1965) and Cupselh (Schulz and Jensen. 1973) ribosottles are free as well as associated with the ER. In Petirrzia ribosomes occur as monosomes and rarely as polysomes (Van Went, 1970a). A striking feature of the central cell cytoplasm is the presence of numerous large and well-developed chloroplasts. The plastids are frequently large and irregular in shape but in Epidendrum (Cocucci and Jensen, 1969b) and Capsellu (Schulz and Jensen, 1973) they are elongated and ellipsoidal. Cup-shaped or amoeboid plastids have been reported in Zcw muys (Diboll, 1968) and cotton (Schulz and Jensen, 1977). The central cell plastids have starch grains that are usually smaller than those found in the egg plastids. Also. the number of plastids found in the central cell is more than in the egg (Jensen, 1972). The plastid matrix apart from starch grains also contains deposition of phytoferritin, carotene droplets (Jenscn, 1973, proteins, and ribosomes (Jensen, 1965; Schulz and Jensen, 1973). In Zeri (Diboll, 1968), phytoferritin could not be identified in plastids. The central cell cytoplasm contains many mitochondria, which may be spherical, oval, or rod-shaped. They are generally scattered throughout the cytoplasm but in L . regale they are clustered together into a large group near the embryo sac wall or the nucleus (Mikulska and Rodkiewicz, 1967). Soon after fertilization, the mitochondria become large and irregular, and show large numbers of randomly distributed cristae in cotton (Jensen, 1965), Petiiniu (Van Went, 1970a), and Capsrlla (Schulz and Jensen, 1973). The mitochondria1 translucent matrix contains ribosomes (Jensen, 1965; Schulz and Jensen, 1973). lntramitochondrial granules and fibrils presumed to contain DNA are reported in the wituchondrial matrix of Cupsellu (Schulz and Jensen, 1973). lntramitochondrial granules are rare or absent in cotton (Jensen, 1965). A single giant (2.6 p m in diameter) mitochondrion appears i n the cytoplasm near the polar nuclei at the time of fertilization in Cappsellu (Schulz and Jensen, 1973). It is not seen after the first division of the primary endosperm nucleus. It has shont vesiculate or tubular cristae, intramitochondrial granules, and ribosomes. Its matrix also has fibrils of DNA. The presence of large amounts of fibriliar material of DNA indicates that it may be involved in the formation of mitochondria during the early development of the endosperm. There is no clear evidence to indicate its origin, fate, and function. The Colgi bodies with three to six cisternae are abundant and appear active in the production of vesicles. These vesicles are commonly grouped into large clusters. The distribution and number of Golgi bodies vary in different plants. They are numerous in cotton (Jensen, 1965), Petunia (Van Went, 1970aj, Hrlianrhus (Newcomb, 1973a). and Stipa (Maze and Lin, 1975), but scarce in Epidendrum (Cocucci and Jensen, 1969a) and Cupsella (Schulz and Jensen, 1973). In Helianthus (Newcomb, 1973a) and Cupsella (Schulz and Jensen,

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1973) they are scattered throughout the cytoplasm, whereas in L . regale (Mikulska and Rodkiewicz, 1967) they are seen around the nucleus. In G . hirsuturn (Jensen, 1965) they are mostly concentrated near the cell wall, and it has been suggested they are involved in the formation of the wail. Two types of Golgi bodies with a variable number of flat cisternae are reported in Petunia (Van Went, 1970a). Microbodies of variable size and shape in association with large lipid droplets are frequently seen throughout the central cell cytoplasm of Gossypium, Helianthus, Petunia, and Stipa. Lipids are absent in Zephyranthes and Lagenaria (Malik and Vermani, 1974), and in Zea (Singh and Malik, 1976). The close association of microbodies and lipid droplets in the central cell indicates the possible degradation of lipid droplets to supply energy for subsequent development (Newcomb, 1973a; Schulz and Jensen, 1973). The central cell is surrounded by a cell wall and plasma membrane. The wall is very complex and highly variable from one part of the embryo sac to another. It is thickest against the nucellus or integuments and is believed to be rich in pectic substances (Jensen, 1965; Bhatnagar and Johri, 1972; Johri and Bhatnagar, 1973). It thins toward the chalazal end of the egg apparatus and in the chalazal region there is no wall between the plasma membrane of the central cell and that of the egg and synergids. The central cell is connected by plasmodesmata with the egg apparatus and antipodals. No such connections exist between the central cell and the adjacent nucellar cells. In E . scutellu (Cocucci and Jensen, 1969a) a gap is observed in the wall in the region of the degenerated synergid so that the plasmalemma of the central cell is in contact with the cytoplasm of the synergid. In Jasione rnontana (Berg and Erdelska, 1973) the embryo sac is surrounded by a cuticle that is discontinuous in the region of the egg apparatus. This indicates the likely pathway for absorption of nutrients from the surrounding tissues. In L . regale (Mikulska and Rodkiewicz, 1967) the wall differs in structure at the micropylar and chalazal end. The wall between three micropylar cells and the central cell is optically empty or contains a certain amount of granular material. However, the wall between the central cell and antipodals is 3-4 p m thick and consists of fine-grained substance. In 2. mays, Diboll and Larson (1966) observed a difference in the origin and structure of the wall limiting the central cell in various regions. The wall of the former functional megaspore extends to form the wall of the central cell where it is in contact with the nucellus. Adjacent to the antipodals it is formed by cytokinesis of the megaspore cytoplasm during early gametophyte development. Wall ingrowths or projections have been observed in the micropylar region in Linum usitatissimum (Vazart and Vazart, 1966), Pisum sativum (Marinos, 1970), Helianthus annuus (Newcomb and Steeves, 1971), Eschscholtzia californica (Negi, 1972), and Lobelia dunii (Torosian, 1972). It is suggested that embryo sac wall projections greatly increase the surface area for the absorption of

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FIG. 2 . (A-C) Cotton. (A) Electron micrograph showing the sperm nucleus fusing with one polar nucleus. X7150. (B,C) Sperm nucleus with fusing polar nuclei; 1.5-pm sections stained with aniline

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metabolites from the surrounding tissue (Vazart and Vazart, 1966;Newcomb and Steeves, 1971; Negi, 1972; Torosian, 1972; Newcomb, 1973a). In Cupsellu (Schulz and Jensen, 1973) the wall projections are absent before fertilization and they develop only after fertilization. The central cell, therefore, probably does not absorb nutrients directly from the integuments before fertilization. Because of this and also due to the presence of many well-developed chloroplasts, it is considered to be autotrophic in nature before fertilization. The central cell, with its extensive ER, numerous and well-developed chloroplasts, mitochondria, Golgi bodies, and polysomes, appears to be engaged in intense metabolic activity (Hu, 1964a,b; Jensen, 1965, 1972; Diboll and Larson, 1966; Vazart and Vazart, 1966; Jensen and Fisher, 1967; Mikulska and Rodkiewicz, 1967; Diboll, 1968; Cocucci and Jensen, 1969a,b; Van Went, 1970a,b; Schulz and Jensen, 1973). In addition to the absorption of nutrients from the surrounding tissue, the metabolism of the central cell is also associated with the breakdown of metabolites into simpler and useful precursors and cellular components, which are utilized by the central cell, egg, and synergids.

111. The Primary Endosperm Nucleus The fusion of the secondary nucleus or the polar nuclei with the sperm nucleus (Fig. 2A-C) gives rise to the primary endosperm nucleus, which is generally triploid. It appears directly below the egg cell (Alvarez and Sagawa, 1965) and divides almost immediately after it is formed. Only the sperm nucleus fuses with the polar nuclei and the sperm cytoplasm plays no role during this process. The primary endosperm nucleus appears convoluted in Petunia (Van Went, 1970b), unlike Cupsellu where it is rounded and slightly flattened on the side facing the zygote (Schulz and Jensen, 1973). The primary endosperm nucleus is associated with aggregates of dense material in the perinuclear cytoplasm. These aggregates have granules about 15-20 nm in size and strongly resemble the peripheral material of the nucleolus and the aggregates present in the nucleoplasm (Fig. 3; Schulz and Jensen, 1973, 1977). These aggregates are believed to be extruded nucleolar material, which is rich in ribosomes, RNA, and proteins and is also suggested to be involved in supporting the initial rapid growth of the endosperm. The membrane of the primary endosperm nucleus is contributed by both the secondary nucleus and the male nucleus (Jensen, 1964). The nucleus gives a faint PAS-positive reaction, indicating low levels of total insoluble polysaccharides (Alvarez and Sagawa, 1965). blue black and seen under the light microscope. X750. Pn, polar nucleus; Sn, sperm nucleus. (From Jensen and Fisher, 1967.)

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S. P. BHATNAGAR AND VEENA SAWHNEY

FIG. 3 . Cupse/lu. A portion of the primary endosperm nucleus showing similarity between the peripheral nucleolar material and aggregates in the nucleoplasm (arrow) and pennuclear cytoplasm (double arrow). X20.125. Inset shows pennuclear aggregate at a higher magnification ~35,460. Pnl. peripheral nucleolar material. (From Schulz and Jensen, 1973.)

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IV. The Endosperm Cell The primary endosperm nucleus divides rapidly to form the endosperm tissue. In many species, following fertilization, several changes occur in the fine structure of the central cell (Diboll, 1968; Cocucci and Jensen, 1969a,b; Marinos, 1970; Van Went, 1970b; Singh and Mogensen, 1976; Schulz and Jensen, 1977). These changes reflect increased metabolic activity and the organization of the protein-synthesizing machinery for the rapid differentiation of the primary endosperm cell (Van Went, 1970b). In Capsella (Schulz and Jensen, 1973) the mature female gametophyte is quite active even before fertilization and therefore does not undergo notable structural reorganization as a result of fertilization. The endospermic cytoplasm is characterized by numerous organelles, that is, nucleus, ER, Golgi bodies, plastids, microbodies, vacuoles, various kinds of vesicles, spherosomes, protein bodies, and starch granules. Protein bodies and starch granules at maturity are abundant and almost completely fill the endosperm cell. The endosperm cytoplasm is rich in RNA, protein, and polysaccharides (Pritchard, 1964). The endosperm nuclei increase considerably in size as the development proceeds. In the young tissue, the nuclei are triploid but later when the endosperm grows in age and size, some of its nuclei become polyploid. This may be due either to fusion of adjacent nuclei before wall formation (Maheshwari, 1950) or to several other processes such as endomitosis, spontaneous C-mitosis, endoreduplication, fusion of adjacent spindles at anaphase, and persistent bridges (Roy and Saran, 1975). The nucleus is highly variable in size and shape and contains dense granular nucleolus. The nucleolus is irregular and basophilic; its size and the basophilic content decrease as wall formation starts between the nuclei (Pritchard, 1964). Often two or more nucleoli may also be present. In Ornithogalum (Stephen, 1978) autoradiogrpahic studies with [ 3H]thymidine reveal selective nucleolar DNA amplification in the maturing endosperm cells, which boost protein biosynthesis in the cells to nourish the developing embryo. Soon after fertilization, there is a notable increase in the ER formation in Epidendrurn (Cocucci and Jensen, 1969a,b), Petunia (Van Went, 1970b), and cotton (Schulz and Jensen, 1977). The strands of ER associated with ribosomes are dispersed throughout the cytoplasm (Cocucci and Jensen, 1969a,b; Marinos, 1970; Van Went, 1970b; Vigil, 1970; Newcomb, 1973b; Weinbaum and Simons, 1974; Schulz and Jensen, 1974, 1977). Apart from rough ER, tubular ER (Cocucci and Jensen, 1969b) and smooth ER (Marinos, 1970; Schulz and Jensen, 1974) are also present. The smooth ER frequently branches and shows connections with the embryo sac wall projections (Schulz and Jensen, 1974, 1977). The direct connection between the smooth ER and the plasma membrane of the endosperm cells may be involved in the deposition of wall material and secretion

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of hydrolytic enzymes and some other substances (Schulz and Jcnsen, 1977) into the degenerating synergid and nucellus. Ribosomes generally occur attached to the ER or as polysomes. They may also occur free (Vigil, 1970; Newcomb, 1973b; Jones, 1974). The cytoplasm of the endosperm cell possesses mitochondria. In Pisum (Marinos, 1970)they are found mainly close to the boundary wall. After fertilization, mitochondria change both in shape and fine structure in Zea (Diboll, 1968) and Prrunicl (Van Went, 197%). They become hypertrophied and the swelling is attributed to the increase in the rate of biochemical activity. The mitochondria show randomly distributed cristae (Buttrose, 1963a; Van Went, I970b; Weinbaum and Simons, 1974; Singh and Mogensen, 1976) that lose their integrity in degenerating cells (Singh and Mogensen, 1976). Golgi bodies actively engaged in vesicle production are distributed throughout the cytoplasm of the endosperm cell (Van Went, 1970b; Newcomb, 1973b; Jones, 1974; Weinbaum and Simons, 1974). Plastids with one or more starch grains are numerous and highly variable in size and shape. The inner membrane of the plastid envelope may protrude into the electron-dense stroma at many places to give rise to internal membrane systems (Buttrose, 1963a; Vigil, 1970; Newcomb, 1973b; Singh and Mogensen, 1976). In Copsella (Schulz and Jensen, 1974). chloroplasts with three to five fused thylakoids are present. The plastid matrix contains many osmiophilic bodies (Vigil, 1970; Schulz and Jensen, 1974; Singh and Mogensen, 1976) and ribosomes (Schulz and Jensen, 1974; Singh and Mogensen, 1975). In Pisum (Marinos, 19701, apart from the usual organelles, an organelle called “organelle A” of uncertain identity is also present in the endosperm cytoplasm. This organelle is larger than the mitochondria and is bounded by a double membrane. The peripheral region is occupied by lacunae and the strorna is exceptionally dense with finely granular material. So far, no information is available that could help in establishing its developmental or functional relationship to mitochondria or plastid. A similar type of organelle is present in the basal cell of suspensor in Capsdlu (Schulz and Jensen, 1969) and has been identified as plastid. Long contractile fibrillar element bundles have also been reported in the cytoplasm. These tubules are thought to be responsible €or the conspicuous motility of the endospermic cytoplasm, which is probably involved in the transport and metabolism of materials coming from the ovular tissue (Marinos, 1970). The cell wall has plasmodesmata and stains strongly with PAS reagent. In lettuce (Jones, 1974) the wall shows peglike projections penetrating the cytoplasm (Fig. 4A,B). In many plants, a peculiar feature is the formation of endosperm nodules or cytoplasmic vesicles, which arise from the general peripheral lining of the embryo sac. The nodules may be nucleated as in S d i x cinerea (Hakansson, 1954),

FIG. 4. (A, B) Lactuca (lettuce). (A) Surface view of PAS-stained endosperm showing darkly stained cell wall protuberances inside cell (arrows) in sections. (B) Electron micrograph showing endosperm cells in cross section. Numerous projections from the wall enter into the cytoplasm, which is rich in organelles. Nu, nucleus; Pb, protein bodies; Sp, spherosomes; Wp, wall protuberances. X7300. (From Jones, 1974.)

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Myriw gale (Hakansson, 1955), Curica p u p u p (Singh, 1960). and Cupsella (Schulz and Jensen. 1974). or nonnucleated as in Stackhousia linririefolia, Penniserum typhoideum. and some cucurbits such as Cyclanthcra e.xp1nden.s. Sechium edule, and a hermaphrodite variety of Lufla acutangulu (Singh, 1955). In Cupsellu the nodular nuclei never lobe, while the nuclei in the rest of the endosperm do so. The nodular cytoplasm is rich in chloroplasts, mitochondria, microbodies. plysornes, Golgi bodies, small vacuoles, and short vesiculate profiles of rough ER that are often continuous with the nuclear envelope. Wall projections and smooth ER are absent (Schulz and Jensen, 1974). Ultrastructural studies do not offer any clue to their function. During subsequent development they either merge with the general cytoplasm of the endosperm or degenerate. The most important storage materials of the endosperm are carbohydrates, fats, oils, and proteins. Starch is the principal carbohydrate source, which occurs as grains. Starch grains are initiated in the plastid stroma (Buttrose, 1960. 1963a,b; Hoshikawa. 1968a). Usually one starch granule develops within each proplastid, but in some plants such as oat (Buttrose, 1960) and rice (Buttrose, 1960; Hoshikawa, 1968a.b) several granules are formed in the young plastid to give rise to a compound starch grain. The initiation of starch synthesis is accompanied by a rapid decline in the concentration of precursor compounds, sucrose and reducing sugars (Jennings and Morton, 1963a). Starch grains are highly variable in number, size, and shape. In cereals, where they are the main storage product, their concentration is highest in the central endosperm and lowest in the peripheral subaleurone layer. Starch grains are embedded in a protein matrix within the cell (Fig. 1 IA). Both large or lenticular starch grains and small spherical grains are encountered. Small grains generally fill the space between the large grains (Fig. I I A). The storage protein is located in distinct membrane-bound organelles called protein bodies. In monocots, the protein bodies generally occur throughout the endosperm, and their concentration decreases from the peripheral layers to the center where they become more scattered and sparse. In dicots, where there is no special storage tissue, these occur in the embryo and cotyledons. There has been a lot of controversy regarding the origin of the protein bodies. They are considered to be formed in vacuoles in wheat (Buttrose, 1963a,c). Yucca spp. (Homer and Arnott, 1965), cotton (Englernan, 1966), Phaseolus (Opik, 19681, Ricinus (Sobolev et ul., 1968), Vicia (Briarty et a f . , 196Y), Sinapis (Rest and Vaughan, 1972), Crurnhe (Smith, 1974), and Linurn (Dhar and Vijayaraghavan, 1979). According to Mottier (192l), protein bodies owe their origin to plastids. Jennings et 01. (1963). Morton and Raison (1963), and Morton et nl. (1964) in wheat and Hoshikawa (1970) in rice have suggested that protein bodies are formed within characteristic structures, the “proteoplasts” or ”protein-forming plastids. Studies of Khoo and Wolf ( 1970) on maize show that the protein granules develop from vesicles produced by ER. Golgi bodies also appear to proliferate protein granule vesicles. ”

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Protein synthesis commences between 12 and 20 days after fertilization (Duvis, 1961; Graham et al., 1963; Jennings and Morton, 1963a) and is accompanied by rapid decline in the concentration of free amino acids (Jennings and Morton, 1963a,b). Protein bodies show variation not only in size, shape, and internal structure, but also in their distribution. The most simple protein body has a granular, homogeneous matrix without any inclusions or subunits (Rost, 1972). The protein bodies may show a concentric pattern of light and dark layers as in rice (Mitsuda et al., 1969; Hoshikawa, 1970; Bechtel and Pomeranz, 1978), Seraria (Rost, 1970, 1971), maize (Khoo and Wolf, 1970), and Sorghum (Seckinger and Wolf, 1973). The concentric layers in wheat (Jennings er al., 1963), rice (Mitsuda er al., 1969), barley (Ory and Henningsen, 1969), and Sorghum (Seckinger and Wolf, 1973) are restricted to the periphery. Protein bodies in maize (Khoo and Wolf, 1970), Seraria (Rost, 1971), Sorghum (Seckinger and Wolf, 1973), and rice (Harris and Juliano, 1977; Bechtel and Pomeranz, 1978) show a dense central core or a nucleus. In rice more than one type of protein body has been reported (Bechtel and Pomeranz, 1978). These are large spherical and small spherical protein bodies with concentric rings and/or radial rays and crystalline protein body. Of the three kinds, the central endosperm has only large spherical protein bodies while the subaieurone region has all three. Protein bodies with one or more inclusions (i.e., globoid and/or crystalloid) embedded within the protein matrix are the most highly structured bodies. Depending on whether the inclusions are present or absent, Rost (1972) has classified protein bodies into three types: ( a ) protein bodies without inclusions; ( b ) protein bodies with only globoids; and (c) protein bodies with both globoid and crystalloid inclusions. In Picea (Simola, 1976), Ricinus (Tulley and Beevers, 1976; Youle and Huang, 1976), and Linum (Dhar and Vijayaraghavan, 1979), protein bodies have both globoid and crystalloid embedded within the proteinaceous matrix.

V. Types of Endosperm Formation In angiosperms there are three types of endosperm development: nuclear, cellular, and helobial. These are briefly described here, since a detailed account of the types of endosperm formation has already been given by several authors (see Maheshwari, 1950; Chopra and Sachar, 1963; Bhatnagar and Johri, 1972). A. NUCLEAR ENDOSPERM In nuclear endosperm dcvelopment, the primary endosperm nucleus undergoes many nuclear divisions that are not accompanied by wall formation. The nuclear divisions may be synchronous or asynchronous. As divisions continue, the

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nuclei are pushed toward the periphery, so that the center is occupied by a large vacuole. Free nuclear condition may persist throughout in Floerkea (Maheshwari and John, 1956), Limnanthes (Mathur, 1956), and Oxyspora (Subramanyam, 1951); or wall formation may take place later. When the latter is the case, the wall formation is usually centripetal, first initiated at the micropylar region and gradually extending toward the chalazal end. In several families such as Cucurbitaceae, Leguminosae, and Proteaceae there is much variation with regard to the relative extent of the free nuclear and cellular phases, and also in size and shape of the haustorium in individual species. In some plants, wall formation does not extend to the central portion, as a result it remains free nuclear. In others, wall formation is restricted to the upper and the middle regions, and the chalazal end remains free nuclear. The chalazal region elongates to form the haustorium. It is generally tubular and contains dense cytoplasm with one to several free nuclei of varying sizes. The chalazal haustorium usually remains coenocytic as in Echinocysris lobatu. Sicyos ungiilnta, and Tric.hosrrnthes cucumerirta (Chopra and Seth, 1977), but it may become cellular as in Cwrnopsis psorulioides and Desmodium pulchtdlum (Rau, 1953), Cirriillus jisrido.~rrs(Chopra, 1955). and Lksrnodiirrn lahirrna~folium(Johri and Garg, 1959). In C . fistitlosits the haustorium becomes cellular by becoming segmented into multinucleate chambers. These chambers finally subdivide to give rise to uninucleate cells. In Cucurbitaceae, the broader upper portion of the endosperm becomes cellular while the lower chalazal haustorium in some plants frequently reaches down to the chalazal region forming a direct channel for the transportation of food materials, Lately, some aspects of endosperm development have been studied in this family by Chopra and Seth (1977). The studies, conducted on 14 species. have revealed that with the exception of Blustania gurcini and E . lobuta, there is no correlation between the length of the seed and that of the haustorium as suggested earlier (Chopra and Sachar. 1963). Also. the survey of fhe haustorial length in the Cucurbitaceae members, so far investigated, has shown that the longest haustorium occurs in E . In&zru. Its length in this plant is 16,165 pm. In Cyperaccae the endosperm lacks haustoria. Recently both micropylar and chalazal endosperm haustoria have been reported in Scleria Juliosa by Nijalingappa and Devaki ( 1979). The wall formation in the nuclear endosperm of this plant begins around the proembryo and is restricted to the upper one-third portion of the endosperm. The chalazal end remains coenocytic and elongates up to the base of the nucellus to form the chalazal haustorium. The chalazal haustorium has dense cytoplasm and, at times, nodule-like aggregations. The micropylar end also gives out tubular extension which functions as the micropylar haustorium. In the haustorium of fresh materials of Crevillea robustu (Kaushik, 1941), Desmrdium tri’orutn (Rau , 1953), and Trichosantfic>sunyuina (Chopra, I955), cytoplasmic streaming has been observed. In GrevillPa the chalazal haustorium occurs in the form of a vermiform appendage.

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The nuclear behavior during the development of the coconut endosperm is very interesting. After fertilization, numerous free amoeboid nuclei ranging in diameter from 10 to 90 p m and with variable numbers of nucleoli form the liquid syncytium (Cutter and Freeman, 1954; Cutter et al., 1955). The exact source of the free nuclei is not known and this has created some difficulty in assigning endosperm development in coconut to any one of the ontogenetic patterns described so far. According to Quisumbing and Juliano (1927), the free nuclei in the embryo sac are partly contributed by the digestion of the nucellus and subsequent release of its nuclei, and partly from the divisions of the primary endosperm nucleus. Later, Cutter and Freeman (1954) and Cutter ef al. (1955) could not find any evidence to support the observations of Quisumbing and Juliano. According to Cutter and Freeman and Cutter et al., the increase in the number of nuclei is by gradual muitiplications of endosperm nuclei. When the fruit is about 100 mm in length, numerous large spherical cells or vesicles are seen in the coconut milk. These vesicles probably arise as a result of coalescence of cytoplasm around free nuclei in the liquid syncytium. The free cells with 1-40 nuclei vary greatly in size and may have a diameter of about 300 pm. As the fruit reaches a length of 150 mm, the free cells settle out of the milk and attach themselves to the ,endothelial surface to form a gelatinous syncytium. The gelatinous deposition increases in thickness and its nuclei undergo mitotic divisions. The syncytium is transformed into a meristem of isodiametric cells, which develops into characteristic coconut meat. In Areca catechu (Datta, 1955) the development of endosperm follows almost the same pattern as described for coconut. The embryo sac cavity in Areca is small and becomes completely filled by the endosperm which finally becomes extremely hard. Recently, in Eranrhis hiemalis, Pacini et al. (1972) have reported the incorporation of integumentary nuclei by the endosperm. These nuclei finally disaggregate into the cytoplasm; as a result the endosperm is formed only from the nuclei contributed by the divisions of the primary endosperm nucleus.

B. CELLULAR ENDOSPERM In the cellular type of endosperm, the first and the subsequent nuclear divisions are followed by cell wall formation. Thus, the endosperm is ab initio cellular. Sedgley (1979) has reported that in avocado after the first division of the primary endosperm nucleus, the wall developed from one edge of the embryo sac to the other. This wall was probably callosic in nature as it fluoresced with aniline blue. Members of the families Acanthaceae, Lobeliaceae, Scrophulariaceae, and several others belonging to the Sympetalae are characterized by this type of endosperm formation. A very interesting feature of the cellular endosperm is the formation of the haustoria. The haustorial character is evident from their hypertrophied nature, densely staining cytoplasm and large nuclei, and the depleted

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contents of the cells in their vicinity. The haustoria in the cellular endosperm are more varied than in the nuclear type. The haustoria may develop at either the micropylar or chalazal end, or at both ends, and show great variation in their structure and organization. Almost all the Scrophulariaceae members are characterized by the presence of both micropylar and chalazal haustoria that exhibit great diversity in the degree of aggressiveness in different plants. The chalazal haustorium is one-celled and binucleate in Pedrcularis (Berg, 1954), Srriga (Tiagi, 1956). and Euphrusiu and OrthocarpuA (Arekal, 1963). In Euphrasia and Orthocarpus the micropylar and chalazal haustoria extend as tubular processes and come in contact with the conducting mands of the seeds. The chalazal haustorium in Euphrasia often branches into two lobes one of which becomes extraovular. The micropylar endosperm haustonum also differs in its structure in different members of the family. In Melompyrum (Arekal, 1963) it becomes branched and the branches penetrate the vascular tissue in the funiculus. One-celled binucleate chalazal haustorium is broader above and narrow below. In Melampvrum (Tiagi, 1965) and Rhinanthus (Tiagi, 1966) some very interesting results have been obtained In Melampyrum when the chalazal endosperm haustorium starts degenerating, some cells of the storage endosperm adjacent to the haustorium become cambium-like. These cambium-like cells produce large, thin-walled cells without any f w d reserves. which fill the cavity created by the degeneration of the haustoriurn. The tissue so formed is termed chalazal endosperm, and its cells are haustorial and conducting in nature. A similar type of chalazal endosperm has also been recorded in Rhinunthus, where micropylar endosperm is also formed from the storage tissue during later stages o f \eed development It i k lesf developed than the chalaza1 endosperm. Mohan Ram and Wadhi (1964) have reviewed the development of endosperm in Acanthaceae. The family is characterized by asymmetric growth of the endosperm. Initially three cells in a linear row ace formed by the divisions in fhe primary endorperm nucleus. The upper and the lower cells develop into haustoria while the middle cell gives rise to the endosperm proper in various way\. Depending on the earlier stages of endosperm development and segmentation of the haustoria, endosperm development in Acanthaceae has been classified under the following four categories: 1. Thunhergia qp..The primary endosperm nucleus divides to form an upper

cell and a lower cell. The upper cell produces micropylar haustorium while the lower develops into the endosperm proper. A chalazal haustorium is absent. In Thuiibergia secondary haustoria develop from the endosperm proper. 2. Acanrhus gpe: The middle cell undergoes cell divisions to form endosperm. The development is a b initio cellular. 3 . Schaueria t-vpa: The nucleus of the middle cell undergoes free nuclear divisions and later endosperm becomes completely cellular by wall formation.

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4. Ruellia type: It is similar to the Schaueria type, but here wall formation is not complete so that the endosperm is partly cellular and partly nuclear. The free nuclear portion of the endosperm is referred to as the basal apparatus which presents variations in its size and shape. Recently in Ruellia (Karlstrom, 1972), Eranthemum, Lankesteria, Pseudoranthemum, Ruspolia (Karlstrom, 1973), Barleria, Crabbea (Karlstrom, 1974a), Asystasia, and Chamaeranthemum (Karlstrom, 1974b) it has been shown that there is no formation of three cells in a linear row by the divisions in the primary endosperm nucleus. In all these members studied, the first division in the primary endosperm nucleus results in the formation of a chalazal cell, which forms the chalazal endosperm haustorium and a larger upper cell. The nucleus of the latter undergoes division that is not followed by wall formation. The two daughter nuclei so formed lie close to each other and divide. This division is accompanied by wall formation, which separates the binucleate central endosperm chamber from the binucleate micropylar endosperm haustorium. The chalazal endosperm haustorium is generally binucleate but it develops four nuclei in Eranthemum and Ruellia. The micropylar haustorium shows great variation in its structure and activity. It is binucleate in Barleria (Phatak and Ambegaoker, 1956; Mohan Ram, 1962; Karlstrom, 1974a), Eranthemum (Mohan Ram, 1959; Karlstrom, 1973), and Ruellia (Mohan Ram, 1960b; Karlstrom, 1972), but multinucleate in Thunbergia (Mohan Ram and Wadhi, 1964). The central endosperm chamber forms the endosperm proper. The nuclei of the central endosperm chamber may undergo many free nuclear divisions except in Chamaeranthemum~(Karlstrom, 1974b) and Ruspolia (Karlstrom, 1973) where the central endosperm chamber remains four-nucleate for a very long time. The free nuclear endosperm finally becomes cellular, though to a variable extent in different members. It may be complete as in Crabbea, Lankesteria, Ruspolia, and Pseudoranthemum, or incomplete. In Asystasia (Mohan Ram, 1958; Karlstrom, 1974b) the chalazal part of the endosperm remains free nuclear to form the basal apparatus. Also, the basal apparatus sends out many small protuberances that are haustorial in nature. In Chamaeranthemum also, the central endosperm chamber puts forth protuberances but this occurs before wall formation is initiated. The protuberances are coenocytic but in Ruspolia (Karlstrom, 1973) these become cellular. In Andrographis echioides and A . serpyllifolia (Mohan Ram, 1960a; Mohan Ram and Masand, 1962) the endosperm is ab initio cellular. Uninucleate, unbranched secondary haustoria are formed from the endosperm proper and these penetrate into the integumentary cells. In Downingia (Campanulaceae) (Kaplan, 1969) the primary endosperm nucleus undergoes transverse division (Fig. 5A) to give rise to micropylar and chalazal endosperm cells (Fig. 5B). The next division in both micropylar and chalazal cells is longitudinal, which results in the formation of two, two-celled

D

E

FIG. 5 . (A-E) Early esdosperm development in Dowwingia harigalupii and D . pukhella. (A) First division of primary endosperm nucleus to form two-celled stage. (B)Two-celled endosperm showing transition to the four-celled condition. (C) Four-celled stage. (D) Four-celled stage in process of division to form an eight-celled stage. (E) Eight-celled endosperm stage with conspicuously differentiated chalazal and micropylar endosperm haustoria. Ch, chalazal haustorium; Mh, micropylar haustorium; Psy, penetrated synergid; Usy, unpenetrated synergid; Z, zygote. (From Kaplan, 1969.)

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endosperm tiers (Fig. 5C), that is, micropylar and chalazal. Longitudinal division is then followed by transverse division in both the tiers to give rise to eight-celled endosperm tissue (Fig. 5D and E). The terminal two cells of the chalazal tier undergo one more transverse division before differentiating into chalazal haustorium. The transverse division in the cells of the chalazal tier is nearly equal, unlike the micropylar tier where unequal cytokinesis forms a pair of smaller vacuolate cells and a larger pair of micropylar derivatives. The micropylar derivatives become densely cytoplasmic and form the micropylar endosperm haustorium. The haustorial cells show nuclear and cytoplasmic hypertrophy. Both micropylar and chalazal haustoria persist in the mature seed. They appear senescent and have cellulosic fibrils. The development of the endosperm in Santalales has been reviewed by Johri and Bhatnagar (1969). The mode of endosperm development in this order is cellular with the exception of Santalum (Bhatnagar, 1959), Mida (Bhatnagar, 1960), and Olax (Agarwal, 1963), where a helobial type of endosperm has been recorded. The chalazal haustorium is common and shows great variation in its structure, behavior, and activity. It is generally single-celled and uninucleate. In Olax (Agarwal, 1963) it has four nuclei, whereas in Comundra (Ram, 1957), Mi& (Bhatnagar, 1960), Exocarpus (Bhatnagar and Joshi, 1965), and Quinchamalium (Johri and Agarwal, 1965) it is multicelled. In Comandra (Ram, 1957) a lateral cecum is formed from the micropylar part of the embryo sac. A small cell is cut off from the cecum, the derivatives of which function as secondary haustoria. The latter elongate in the same direction as the primary haustorium. Both primary and secondary haustoria branch profusely and invade the vascular tissue. In Zodina (Bhatnagar and Sabharwal, 1966) also the haustorium is highly branched. In Loranthaceae, composite endosperm is formed by the fusion of endosperms formed in many embryo sacs of the same ovary. The formation of composite endosperm is restricted to this family.

C. HELOBIAL ENDOSPERM The helobial type of endosperm shows intermediate condition where the primary endosperm nucleus divides into two nuclei with different divisional potencies. Swamy and Parameswaran (1963) and Swamy and Krishnamurthy (1973) have reviewed helobial endosperm in angiosperms. The division of the primary endosperm nucleus, followed by wall formation, results in a larger micropylar chamber and a smaller chalazal chamber. The micropylar chamber undergoes several free nuclear divisions and ultimately becomes cellular. The nucleus of the chalazal chamber may or may not divide. If it divides, it usually remains coenocytic. In some instances, the chalazal chamber may become cellular, but this always occurs after the micropylar

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chamber (see Swamy and Parameswaran, 1963), with the exception of certain genera belonging to families Pbilydraceae (Kapil and Walia, !965), Bromeliaceae (Lakshmanan. 1967). and Sparganiaceae (Muller-Dobbis, 19h9), where the reverse case has been reported. These exceptions have been considered as aberrant conditions of the helobial type of monocotyledons by Swamy and his co-workers, Almost all the monocotyledons are characterized by the helobia! type of endosperm formation. Swamy and Parameswaran state that the helobial type of endosperm is an exclusive feature of monocotyledons, which is perhaps as exclusive as other primary exomorphis and endomorphic features by which this group is recognized. Swamy and Krishnarnurthy reaffirmed the above statement in 1973. The helohial endosperm recorded in some of the dicotyledons such as Santulum (Bhatnagar, 1959), Mida (Bhatnagar, 1960) of Santalaceae; Ofax (Agamal. 1963) of Olacaceae; and Saxifragu (Rau and Shanna, 1967) of Saxifragaceae is considered as representing modified aberrant ontogeny of the general condition-that is. cellular or nuclear4ccurring in the respective families.

VI. Ruminate Endosperm The mature endosperm in some plants shows rumination, which may be caused by the seed coat activity or by the endosperm itself (see Periasamy, 1962). This type of endosperm has been termed ruminate endosperm and it may belong to any one of the three types of endosperm described so far. In the case of seed coat activity. the rumination is caused by the ingrowths or irregularities on the seed coat, which may arise either by ( a ) unequal radial elongation of the cells of any one layer or the only layer of the seed coat, or by ( h )a definite ingrowth or infolding in the seed coat. These infoldings may arise either by the excessive elongation or enlargements of the cells of the seed coat as in Annonaceae and Aristolochiaceae, or by the localized meristematic activity that results in the formation of ingrowths from separate meristems as in Vitaceae and Palmae. Rumination by the activity of the endosperm itself may be due to (a) increase in the volume of endosperm along with the increase in seed volume, which results in the absorption of the nucellus and hence direct contact of the endosperm with the irregularities on the inner seed coat surface, as in Coccofoba uvifera, Mvristica frugrans, and Shorea talura; or ( b )unequal peripheral activity exhibited by the endosperm during later stages of development. This causes the seed coat to attain an irregular configuration as in Androgruphis echioides, A . serpyllifolia, and Elytraria ucaulis.

VII. Cell Wall Formation The coenocytic nuclear endosperm in most of the plants finally becomes cellular, but the manner in which the transition occurs is little understood. Re-

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cently , the mechanism of cellularization of nuclear endosperm at the fine structural level has been studied in four plants, namely, Helianthus annuus (Newcomb, 1973b), Stellaria media (Newcomb and Fowke, 1973), Triticum aestivum (Morrison and O’Brien, 1976; Mares et al., 1975, 1977), and Haemanthus katherinae (Newcomb, 1978). The sequence, the overall timing, and the manner of cell wall deposition in wheat given by Mares et al. differ in a number of respects from the one given by Momson and O’Brien. Mares et al. (1977) state that in wheat the transformation from the nuclear condition to the cellular condition is accomplished by centripetal growth of the wall projections from the central cell wall to form highly vacuolate cylindrical alveoli (Fig. 6A). The alveoli are not complete cells, because they lack wall

FIG. 6. (A, B) Wheat. (A) Alveolus is bounded on the dorsal side by the central cell wall and disorganized nucellar cells (at the bottom of figure). On the inner side it is bounded by thin strand of cytoplasm and central cell vacuolar membrane. The cell wall projections forming the side walls of alveoli are distinct (arrows) and terminate in arrowhead-shaped mass of cytoplasm. X875. (B) Electron micrograph of a cell wall projection in a section adjacent to that shown in A . A continuous wall traverses the cytoplasm forming the wall of alveolus and ends in an array of vesicles in the arrowhead portion. Cell wall material is absent in the cytoplasm bridging in the innermost end of the alveolus (large arrows). On one side the cytoplasm is bounded by central cell vacuole and its membrane, and on the other by the alveolus vacuolar membrane. Golgi apparatus is seen near the legs of wall projection. X6880. av, alveolus vacuolar membrane; cv, central cell vacuole; g , Golgi apparatus; vm, central cell vacuolar membrane; w, wall. (From Mares ef a / . , 1977.)

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5 P EHATNAGAK AND VEENA SAWHNEY

FIG. 7 . ( A . B) Wheat. (A) Electron micrograph of wall projection tip in which an array of microtubules aligned at right angles to the planar array of vesicles is clearly visible. X 17,500. (B) A single layer of endosperm cells formed from the basal portions of alveoli (arrows). This layer is separated from the central cell vacuole by alveoli chat appear similar to those in Fig. 6A. X600. av. alveolus vacuole; c v , central cell vacuole: mt. misrotubules. (From Mares el NI.. 1977.)

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material on the side facing the vacuole (Fig. 6A and B). The tip of the wall projections consists of a planar array of vesicles (Fig. 6B). Large numbers of microtubules aligned at right angles to these vesicles were evident (Fig. 7A), and probably play some role in orienting vesicles into the plane of developing wall projections. The alveolar nucleus divides to give rise to proximal and distal nuclei which are set apart by cross walls formed by normal cytokinesis. A peripheral layer of endosperm is thus formed from the basal portions of the alveoli (Fig. 7B). The continued centripetal growth of the alveoli, along with further divisions of the distal nuclei that remain a part of the nuclear syncytium and cross-wall formation from opposite sides of the embryo sac, completes the process of cellularization by 4-5 days after anthesis (Fig. 8). The alveoli arising from opposite sides of the embryo sac also join. According to Morrison and O'Brien (1976), however, the cellularization of the peripheral layer of endosperm cytoplasm is completed after 2 days of anthesis by cw vrn

FIG. 8. Wheat. Diagrammatic sketch of the sequence of events leadin to the dlularization of the free nuclear endosperm. 0 ,Endosperm nucleus; -0, free end of a growing wall partition or a phragmoplast, which consists of a planar array of vesicles and transversely oriented microtubules. For explanation see text. am, alveolar vacuolar membrane; av, alveolar vacuole; c, dorsal cytoplasm of central cell; cv, central cell vacuole; cw, central cell wall and plasma membrane; d, dorsal surface of the endosperm; v, ventral surface of the endosperm; vm, central cell vacuolar membrane; w, wall partition and accompanying plasma membrane. (From Mares er al., 1977.)

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FIG. 9 . (A-D) Hurtnunrhus. ( A ) Light micrograph (LM)showing an early stagc of cellular endosperm in the micropylar chamber where three to four layers of cells have been formed. Freely growing walls grow toward the central vacuole and finally terminate there. The cross walls derived

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freely growing walls, which frequently branch and eventually meet on the side nearest the central vacuole. At this stage microtubules are not involved. Further cellularization occurs by radial and tangential divisions in the peripheral layer of cells involving cell plate formation and microtubules. Studies of Buttrose (1963a) in wheat also indicate the absence of cell plate formation during the initial stage. In Helianthus (Newcomb, 1973), Stellaria (Newcomb and Fowke, 1973), and Haemanthus (Newcomb, 1978) the cellularization of the nuclear endosperm occurs in the same manner (Fig. 7A,B) as reported in wheat by Morrison and O’Brien (1976). Freely growing walls during the early cellularization process have also been reported in Quercus gambelii (Singh and Mogensen, 1976) and in the cultured unfertilized cotton ovules (Jensen et a l . , 1977). During early development of endosperm, wall ingrowths or projections of transfer cell type occur on the embryo sac wall. These ingrowths help in the translocation of assimilates that are required for the growth of endosperm. In Haemanthus (Newcomb, 1978), where the helobial type of endosperm occurs, wall ingrowths are more developed in the small dome-shaped chalazal chamber than in the micropylar chamber. Freely growing walls are initiated first at the micropylar region of the embryo sac, from where they progress toward the chalazal end (Newcomb and Steeves, 1971; Newcomb and Fowke, 1973; Newcomb, 1973b, 1978) and inward toward the central cell vacuole (Fig. 9A). There is thus a direction of growth with no apparent set pattern of development. In Helianthus and Haemanthus, the freely growing walls are irregularly thickened, crooked, and discontinuous with few plasmodesmata (Fig. 9B and C). The wall stains intensely with aniline blue, which suggests the presence of callose as the principal wall component in wheat (Morrison and O’Brien, 1976; Morrison et al., 1978). During later stages, the wall shows uneven electron density and nonuniform distribution of aniline-bluepositive material, thereby suggesting the incorporation of components other than callose in the wall. The cytoplasm near the tips of the freely growing walls, which frequently appears fenestrated, is rich in Golgi bodies and dilated ER. Golgi bodies are associated with freely growing wall formation in Helianthus (Newcomb, from the freely growing walls have a convex shape. ~ 2 3 0 (B) . LM illustrating cellular endosperm, cells of which are undergoing cytokinesis. Two cell plates (small arrows) associated with karyokinesis and the ends (large arrows) of the freely growing walls are seen. The freely growing walls are thin, irregularly thickened, and crooked. X390. (C) LM showing an advanced stage of endosperm in the micropylar chamber when many layers of cells have been formed and a binucleate free cell in the central vacuole. X120. (D) LM showing a portion of endosperm in the micropylar chamber where one layer of endosperm having convex-shaped cell walls (single arrow) has been formed from the freely growing walls. Coenocytic endosperm with several nuclei and a free cell in the central vacuole are also visible. Wall ingrowths of the embryo sac wall (double arrows) are also seen. X460. CV, central vacuole; FC, free cell. (Reproduced from Newcomb, 1978.)

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1973b). Srdkiriri (Newcomb and Fowke, 1973). and Hocnrcrnihi4s (Newcomb, 1978). In Srellcwirr. ER also contribute during wall formation. I n wheat (Morrison and O'Brien, 1976). groups of hypcrtrophied vesicles of uncertain origin are seen near the developing wall at the closing stage of cell formation. The cytoplasm at this stage is also rich in free ribosomes, mitochondria especially near thc wall ingrowths, proplastids, numerous vacuoles, and profiles of ER. In Heliurrrhus and Srelluria, microbodies are also present. In wheat (Morrison and O'Bricn, 1976). some electron-dense, amorphous substance is seen around the freely growing ends and gaps in the wall. This substance is believed to be involved in wall synthesis since microtubules at this stage we absent. I n Hactnutithir.s the chalaza1 chamber has numerous large nuclei (which may arise due to nuclcar fusion), smooth ER. lipid droplets, proplastids, and mitochondria. Later, endosperm nuclei increase by karyokinesis and are separated by cell plate formation between the freely growing walls to add to the cells of endosperm tissue (Fig. 9B). In Stellarirr (Newcomb and Fowkc, 1973) the innermost cells bordering the central vacuole are convex-shaped and do not have wall material like the other cells. Later. wall material gets deposited along the plasma membranc to enclosc them entirely (protoplast-likc wall formation). The source of wall material is not known. in Hwmuntlius (Newcomb, 1978) a special type of wall-less cells known as free cells are formed from the nuclei of the innermost layer of endospcrm in the micropylar chamber. These free cells move into the central vacuole and may become binucleate (Fig. 9C). So far, nothing much is known about their probable role. The free cells are generally observed when 10 or more layers of endosperm cells have been formed. though occasionally they may also be present in younger stages of endosperm (Fig. 9D).

VIII. The Endosperm Haustoria The endosperm haustoria are of widespread occurrence in angiosperms. Studies on haustorium i n terms of embryogenetic development are many, but in terms of ultrastructure and histochemistry these are few. All the evidences for the haustonal behavior of the haustorial cells are circumstantial and come from morphological data (Subramanyam, 1960; Kaplan, 1969), such as their association with integument, vascular supply, dark staining of the cytoplasm, and the presence of hypertrophied nuclei (Maheshwari, 1950). The haustorial cells undergo considerable enlargement and aggressive growth during development, which results in the destruction of ovular tissues adjacent to the invading haustoria. The nutrients are absorbed by the haustorium from the

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surrounding tissue and after getting metabolized in the haustorial cells are passed on to the central endosperm cells for their growth. The strongest evidence for their absorptive nature comes from the fine structural studies on Lobelia dunii (Torosian, 1971), where haustorial cell walls develop many protuberances, which results in invaginations in the plasma membrane. According to Torosian, these ingrowths act like transfer cells. Similar finger-like projections extending into the cytoplasm have also been observed in the micropylar and chalazal endosperm haustoria of Vaccinium macrocarpon (Brisson and Peterson, 1975) by scanning electron microscopic studies. In Plantago lanceolata (Vannereau and Mestre, 1975) no such projections are sgen in the micropylar haustorium. The ceils have smooth walls. In Lobelia (Torosian, 1971) the cytoplasm of the haustorial cells contains mitochondria, spherosomes, and densely osmiophilic membrane-bound organelles around the cell periphery, indicating high metabolic activity of the cells. Also, rough ER oriented parallel to the long axis of the cell gives an important clue to its nutritive and polarized transport role. The nuclear size is variable and polyploidy and hypertrophy are common in the haustoria1 cells. In Downingia (Kaplan, 1969), small strands of fibrillar material are present in the cytoplasm. Cytochemical studies in Pedicularis silvatica (Berg, 1954) and Downingia suggest they are cellulosic in nature. Histochemical studies on the haustorial cell in Linaria bipartita (Kallarackal, 1976) show that its nucleus is rich in DNA and proteins. The cytoplasm stains intensely for proteins, RNA, histones, and starch (Bhatnagar and Kallarackal, 1976). The intense staining for RNA and proteins suggests increased protein production and hence increase in enzymes, required by a very active cell. In most angiosperms, the primary endosperm nucleus is triploid and is formed by the fusion of two polars and a sperm. But it may be diploid as in Onagraceae or 15 n as in Peperomia hispidula (Maheshwd, 1950). This is due to the difference in the number of polar nuclei contributed by the female gametophyte during fertilization in each case. With age and increase in size, the nuclei of the endosperm tissue tend to become polyploid. Erbrich (1965) has extensively investigated the behavior of the endosperm nuclei and haustorium in several angiosperm families. In Thesium alpinum and T . liizophyllon (Santalaceae) the nuclei of the chalazal haustorium become 384-ploid. In the micropylar haustorium af Codonopsis alematidea (Campanulaceae) the ploidy level varies from 96 n to 192 n, while in Melampyrum lineare (Scrophulariaceae) ploidy level of the order of 1536 n is attained by each nucleus of the micropylar haustorium. In Salvia the endosperm haustoria disappear very early during development and the endosperm gives out cellular processes of the haustorial nature along the funiculus. The nuclei of this haustorium and the neighboring cells become 48-ploid. The highest ploidy level of 24576 n has been reported in the haustorium of Arum maculatum (Araceae).

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The increased ploidy level seems to be intimately associated with the haustorial and the endosprmic aggressiveness, which is significant in the nutrition of the embryo. The endosperm is generally nonchlorophyllous. In certain cruciferous plants, the presence of chloroplasts has been recorded (Yoffe. 1952). According to Yoffe the chloroplasts have a physiological significance, since they aggregate at the micropylar and the chalaza1 ends of the young endosperm where the metabolic activity is quite intense. In Mathiola and Raphanus the chloroplasts are present both in the nuclear and the cellular phases of the endosperm. The chlaroplasts have also been reported in Lysiuna (Rigby, 1959) of Loranthaceae and certain mistletoes (Kuijt, 1960). In Crinum (Dutt, 1962) during seed development, the seed coat and fruit wall are absorbed; as a result, endosperm is exposed to sunlight and becomes green. The exposed endosperni cells also behave like phellogen and give rise to cork tissue. The chloroplasts generally occur i n the peripheral layer of endosperm cells below the phellogen, but i n some rare cases they may extend to the inner layers of the endosperm as well.

IX. Aleurone Tissue A number ot studies have been conducted on the endosperm development in mtinocots. These studies indicate that the outermost layer of the endosperm cells. which IS cut from the free nuclear endosperm by the process of cell wall formation, differentiates into highly specialized tissue known as aleurone tissue. The aleurone cells wrrounding the starchy endosperm have been extensively studied from the developing and germinating caryopses at the fine structural level (Buttrose, 1 9 6 3 ~Paleg ; and Hyde, 1964; Eb and Nieuwdorp, 1947: Jones. 196Yb.c; Evers, 1970, Jones and Pnce, 1970: T a u and Jones, 1970; Jacobsen e t d . , 1971; Mortison rr csl., 1975; Colbome cia1 , 1996). Some work has also been done on the unirnbibed and ungerminating caryopses of barley (Jones, 1969a; Buttrose, 1971; Taiz and Jones, 1973). Brurnus and barley (Macleod e t a / . , 1964), Snrghum (Adams and Novellie, 1975a,b), and rice (Bechtel and Pomeranz, 1977). Ontogeny of the aleurone tissue at the electron-microscopic level have been done only in wheat (Buttrose, 1943c, Momson et a l . , 1975, 1978) and maize (Kyle and Styles, 1977). The aleurone tissue consists of one layer, as in most cereals, or as many as four cell layers, as in barley. The size and shape of the aleurone cells are not uniform throughout the whole grain. Many morphologically distinct types of aleurone cells have been dewribed. In Seraria (Rost and Lersten, 1970; Rost, 1970, 1973) aleurone cells that occur adjacent to the placental vascular bundle are columnar in shape with ingrowths on their walls These ingrowths aid in transfer of sub-

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stances from the vascular tissue to the developing embryo and endosperm. These cells are called “transfer aleurone cells” and have also been reported in Echinochloa utilis (Zee and O’Brien, 1971) and Zea mays (Kiesselbach and Walker, 1952), but are absent in wheat (Zee and O’Brien, 1971) and rice (Bechtel and Pomeranz, 1977). According to Bechtel and Pomeranz, the absence or presence of transfer aleurone cells may be of taxonomic significance or may be related to embryo-caryopsis size. In rice (Bechtel and Pomeranz, 1977), two types of aleurone cells are present: ( a ) aleurone cells (rectangular in shape with less-dense cytoplasm) surrounding the entire portion of the embryo (Fig. 10B); and ( b ) cuboidal aleurone cells with dense cytoplasm surrounding the starchy endosperm (Fig. 10A). The former have been termed modified aleurone cells by Bradbury et al. (1956). Since the two are structurally different, they are believed to have different functions (Bechtel and Pomeranz, 1977). In wheat also the aleurone cells differ structurally in the dorsal and ventral region of the grain (Morrison et al., 1978). The cells in the ventral region have been variously referred to as modified aleurone cells (Bradbury et al., 1956), thick-walled cells (Evers, 1970), and groove aleurone cells (Fulcher, 1972; Morrison et al., 1978). The cells in the dorsal region are meristematic and undergo divisions before differentiating into aleurone tissue, while those in the ventral region do not undergo divisions and directly function as the groove aleurone cells (Morrison et al., 1978). The aleurone cells can be distinguished from the rest of the endosperm after about 10 days of anthesis as small, highly vacuolate thin-walled cells with few organelles and large nuclei (Morrison et al., 1975; Kyle and Styles, 1977). At maturity, the cytoplasm of the aleurone cell is characterized by aleurone grains and also organelles such as plastids, mitochondria, spherosomes, Golgi bodies, ER, and microbodies. Aleurone grains and spherosomes are abundant and they often mask other organelles. The cell wall, which is initially thin, becomes uniformly thick and distinctly two-layered at maturity in wheat (Fulcher et al., 1972; Pomeranz, 1973; Morrison et al., 1975, 1978), barley (Jones, 1969a; Jacobsen et a f . , 1971; Taiz and Jones, 1973; Pomeranz, 1973; Ashford and Jacobsen, 1974), rye, and oat (Pomeranz, 1973). In wheat (Morrison et al., 1978), the groove aleurone cells have pitted and irregularly thickened walls. Plasmodesmata are few as compared to aleurone cells in the dorsal and flank regions of the grain. The adjacent aleurone cells are connected by plasmodesmata. Plasmodesmatal connections are also found between the aleurone cells and the starchy endosperm. The bulk of the storage proteins in cereal grains is confined to aleurone cells in membrane-bound organelles known as aleurone grains (Figs. 11B and 13B). Aleurone grains have electron-dense and electron-opaque inclusions (Buttrose,

FIG. 10. (A-C) Rice ( A ) Aleurone cells external to starchy endosperm with centrally located nucleus. x2100. ( 8 ) Modified aleurone cells opposite lateral scale of scutellum. The cells are rectangular in shape with less-dense cytoplasm ~ 3 1 5 0 (C) . Aleurone cell cytoplasm showing plastid 86

FIG. 11. (A, B) Triticale. (A) Scanning electron micrograph of starchy endospem of dehydrated mature kernel showing small spherical and large or lenticular starch grains within the proteinaceous matrix. x 7 185. (B)Scanning electron micrograph of transversely cut caryopsis showing aleurone cells with tightly packed aleurone grains. Aleurone grains have been shown to have lipid material by histochemical tests. ~ 9 3 0 0Ag, . aleurone grain; Pm, proteinaceous matrix. (Courtesy Arati Saxena.) with numerous tubules and vesicles that are invaginations of the plastid envelope (arrow). Cytoplasm also shows lipid body, microbody, mitochondria, a portion of nucleus, and small vesicle. X 14,000. L, lipid body; M, mitochondria; Mi, microbody; N, nucellus; Nu, nucleus; P, pericarp; PI, plastid; S, seed coat; Ve, small vesicle. (From Bechtel and Pomeranz, 1977.) 87

F w . 12. (A-E1 Barley. Light micrographs of aleurone tissue to show various inclusions after different staining procedures. ( A ) Toluidine blue-stained aleurone tissue showing many aleurone grains, Aleumne grains show globoid and pmtein-cahhydmte body. X 1430. (B) The section shows only protein-carbohydrate bodies after 10 minutes incubation in 0.05 M acetate buffer at pH 4. Globoids have been dissolved. x 1430. (C) Aleurone cells stained in 0.2% toluidine blue at pH 4 showing red-staining tubules (microchemical gardening) arising from ploboids. x 1430. (D) Aleurone grains with globoids within the globoidal cavity as seen after toluidine staining and mount. Sudan IV-Stained aleurone cells showing lipid in the ing in water without dehydration. ~ 9 3 0 (E) aleurone grains and lipid pool above the sections. x930. G, g l o b i d ; Gc, globoidal cavity; L, lipid, Lp, lipid pool; Pcb. protein-carbohydrate body. (From Jacobsen e r a / . , 1971.)

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1963c; Paleg and Hyde, 1964; Macleod et al., 1964; Jones, 1969a) that suggest the heterogeneous nature of the aleurone grain contents. Jacobsen et al. (1971) report two types of inclusions in barley: a phytin globoid and a protein-carbohydrate body (Figs. 12A-D and 13B). The proteincarbohydrate body is similar to the crystalloid described by Pfeffer (1872). The protein bodies isolated from Sorghum aleurone tissue also contain a carbohydrate, which occurs as a protein-carbohydrate mixture (Adams and Novellie, 1975a). No special substructures have been seen in the isolated protein bodies. In wheat aleurone grains, Fulcher (1972) and Morrison et al. (1975) describe two types of inclusions (Fig. 13A). Type I inclusion is similar to the phytin globoid seen in barley, and type I1 inclusion has a significant amount of protein. The groove aleurone cells in wheat caryopsis have only type I inclusion, unlike aleurone cells in the dorsal and flank regions where they have both types of inclusions (Morrison et al., 1978). In rice (Bechtel and Pomeranz, 1977)the aleurone cells surrounding the starchy endosperm have aleurone grains, whereas the modified aleurone cells surrounding the embryo lack them. The authors consider aleurone grains to be a type of protein body. Modified aleurone cells probably have a simple kind of protein body with a flocculent material, which lacks globoids typical of aleurone grains. In Setaria globoids resembling those of other Gramineae are present in protein bodies (Rost, 1970). In maize (Kyle and Styles, 1977) aleurone protein bodies (APB), which appear as vacuolar precipitates after 15 days of anthesis, attain full maturity by 35 days of anthesis. The dense body of the APB shrinks away from the vacuole wall at maturity; as a result a less-dense ground substance in the peripheral matrix is seen. A large electron-transparent core is also seen within the dense body of the grain. Besides size difference, the maize aleurone protein body differs from that of wheat and barley in internal structure also. The globoids consist of storage phosphate compounds, the major component being phytin, an insoluble calcium-magnesium salt of inositol hexaphosphoric acid. The globoids occur inside the transparent area referred to as globoid cavity (Figs. 12D and 13B) (Jacobsen et al., 1971) or internal cavity (Eb and Nieuwdorp, 1967; Jones, 1969a). The aleurone grains stain intensely with Sudan IV, thereby indicating high lipid content (Fig. 12E). The globoids appear to have most of the lipid of the aleurone grain (Jacobsen et al., 1971). The crystalloid is a crystalline protein deposit. Freeze-etched replicas of barley aleurone grains show a crystalloid, irregular in outline (Buttrose, 1971). The protein bodies from the aleurone tissue have protein carbohydrate, and polyphenolic materials (Adams and Novellie, 1975a). They are also rich in metals such as Ca, Mg, Fe, and K (Pomeranz, 1973; Adams and Novellie, 1975a). The components tend to be localized in discrete particles, protein confined to matrix and crystalloid, phytin to globoids, and carbohydrate to proteincarbohydrate body.

FIG. 13. (A) Wheat. Portion of aleurone cell showing tightly packed aleurone grains surrounded by lipid droplets. The lipid droplets also line the inside periphery of the plasma membrane. The aleurone grains have type 1 inclusions (arrow) and type I1 inclusion (double arrows) embedded in an amorphous matrix. Cytoplasm also shows plastids and mitochondria with indistinct cristae. ~4400. (B) Barley. Picture shows membrane-bound aleurone grain with globoid cavity. proteincarbohydrate body, and the ground substance. Aleurone grain is completely surrounded by sphero-

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The cytoplasm of the aleurone cell is characterized by many small to large electron-transparent (after KMNOl fixation) osmiophilic lipid bodies/droplets, also known as spherosomes. Most of the spherosomes, at maturity, aggregate around the aleurone grains and completely surround them (Fig. 13A and B). They also line the inside periphery of the plasma membrane (Fig. 13A). Spherosomes in aleurone cells may have a membrane as in Bromus (Macleod et al., 1964), barley (Buttrose, 1963c, 1971; Macleod el al., 1964; Paleg and Hyde, 1964; Jones, 1969a,b), Seturia (Rost, 1970), and maize (Kyle and Styles, 1977), or lack membrane as in barley (Jac.obsen et al., 1971) and Sorghum (Adams and Novellie, 1975a). Lately, Kyle and Styles (1977) have shown that the spherosomes in maize aleurone cells are surrounded by a distinct membrane that lacks the normal tripartite structure where the spherosomes are adjacent to the unit membrane of the aleurone grain. This concept of a half-unit membrane was first described by Yatsu and Jacks (1972) in spherosomes isolated from peanut seeds. They interpret that these membranes represent a phospholipid monolayer with the apolar region toward the center of the lipid containing spherosomes. The nucleus is generally centrally located in the cell and possesses from one to several nucleoli. Maherchandani and Naylor (1971) and Keown et al.. (1977) studied the variation in nuclear DNA content in aleurone cells of oat and barley, respectively. With Feulgen cytophotometry, they found that both the plants exhibit heterogeneity with respect to DNA in aleurone cells. At no stage during development is a single peak, corresponding to the triploid amount of DNA, observed. In barley aleurone cells (Keown et al., 1977) two classes of DNAlow and high-are present. The specific region of their location in the aleurone tissue is not known, because of the loss of in vivo arrangement during isolation of cells. Plastids, generally associated with osmiophilic globules, may be uniformly distributed-as in modified aleurone cells in rice-or restricted to regions where aleurone grains are not present. In rice (Bechtel and Pomeranz, 1977) a unique type of plastid is present, which has so far not been reported in aleurone cells of other plants. These plastids lack grana and their membranes invaginate to form vesicles and tubules within the cytoplasm (Fig. 1OC). Their function is not understood. In barley (Jones, 1969a), leukoplasts with stromal lamellae, without any distinct organization, are present. The cytoplasm of the aleurone cell has mitochondria, which become distinctly visible only after about 5 days of anthesis. In barley (Eb and Nieuwdorp, 1967) the cristae become distinct after 6-8 days of anthesis, unlike wheat where they somes. X21,OOO. Ag, aleurone grains; Am, aleurone grain membrane; Gc, globoid cavity; Cis, ground substance; L, lipid droplets; M, mitochondria;P, plastid; Pcb, protein-carbohydrate body; Sp, spherosome. (A, from Momson et al., 1975; B, from Jacobsen et at., 1971.)

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are indistinct (Fig. 13A) even after 6 weeks of anthesis (Morrison et a1 , 1975). According to Morrison et nl., mitochondna may be involved in the synthests of phytate, which is transported to the developing aleurone grains. Rough ER, which is abundant, forms a stack of lamellae and is probably involved in the production and secretion of proteins (Eb and Nieuwdorp, 1967). Microbodies are also present in the cytoplasm and these increase in number after GA, treatment In barley (Jones, 1969a), microbodies are seen as small membrane-bound vesicles with granular matnx and amorphous inclusions. The similarity in the nature of the amorphous substance in the ER cisternum and microbodies, and the close association of the two, suggest the possible origin of microbodies in the ER (Jones, 1969a). Golgi bodies with distinct cisternae, associated with vesicle production, are present (Buttrose, 1 9 6 3 ~Paleg ; and Hyde, 1964; Kyle and Styles, 1977). In rice (Bechtel and Pomeran7, 1977) Golgi bodies are absent. The transfer aleurone cells of Scvurra have glyoxysomes. which bring about the lipid mobilization. In aleurone cells, starch has been reported to be present as early as 10 days after anthesis (Buttrose, 1963~;Morrison ef ul.. 1975). These starch granules are never present at maturity Aleurone cells are nch in phytin Spherosomes and aleurone grains are the major site of accumulation In harley (Jacobsen €I nl . 1971) the sells nearest to the suture (groove) close to the vascular supply have maximum amounts of phytin, whereas those located away from the suture have a low phytin content. This distribution of phytin is considered to reflect the way in which phosphate is supplied tn the seed In wheat (Mornson et ( I ! . 1978) the phytin distribution cannot be explained In similar terms since the case is reversed

FuNcrroN

OF

ALEURONE TISSUE

The aleurone tissue has been assigned great importance during the last few decades in the plant hormone-action studies. During germination the cereal aleurone cells can secrete almost a!! the hydrolytic enzymes involved in the complete degradation of the starchy endosperm. Studies indicate that G a g from the embryo either induces the de ttovo synthesis of hydrolytic enzymes such as L-amylase by causing the production of specific mRNAs (Paleg and Hyde, 1964; Varner and Ram Chandra, 1964) or it enhances activity of certain other enzymes (Briggs, 1963; Taiz and Jones, 1970). These hydrolytic enzymes bring about the breakdown of reserve food materials in the endosperm tissue that are utilized by the embryo during its growth and development. Aleurone vacuoles or aleurone grains have several functions that have been summarized by Matile (1968). First. they store certain reserve substances such as proteins, metals, and phytin. Second, during germination, the aleurone vacuoles are transformed to a lysosome and their food reserves are mobilized. Finally,

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they may form a cell compartment in which other constituents of the cell are broken down.

X. Functions of Endosperm and Endosperm Haustoria There are several reports that support the concept of a nutritive role of endosperm for the developing embryo. Precisely at what stage the embryo begins to draw on the food substances of the endosperm is highly disputed. In Podelepis jaceoides (Davis, 1961) and Minuria denticulata (Davis, 1963), it has been shown that during early stages of development no food materials are drawn by the growing embryo. It is only after the embryo has reached the heart-shaped stage that the digestion of the neighboring endosperm cells occurs. Similar results have also been obtained in Capsella (Schulz and Jensen, 1969), Helianthus (Newcomb, 1973b), Linaria (Kallarackal, 1976), and Nicotiana (Sehgal and Gifford, 1979). According to Newcomb and Kallarackal, when the embryo is young, the endosperm cells are in an active state of division and, therefore, seem to be of limited nutritional value to the embryo. During this period, endosperm cells themselves require various cellular constituents and precursors to produce nucleic acids, proteins, and carbohydrates for rapid growth and differentiation. Recently, Kallarackal(l976) has shown that in Linaria, when endosperm cells are actively dividing, the haustoria become involved in the nutrition of the endosperm cells. They do not provide nutrition to the embryo directly, since no close association between the proembryo and the haustorial cells is seen. Also, the haustoria show signs of senescence when the zygote starts dividing. Contrarily, in Lobelia (Torosian, 1971) wall protuberances in the basal cell of embryo suspensor are present in areas that are in contact with the haustorium. During initial stages of endosperm development, storage materials such as proteins and starch are less abundant, and their accumulation takes place later. It is thus obvious that the endosperm provides nutrition to the embryo when the latter has reached the heart-shaped stage. The young embryo is therefore presumed to be dependent on other structures such as suspensor, persistent and degenerating synergids, and antipodals for its nutritive requirements. The biosynthetic activity of suspensor has been proved by several ultrastructural, biochemical, and cytochemical studies. In Linaria, Kallarackal(l976) has proposed a path of nutrition to the embryo sac before and after fertilization. Before fertilization antipodals absorb nutrients from the chalazal end from the vascular supply and synergids from the integumentary tapetum and pass them on to the central cell, which later develops into the endosperm (Fig. 14). After fertilization the chalazal and the micropylar haustoria function in place of antipodals and synergids, respectively, to provide nutrition to the embryo sac (Fig. 15).

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Fib 14 Litiurru hrpurrrro Diagrammatic representation lo \how the path ot nutrition into the en1bijw \aL hefufe feflilir.~lron Ant, anrip#al\. Cr. cenfrdjl dl.E. egg. s y . synefgids (Rum

Kallarackdl. 1976

)

The reserve materials of endosperm tissue undergo autolysis to yield simpler components, which are finally used up during the growth and germination of embryo. The endosperm, in addition to carbohydrates, fats. oils, and proteins, also contains growth substances such as auxins, cytokinins, and gibberellins. It has been shown that the liquid endosperm or endosperm extract from imnmture fruits not only r;upports the growth of i t s o w n embryo but can also nourish the embryos of other angiosperms. Coconut milk frQm the green fruits, corn endosperm extract in the milk stage, and liquid endosperm of horsechestnut and walnut have all been used to a great extent in basic media to induce the differentiation of embryoids and plantlets from various plant tissues. This stimulatory effect has been correlated with the presence of growth substances in the young endosperm. The mature endosperm lacks or has very little of these growth substances and hence cannot induce differentiation of embryoids and plantlets. Thus. apart from its nutritive role, the endosperm tissue-along with a variety of growth-regulating substances--at least partially controls the growth and differentiation of the embryo.

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FIG. 15. Linaria bipartita. Diagrammatic representation to show the path of nutrition into the embryo sac after fertilization. Ch, chalaza1 haustorium; En, endosperm cells; It, integumentary tapetum: Mh, micropylar haustorium; Z, zygote. (From Kallarackal, 1976.)

XI. Conclusions and Prospects Fine structural and histochemical studies have revealed the central cell with its numerous organelles to be a very active cell (Hu, 1964a,b; Jensen, 1965, 1972; Diboll and Larson, 1966; Vazart and Vazart, 1966; Jensen and Fisher, 1967; Mikulska and Rodkiewicz, 1967; Dibofl, 1968; Cocucci and Jensen, I969a,b; Van Went, 1970a,b; Schulz and Jensen, 1973), but a host of questionsespecially those concerning the role of the central cell during embryogenesisremain unsolved. Further studies on the embryo sac structure in association with its neighboring tissues may help in a better understanding of the relationship of the central cell with other cells of the female gametophyte. There are three general modes of endosperm development: nuclear, cellular, and helobial. It has been shown that the free nuclear endosperm in most of the

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plants finally becomes cellular. At the fine structural level, the cell wall formation during the cellularitation o f nuclear endosperm has been studied by Newcomb (1973b), Newcomb and Fowke (1973), Morrison and O’Brien (1976), Mares et nl. ( 1475, 1977), and Newcomb (1978). The sequence and the manner of cell wall deposition-as reported by Morrison and O’Brien (1976) and Mares et ~ i l (1975. . 1977) in wheat-differ significantly. It is quite possible that more than one mechanism is operating within a plant for the cellularization of the free nuclear endosperm. Extension of these investigations to other plants would surely prove interesting and rewarding. Another problem regarding cell wall formation. as raised by Newcomb (19781, concerns the factor(s) that might influence the linkage of karyokinesis with cytokinesis during the cellularization of the nuclear and helobial endosperms. Information about the coupling factor(s) could be useful in controlling the morphogenesis of other plant systems. The endosperm tissue at maturity is rich in storage materials (proteins, fats, oils. and carbohydrates), which are used by the developing embryo. The stage at which the embryo begins to draw on the endosperm food is highly disputed. Recently. in some plants it has been shown that the endosperm gives food to the embryo only when the latter is at about the heart-shaped stage (Davis, 1961, 1963; Schulz and Jensen, 1969, 1974; Kallarackal, 1976; Sehgal and Gifford, 1979). Studies on the role of endosperm nutrition therefore need to be extended further in different plants to know the exact time the embryo starts drawing nutrition from the endosperm. and also the extent to which the latter acts as a reservoir of nutrients, since in certain plants such as Q u ~ r c u s(Singh and Mogcnsen. 1976) the endosperm is shown to have only a minor role as a foodstorage tissue. Most of the food substances in this plant occur in the integuments, and the endosperm plays a major role as a medium for the translocation of food material from other parts of the ovule to the embryo. The form in which the food substances are taken up by the embryo sac and embryo is still not clear. The use of histochemical techniques coupled with radioactive isotopes will surely prove useful in identifying the nature of the nutrients. Our knowledge regarding the fine structure and histochemistry of the endosperm haustorial cells is meager. Intensive studies along these lines in the future will not only provide information on the nature and behavior of these cells hut will also throw some light on the exact time of haustorial activity during embryo and seed development. In monocots the outermost layer of the endosperm differentiates into a highly specialized tissue called aleurone tissue. The aleurone tissue is rich in protcinstorage organelles, and since these were first identified in the aleurone tissue, they were termed aleurone grains. Later, similar organelles were found in other tissues such as endosperm, embryo, and cotyledons, and were called protein

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bodies. Since then, protein-storage organelles have been variously referred to as protein vacuoles, protein granules, aleurone vacuoles, aleurone bodies, and aleurone grains. These terms have been used liberally and indiscriminately by some authors. All this has created some confusion, since some authors (see Bechtel and Pomeranz, 1977) consider protein bodies to be different from aleurone grains. According to them, aleurone grain is only a kind of protein body with a globoid inclusion. Rest and Vaughan (1972) consider the protein-storage organelles with inclusions to be aleurone grains, and those without inclusions, as myrosin grains. If we consider aleurone grains to be the ones with inclusions, as suggested by Rest and Vaughan (1972) and Bechtel and Pomeranz (1977), then all those protein-storage organelles that have inclusions should be called aleurone grains, irrespective of where they are located. This again creates a perplexing situation, since one usually associates the aleurone grain with the aleurone tissue. According to Rost (personal communication), the term protein body should be used for all the protein-storage organelles to avoid any confusion. In Sorghum (Adams and Novellie, 1975a) and maize (Kyle and Styles, 1977), no special substructures or inclusions have been reported within the protein-storage organelles from the aleurone tissue. These organelles, therefore, differ from the aleurone grains of Rest and Vaughan (1972) and Bechtel and Pomeranz (1977), and fall into the category of myrosin grains of Rest and Vaughan. Rost (1972) has classified protein bodies into three categories: ( a ) protein bodies without inclusions; ( 6 ) protein bodies with globoid inclusions; and (c) protein bodies with both globoid and crystalloid inclusions. Thus there are various kinds of proteinstorage organelles present within a tissue. We tend to agree with Rost that it is best to use the term “protein body” for various protein-storage organelles. This, in our opinion, will be more meaningful. So far most of our knowledge concerning protein bodies is from studies on oil seed plants and Gramineae. It would be worthwhile to extend such investigations to other families as well.

ACKNOWLEDGMENTS .

We are extremely grateful to Drs. Donald B. Bechtel (U.S. Grain Marketing Research Laboratory, USDA, New York), William A. Jensen and Russell L. Jones (University of California, Berkeley, California), John V. Jacobson (CSIRO, Canberra City, Australia), D. Mares (University of Sydney, PBI, Narrabri, N.S.W., Australia), I. N . Momson (Monash University, Clayton, Victoria, Australia), and Jose Kallarackal (University of Delhi, Delhi, India) for providing illustrations from their publications. We are thankful to the National Research Council of Canada (Can.J . Bof. 56,483-501, 1978); The Company of Biologists Ltd, Cambridge, England ( J . Cell Sci. 12, 741-763, 1973); CSIRO Editorial and Publications SVC., Australia (Azcs. J . Bot. 25, 599-613, 1977); The Intemational Society of Plant Morphologists, Delhi, India (Phytomorphology 17, 261-269, 1967; 19, 253-278, 1969), and A m . J . Bot. (64, 966-973, 1977). for permission to reproduce some photo-

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graphs. We also wish to express our appreciation to Dr. Thomas L. Rost (University of California, Davis. California) for his valuable suggestions.

REFEKt.N(-I(!i

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Karlstrom, P. 0.(1974h).S i w i s k Ror. Tidskr. 68, 315-328. Kaujhik. S, B. (1941). Proc-. lrirliun Arud. Sci.B 14, 133-140. Keown, A. C . , Tarz. L.. and Jones. R. L. (1977).A m . J . But. 61, 1248-1253. Khoo, LJ., and Wolf, M. J. (1970).A m J . But. 57, 1042-1050. Kiesselbach, T. A , . and Walker, E. R. 11952).A m . J . Eor. 39, 561-569. Kuijt, J. (1960). L’niv. Culif. Berkeley Puhl. Bot. 30, 337-436. Kyle, D. 1.. and Styles. E. D.(1977).Pluntu 137, 185-193. Lakshmanan. K . K.(1967).Proc. lndiun Arud. Sci. E 65, 49-55. List. A , . Jr.. and Steward. F. C . (1965).Atin. B o f . 29, 1-15. Luxova. M (10681.B i d Plunr. 10, 10-14. Macleod, A . M . . Johnsten. C. S;. and Duffus. I . I=. (19641.1. /PLW # r m . 70, 303=307. MacCiregw. H. C . (1972).Hiol. Rev. 47, 177-210. Maherchandani, N. J.. and Naylor. J. M. (1971).Can. J. Genet. Cytol. 13, 578-584. Maheshwari. P. ( 1950). ”An Introduction to the Embryology of Angiosperms.” McGraw-Hill, New York. Mheshwari, P.. and Johri. 8 , M , (1956).O g u m Cornm. Vol.. #of. Mug T o b o 69, 410-423. Malik. C. P.. and Vermani. S. (1975).Actu Hisrochem. 53, 244-280. Mares. D.J., Norstog, K., and Stone, B. A. (1975).Aust. J. Bnt. 23, 31 1-326. Mares. D. J . . Stone. 8 . A.. Jeffery. C.. and Norstog. K. (1977).Ausr. J. B o t . 25, 599-613. Marinos, N . G. (1970).Protriplusmu 70, 261-279. Mathur. N. (1956). P/iyrcimutpholog~6, 41-51. Matile. P. ( 1968).fflun:criphysiologie 58, 365-368. Maze, J . , and Lin. S. C. (1975). Can. J . Bot. 53, 2958-2077. Mikulska, k,,and Rodkiewicz, B. (1967).Actct SOL..Bor. Pal, 36, 555-566. Mitsuda. H.. Murakami. K . . Kusano. T.. and Yasumata, K. (1969).Arch. Biorhem. Biophys. 130, 678- 680. Mohan Ram. H. Y . (1958). Ph.D. Thesis, clriiv. offlelhi. India. Mohan Ram. H. Y. (1959).Curr. Sri. 28, 169-170. Mohan Ram, H.Y. rlY6Oa). Am. J. Bor. 47, 215-219. Mohan Ram. H. Y. 11960b). Llowfiu 23, 21-27. Mohan Rani, H. Y. (1962).J. lndiun Bot. Soc. 41, 288-296. Mohan Ram. H.Y..and Masand, P. (1962).Curr. Sci. 31, 7-8. Mohan Ram, H. Y.. and Wadhi. M. (1964). fhytomorphology 14, 388-413. Morrison, I. N . . Kuo. J.. and O’Brien, T. P. (1975). Plunra 123, 105-1 16. Morrison, I . N..and U’Brien. T. P. ( 1976). Plunru 130, 57-67. Morrison. I . N . , O’Brien. T. P.. and Kuo. J. (1978).Planru 140, 19-30. Monon. R. K., and Raison, J . K. (1963).Nufure ( L o n d t ~ n200, ) 429-433. Morton, R. K . , Palk, B. A , , and Raison. J. K. (1964).Eiochern. J. 91, 522-528. Mottier. D M. (1921). Ann. Eor. 35, 349-364. Miiller-Dobbis. U. (1969).But. Zenfrulbl. 89, 359-450. Nawaschin. S. G. (1898).Bull. Acod. Imp. Sci. St. Petersburg 9, 377-382. Negi. I). (1972).Ph.D. Dissertation, Univ. of California, Berkeley. Newcomb. W. (l973a). Curl. 1.Bor. 51, 863-878. Newcomb, W. (1973h).Can. J. Bof. 51, 879-890. Newcomb, W. (1978).Can. J. Bot. 56,483-501. Newcomb. W., and Fowke. L. C. (1973). Bor. Gaz. 134, 236-241. Newcomb. W.. and Steeves, T. A. (1971). Eor. Go;. 132, 367-371. Nijalingappa. B . H.M . . and Devaki, N. (1979). Curr. Sci. 48, 451-452. Opik, H . (1068). J. E i p . Bur. 19, 64-76. Ory. R. L.. and Henningsen. K . W. (1969).Plunr Physiul. 44, 1488-1498.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 73

The Role of Phosphorylated Dolichols in Membrane Glycoprotein Biosynthesis: Relation to Cholesterol Biosynthesis JOANTUGENDHAFT MILLS^

AND

ANTHONY M. ADAMANY

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York I. Introduction . . . . . . . . . . . . . . . . . . 11. Structure and Biosynthesis of Carbohydrate Units of Animal

111. 1v.

V.

VI .

VII.

VIII.

.

.

104

Glycoproteins . . . . . . . . . . . . . . . . . . . A. The Glycopeptide Bond . . . . . . . . . . . . . . B. Structure of Carbohydrate Units . . . . . . . . . . . C. Biosynthesis of Carbohydrate Units of Glycoproteins . . . . Lipid Intermediates in Bacterial Systems . . . . . . . . . Lipid Intermediates in Eurkaryotic Systems . . . . . . . . A. Formation of Dolichylmonophosphoryylmonosaccharides: Subcellular Systems . . . . . . . . . . . . . . . . B. Formation of Dolichylpyrophosphorylsaccharides:Intact Cell Systems . . . . . . . . . . . . . . . . . . . . C. Structure of the Carbohydrate Portion of Dolichylsaccharides . Relationship of Dolichylsaccharides to Asparagine-Linked Carbohydrate Units of Glycoproteins: Processing and Maturation of Protein-Bound Saccharide Units . . . . . . . . . . . . . A. Regulatory Role of Glucose . . . . . . . . . . . . . B. “Trimming” and “Maturation” of Asparagine-Linked Saccharides . . . . . . . . . . . . . . . . . . . Cellular Location of Dolichol-Dependent Glycosylation Reactions A. Spatial and Temporal Relationships of Glycosylation to the Ribosomal Machinery . . . . . . . . . . . . . . . B , Disposition of Polyisoprenol Derivatives in Membrane Systems C. Cell Surface-Associated Dolichol-Dependent Mannos yltransferases . . . . . . . . . . . . . . . Regulation of Glycoprotein Biosynthesis: Interdependence of Glycoprotein and Cholesterol Biosynthetic Pathways . . . . . A. Biosynthetic Sequences Common to Phosphorylated Dolichol and Cholesterol-Regulation of Cholesterol Synthesis . . . . . B. Interdependence of Cholesterol Synthesis and Glycoprotein Assembly . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

105 105 107 108 110 112 112 114 118

122 123

125 127 127 130 131 133 134 137 143

144

‘This article is dedicated to Dr. Danielli on the occasion of his retirement: we continue to build on your science. Tresent address: Department of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021. 103 Copyright 0 1981 by Academic Press, Inc. AIL rights of reproduction in any form reserved. ISBN 0-12-364473-9

104

JOAN TUGENDHAFT MILLS AND ANTHONY M. ADAMANY

I. Introduction The participation of certain phosphorylated long-chain polyisoprenols (dolichols) as saccharide carriers and donors in the biosynthesis of complex carbohydrates has been among the most fascinating and actively pursued discoveries in the field of membrane biosynthesis and glycoprotein metabolism in the past decade.' The discovery that a general class of phosphorylated polyisoprenols, dolichols in eukaryotes, and undecaprenols in prokaryotes, function as cofactors in the assembly of such varied macromolecules as peptidoglycan (Anderson rt d . . 1967: Katz et al., 1967; Matsuhashi et al., 1967; Dietrich rt al., 1967: Siewart and Strominger, 1968; Higashi et al., 1967) and lipopolysaccharides (Osborn and Tze-Yuen, 1968; Osborn, 1969) of bacterial cell walls, and glycoproteins of animal cell membranes (Lucas et al., 1975; Mills and Adamany, 1978; Olden et ul., 1978) and secretions (Struck and Lennarz, 1977; Housley et d.. 1980) has furthered our understanding not only of the mode of assembly of such macromolecules, but also (particularly in animal cells) of the spatial dictates of peptide translationhegregatiordglycosylation events and the control mechanisms of their metabolic regulation. It has become apparent that, because these cofactors are also synthesized by the cell, their availability for participation in complex biosynthetic pathways may provide an example of cofactor regulation that modulates the flux of substrates through those pathways. The dolichols, the major long-chain polyisoprenols found in animal cells, mediate the assembly of asparagine-linked oligosaccharide units of exported and intracellular glycoproteins (M. J . Spiro ef al., 1976a; Mills and Adamany, 1978; Baynes rt a / . , 1973; Parodi et al., 1972b: Lucas et al., 1975; Herscovics etal., 1977; Chambers et d . , 1977). Although the mode of synthesis of such glycoproteins appears to be a well-ordered process of considerable precision, relatively little is known as yet of the energetics or regulatory aspects of these pathways. However, the extensive advances over the past decade have provided a sound and necessary background for metabolic regulation studies. The polyisoprenoid nature of the dolichols and their biosynthesis from acetate or mevalonate suggested the intriguing possibility that their availability may be regulated by certain enzymes also involved in the synthesis of other polyisoprenols, particularly cholesterol. 'Trivial names and abbreviations used: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; 25-hydroxycholesterol. 5-cholesten-3P-25 diol: pregnenolone, 5-pregnen-3P-ol-20-one; tigogenin. Sa.20a.22a-@spieosten-3~-ol; sitosterol, S-cholesten-24P-ethyI-3~-ol; 7-ketocholesteroi, 5-cholesten-3P-ol-7-one; stigmasterol, 5,22-cholestadien-24~-ethyl-3P-01: triparanol (MER-29). 4-chloroa-[4-[ 2-(diethylamino)ethoxyjphenyll-a-(methylphenyl)benzeneethanol; Dol-P, dolichylphosphate; Man. o-mannose; Glc. @glucose; GlcNAc. BN-acetylglucosamine; GalNAc, o-N-acetylgalactosamine; AcNeu, N-acetylneuraminic acid; Fuc, ~-fUcose;Gal, &galactose; MurNAc, N-acetylmuramic acid. All sugars are in the pyranose form.

ROLE OF PHOSPHORYLATED DOLICHOLS

105

We have investigated the interrelationship of the pathways of biosynthesis of cholesterol, dolichol, and dolichol-dependent glycoprotein biosynthesis in the cultured aortic smooth muscle cell and found that they are coordinately regulated. Inasmuch as cellular glycoproteins and cholesterol are found primarily in association with plasma membranes, the cell might well coordinate the synthesis of these important components of its surface membrane. Such regulation may be of particular importance to the aortic smooth muscle cell, for the accumulation of cholesterol (Porter and Knight, 1973) and the proliferative response (Stary and McMillan, 1970) of this cell type have been implicated in the development of the atherosclerotic plaque. The involvement of surface carbohydrates in cell proliferation has been the subject of numerous investigations (Gahmberg, 1977) and may provide new insights into the molecular processes involved in atherogenesis. In this article we focus on the animal cell, particularly the cultured vascular smooth muscle cell, and review in a limited form: (1) aspects of glycoprotein structure; (2) the participation of phosphorylated dolichols in glycoprotein biosynthesis; and (3) the interdependent regulation of biosynthesis of phosphorylated dolichols and cholesterol, particularly as pertains to dolichol-dependent membrane glycoprotein assembly. A thorough and excellent review tracing the development of the dolichol story has appeared recently (Parodi and Leloir, 1980). 11. Structure and Biosynthesis of Carbohydrate Units of Animal

GIycoproteins

A. THEGLYCOPEPTIDE BOND Although a vast array of glycoproteins has been identified in mammalian tissues, the saccharide units of many of these proteins share a number of common features and, therefore, can be classified on the basis of structure. A primary distinction among the major classes of carbohydrate units lies in the type of carbohydrate-peptide linkage. Only three such linkages have been identified in animal glycoproteins: the 0-glycosidic bonds to serine (or threonine) and to hydroxylysine; and the N-glycosylamine linkage to asparagine. The 0-glycosidic bond between the 8-hydroxyl of hydroxylysine and @-galactoseis characteristic of collagens (Spiro, 1969) and basement membranes (Spiro, 1967a,b) and also occurs in certain proteins, such as Clq of the complement system (Yonemasa et al., 1971). The @-hydroxyl group of serine and threonine is involved in the 0-glycosidic linkage to two types of sugar residues in animal cell proteins. These amino acids are attached to the anomenc carbon of a-N-acetylgalactosamine in a number of glycoproteins (Spiro, 1973), and to xylose in the linkage region of the proteoglycans of animal cell matrices (Spiro, 1973).

106

JOAN TUGENDHAFT MILLS AND ANTHONY M. ADAMANY

The N-glycosylamine bond to asparagine is found in a variety of glycoproteins including those of the plasma (Spiro, 1962; Satake e t a / . , 1965), cell membranes (Thomas and Winzler, 1969). and hormones (Liao and Pierce, 1970), as well as in procollagen (Clark and Kefalides, 1976) and basement membranes (Spiro, 1967a.b). This type of linkage occurs between the anomeric carbon of N-acetylglucosamine and the amide nitrogen of asparagine. It is of some interest that this glycopeptide bond always occurs within the specific amino acid sequence, Asn-X-Ser (Thr), where X can be any amino acid (Neuberger and Marshall, 1969). However, because this amino acid sequence also occurs in a nonglycosylated form in a number of proteins (Hunt and Dayhoff, 1970), it may not be the sole determinant specifying the assembly of asparagine-linked carbohydrate units. The major types of glycopeptide bonds are most easily identified by their different susceptibility to alkaline conditions. The 0-glycosidic bond to the P-hydroxyl of serine or threonine is readily split under mild alkaline conditions (e.g., 0.1 N NaOH, 40"C, 48 hours), by the process of p-elimination (Spiro, 1973). Such treatment results in the release of a reducing saccharide and the formation of an unsaturated amino acid. When performed in the presence of sodium borohydride, mild alkaline treatment of glycoproteins containing 0-glycosidic linkages to serine or threonine results in the release of an intact oligosaccharide containing a sugar alcohol at the reducing terminus. This type of elimination reaction does not hold for the 0-glycosidic linkage to hydroxylysine, for the 6-hydroxyl of this amino acid is not properly positioned to provide a good leaving group. Thus, this type of bond is extremely stable even to harsh alkaline treatment. Indeed, such treatment of glycoproteins containing this linkage results in the release of intact Glc-Gal-Hyl (Spiro, 1969), which can be purified from alkaline hydrolysates. In contrast, the N-glycosylamine bond to asparagine is susceptible to harsh alkaline treatment, which results in the hydrolysis of the glycopeptide bond. Treatment of glycoproteins containing this type of linkage with 2 N NaOH containing 2 M sodium borohydride at 80°C for 16 hours results in the hydrolysis of the N-acetylglucosaminyl asparagine bond and deacetylation of the Nacetylglucosamine residues, with release of intact oligosaccharides terminating in glucosaminitol (Spiro, 1972). The differential responses of the glycopeptide bonds to alkaline treatment have proved useful in identifying the types of linkages present on a glycoprotein. More importantly, however, they have permitted the isolation and structural characterization of peptide-free oligosaccharides as well as Glc-Gal-Hyl glycopeptides (Spiro, 1973). Indeed, the harsh alkaline sodium borohydride conditions have proved useful for the structural studies on dolichylsaccharides and membrane glycopeptides of the smooth muscle cell noted in a later section.

ROLE OF PHOSPHORYLATED DOLICHOLS

107

B. STRUCTURE OF CARBOHYDRATE UNITS Although only a limited number of carbohydrate-peptide linkages are known to occur in mammalian glycoproteins, the great variety in structural and functional specificities of carbohydrate chains present on such proteins derives from the numerous types of monosaccharides found outside these linkages, from the position of glycosidic linkages between sugar residues, and from the usual occurrence of branching in the carbohydrate chains. Such variety, however, is not prevalent in the hydroxylysine-linked units of collagen and basement membranes. These units contain only glucose and galactose (Spiro, 1969) and exist either as mono- or disaccharides: P

Gal

-+

al,Z

p

Hyl or Glc +Gal+Hyl

The 0-glycosidic linkage between N-acetylgalactosamine and serine or threonine forms the core of carbohydrate units present on numerous serum glycoproteins (Spiro, 1973) and the erythrocyte membranes (Thomas and Winzler, 1969; Adamany and Kathan, 1969). Although a vast array of such units has been identified, their sugar composition is relatively limited and generally consists only of N-acetylgalactosamine, galactose, sialic acid, and fucose. The tetrasaccharide units of MN glycoproteins (glycophorins) of the human erythrocyte membrane (Thomas and Winder, 1969; Adamany and Kathan, 1969) are an example of this type of carbohydrate unit: a2 3

ACNWL ~ a i I AcNeu

a2 6

GalNAc

-

Ser (Thr)

The P-xylosylserine bond is found in a number of proteoglycans including the chondroitin sulfates, dennatan sulfate, heparin, and heparin sulfate. These units are considerably larger than those of other glycoproteins, and consist of long chains of repeating disaccharide units in which hexuronic acid and N-acetylhexosamine residues alternate, and which also contain variable amounts of 0- and N-sulfated sugar residues. Chondroitin sulfate, dermatan sulfate, and heparin contain identical core structures (Roden, 1968): 01.3

GlcUA

P1.3

PL.4

+ Gal + Gal + Xyl

P ---*

Serine

The variety in carbohydrate units of proteoglycans lies in the type of repeating disaccharide unit linked to this core and in the degree of sulfation of the sugar residues. A common core of sugar residues also occurs on the two types of asparaginelinked units, which are generally referred to, trivially, as simple and complex

I08

JOAN TUGENDHAFT MILLS AND ANTHONY M . ADAMANY

units, as exemplified by thyroglobulin unit A (Ito ef al., 1977) and fetuin glycopeptide (Nilsson et al., I979), respectively: AcNeu

AcNeu

AcNeu

l a 2.3 Man

Man

1 al,Z

hlan

lal.2

f

1111,2

Man

Man

Asn Thyroglobulin unit A

I

Gal

Gal

1J1.4 GlcNAc

Man

Man

Gal

1$1.4

GlcNAc

I/

GlcNAc

//

Man /

\

Man

I

/

Asn Fet uin gl yc opept ide

Despite their obvious differences, these two types of unit share a number of common features. First, both contain mannose, a sugar that is found only in asparagine-linked units of mammalian glycoproteins and thus serves as a marker for such units. Second, both units contain a common core, as indicated by the dashed lines, consisting of N,N'-diacetylchitobiosylasparagine,which is linked to the P-mannosyl branch point of a characteristic trimannosyl sequence. The marked similarities between these two structures suggest a common mode of biosynthesis for the core portion of the two units. Indeed, since both units have been found linked to the same peptide chain in some proteins, such as thyroglobulin (Arima et al., 1972), it has been suggested (M. J . Spiro rt al., 1976a; Robbins et ul., 1977a,b; Hunt et ul., 1978) that the simple high-mannosecontaining unit may serve as a precursor for the more complex unit, a concept that will be discussed in greater detail later (Section V). C. BIOSYNTHESIS OF CARBOHYDRATE UNITSOF GLYCOPROTEINS Most of the early biosynthetic studies concerned the transfer of sialic acid onto proteins, since exogenous acceptors of this sugar could be easily prepared. Treatment of glycopeptides or peptide-free oligosaccharides with mild acid (e.g., 0.05 N sulfuric acid, 8OoC, 60 minutes) results in the release of sialic acid

109

ROLE OF PHOSPHORYLATED DOLICHOLS

exclusive of other sugars and permits the asialoglycoprotein to function as an exogenous acceptor for the action of sialyltransferases. Such studies have demonstrated that CMP-NANA serves as the glycose donor for these reactions (Roseman, 1968). With the advent of specific glycosidases, other sugars could be released from glycopeptides and oligosaccharides and replaced with glycoses from specific nucleotide sugar donors. This approach elucidated the complete pathway for the assembly of 0-glycosidically linked units exemplified, most simply, by the synthesis of Glc-Gal-Hyl (R. G. Spiro and Spiro, 1971; M. J. Spiro and Spiro, 1971):

-

+ Collagen-Hyl + MnZ+ Glc transferase Collagen-Hyl-Gal + UDP Gal transferase UDP-Glc + Collagen-Hyl-Gal + Mn2+ + Collagen-Hyl-Gal-GIc + UDP UDP-Gal

Similarly, the outer segments of the complex units linked to asparagine in several glycoproteins are assembled as follows (Spiro et al., 1974): (AcNeu),

I

(Gal),

I

GlcNAc- (Man), 2UDP-G1cNac (GlcNAc), Asn

2UDP

I Asn

ireTf

(GlcNAc),

3 CMP- AcNeu

(y),

I I

3 UDP

Asn

The elucidation of the modes of carbohydrate chain assembly noted above provided several important concepts. (1) Nucleotide sugars served as glycose donors in chain-elongation reactions. (2) Each residue was added singly and sequentially with the product of one enzymatic reaction serving as the substrate and determinant for the next. (3) Most of the glycosyltransferases required a divalent cation for activity, with manganese serving as the most effective cation. One notable exception was sialyltransferase (Roseman, 1968), which utilized the nucleoside monophosphate derivative, CMP-AcNeu and did not require a divalent cation. Interestingly, the sialyltransferases catalyze a chain-termination event, which bears some similarities to the addition of outer a1,2-mannose sequences of the simple asparagine-linked unit. As described below, these terminal sequences are formed from donor dolicholmonophosphorylmannose by a specific transferase that docs not require a divalent cation. Attempts to decipher the requirements for addition of core mannose sequences using appropriate glycosidase-degraded acceptors and conventional assay procedures, as above, were not fruitful. However, new insights were gained when enzymes involved in the addition of mannose residues were studied by use of specially modified exogenous acceptors (Adamany and Spiro, 1975a). Thus, it was noted that certain mannosides that bore lipophilic aglycone moieties (e.g . ,

110

JOAN TUGENDHAFT MILLS AND ANTHONY M. ADAMANY

4-methylumbelliferylmannosidesand N-dinitrophenylated thyroglobulin Unit A glycopeptides) served as excellent acceptors of mannose from GDP-mannose in reactions catalyzed by calf thyroid particulate enzyme preparations. Unmodified glycopeptides and oligosaccharides did not serve this function (Adamany and Spiro, 1975a). As will be discussed later, the natural substrate for this reaction was in fact dolichylsaccharide, rather than a glycoprotein, and thus the lipophilic features of the exogenous acceptor molecule, the aglycone portion, mimicked the natural acceptor and fooled the enzyme. Nevertheless the structural specificities recognized by this transferase were distinctly different from those recognized by other glycosyltransferases described above. In addition, the reaction proceeded in two steps (see Section 1V.B) with a lipid intermediate, dolichylphosphorylp-D-mannose, serving as the immediate donor of the chain-elongating sugar residue (Adamany and Spiro, 1975b). These findings provided the first direct evidence for the involvement of dolichylphosphorylmannose as the most immediate glycose donor for chain-elongation purposes, and fit within the developing concept of lipid intermediates in glycosylation reactions as discussed next. The enzymes involved in the assembly of the core region of asparagine-linked carbohydrate units could not be identified by the use of simple glycoside acceptors. The mode of assembly of these units remained a mystery for many years until the discovery of lipid-linked intermediates in the glycosylation of bacterial complex carbohydrates (Anderson et al., 1967; Katz er a / . , 1967; Matsuhashi et a/., 1967; Dietrich et al., 1967; Siewart and Strominger, 1968; Higashi et al., 1967; Osborn and Tze-Yuen, 1968; Osborn, 1969) led investigators to propose that similar reactions might occur in animal cells (Spiro ef al., 1976; Parodi et al., 1972a,b; Lucas ef a ) . , 1975; Herscovics et al., 1977; Chambers et al., 1977). Tt is now known that the assembly of the core region of asparagine-linked units occurs by the en bloc transfer of a preassembled oligosaccharide from a dolichyl(pyro)phosphate carrier to the protein acceptor (Parodi and Leloir, 1980). Unlike the transfer of sugars to the 0-linked units or to the outer region of asparagine-linked units, this en bloc transfer occurs on the nascent polypeptide before it is released from the ribosome (Molnar and Sy, 1967). Although the dolichol story evolved only i n the past decade, much elegant work was carried out in bacterial systems in the mid-I960s, which set the stage for defining the role of dolichylphosphate in the assembly of glycoproteins of animal cell membranes and secretions. We believe it is altogether appropriate to describe briefly these advances in the context of this article.

111. Lipid Intermediates in Bacterial Systems

During the course of their studies on the synthesis of the peptidoglycan of bacterial cell walls, Anderson and colleagues (1976) discovered that during

ROLE OF PHOSPHORYLATED WLICHOLS

111

incubations containing UDP-[14C]M~rNAc-pentapeptideand particulate enzyme fractions from Staphylococcus aureus or Micrococcus lysodeikticus, two lipophilic products were formed: [14C]MurNAc(pentapeptide)-P-P-lipid and GlcNAc-MurNAc(pentapeptide)-P-P-lipid.Both substances were bound to the enzymes and, through a series of elegant experiments, were shown to be involved in the assembly of the repeating disaccharide-pentapeptide unit of the peptidoglycan (Katz e t a l . , 1967; Matsuhashi e t a l . , 1967; Dietrich e t a l . , 1967; Siewart and Strominger, 1968). The formation of the intermediates was readily reversible and yielded UMP from UDP-MurNAc-pentapeptide and UDP derived from UDP-GlcNAC. Based on these results, Anderson and co-workers (1967) proposed a cyclic reaction whereby the disaccharide-pentapeptide unit was assembled on the phospholipid carried prior to its en bloc transfer to the peptidoglycan acceptor. The products of this transfer were elongated peptidoglycan and lipid pyrophosphate. The elucidation of this mechanism introduced the concept of en bloc transfer of simple repeating saccharide units from lipid carriers to protein acceptors. In addition, the cyclic nature of this reaction sequence suggested a catalytic role for the lipid, the active form of which could be regenerated by cleavage of the pyrophosphate bond (Anderson et al., 1967). The phospholipid carrier involved in this process was purified by Higashi et al. (1967), and its structure was determined by a combination of chemical and physical techniques. Mass spectrum analysis was used to identify the lipid as a polyisoprenyl alcohol composed of 11 isoprene units and one alcoholic function and, unlike the dolichols, this lipid had no saturated isoprene unit. The lipidsaccharide contained 2 moles of inorganic phosphate per mole of lipid disaccharide-pentapeptide, both of which were extremely acid-labile. The purified lipid-disaccharide-pentapeptide was biologically active when incubated with a particulate fraction from M . lysodeikticus, serving as an acceptor of glycine from glycyl-tRNA, as well as a donor of glycopeptide for peptidoglycan synthesis (Higashi et al., 1967). Shortly after the discovery of lipid intermediates in peptidoglycan biosynthesis, a similar pathway was identified in Salmonella typhimurium for the assembly of the 0-antigen (Osborn and Tze-Yuen, 1968; Osborn, 1969). An analogous pathway has been demonstrated for S. newington by Wright et a / . (1965). The carrier lipids involved in these systems were apparently similar or identical to those utilized for the synthesis of peptidoglycan based on the following observations: (1) Bacitracin, an antibiotic that inhibited peptidoglycan synthesis by preventing the dephosphorylation of lipid pyrophosphate (Siewart and Strominger, 1967), blocked 0-antigen synthesis in the same manner (Osborn, 1969). (2) Lipid carrier preparations for S. aureus and M . lysodeikticus served as effective substrates for the synthesis of 0-antigen in lipid-dependent enzyme preparations from S. typhimurium (Osborn, 1969). Lipid intermediates appear to be involved in the assembly of other complex

112

JOAN TUGENDHAFT MILLS AND ANTHONY M. ADAMANY

polysaccharidcs in bacteria including the membrane-bound mannan of M . lysodeikticus (Scher et ul., 1968), the capsular polysaccharide of Aerobucter ueroyenes (Troy and Heath, 1968), and the teichoic acid of Sraphylococcus lncris (Douglas and Baddiley , 1968). Unlike the lipids involved in peptidoglycan and lipopolysaccharide synthesis, the mannose-containing intermediate in mannan synthesis was a monophosphoryl derivative (Scher et al., 1968) and served as the donor of a single sugar residue for chain-elongation reactions (Scher et al., 1968). Thus, the excellent studies describing the assembly of complex saccharides in microorganism carriers provided a simple framework of reference, a unifying theme involving lipid carriers. to elucidate the complex reactions involved in protein glycosylation in higher organisms.

IV. Lipid Intermediates in Eukaryotic Systems Polyisoprenols varying in length, configuration, and degree of saturation were identified in a number of eukaryotic cells prior to the discovery of their roles in oligosaccharide biosynthesis. The unsaturated all-trans C5, Cl0, and C,, pyrophosphate alcohols were known to be involved in sterol synthesis in animal cells (Bloch, 1965). However, larger polyisoprenols of unknown function were reported to occur in both animal and plant cells. These included a series of polyisoprenoid alcohols ranging in length from C,, to Cs0 isolated from plant tissues (Ness and McKean, 1977), and the longer polyisoprenol, dolichol, from animal tissues (Pennock et al., 1960). Dolichol was first identified in the unsaponifiable lipid fraction from pig liver by Pennock et af.,(1960), and was characterized as a long-chain polyisoprenol (Pennock et al.. 1960; Burgos ef al., 1963) consisting of 19-2 1 isoprene units, predominantly in the cis configuration (Burgos et al., 1963). Although these workers did not isolate any phosphorylated derivatives, the structural similarities between dolichol and the shorter polyisoprenols, previously shown to be involved in bacterial cell wall biosynthesis (Anderson er ul., 1967; Katz et al., 1967: Matsuhashi et ul., 1967; Dietrich et al., 1967; Siewart and Strominger, 1968; Higashi et al., 1967), served as a background to the discovery, several years later, that dolichol participates in carbohydrate-transfer reactions in mammalian cells.

A. FORMATION OF DOLICHYLMONOPHOSPHORYLMONOSACCHARIDES SUBCELLULAR SYSTEMS

In 1969 Caccam et ul. isolated a mannosyl lipid from a variety of mammalian tissue homogenates incubated in the presence of GDP-['4C]rnannose. This compound was similar in solubility properties to the mannosyl- 1-phosphoryl

ROLE OF PHOSPHORYLATED DOLICHOLS

113

polyisoprenol involved in bacterial mannan synthesis (Scher et a/., 1968). Pulse-chase studies revealed the transient appearance of this compound, and suggested that it may function as an intermediate in glycoprotein biosynthesis in those tissues. The results of these studies were confirmed by work from a number of laboratories within the next several years. During the course of their studies on the synthesis of plasma glycoproteins by liver, Tetas et al. (1970) observed the transfer of radioactively labeled galactose, mannose, and N-acetylglucosamine from their respective sugar nucleotides to endogenous lipid as well as protein by rat and rabbit liver microsomes. The transfer to lipid was readily reversed by the addition of UDP or GDP, and pulse-chase experiments suggested that the sugarlipids might serve as precursors of the protein-bound sugars. Because their previous isolation of ['4C]glucosamine-labeled puromycin-terminated peptides (Molnar and Sy, 1967) had suggested that the initial steps of protein glycosylation occurred on the nascent polypeptide chains, the authors put forth the provocative suggestion that the lipid intermediates may serve to carry sugars from the cytosolic nucleotide sugar pools to the lumen of the rough endoplasmic reticulum (Tetas et al., 1970). At about the same time, studies by Behrens and Leloir (1970) first identified the lipid carrier molecule as a phosphorylated dolichol. In these studies, however, the endogenous lipid from rat liver microsomes was found to accept glucose from UDP-glucose to form dolichylmonophosphoryl glucose. Analogously to the mannose-containing derivatives described previously (Caccam ef al., 1969), the glucosylated lipid was labile to mild acid, required a divalent cation for its formation, and functioned as a glucose donor for the synthesis of glucosylated protein. Importantly, as will be discussed later, it was observed that the radioactive glucose was eventually lost from the protein (Behrens and Leloir, 1970). The acceptor lipid was identified as dolichylphosphate based on its chromatographic properties and response to hydrolytic conditions (Behrens and Leloir, 1970), as well as the ability of authentic dolichylphosphate to accept glucose from UDP-glucose in the presence of rat liver microsomes. That the phosphate group did not derive from the nucleotide sugar was proved by the observation that the glucosylated acceptor was not labeled when [P-32P]UDPglucose was used as the donor (Behrens and Leloir, 1970). On the basis of these studies, the authors postulated the following sequence: 1. U:DPGLc + DO]-P Dol-P-Glc + UDP 2. Dol-P-Glc + protein + Dol-P + Glc-protein 3. Glc-protein -+ Glc + protein

The identity of the lipid acceptor as a phosphorylated dolichol has been confirmed in a number of laboratories (Adamany and Spiro, 1975b; Richards et al., 1971; Baynes et al., 1973; Tkacz et al., 1974; Herscovics et al., 1974).

114

JOAN TUGENDHAFT MILLS AND ANTHONY M . ADAMANY

Richards et al. ( 197 1) prepared [3H]dolichol from the fungus Phytophfhera cuesforurn grown in medium containing [3H]mevalonate. When this 13H]dolichol was phosphorylated by chemical means and incubated with pig liver microsomes in the presence of GDP-[’4C]mannose, a doubly labeled mannolipid was formed. This product had chromatographic properties characteristic of dolichylmonophosphorylmannose and, after mild acid hydrolysis, released [14C]mannose and [3H]dolichylphosphate. To support further the identity of the endogenous mannose acceptor as dolichylphosphate, fertilized chicken eggs were injected with [3H]mevalonateduring the first day of development (Richards et al., 1971). When liver microsomes prepared from 1 -day-old chicks were incubated with GDP-[’*C]rnannose, a doubly labeled mannolipid was formed that was chromatographically identical to dolichylphosphorylmannose (Richards et al., 19711. Similarly, dolichylphosphate has been identified as the endogenous mannose acceptor in pancreatic (Herscovics et al., 1974) and thyroid (Adamany and Spiro. 1975b) tissue homogenates. A more direct identification of the endogenous lipid acceptor as dolichylphosphate was achieved by Baynes et al. (1973). These investigators purified large quantities of dolichylphosphorylmannose from bovine liver homogenates labeled with GDP-[14C]mannose. and, by mass spectrum analysis, characterized the lipid portion of the molecule as a dihydropolyisoprenol consisting of at least 18 isoprene units, one of which was saturated. Therefore. compared to the bacterial undecaprenols, the dolichols were about twice as large and contained a saturated isoprene unit. Formation of Dol-P-Man and Dol-P-Glc is easily detected in subcellular particles using appropriate sugar nucleotide donors, but not in intact cell systems whether using monosaccharide precursors (Spiro et a / . , 1976a,b; Adamany, 1976; Mills and Adamany, 1978) or GDP-Man, when mannosyltransferases of the cultured aortic smooth muscle cell surface were assayed (see Section V1,C). It would be expected that a good intermediate would be difficult to trap in an efficiently metabolizing system as the intact cell. Indeed, the formation of these substances in subcellular particles occurs very rapidly and is transient, as shown in Fig. 1. An initial rapid “burst” of formation of Dol-P-Man and hi-P-Glc is observed, which decays rapidly before achieving a steady rate of formation. It is tempting to speculate that this initial “burst” may represent glycosylation of enzyme-bound Dol-P, which products then equilibrate with endogenous or exogenous nucleosides diphosphate formed from the sugar nucleotide donors. The formation of Glc and Man products requires manganese, and the sugars of both products are in the P-configuration (see Section IV,B). B . FORMATION OF DOLICHYLPYROPHOSPHORYLSACCHARIDES: INTACTCELL SYSTEM A second giycosylated dolichol derivative was discovered in a liver microsome system by Parodi et al. (1972a,b), and was implicated as the most immediate

ROLE OF PHOSPHORYLATED DOLICHOLS

115

6

?0 -x z a P

4

U

> 3 i= t-

0

s25 2

a

a

0

TIME (min.) FIG. 1. Time course of formation of dolichylmonophosphoryl-~-~-mannoseand dolichy1monophosphoryl-@-Lhglucose in aortic smooth muscle cell particles. Membrane particles sedimenting between 650 and 100,OOO g were incubated with GDP-[14C]Man or UDP-["C]Glc and the products isolated as described. (Adamany and Spiro, 1975.)

glycose donor for protein glycosylation. These investigators discovered that the endogenous acceptor of Glc form Dol-P-Glc was not protein, but rather a lipid saccharide that was insoluble in chloroform/methanol (2: l), but could be extracted in chloroform/methanol/water (10 103). The oligosaccharide portion of the molecule was rather large, and was later (Parodi et al., 1973) found to contain mannose and N-acetylglucosamine as well as several glucose residues. The lipid portion of the molecule was tentatively identified as dolichyl(pyro)phosphatebased on its behavior on ion-exchange chromatography (Parodi et al., 1972a) and response to alkaline hydrolysis and E. coli phosphatase treatments (Behrens et al., 1971). The compositional similarity of the lipid-bound oligosaccharide to that found linked to asparagine residues in a number of glycoproteins (Spiro, 1973) led

116

JOAN TUGENDHAFT MILLS AND ANTHONY M. ADAMANY

Behrens et al. (1973) to propose the following scheme for the assembly of asparagine-linked oligosaccharide units: 1 ) UDP-GIcNAc + Dol-P % Dol-PP-GIcNAc + UMP (2) &I-PP-GIcNAc + UDP-GIcNAc * Dol-PP(GlcNAc), + UDP ( 3 ) Dol-PP(GlcNAc), + GDP-Man and/or Dol-P-Man + Dol-PP-(GlcNAc)l(Man), (4) hl-PP(GlcNAc), (Man), + Dol-P-Glc -+ Dol-PP(GlcNAc), (Man),(Glc), (5) Dol-PP(GlcNAcj2(Man),(Glcj, + Asn-protein protein-Asn(GlcNAc),(Man),(Glc), (

-+

+ Dol-PP

Although this proposal may have been somewhat premature considering the limited data available at the time, numerous studies over the course of the next 6 years supported and extended it. Tetas et al. (1970) had already isolated DolPP-GlcNAc from rat liver microsomes, and Molnar et al. (1971) had confirmed reaction (1). That the dolichylpyrophosphoryl-GIcNAc derived one of its phosphates from the nucleotide donor was demonstrated by the incorporation of label from @-:32P]UDP-GlcNAcinto the lipid product (Molnar ef a l ., 1971). Thus, analogously to the situation in prokaryotic systems, dolichylmonophosphate served as substrate for reactions (1) through (5). Once relieved of its carbohydrate [reaction ( 5 ) ] product, dolichyl(pyro)phosphate must then be converted to Dol-P in order to achieve a cyclic reaction sequence. Indeed, Wedgewood and Stominger recently identified dolichyl(pyr0)phosphatase activity in homogenates of cultured human lymphocytes (Wedgewood and Strominger, 1980). Leloir and his colleagues ( 1973) isolated the N,N'-diacetylchitobiose-containing lipid from rat liver microsomes and provided evidence for the existence of reaction (2). The enzymes involved in reactions (1) and (2) were later solubilized and partially purified by Heifetz and Elbein (1977), as was the enzyme responsible for the formation of Dol-P-mannose (Heifetz and Elbein, 1977; Chambers et al., 1977). The transfer of several mannose residues to the lipid-linked N,N'-diacetylchitobiose core [reaction (3)] has been confirmed in a number of laboratories. The innermost mannose, which is /3-linked to this core, was transferred directly from GDP-mannose with inversion of configuration and without the involvement of a lipid carrier (Levy et al., 1974). The outer mannose sequences, however. which are all present in the a-configuration, may be formed by the transfer of mannose either directly from GDP-mannose (Chambers et al., 1977) or from a Dol-P-P-mannose intermediate (Heifetz and Elbein, 1977; Chambers et al., 1977; Levy et ul., 1974: Forsee and Elbein, 1975, 1977; Waechter et d.,1973; Adamany and Spiro, 1975b; Adamany and Mills, 1977). Because the two types of transfers could be distinguished by cation requirement for the nucleotide sugar, but not for the lipid mannose, Chambers et al. (1977) found that in pig aorta particles Dol-P-mannose served as a better mannose donor for the formation of large oligosaccharides (8-10 residues). Conversely, GDPmannose was the better substrate in forming short oligosaccharides containing about 7 sugar residues. The larger saccharide contained 2- 0-a-D-mannosylmannose sequences.

117

ROLE OF PHOSPHORYLATED WLICHOLS

The enzymes involved in the formation of 2- 0-a-D-mannosylmannose sequences, as occurs in dolichylsaccharides, were defined by the work of Adamany and Spiro (1975a,b). It was observed that certain hydrophobic glycosides could serve as exogenous acceptors of mannose from GDP-mannose in reactions catalyzed by particulate enzyme fractions from calf thyroid through the obligatory participation of dolichylphosphory1-P-D-mannose. Indeed when assayed as substrate, the latter was the best donor. From the results of these studies it appeared that the transfer of mannose proceeded through a two-step reaction catalyzed by two different mannosyltransferases, only the first of which required manganese activity: Mn”

Dol-P= Dot-P-P-o-Man + GDP (2) Dol-P-P-o-Man + Man-R 3 2-0-a-D-Man-Man-R Dol-P Sum: GDP-Man + Man-R% 2-0-a-D-Man-Man-R + GDP

(1) GDP-Man

f

+

That the product of step (1) was in the P-configuration was deduced when alkaline hydrolysis of the lipid-mannose released mannose-2-phosphate. This product was formed through a cyclic 1,Zphosphate, which would occur only if the mannose were present in P-linkage to the phosphate. In contrast, the product formed in step (2) was found to be in the a-configuration since all of the radioactivity could be released by treatment with jack bean a-mannosidase (Adamany and Spiro, 1975a). Clearly, a double inversion of anomeric configuration occurred during the transfer of mannose from the a-linked sugar nucleotide to the final a-linked product. That the a 1,2-linkage formed by this reaction is identical to that found in the outer region of the dolichylsaccharide (R. G . Spiro er al., 1976; Li er al., 1978; Liu et al., 1979), and that the enzyme required a lipophilic moiety on the acceptor molecule for activity, suggested that the natural substrate for step (2) may be the dolichylsaccharide. Although the initial studies from Leloir’s laboratory suggested the involvement of one or more glucose residues in the assembly of the dolichyl(pyro)phosphate-linked oligosaccharide (Behrens el al., 1973), the presence of glucose was not firmly established until several years later. This may have been due to the absence of glucose from any known glycoproteins other than collagen (Spiro, 1973). Nevertheless, Spiro et al. (1974) reported the isolation of a glucose-containing dolichol-bound oligosaccharide from calf thyroid slices, and later M. J. Spiro et al. (1976b) reported the same using slices from a number of other tissues including calf kidney, thymus, and liver and hen oviduct. The saccharide moiety was prepared in chemical amounts and contained about 10 mannose, 2 glucose, and 2 N-acetylglucosamine residues. This composition was consistent with an observed molecular weight of about ,2400 for the saccharide. Similarly (Adamany, 1976; Mills and Adamany, 1978), when cultured calf aortic smooth muscle cells were incubated as single cell suspensions in the presence of labeled free monosaccharides, a dolichylsaccharide was isolated

118

JOAN TUGENDHAFT MILLS AND ANTHONY M. ADAMANY

whose carbohydrate size and composition were nearly identical to the saccharide from thyroid slices. Clearly, dolichylsaccharides from intact cells differed markedly in size and composition from those isolated from subcellular particle incubations containing a labeled sugar nucleotide, usually GDP-Man. Moreover, the yield of dolichylsaccharides from intact cell systems were orders of magnitude greater than from subcellular particles when normalized per unit of protein or DNA. The size difference was due in part to the absence of glucose residues from dolichylsaccharides obtained from cell-free systems (Chen er al., 1975; Lucas er al., 1975; Hsu et a l . , 1974). This important difference emphasized the advantage of intact cell incubations, and led Spiro et al. (1976a,b) to suggest that certain key enzyme sequences may not occur in subcellular systems for lack of appropriate substrates or because sequential reactions had become uncoupled. In support of this suggestion, Robbins et al. (1977a) found that the addition of UDPglucose to fibroblast cell-free systems resulted in the formation from GDP['*C]mannose of a larger, glucose-containing dolichylsaccharide. Subsequent to these studies, glucose-containing oligosaccharides were isolated from numerous other intact (Li er al., 1978; Liu et al., 1979) and cell-free (Herscovics et al., 1977) systems. The addition of this residue appeared to be mediated by a dolichylphosphoryl glucose intermediate as demonstrated in rat liver (Parodi et af.,1973), calf pancreas (Herscovics el al., 1977), and aortic smooth muscle cell-free systems.

C. STRUCTURE OF THE CARBOHYDRATE PORTION OF DOLICHYLSACCHARIDES A complete structure of the dolichol-linked oligosaccharide from VSVinfected CHO cells has recently been proposed by Li et al. (1978): 01.2

Man -Man 1 1 , 6

0

? Glc

Glc

Man

13

13

Glc -Man-

a 1.2

at 2

Man

a1 2

1.3 ,,/"\a

Man

Man

1,6 131 3(4) Man A GICNAC

i

GlcNAc

This structure was similar or identical to those of dolichylsaccharides isolated from calf thyroid slices (R. G. Spiro et al., 1976), aortic smooth muscle cells (Mills and Adamany, unpublished), and NIL fibroblasts (Liu et al., 1979). Many important features of the above structure were first reported by R. G. Spiro et al. (1976). Except for the presence of glucose residues, this oligosaccharide was

ROLE OF PHOSPHORYLATED WLICHOLS

119

identical to the thyroglobulin unit A-type asparagine-linked saccharide (It0 et al., 1977) depicted earlier. Indeed, we used the latter as carrier during our structural studies on the uniformly labeled dolichylsaccharide isolated from cultured aortic smooth muscle cells, and this usage permitted better yields and more precise identification of products after chemical analyses such as acetolysis and permethylation. The structure of the saccharide just shown was deduced from information obtained from some enzymatic and numerous chemical degradation procedures. Because such approaches have been common to many laboratories (R.'G. Spiro et al., 1976; Li et al., 1978; Liu et al., 1979) including our own, and because of their ease and reproducibility, we shall briefly outline the types of information they yield. Although structural studies are most informative when performed on chemical quantities of saccharide (R. G. Spiro et al., 1976), this has generally not been feasible because, expectedly , dolichylsaccharides are found in minute amounts in the cell. For example, the calf aortic smooth muscle cell in culture contains about 1-2 nmoles dolichylsaccharide per lo9 cells (Adamany, 1978) and the calf thyroid about 1 nmole per gram of wet tissue (R. G. Spiro et al., 1976). Therefore most studies used labeled saccharides obtained from intact cells that were incubated with precursor monosaccharides for various lengths of time. In order to ensure nearly uniform and randomized labeling of all sugars, we grew the smooth muscle cells in the presence of ['4C]glucose for at least two population doubiings and performed structural studies on the isolated dolichylsaccharide. When the saccharide portion was released intact from the lipid carrier by mild acid hydrolysis (0.01 N HCl in 20% methanol, 10 minutes 100°C), purified by gel filtration, and treated exhaustively with a-mannosidase from jack bean, only 36% or about four of the mannose residues were released. The remaining mannose residues were found associated with a resistant core that contained all the Glc and GlcNAc of the parent structure. A mixture of a- and P-mannosidases released the same amount as a-mannosidase alone. The residues so released corresponded to those found external to the a 1,6-branch point extending from the core P-mannosyl residue in the structure just shown. Acetolysis cleaves 1,6-1inkage between sugar residues (It0 et al., 1977). When applied to the intact saccharide, this procedure released that portion of the molecule that was susceptible to mannosidase treatment as a mixture of mannobiose, mannotriose, a core containing some Man in addition to all the Glc and GlcNAc of the parent structure, and some free Man. The low-molecular-weight products were separated from the core by gel filtration and resolved by paper chromatography, as shown in Fig. 2. Mannobiose was the major species observed, and this puzzled us for the following reasons. If the di- and trisaccharides were released from a homogeneous oligosaccharide, and if the latter were uni-

FIG. 2. Radioscan of chromatogram of low-molecular-weight products obtained from acetolysis of the [ I 4 C]oligosaccharide portion of d~lichyl-[~~C]saccharide from aortic smooth muscle cells cultured in the presence of [U-'4C]glucose for two generations. Descending chromatography system was ethyl acetate/pyridine/water/acetic acid (5:5:3:1), Whatman I paper, 16 hours. Guide strip shows position of migration of low-molecular-weight acetolysis products of bakers' yeast mannan: mannose, mannobiose, and mannotriose.

FIG. 3. Radioscan of chromatogram o f neutral sugar methylation products of [I4 C]oligosaccharide (scan 1) and C]oligosaccharide acetolysis-resistant high-molecular-weight fraction (dehranched saccharide) (scan 2). Descending chromatography system was water-saturated n-hutanol, Whatman 1 paper, 12 hours. Guide strip shows position of migration of standard methylated mannose derivatives obtained from permethylation of calf thyroglobulin unit A . [I4

122

JOAN TUGENDHAFT MILLS AND ANTHONY M. ADAMANY

formly labeled, equimolar yields of the two products should result. Consequently the isotopic distribution in the disaccharide should equal 2/3 of that observed for the trisaccharide, but the opposite was the case. However, after elution of these substances and reduction with NaBH,, the ratios of mannose to mannitol in the di- and trisaccharides were close to theoretical, and indicated that within each saccharide the distribution of radioactivity was uniform. Importantly, when the eluted substances were degraded with alkali (0.1 N NaOH, 10 minutes, 95"C), about 50% of the di- and 70% of the trisaccharides underwent @-elimination, indicating the presence of 1,3-substituted reducing termini; 1,Zreducing termini are resistant to this treatment. Clearly, in our hands the saccharide was heterogeneous in structure. Permethylation and analysis of the neutral sugar fraction of the parent and acetolysis-resistant core indicated that indeed the parent contained 1,6-branch points (Fig. 3, upper scan), which were no longer present in the acetolysis core product (Fig. 3, lower scan). Except for the glucose derivatives, which could not be identified in this chromatography system, the mannose derivatives comigrated with neutral sugar derivatives obtained from the calf thyroglobulin unit A (It0 et 01.. 1977) used both as carrier and standard for these studies. Based to a large extent on comparison with results obtained for thyroglobulin unit A glycopeptide, and except for some heterogeneity, the saccharide of the smooth muscle cell has many of the features of, and is perhaps identical to, those described for other systems (see foregoing discussion). The anomeric configuration of the glucose residues in the oligosaccharide has proved difficult to determine, since they were not susceptible to any of the known a- or P-glucosidases (R. G. Spiro er al., 1979). However, these authors also reported that these residues are resistant to oxidation with chromium trioxide, and have concluded that they were present in the a-configuration. Such treatment would be expected to oxidize p-linked residues (Hoffman er al., 1972). Therefore if these glucose residues derive from dolichylmonophosphoryyl-PD-glucose, then, analogously to dolichylmonophosphoryyl-@+-mannose, the transfer to the lipid saccharide is accompanied by an inversion of configuration.

V. Relationship of Dolichylsaccharidesto Asparagine-linked Carbohydrate Units of Glycoproteins: Processing and Maturation of Protein-Bound Saccharide Units The apparent structural similarities among the lipid-bound saccharide and the two types of asparagine-linked units (Ito et af., 1977; Nilsson et clf., 1979; Li et al., 1978; Liu er a!. , 1979) suggested that the former might serve as a precursor for both of these units. The en bloc transfer of the saccharide onto endogenous and exogenous substrates has been shown to be effected by microsomes from

ROLE OF PHOSPHORYLATED WLICHOLS

123

various tissues (Parodi and Leloir, 1980). The transfer reaction is cationdependent and requires the presence of detergent. Although there is some controversy as to the size and composition of the saccharide portion of the dolichylsaccharide donor (see subsequent discussion), there is little disagreement that the preassembled oligosaccharide is indeed transferred en bloc onto certain asparagine residues of polypeptides. The provisions of such transfer and subsequent “processing” of the transferred product so as to yield the various Asn-linked saccharides as found in proteins necessitate that considerable glycose excision and remodeling take place, as initially suggested by Hunt et al. (1978). Several laboratories have contributed to the clarification of these “processing” or “maturation” events. Because of the apparent structural identity of the unit A-type of Asn-linked saccharides and the glucose-free lipid-bound oligosaccharide, it might be anticipated that excision of the glucose residues would represent the initial processing event. Indeed it has become apparent from the work of M. J. Spiro et al. (1979), Liu et al. (1979), and Hubbard and Robbins (1979) that glucose was removed first, and exclusively of any other monosaccharide, after the glucosylated saccharide was transferred to the peptide. Such an action necessitated, of course, that all processing and maturation events would take place while the saccharide was peptidebound. ROLEOF GLUCOSE A. REGULATORY The presence of two to four glucose residues on dolichyl-bound saccharides, their transient appearance on peptide-bound saccharides, and their loss during maturation of certain glycoproteins, suggest a significant and special function(s) for this sugar. Some of these functions may include 1. A “capping” signal for terminating the synthesis of dolichylsaccharides of appropriate size. 2 . A recognition marker sequence that specifies the transfer of the saccharide to the polypeptide. 3. A recognition marker that may initiate “processing” of the saccharide after its transfer to the polypeptide. 4. A specifier sequence that, if conserved on the carbohydrate of certain glycoproteins, may aid their migration to their position of residence in the cell. These are intriguing possibilities, some of which (2 and 3 above) have gathered considerable experimental support, which will be discussed. In contrast, possibilities 1 and 4 have little or no experimental support. Certain relevant observations are worthwhile noting. Although it is now evident that cell-free systems can synthesize dolichylsaccharides that contain a nearly full complement

I24

JOAN TUGENDHAFT MILLS AND ANTHONY M . ADAMANY

of mannose residues, it is not yet clear that the sequence of these residues is identical to that of glucosylated saccharide from intact cell systems. Conceivably, addition of glucose residues may influence the ordered addition of mannose residues and/or terminate further chain-elongation events. In sense, if added last, glucose may “cap” the dolichylsaccharide and orient it to its next function. Certain lysosomal glycosidases that are glycoproteins appear to contain some glucose residues in their saccharide moieties. Although these structures have not been elucidated, it is likely that the glucose may derive from glucosylated dolichylsaccharide. If such turns out to be the case, then these residues may not be subject to normal “processing” events as described below. Conceivably, they may aid in directing those enzymes to their destination in the lysosome. As already noted, there is substantial evidence in support of possibilities 2 and 3. We shall describe this evidence. R. G . Spiro et d.(1979a) have recently identified glucosidase activity in calf thyroid homogenates, which appeared most suitable for initiating the described processing reactions (M. J . Spiro et al., 1979). The glucosidase was solubilized from a microsomal preparation with Triton X-100. The neutral pH optimum of the enzyme, as well as its substrate specificity, clearly distinguished it from previously identified thyroid glucosidases. Although this enzyme did not act on any of the commercially available substrates, it released glucose residues from glucosylated glycopeptides and, unexpectedly, from dolichylsaccharides. Interestingly M . J . Spiro et a/.( 1979) have observed that, quite in contrast to the native dolichylsaccharide, the glucose-free lipid-saccharide could not be transferred to any protein acceptor during subcellular assays. This last finding, confirmed by Hubbard and Robbins ( 1979), focused on an important ongoing discussion among three groups (Hubbard and Robbins, 1979; M. J . Spiro et ul., 1979; Chen and Lennarz, 1977; Pless and Lennarz, 1977) concerning the size and glucose content of the saccharide involved in the en bloc transfer to the protein. Chen and Lennarz (1977) and Pless and Lennarz (1977) found that nonglucosylated dolichylsaccharides of varying mannose content could serve as substrates for the transfer to exogenous modified protein acceptors. In the latter case (Pless and Lennarz, 1977), denatured S-carboxymethylated lactalbumin, which contains Asn-X-Ser(Thr) sequences, was utilized as acceptor and glucose-free dolichylsaccharide as donor. However, only about 7% of the substrate was transferred under optimal conditions. In contrast, M. J . Spiro et d. ( 1979) used a glucosylated dolichylsaccharide as donor and were able to accomplish a 50-60% transfer of saccharide to endogenous protein but only 2-3% to reduced alkylated lactalbumin. Whereas the use of exogenous acceptors has often been valuable in characterizing transferase reactions (Roseman, 1968; Adamany and Spiro, 1975a), in this case the reaction with endogenous substrates was apparently more efficient. Indeed, in a more recent report, Chen and Lennarz

125

ROLE OF PHOSPHORYLATED DOLICHOLS

( 1978) have demonstrated the transfer of glucose-containing oligosaccharides to endogenous hen oviduct proteins with a significantly greater yield than had previously been accomplished with the nonglucosylated substrate (Chen and Lennarz, 1977). Although these differences in the literature have not been fully resolved, M. J. Spiro et al. (1979) have suggested that the glucose residues function as important recognition sites for the enzyme concerned with the transfer of the oligosaccharide to protein. In addition, these authors have postulated that under certain conditions, the enzymatic removal of glucose from the dolichylsaccharide could prevent the transfer reaction, thus serving as a control point in the glycosylation process.

B. “TRIMMING” A N D “MATURATION” OF ASPARAGINE-LINKED SACCHARIDES The enzymatic removal of glucose residues from newly glycosylated proteins resulted in the formation of an oligosaccharide identical in structure to that of thyroglobulin unit A as depicted above. Further processing of this unit could transform it into a complex type of asparagine-linked unit, as occurs in thyroglobulin unit B (Spiro, 1973) or fetuin (Nilsson et al., 1979). Although it has not yet been established whether the formation of the simple, unit A-type saccharide represents an incomplete intermediate in this processing reaction or is, in fact, assembled by a unique pathway, it has been suggested that the dolichylsaccharide serves as a common intermediate in the assembly of both types of asparaginelinked units. This idea, while originally postulated by Behrens et al. (1 973) and suggested by M. J . Spiro et al. (1976a) has gathered much support. Hunt et al. (1978) were the first to provide kinetic evidence that the dolichylsaccharide did, indeed, serve as a precursor for both unit A and unit B types of protein-bound saccharides in VSV-infected CHO cells. Based on pulse-chase experiments performed with the same system, Kornfeld et al. (1978) have postulated the following sequence of events for the synthesis of the complex saccharide units of VSV G-protein: Dol-PP-(GlcNAc)2(Man)s(Gk)3 -+ Protein-Asn(GlcNAc),(Man)s(Glc), -+ Protein-Asn(GlcNAc), (Man)s (Glc) + Asn-(GlcNAc), (Man), -+ Asn-(GlcNAc)2(Man) + Asn(GIcNAc),(Man),GlcNAc + Asn-(GlcNAc),(Man) ,GlcNAc + Asn(GlcNAc),(Man),(GlcNAc) -+

,

Asn-(GlcNAc),(Man),(GlcNAc), -+ Asn(GlcNAc),(Man),(GlcNAc),(Gal), -+ Asn-(GIcNAc) 2(Man)3(Gal)3(NANA)3(Fuc)

Whereas the initial en block transfer takes place onto nascent polypeptide chains (Molnar and Sy, 1967), the later processing events and addition of the outer sugar residues presumably occur in the smooth endoplasmic reticulum (Ikehara er al., 1977) as the protein is trabported toward the cell surface. A similar type of process has recently’been reported to occur in uninfected chick embryo.

126

JOAN TUGENDHAFT MILLS AND ANTHONY M . ADAMANY

The enzymes involved in the processing reactions have been partially characterized by several laboratories (R. G. Spiro er al., 1979; Tabas and Kornfeld 1978; Michael and Kornfeld, 1980; Grinna and Robbins, 1979). Important information about the progression of processing was obtained when, in characterizing an a-mannosidase involved in a late stage of processing, Tabas and Kornfeld ( 1978) made use of a ricin-resistant mutant of Chinese hamster ovary cells. Due to a defect in one of the two UDP-G1cNAc:glycoprotein N-acetylglucosaminyl transferases normally present in CHO cells (Narasimhan ef al., 1977), this mutant cell line accumulated a glycoprotein with a partially processed oligosaccharide unit:

Tabas and Kornfeld found that, in addition to the defect in N-acetylglucosamine transferase activity, this cell type was incapable of removing the two outermost mannose residues (D and E). Their evidence suggested that addition of an N-acetylglucosamine residue to the innermost a 1,3-mannose (B) could serve as a signal for the a-mannosidase in the following manner: M

M

M

M

T . y

M

UDP-

\1.6

M-(GlcNAckAsn

M

\1,6

,M-~GlcNAc),--Asn

1 , 3

M

M -(GlcNAc),-Asn

GlcNAc -M

At this point the second N-acetylglucosaminyltransferase (Narasimhan et a l . , 1977) could act to add an N-acetylglucosamine residue to the a 1,Blinked mannose, and thus provide the proper substrates for the subsequent addition of

ROLE OF PHOSPHORYLATED DOLICHOLS

127

galactose and sialic acid (Ikehara et al., 1977). Thus, under normal circumstances, this tightly coupled reaction would prevent the accumulation of intermediates, and ensure the complete assembly of a unit B-type oligosaccharide (Tabas and Kornfeld, 1978).

VI. Cellular Location of Dolichol-Dependent Glycosylation Reactions Dolichol-dependent glycoprotein assembly involves two primary events: peptide translation and peptide glycosylation. The questions whether peptide translation is completed prior to glycosylation or whether both events are cotranslational have been actively investigated. Along with the temporal relationship, there remains a spatial component relating to compartmentalization of dolichylsaccharide assembly and delivery of the saccharide to the protein-that is, whether both events occur on the same or opposite sides of the endoplasmic reticulum. Again we focus on asparagine-linked glycosylation. AND TEMFVRAL RELATIONSHIPS OF GLYCOSYLATION TO THE A. SPATIAL RIBOSOMAL MACHINERY

Considerable evidence has now accumulated indicating that, after the nascent polypeptide interacts with and crosses the endoplasmic reticulum membrane (Blobel and Dobberstein, 1975), Asn-linked glycosylation is observed cotranslationally while the peptide is still attached to the ribosome (Kiely et al., 1976; Bergman and Kuehl, 1977; Rothman and Lodish, 1977; Davis and Tai, 1980). However, the experimental procedures used in these studies could not distinguish conclusively whether the observed glycosylation reflected en bloc transfer of carbohydrate from dolichylsaccharide or whether it occurred vectorially on either side of the endoplasmic reticulum. Hanover and Lennarz (1979) found that the N,N’-diacetylchitobiosyl portion of the dolichyl derivative was segregated exclusively to the luminal side of hen oviduct microsomes. In a subsequent report, these investigators reported that dolichol-dependent glycosylation of protein in isolated hen oviduct microsomes was also segregated to the luminal side of these vesicles, and that the glycosylated endogenous products, although membrane associated, were not ribosomebound nascent chains (Hanover and Lennarz, 1980). We have observed that a rough endoplasmic reticulum-enriched fraction isolated from the cultured vascular smooth muscle cell transfers saccharide units from exogenous dolichylsaccharide to endogenous protein (Table I). Although the extent of transfer may be limited by availability of acceptor, those data, nevertheless, indicated close proximity of the key oligosaccharide transferase to the ribosome.

128

JOAN TUGENDHAFT MILLS AND ANTHONY M. ADAMANY TABLE I TRANSFER OF DOLICHOL-BOUND OLIGOSACCHARIDE ONTO ENDOGENOUS PROTEIN

Su bcel I ul ar fraction"

Specific activity" ( d p d m g proteidhour)

Conversion to product (8)

Homogenate Nuclear pellet Postnuclear sup. Mitochondria1 lysosomes Plasma membrane Rough ER-enriched particles

665 440 725 1070 670 2770

1.9 0.7 I .8 2.4 0.5 3.5

" Subcellular particles were dialyzed free of sucrose and incubated with purified dolichylpyrophosphoryl-["C]oligosaccharide for 2 hours. Assay mixtures contained 10 mM manganese acetate. 10 mM calcium acetate, 4 mM mercaptoethanol, and 0.6% Triton X-100 in 0.1 M Tris acetate buffer pH 7.0. Reaction was terminated with CIM 3:2. Liver microsomes were added as carrier and, after extraction of unused substrate and free [ "C Joligosaccharide,particles were digested with pronase and glycopeptides isolated by column chromatography of digests on Bio-Gel P-6. Enzyme activity is expressed in disintegrations per minute of glycopeptide formed per milligram particle protein per hour.

We examined the relationship of dolichylsaccharide synthesis to nascent peptide glycosylation in the intact cell system by incubating intact cells in the presence of [ U-14C]Glcand protein-synthesis inhibitors (Table 11). We found that cycloheximide and puromycin inhibited glycoprotein biosynthesis as monitored by measurement of incorporation levels into glycopeptides of the oligomannosyl (unit A ) and complex (unit B) types (see Section II,B) derived from pronase TABLE I1 EFFECT OF ANTIBIOTICS O N T H E ACCUMULATION OF ~ L I C H Y L P Y R O P H O S P H O R Y L - 14c]SACCHARlDES [ A N D SYNTHESIS OF MEMBRANE GLYCOPEPTIDES

Experiment"

Dol-P-P-Olig. ( d p d 2 x lo7cells)

Complete system Plus cycloheximide (200 pgjml) Plus bacitracin (300 pg/ml) Plus puromycin (300 pgjml) Plus chloramphenicol (300 pgjml)

64.600 126,200 44,400 7 1,700

85.000

Glycopeptide ( d p d 2 x lo7cells) Unit A

Unit B

407,800 82,900 9 12,700 40,100 4 14,400

107,600 98,300 227.600 63,700 76,600

"Aortic smooth muscle cells, in suspensions, were incubated in the presence of indicated inhibitors and IU-"Cjglucose for I hour. after which dolichylsaccharides and glycopeptides were prepared (Mills and Adamany, 1978).

129

ROLE OF PHOSPHORYLATED DOLICHOLS

digests of labeled cell membranes. Chloramphenicol had minimal effect and bacitracin was inexplicably stimulatory. Unexpectedly, no accumulation of dolichylsaccharide occurred in these studies. That inhibition of formation of unit A-type saccharide was more pronounced (80-90%) than unit B was consistent with the notion that the former was an immediate product of transfer from the lipid saccharide and that the latter was a "processed" product. We then measured the turnover profile of dolichylsaccharide in the presence of protein-synthesis inhibitors (Table 111). When cells were pulsed with [ C]glucose in the presence of antibiotics and chased with unlabeled glucose, only in the case of puromycin was a total absence of lipid-saccharide turnover evident, suggesting an absence of protein acceptors. It is of considerable interest that although cycloheximide prevents the appearance of glycoprotein in the cell membrane, it does not prevent the turnover of the lipid oligosaccharide during the short chase period employed; this is in marked contrast to the effect of puromycin. Drawing on the postulated action of puromycin and cycloheximide on the protein-synthesizing machinery of the mammalian cells, it would appear that glycosylation reactions involving polyisoprenyl oligosaccharide transfer may occur onto nascent peptides still attached to the ribosomes, but not onto released incomplete peptides. Recently, Glabe et al. (1980) reported that glycosylation of ovalbumin occurs as the nascent chain glycosylation site exits the ribosomal cleft and extends about 30 amino acid residues beyond it into the lumen of the endoplasmic reticulum. It is therefore likely that all the chain-elongation events concerned with the assembly of the saccharide moiety of dolichylsaccharides occur at the luminal sides of the endoplasmic reticulum. There remain, among many others, the

TABLE 111 TURNOVER OF DOLICHYLPYROPHOSPHORYL-[ ''c]SACCHARIDE I N THE PRESENCE OF SEVERAL ANTIBIOTICS Experiment (dpm/lO' cells) Incubation" Complete system Plus cycloheximide (200 pg/ml) Plus bacitracin (300 pg/ml) Plus puromycin (300 pglml) Plus chloramphenicol (300 pglml)

Turnover of dolichyl-P-P-oligosaccharide

Pulse

Pulse-chase

(%)

24,750 16,800 32,100 12,750 26,550

16,875 13,500 16,650 12,600 16,650

32 20 48 1 37

'

"Aortic smooth muscle cells, in suspension, were pulsed with [U-'4C]glucose in the presence of inhibitors as in Table 11, then chased with complete Dulbecco's MEM containing inhibitors and unlabeled glucose for an additional 30 minutes.

130

JOAN TUGENDHAFT MILLS AND ANTHONY M . ADAMANY

questions of how sugars are transported from the cytoplasmic compartment across this membrane system, and whether separate nucleotide sugar pools exist on both sides.

B . DISPOSITION OF POLYISOPRENOL DERIVATIVES I N MEMBRANE SYSTEMS As might be anticipated from their structure, phosphorylated and glycosylated dolichols, although amphipathic, do not partition into aqueous solutions. In the cell they appear to reside in the hydrophobic environment of the cellular niembrane systems, and have not been found in the cell sap. However, their amphipathic nature, a major contributor being the carbohydrate, may govern their specific orientation and mobility within the membrane relative to the cytosol or the extracellular space. This is of critical importance if these substances accept sugars from nucleotide sugar pools in the cytosol and transport them across the membrane to the external environment, where they may participate in peptide glycosylation or carbohydrate chain-elongation events. Such a “shuttle” role has long been postulated for the undecaprenols of the bacterial system (Anderson et a/., 1972) and dolichols of the eukaryotic system (Tetas et al., 1970; Richards and Hemming, 1972b; Parodi and Leloir, 1980). However, there are few available data in support of this suggested transverse mobility of these substances across biological membranes. Recently some investigators (Weppner and Neuhaus, 1978; Hanover and Lennarz, 1979; McLoskey and Troy, 1980a,b) have begun probing those questions using synthetic and natural membrane systems. Weppner and Neuhaus (1978). using membrane particles from S. aureus Copenhagen and a fluorophor, UDP-MurNAc-(NE-Dns)pentapeptide, were able to monitor the transfer of the phospho-MurNAc-(N‘-Dns) pentapeptide portion from the nucleotide parent to endogenous undecaprenylphosphate. They were able to conclude that this fluorophore had undergone a phase transition from an aqueous to a hydrophobic environment close to the membrane surface, and that this transition was catalyzed by a translocase in the membrane. Further lateral or transverse mobilities were thought to be limited by other enzymes or lipids of the membrane concerned with peptidoglycan synthesis. McCloskey and Troy (1980a,b) used a similar physical approach. They incorporated spin-label analogues of polyisoprenol phosphodiesters into synthetic liposomes and measured their translocation by electron paramagnetic resonance (EPR). These investigators observed considerable segregation of these substances in the lipid bilayers and obtained rate values of “flip-flop” or transverse diffusion in the order rt = 5 hours. Understandably, and considering biosynthetic rates, they concluded that these rates were too slow an approximation of efficient biological systems. Because these observations reflected “unassisted” diffusion iu \*itro, the transposition of these substances in rivo may be “assisted” either by

ROLE OF PHOSPHORYLATED WLICHOLS

131

the specific glycosyltransferases (translocases) of which these compounds are cofactors, or by ancillary carrier proteins. Hanover and Lennarz ( 1979) approached the question of transverse mobility of endogenous N,N’-diacetylchitobiosylpyrophosphoryldolichol [Dol-P-P(GlcNAc).‘I in oviduct microsomes and in synthetic liposomes containing this glycosylated dolichol, by taking good advantage of a reaction that does not normally occur in vivo, that is, the capacity of this compound to serve as a good acceptor for galactosylation by galactosyltransferase. They found that, when incorporated into unilamellar phosphatidylcholine vesicles, Dol-P-P-(GlcNAc), distributed randomly between the inner and outer surfaces of the liposomes. Only that portion residing at the surfaces, and predicted by outer surface measurements, became galactosylated when incubated with UDP-Gal and galactosyltransferase for a 3-hour period. Complete galactosylation of all Dol-P-P(GlcNAc), was achieved in the presence of detergent. Similar studies utilizing hen oviduct microsomes, which contained endogenously generated Dol-P-P(GlcNAc),, indicated that this compound could not be galactosylated in the absence of detergent. Because gaiactosylation occurred only in the presence of detergents, it was concluded that Dol-P-P-(GlcNAc), was present on the luminal side of the microsomes. Taken together, the data derived from lysosomes and microsomes indicated that only those molecules exposed to the galactosyltransferase were modified, and those that were protected by the membrane system remained so after a 3-hour incubation period. Therefore no transverse mobility, or “flip-flop,” was observed in this time period. It would be of some interest to assess in microsomes the vectorial translocation of monophosphoryl monoglycosyl derivatives of dolichol such as Dol-P-Man or Dol-P-Glc or particularly the pyrophosphorylmonoglycosyl compound, Dol-PP-GlcNAc. It is conceivable that once a second GLcNAc is added irreversibly to the latter, its further mobility may be arrested by its binding to or association with certain mannosyltransferases concerned with its elongation. It may be anticipated that the monoglycosyl derivatives are more suitable for a “shuttle” role because they are formed reversibly and function both as acceptors and donors. C. CELLSURFACE-ASSOCIATED DOLICHOL-DEPENDENT MANNOSYLTRANSFERASES In light of the preceding discussions, dolichol phosphate may accept glucose or mannose from UDP-Glc or GDP-Man to form monoglycosyl derivatives, which, in turn, acquire a glycose-donor function in chain-elongation reactions as noted above. On the other hand, dolichol phosphate may accept N-acetylglucosamine-1-phosphate and the product acquires, except for the reverse course, a glycose-acceptor function and serves as a locus for the assembly of the saccharide. Once committed to this role, it becomes trapped or segregated

I32

JOAN TUGENDHAFT MILLS AND ANTHONY M . ADAMANY TABLE IV OF THE VASCULAR SMOOTHMUSCLECELLSURFACE GLYCOSYLTRANSFERASES

Enzyme Mannosyltransferase Galactosyltransferase Sialytransferase

Exogenous acceptor 4-CH -Umbelliferyls-*Man N-acetylglucosamine AcNeu-free fetuin glycopeptide

Glycose donor"

Enzyme activityb (dpm/lO' cells)

GDP-[ "C]Man UDP-[ "C]Gal CMP-[ "CIAcNeu

33,000 7,730 34,200

"In addition to glycose donor. incubation mixtures contained 5.6 m M Man. Gal, or AcNeu when mannosyl-, galactosyl-. or sialyltransferase was assayed. Enzyme activity is expressed as d p d 1 0 ' cells per hour per ymole available acceptor site.

vectorially and purposefully in the external face of the endoplasmic reticulum. Dol-P-Glc and Dol-P-Man may not be so restricted, and may therefore be free to move laterally and transversely in the membrane. In a sense they may constitute a pool separate from the pyrophosphoryl products. We looked for dolichol-dependent mannosyltransferases on the surfaces of cultured smooth muscle cells, and found that, whether assayed in monolayer or as single cell suspensions, these cells had substantial activity (Table IV). The assays, employing GDP-[I4C]Man as donor and various hydrophobic mannosides (see Section IV,B) as acceptors, resulted in the formation of the mannobioside products, which were isolated from the cell-free supernatants. To negate the possibility that hydrolysis products of GDP-[I4C]Man entered the cell along with the acceptor, and that mannosylation occurred within the cells, 5.6 mM free mannose was included in the incubation medium. We were unable to isolate d~lichyl-['~C]oligosaccharidesfrom such incubations. When assayed with appropriate donors and acceptors, the smooth muscle cell, as in other cell systems, possessed surface sialyl- and galactosyltransferases. It should be noted that the products of all transferases were purified by paper chromatography in the case of 4-methylumbelliferylMan-[ I 4 C]Man and [ l4 C]Gal-GlcNAc, and by gel filtration in the case of [ l4 C ]AcNeu-fetuin glycopeptide. While the function of surface transferases is not yet understood, it is tempting to speculate that the dolichol-dependent transferases may scavenge exogenous GDP-Man and shuttle the sugar into the cell, or transmannosylate analogous systems of adjacent cells (intercellular "shuttle"), or may simply represent activity that appears on the cell surface as a result of membrane flow. On the other hand. it is not altogether proved that all mannosylation occurs during the synthesis of dolichylsaccharides in the rough ER. It is possible that additional mannosylation of the protein-bound unit A-type of saccharide may occur at sites other than the endoplasmic reticulum.

133

ROLE OF PHOSPHORYLATED DOLICHOLS

VII. Regulation of Glycoprotein Biosynthesis: Interdependence of Glycoprotein and Cholesterol Biosynthetic Pathways It is now clear that the assembly of dolichol-dependent glycoproteins is a process of great complexity and precision, certain features of which are shown in Scheme 1. The numerous biosynthetic and structural studies that have led to the elucidation of the pathway just formulated have provided a necessary and comprehensive background for investigations of its metabolic regulation. Recent studies by Lucas and Levin (1977) have suggested that dolichylphosphate levels rise in chick oviduct as a result of stimulation with estrogenic hormones. Similarly, Harford et al. (1977) have found dolichylphosphate levels to be about threefold higher in membranes from actively myelinating pig brain white matter than in preparations from nonmyelinating adult brains. In both cases, measurement of dolicholphosphate levels were indirect and depended on the formation of dolicholmonophosphate-['4C]mann~~e by subcellular particles incubated in the presence of GDP-[I4C]Man. Although these observations emphasized the requirements for increased dolichylphosphate levels in proliferative tissues, they did not differentiate whether such increases resulted from de now synthesis of dolichylphosphate, mobilization of the latter as a result of phosphorylation of endogenous dolichol or dolichol ester pools, or a generalized activation of tissue-anabolic pathways. GDP-Man GDP &DOI-PP-(GICNAC)~

J Dol-PP-(GlcNAc)2 Man

/

U

D

UDP-GlcNAc

A

J\ Dol-PP-GlcNAc

Dol-P- Man

1/ GDP-Man

GDP

UOP-GlcNAc

Dol-P-

-

Do1 -P

Protein-(ClcNAcJ2(Man)g_ll(Glc)z-~

i

GLYCOPROTEINS SCHEME I

134

JOAN TUGENDHAFT MILLS AND ANTHONY M. ADAMANY

In our attempts to identify control points in the dolichylphosphate-dependent glycosylation pathway (Mills and Adamany, 1978), we have focused on the biosynthesis of phosphorylated dolichols, and because of their polyisoprenoid structural features, have evaluated the relationship of their synthesis to steroidogenesis. We reasonsed that the regulation of their biosynthesis might also modulate glycoprotein assembly. A. BIOSYNTHETIC SEQUENCES COMMON TO PHOSPHORYLATED DOLICHOL AND CHOLESTEROL-REGULATION OF CHOLESTEROL SYNTHESIS As would be expected for a polyisoprenol, dolichol is synthesized by the head-to-tail condensation of isoprene units derived from mevalonate (Richards and Hemming, 1972a; Butterworth et al., 1963; Daleo and Pontlezica, 1977). This has been demonstrated in pig liver (Butterworth et al., 1963) and regenerating rat liver (Martin and Thorne, 1974), as well as calf thyroid slices (R. G. Spiro et al., 1976) and aortic smooth muscle cells in culture (Adamany, 1976; Mills and Adamany, 1978). Martin and Thorne have found that regenerating rat livers used [4-S-3H]mevalonate for the synthesis of dolichol to a greater extent than the [4-R-3H] isomer. These results were consistent with those of Burgos et al. (1963), which suggested that most of the isoprene units are present in the cis configuration. Both groups have noted that three of the isoprene units, two of which are o-terminal, are in the trans configuration (Martin and Thorne, 1974; Burgos et al., 1963). Thus, it appears that dolichol may be synthesized from farnesylpyrophosphate by the continuous addition, head-to-tail, of cis-isoprene units. Farnesylpyrophosphaterepresents, therefore, the most complex metabolite common to the pathways of dolichol and cholesterol biosynthesis. The two pathways bifurcate subsequent to its formation. Mevalonate. long considered unique to the pathway of sterol biosynthesis in animal cells (Bloch, 1965), is formed from HMG-CoA by the inducible regulatory enzyme HMG-CoA reductase, and its formation represents the primary rate-controlling event in the cholesterol biosynthetic pathway (Brown er a / ., 1974). Because mevalonate has been implicated also as a precursor for dolichol (Richards and Hemming, 1972a; Butterworth et a / . , 1963; Daleo and Pontlezica, 1977; Mills and Adamany, 1978; R. G. Spiro et al., 1976), as well as ubiquinone and other isoprenoid derivatives (Wright, 1961), it might be anticipated that HMG-CoA reductase would ultimately regulate the formation of these compounds as well. The regulation of this enzyme is extremely complex and, although it has been the subject of extensive research for a number of years, is still not clearly understood. Despite its importance to dolichol synthesis, its regulation has been studied exclusively from the point of view of cellular requirements for choles-

ROLE OF PHOSPHORYLATED DOLICHOLS

135

terol. We wish to discuss this type of regulation because it has contributed to the rationale underlyfng our studies. HMG-CoA reductase responds to diurnal variation (Siperstein, 1970; Rodwell et al., 1976) and to feeding of cholesterol and cholesterol derivatives to experimental animals. This response is apparently due to modulation of synthesis of the reductase, which has a short half-life of about 4-6 hours. In addition, recent evidence suggests that reductase activity is modulated by phosphorylatiod dephosphorylation reactions (Beg e t a l . , 1973; Brown et al., 1979; Nordstrom et al., 1977). Brown et al. (1979) have observed that in rat liver, under steady-state physiological conditions, 75 to 90% of the reductase is in the phosphorylated, or inactive state. In contrast, the reductase of cultured human fibroblasts is found to be in the dephosphorylated, active state, regardless of cultural conditions. In light of these results, the authors have suggested that this covalent form of enzyme regulation may occur in the liver, an organ that may be faced with sudden demands for cholesterol, but not in cells that have a more constant, albeit lower, demand for cholesterol. The study of cholesterol metabolism in cultured cells has provided a great deal of information on the regulation of HMG-CoA reductase. From such studies it has become apparent that cells can utilize cholesterol, triglycerides, and fatty acids obtained from the culture medium for membrane assembly (Bailey, 1973; Rothblat and Kritchevsky, 1968; Kritchevsky and Howard, 1970). Indeed, Brown and Goldstein have suggested that the cell depends on exogenously supplied cholesterol, in preference to that synthesized endogenously (Brown et al., 1973, 1974; Goldstein and Brown, 1973, 1974). Through an extensive series of biochemical and genetic studies, Brown and Goldstein proposed a receptor-mediated mechanism for the uptake and regulation of cholesterol metabolism in cultured fibroblasts (Brown et a f . , 1973; Goldstein and Brown, 1973, 1974, 1975; Brown and Goldstein, 1974, 1975; Goldstein et al., 1974, 1976). Cultured cells obtain most of their cholesterol from low-density lipoproteins (LDL) present in the culture medium. LDL binds to a specific high-affinity LDL receptor located in coated pits (Anderson et al., 1976, 1977) on the cell surface, and subsequently becomes internalized in endocytic vesicles. The vesicles fuse with lysosomes where the protein portion of the LDL molecule is degraded, releasing the lipid core composed of cholesteryl esters and phospholipids. The cholesteryl esters are hydrolyzed by the lysosomal cholesterol esterases and free cholesterol diffuses into the cytosol, where it exerts several regulatory influences (Goldstein and Brown, 1975): It suppresses the activity of HMG-CoA reductase, causing cessation of de nova synthesis of cholesterol; it activates fatty acyl-CoA:cholesteryl acyltransferase, stimulating its own reesterification; and it causes suppression of the synthesis of the cell-surface LDL receptor, minimizing further entry of LDL-bound cholesterol. The proposed

136

JOAN T U G E N D H A m MILLS A N D ANTHONY M . ADAMANY

pathway allows the cell to monitor and control carefully the amounts of free cholesterol present in the cell at any time. When grown in the absence of an exogenous cholesterol source, as in serum-free medium, HMG-CoA reductase is induced and cellular synthesis of cholesterol is stimulated. Absence of the LDL receptor and the concomitant loss of regulation of cholesterol synthesis are hallmarks of the recessive genetic disorder familial hypercholesterolemia (FH) and may contribute to the tendency of FH-homozygous individuals to become atherosclerotic at a very young age. Kandutsch and his co-workers (Kandutsch et al., 1978; Kandutsch and Chen, 1973. 1974; Breslow et al., 1975: Chen et a/., 1974). have suggested that compounds other than cholesterol may be better suited for regulation of HMGCoA reductase. They found that addition of highly purified cholesterol to L-cell cultures had no effect on cholesterol synthesis or HMG-CoA reductase activity. However. certain oxygenated steroids, some of which are autooxidation products of cholesterol, were extremely potent inhibitors of cholesterol synthesis in these and other cultured cell lines. This finding led the authors to propose that certain metabolites of cholesterol were the physiological regulators of HMG-CoA reductase, and that minute quantities of oxygenated sterols present in most preparations of cholesterol were sufficient to effect the changes noted in earlier studies from other laboratories. Kandutsch et al. (1978) have recently pointed out certain advantages of a feedback regulatory system involving derivatives of cholesterol. Because in some tissues, cholesterol is used for the synthesis of tissuespecific steroids, HMG-CoA reductase of different organs could respond to feedback regulation by tissue-specific cholesterol metabolites. For example, HMG-CoA reductase of endocrine organs may be regulated by 2 0 4 hydroxycholesterol, an intermediate in steroid hormone production, while the liver enzyme may be more responsive to regulation by 7-a-hydroxycholesterol. the first hydroxylated derivative in bile acid formation. Most other organs do not metabolize cholesterol, and in these systems, oxygenated precursors of cholesterol. such as the 14-hydroxymethyl derivative may serve as regulators. Indeed, this compound has been shown to inhibit sterol synthesis in cultured neuronal cells (Kandutsch et a/., 1978). The mechanism by which oxygenated sterols alter HMG-CoA reductase activity is still uncertain. That these sterols do not inhibit enzyme activity when added to microsomal preparations (Kandutsch and Chen, 1973, 1974) rules out any activity-modulating interactions between the sterols and the enzyme. Thus the sterols do not inhibit enzyme activity directly (Kandutsch and Chen, 1973, 1974), but by either inhibiting its synthesis, promoting its degradation, or activating a specific phosphatasekinase system (Beg et a / . , 1973; Nordstrom rt a / . , 1977; Brown e t a / . . 1979). In this regard, the studies of Bell e t a l . (1976) are particularly noteworthy. These investigators have found that whole serum and cholesteryl succinate specifically decrease the rate of synthesis of HMG-CoA

ROLE OF PHOSPHORYLATED WLICHOLS

137

reductase, while 25-hydroxycholestero1, 7-ketocholesterol, and cholestenone act by increasing the rate of inactivation of the enzyme, presumably by increasing its rate of degradation.

B. INTERDEPENDENCE OF CHOLESTEROL SYNTHESIS AND GLYCOPROTEIN ASSEMBLY Drawing on the extensive documentation (reviewed above) of the inhibitory effect of several oxygenated sterols on cholesterol biosynthesis, we explored their effects on the synthesis of dolichylsaccharides and glycoproteins in the cultured aortic smooth muscle cell. We found (Mills and Adamany, 1978) that, of many sterols examined, 25-hydroxycholestero1 and diosgenin were inhibitory (Table V). We focused on 25-hydroxycholestero1 and investigated its site of action in the intact cell. As expected, the oxygenated sterol inhibited the synthesis of cholesterol from [I4C]acetate but not from [3H]mevalonate, and importantly, also inhibited de n o w synthesis of dolichylsaccharide from acetate but not from mevaionate (Table VI). When calculated on the basis of ['4C]acetate incorporation into the dolichyl moiety, the extent of inhibitory effect was about 90%. Clearly, a site of action of the inhibitor was at a step in the conversion of acetate to mevalonate, possibly that catalyzed by HMG-CoA reductase, which was common to the synthesis of both lipids. The data provided us with the

TABLE V THEEFFECT OF SEVERAL STEROLS ON DOLICHYLSACCHARIDE A N D GLYCOPROTEIN BIOSYNTHESIS BY CULTURED AORTICSMOOTH MUSCLE CELLS

Addition

Dolichylsaccharide (dpm/lOs cells)

Glycopeptide 111" (dpm/106 cells)

None Cholesterol 7-Ketocholesterol 20-Hydroxycholestero1 p-Sitosterol 25-Hydroxycholesterol 5-Pregnen-3P-2Ck-diol F'regnenolone Diosgenin Tigogenin

4,130 4,050 (98) 2,650 (64) 3,030 (73) 4,584 (1 11) 1,890 (46) 5,700 (138) 4,460 (108) 1,360 (33) 4,179 (99)

57,300 (100) 63,190 (110) 40,200 (70) 38,940 (68) 49,278 (86) 25,220 (44) 79,020 (138) 57,300 (107) 21,100 (37) 71,625 (125)

"Cells were incubated with sterols (10 pglml) for 20 hours then with [U-14C]glucosefor 1 hour. * Values in parentheses represent activities relative to controls.

138

JOAN TUGENDHAFT MILLS AND ANTHONY M. ADAMANY TABLE VI EFFECT OF 25-HYDROXYCHOLESTEROL ON THE FORMATION OF CHOLESTEROL A N D DOLICHYLSACCHARIDE FROM ACETATE A N D MEVALONATE” ~~~

Cholesterol (dpmilO’ cells) Precursor [ 1 -’*CjAcetate

HJMevalonate

Dolichylsaccharide (dpmilO’ cells)

Control

25-OH-Cholesterol

Control

25-OH-Cholesterol

19,667 6,300

3,611 (IS)* 6,804 (108)

5,727 3,780

1,388 (24) 3,753 (99)

“Cells were incubated in the presence or absence of 25-hydroxycholestero1 (10 pg/ml) for 20 hours, then with [I-’4C]acetateor [5-3HH]mevalonatefor 1 hour. Values in parentheses represent activities relative to controls.

suggestion that, at least during early steps, the synthesis of both types of lipids were coordinately regulated. Although the inhibition of synthesis of both compounds was similar in magnitude, the amount of labeled substrate incorporated into each differed markedly. Control culture synthesized from acetate 25.2 and 0.72 pmoles of labeled cholesterol and dolichol, respectively, per hour per lo7 cells. However the flux of labeled acetate toward cholesterol was 168 pmoles per hour per lo7 cells and toward dolichol was 27 pmoles per hour per to7 cells. Maximal inhibition of [I4C]dolicholsynthesis from [I4C]acetatewas achieved at concentration of oxygenated sterol of 1 pg/ml of culture medium. and occurred quite rapidly becoming nearly complete about 6 hours after exposure of the cells to the inhibitor (Fig. 4). However, inhibition of doli~hyl-[~4C]saccharide formation from [14C]glucoseexhibited a lag period of about 14 hours, and was maximal after 20 hours of incubation. We interpret this time course to mean that the incorporation of acetate reflected de novo synthesis of dolichyl(pyro)phosphate, while that of glucose into the carbohydrate moiety represented the glycosylation of total endogenous dolichyl(pyro)phosphate pools. The oxygenated sterol inhibited glycoprotein synthesis in parallel (Fig. 4) with that observed for dolichylsaccharide, consistent with a product/precursor relationship between the two species. 25-Hydroxycholesterol inhibited glycoprotein synthesis rather specifically. When the extent of [14C]glucosamineand [3H]leucineincorporation was examined in control and inhibited culture, glucosamine incorporation was about 20% of control while that of [3H]leucine was about 70%. In comparison, analogous experiment with tunicamycin showed that incorporation of glucosamine and leucine were 42 and 56% of controls, respectively. Although monitoring of glycopeptides, containing labeled Man and GlcNAc, which were isolated from pronase digests of cell membrane particles (Mills and

ROLE OF PHOSPHORYLATED DOLICHOLS

A

139

Dolichylpyrophosphate

0 Dolichylsaccharide

0 Glycopeptide IU

z 0

80

k

I

I 40

z s

0

0

20

40

TIME (hr) FIG.4. Effect of time of exposure to 25-hydroxycholestero1 on the inhibition of dolichylsaccharide and glycoproteinformation. Cells were incubated with 25-hydroxycholestero1(2.5 pg/ml) for varying lengths of time, harvested and incubated under stringent conditions using [Z'T]acetate (A) or [14C]glucose(0,0 )as the labeled substrate.

Adamany, 1978), was a sufficiently sensitive index of asparagine-linked glycoprotein biosynthesis, we nevertheless examined the profiles of [14C]glucosamine-labeledglycoprotein species synthesized in smooth muscle cell control and inhibited cultures. Figure 5 shows the glycoprotein patterns obtained by slab-gel electrophoresis of plasma membrane-enriched particles. These particles were separated by discontinuous sucrose gradient fractionation from lysates of control and inhibited cultures of smooth muscle cells labeled with [14C]glucosamine. All solutions used for celI lysis and fractionation procedures contained proteolytic and glycosidase inhibitors. As expected from the glycopeptide data, the sterol-treated cells showed extensive inhibition of synthesis of glycoprotein subunits, and this inhibition was general to all glycoproteins containing asparagine-linked saccharides. That these glycoproteins observed in Fig. 5 contained such units was discerned by eluting regions from the gel corresponding to the observed peaks, reacting them with hot alkaline borohydride to release Asn-linked units from the peptide, fractionating the released saccharides on Bio-Gel P-6, and analyzing the peaks for [14C]glucosamine and [14C]glucosaminitolafter acid hydrolysis (Mills and Adamany, 1979, 1980). All the glycoprotein regions observed on the gel contained Asnlinked units that corresponded in size and glucosamine content to the units A and B type of saccharides (see Section 11,B). Clearly, 25-hydroxycholestero1 was inhibiting glycoprotein assembly, and, disturbingly, its action was not easily reversed (Mills and Adamany, 1980).

140

JOAN TUGENDHAFT MILLS AND ANTHONY M . ADAMANY Y

0 0

(u

I 2 5 - O H Cholesterol

0

20

40

60

8(

SLICE NUMBER FIG. 5 . Sodium dodecyl sulfate 5% polyacrylamide gel electrophoresis of “C-labeled plasma membrane-enriched particles from control and 25-hydroxycholesterol-treatedsmooth muscle cells. Cell monolayers were incubated in complete medium with (shaded areas) or without (unshaded areas) 25-hydroxycholesterol (2.5 pg/rnl) for 20 hours and with SO p C i of [“C]glucosamine for an additional 8 hours. Gels were sliced and the radioactivity measured by scintillation counling. Molecular weight markers: icthyocol-200.000. 100,000. and 95.000. bovine serum albumin67.000.

During the course of these studies, compactin (ML 236B) was reported to be a specific competitive inhibitor of HMG-CoA reductase (Kaneko et id., 1978; Endo et al., 1976), and was therefore a potent inhibitor of cholesterol synthesis in animal cells. We investigated the effect of this compound on dolichylsaccharide and glycoprotein syntheses and found that it also inhibited formation of these compounds (Table VII), emphasizing again the importance of HMG-CoA reductase to the synthesis of dolichol-dependent glycoproteins. Its course of action was more rapid than the oxygenated sterol, and its inhibition could be relieved once it was removed from the incubation medium (Mills and Adamany, 1980). It was of some interest to try to uncouple the synthesis of the dolichol and cholesterol, by inhibiting a step after bifurcation of their pathways at farnesylpyrophosphate. Ttiparanol, an inhibitor of AZ4-sterolreductase (Gibbons and Pullinger, 1977). was used for this purpose and was found, at concentrations that inhibited cholesterol by 80%. to inhibit glycoprotein synthesis by only about 20-30% (Table VII). Although we believe the latter inhibition to be significant,

ROLE OF PHOSPHORYLATED DOLICHOLS

141

TABLE VII OF SEVERAL INHIBITORSow DOLICHYLSACCHAIUDE AND EFFECT GLYCOPROTEIN BIOSYNTHESIS

Addition" None 25-OH-Cholesterol (2.5 pLg/rnl) Cornpactin (2.5 &ml) Triparanol (5 pdrnl)

Dolichylsaccharide Glycopeptide I11 ( d p d 10' celldhour) ( d p d 10' celldhour) 25,200 10,400 (41) 2,300 (9)

17,700 (70)

319,400 (100) 110,200 (34) 38,400 (12) 227,800 (71)

Monolayers were incubated in the presence of indicated concentration of inhibitors for 20 hours, after which single cell suspensions were prepared and incubated with [U"-CJglucose for 1 hour. Values in parentheses represent activities relative to C O R ~ ~ O I S .

it is considerably lower than observed with compactin and 25hydroxycholesterol. Taken together the data allow us to propose Scheme 2, which outlines the relationship of dolichol-dependent glycoprotein biosynthetic pathways to those of dolichol(pyro)phosphateand cholesterol. In this scheme the flux of substrates, through the sequence catalyzed by HMG-CoA reductase, toward dolichol(pyro)phosphate and cholesterol may be regulated by this enzyme. By extension the availability of dolichol(pyro)phosphate, as a catalytic cofactor, may regulate the efficiency of glycoprotein assembly. That perturbations of HMG-CoA reductase activity modulated glycoprotein synthesis has more recently been demonstrated by the studies of Carson and Lennarz (1979). These investigators showed that compactin arrested gastrulation in arbacia eggs and that this inhibition was overcome by exogenously supplied dolichol. This finding clearly supports our suggestion that the reductase regulates the availability of phosphorylated dolichols. Other studies (Keller et al., 1979; and James and Kandutsch, 1979, 1980) have shown that when liver or cultured cells are subjected to non-steady-state conditions that induce HMG-CoA reductase, they synthesize increased amounts of cholesterol and dolichol. Importantly, these investigators observed that the increases were disproportionately in favor of cholesterol synthesis. They reasoned that additional control points specific to dolichols and sterols could explain the observed disproportionate rates. It is important to note that we have not been able to observe the uptake of exogenous dolichol or dolichol phosphate by the cultured smooth muscle cell. However, we have noted (Mills and Adamany, 1978, 1980) that addition of mevalonate rescues synthesis of dolichylsaccharidecompletely, and of glycoprotein partially in sterol-inhibited cells.

ACETYL-CoA

4 t HYDROXYMETHYLGLUTARYL-COA

GDP-Mon

Reducfose

MEVALONIC ACID I I

Dol-P-Mon

Dol-P-

CHOLESTEROL ESTER

FA

I I I

Protein-(GlcNAc)p(Mon)g-II(GIc)2-3

i

GLYCOPROTEINS SCHEME 2.

ROLE OF PHOSPHORYLATED DOLICHOLS

143

VIII. Conclusion Our assessment of the relationships of cholesterol and glycoprotein biosyntheses in the aortic smooth muscle cell was carried out using steady-state cultural conditions. This is particularly important when one evaluates the role that regulatory enzymes play in controlling the flux or availability of substrates to a metabolic pathway. HMG-CoA reductase is an inducible enzyme that, under steady-state cultural conditions, is in the suppressed state and behaves as a classic nonequilibrium enzyme. It has a rapid turnover rate and its activity is barely detectable in vitro. In addition to induction, in some tissues it undergoes covalent modification by phosphorylatioddephosphorylation as a means of modulating its activity. These characteristics contribute to its position as a rate-controlling enzyme in polyisoprenol biosynthesis. When the reductase is induced and its levels increase by orders of magnitude as usually observed in severe non-steady-state conditions, the regulatory, nonequilibrium function of this enzyme ceases. The increased flux of substrates through this enzyme has been useful in discerning regulatory enzymatic steps secondary to the reductase and, perhaps, specific to the pathways of cholesterol or other polyisoprenols such as the dolichols (James and Kandutsch, 1979; Keller et al., 1979). We have observed that the smooth muscle cell, maintained under steady-state conditions of culture where a plentiful exogenous supply of cholesterol is available to it in the form of serum LDL, continues to synthesize from acetate about 10-15% of its total cholesterol complement (Blurnenfeld et al., 1979). We believe that the suppressed level of synthesis is important to maintaining proper levels of synthesis of certain isoprenyl-containing compounds, such as dolichol and ubiquinone, as well as a pool of cholesterol that is quite distinct from that supplied exogenously. The supposition that the biosyntheses of these compounds are coordinately regulated invokes a special function for endogenously synthesized cholesterol that approximates the catalytic cofactor role of the ubiquinones and the phosphorylated dolichols. We have recently suggested (Adamany and Mills, 1980; Mills and Adamany, 1980) that endogenously synthesized cholesterol is, in addition to phosphorylated dolichols, essential for the proper assembly of membrane glycoproteins.

ACKNOWLEDGMENTS We wish to express our appreciation to Drs. Olga 0 . Blumenfeld and Harvey Wolinsky for so many stimulating discussions, to Fred G. Samuels, Ruth Silver and Dr. Mark Mehler for participating in and extending aspects of the reported studies, and to E. Fay Ricksy for skillful and patient preparation of this manuscript. This work was supported by NIH grants HL 19011 and AG00374, and by March of Dimes Basil O’Connor Starter Award 5-120 and grant 1-661.

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Kiely. M. L.. McKnight, G . S., and Schimke, R. T. (1976). J. B i d . Chrm. 251, 5490. Kornfeld. S.. Li. E.. and Tabas, I. (1978). J. Biof. Chem. 253, 7771. Kritchevsky, D.,and Howard, B. V. (1970). In “Aging in Cell and Tissue Culture”(E. Holeckova, and V . J. Cristofalo. eds.). p. 57. Plenum, New York. Leloir. L. F . . Staneloni, R. J., Caminatti, H., and Behrens. N. H. (1973). Biochrm. Biophvs. Res. Cornmun. 52, 1285. Levy, J . A., Carminatti. H.. Cantarella, A. I . , Behrens, N. H., Leloir, L. F., and Tabora, E. ( I 974). Biochem. Biophvs. Res. Commun. 60, 118. Li, E., Tabas, I . , and Kornfeld. S. (1978). J. B i d . Chem. 253, 7762. Liao. T. H., and Pierce. J. G. (1970). J. B i d . Chrrn. 245, 3275. Liu, T . , Stetson, 3..Turco, S. J.. Hubbard, S . C . , and Robbinson. P. W. (1979). J. B i d . Chem. 254,4554. Lucas. J. J.. and Levin. E. (1977). J. Biol. Chem. 252, 4330. Lucas. J. J . , Waechter. C . J., and Lennarz, W. J. (1975). J. Biol. Chem. 250, 1992. McCloskey, M. A,. and Troy. F. A. (1980a). Biochemistry 19, 2056. McCloskey. M. A,. and Troy. F. A . (198Ob). Biochemistry 19, 2061. Martin. H. G.. and Thorne, K. J . I. (1974). Biochem. J. 138, 277. Matsuhashi. M., Dietrich, C. P., and Strominger. J. L. (1967). J . B i d . Chem. 242, 3191. Michael, J . M., and Kornfeld, S. (1980). Arch. Biochem. Biophys. 199, 249. Mills. J. T., and Adamany. A. M. (1978). J. Biol. Chem. 253, 5270. Mills. J. T., and Adamany. A. M. (1979). Fed. Proc. Fed. Am. Soc. Exp. B i d . 38, 351. Mills, J. T.. and Adamany, A. M. (1980). J. Biol. Chrm. (in press). Molnar, J . , and Sy, D. (1967). Biochemisfy 6, 1941. Molnar. J.. Chao, H.. and lkehara. Y. (1971). Biochim. Biophys. Ac-ta 239, 401. Narasimhan. S., Stanley, P., and Schacter. H. (1977). J. Biol. Chem. 252, 3926. Ness, W. R., and McKean. M. L. (1977). “Biochemistry of Steroids and Other Isopentenoids.” University Park. Baltimore, Maryland. Neuberger, A., and Marshall, R. D. (1969). In “Symposium on Foods: Carbohydrates and their Roles” (H. W. Schultze, R. G. Cain, and R. W. Wrotstad, eds.), p. 115. Avi, Westport, Connecticut. Nilsson, B.. Norden. N.. and Svensson, S. (1979). J. Biol. Chem. 254, 4545. Nordstrom. J . L.. Rodwell. V. N., and Mitchelson, J . J. (1977). J . Biol. Chem. 252, 8924. Olden. K., Pratt. R. M., and Yamada, K. M. (1978). Cell 13, 461. Osborn, M. J. ( 1969). Annu. Rev. Biorhem. 38, 501. Osborn, M. J., and Tze-Yuen. R. Y. (1968). J. B i d . Chem. 243, 5145. Parodi, A. J., and Leloir. L. F. (1980). Biochim. Biophys. Acra 557, 1. Parodi, A. J.. Behrens. N. H.. Leloir, L. F.. and Caminatti. H. (1972a). Proc. Natl. Acad. Sci. U.S.A. 69, 3268. Parodi, A. J. Behrens, N. H., Leloir, L. F.. and Dankert, M. (1972b). Biochim. Biophys. Acra 270, 529. Parodi, A. J., Staneloni, R., Cantarella, A. I . , Leloir, L. F., Behrens, N. H., Carminatti, H., and Levy, J. A. (1973). Carbohydr. Res. 26, 393. Pennock, J. F.. Hemming. F. W., and Morton, R. A. (1960). Nuture (London) 186, 470. Pless, D. D., and Lennarz, W.J. (1977). J. B i d . Chem. 252, 134. Porter, R . , and Knight, J. (1973) “Atherogenesis: Initiating Factors.” Assoc. Scientific Publ., Amsterdam. Richards, J. B., and Hemming, F. W . (1972a). Biochem. J. 128, 1345. Richards, J. B., and Hemming, F. W.(1972b). Biochem. J. 130, 77. Richards, J . B., Evans, P. J., and Hemming, F. W.(1971). Biochem. J. 124, 957. Robbins. P. W..Hubbard, S. C . , Turco, S. J., and Wirth, D. F. (1977a). Cell 12, 893.

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Robbins, P. W., Krag, S. S., and Liu, T. (1977b). J. B i d . Chem. 252, 1780. Roden, L. (1968). In “Biochemistry of Glycoproteins and Related Substances, Cystic Fibrosis (E. Rossi and E. Stoll, eds.), Part 11, p. 185. Karger, Basel. Rodwell, V. W., Nordstrom, J. L., and Mitschelen, J. J. (1976). Adv. Lipid Res. 14, 1. Roseman, S. (1968). In “Fourth International Symposium on Cystic Fibrosis of the Pancrease (Mucoviscidosis)” (E. Rossi and E. Stoll, eds.), Part 11, p. 244. Karger, Basel. Rothblat, G., and Kritchevsky, S. (1968). Exp. Mol. Puthol. 8, 314. Rothman, J. E., and Lodish, H. F. (1977). Nature (London) 269, 775. Satake, M., Okuyama, T., Ishihara, K., and Schmid, K. (1965). Biochem. J . 95, 749. Scher, M., Lennarz, W. J., and Sweeley, C. C. (1968). Proc. Nutl. Acud. Sci. U.S.A. 59, 1313. Siewart, G., and Strominger, J. L. (1967). Proc. Nutl. Acud. Sci. U.S.A. 57, 767. Siewart, G., and Strominger, J. L. (1968). J. B i d . Chem. 243, 783. Siperstein, M. D. (1970). Curr. Top. Cell. Reg. 2, 65. Spiro, M. J., and Spiro, R. G. (1971). J. B i d . Chem. 246, 4910. Spiro, M. J., Spiro, R. G., and Bhoyroo, V. D. (1976a). J . Biol. Chem. 251, 6400. Spin, M. J., Spiro, R. G., and Bhoyroo, V. D. (1976b). J. B i d . Chem. 251, 6520. Spiro, M. J., Spiro, R. G., and Bhoyroo, V. D. (1979). J . B i d . Chem. 254, 7668. Spiro, R. G. (1962). J . Biol. Chem. 237, 382. Spiro, R. G. (1967a). 1.Biol. Chem. 242, 1915. Spiro, R. G. (1967b). J. B i d . Chem. 242, 1923. Spiro, R. G. (1969). J . Biol. Chem. 244, 602. Spiro. R. G. (1972). Methods Enzymol. 28, 3. Spiro, R. G. (1973). Adv. Protein Chem. 27, 349. Spiro, R. G., and Spiro, M. J. (1971). J. Biol. Chem. 246, 4899. Spiro, R. G., Spiro, M. J., and Adamany, A. M. (1974). Biochem. Soc. Symp. 40,37. Spiro, R. G., Spiro, M. J., and Bhoyroo, V. D. (1976). J. Biol. Chem. 251, 6409. Spiro, R. G., Spiro, M. J., and Bhoyroo, V. D. (1979). J. Biol. Chem. 254, 7659. Stary, H. C., and McMillan, G. C. (1970). Arch. Puthol. 89, 173. Struck, D. K., and Lennarz, W. J. (1977). J. Biol. Chem. 252, 1007. Tabas, I., and Komfeld, S. (1978). J. Biol. Chem. 253, 7779. Tetas, M., Chao, H., and Molnar, J. (1970). Arch. Biochem. Biophys. 138, 135. Thomas, D. B., and Winzler, R. J. (1969). J . Biol. Chem. 244, 5943. Tkacz, J. C., Herscovics, A., Warren, C. D., and Jeanloz, R. W. (1974). J. l$d. Chem. 249,6372. Troy, F. A., and Heath, E. C. (1968). Fed. Proc. Fed. Am. SOC. Exp. Biol. 27, 345. Waechter, C. J., Lucas, J. J., and Lennarz, W. J. (1973). J . Biol. Chem. 248, 7570. Wedgewood, J. F., and Strominger, J. L. (1980). J. Biol. Chem. 255, 1120. Weppner, W. A., and Neuhaus, F. C. (1978). J. Biol. Chem. 253, 472. Wright, A., Dankert, M., and Robbins, P. W. (1965). Proc. Natl. Acud. Sci. U.S.A. 54, 235. Wright. L. D. (1961). Annu. Rev. Biochem. 30, 525. Yonemasa, K., Stroud, R. M., Niedermeir, and Butler, W. T. (1971). Biochem. Biophys. Res. Commun. 43, 1388.

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INTERNATIONAL REVIEW OF CYTOLOGY,VOL. 73

Mechanisms of Intralysosomal Degradation with Special Reference to Autophagocytosis and Heterophagocytosis of Cell Organelles HANSGLAUMANN,* JAN L. E.

ERICSSON,t AND

LOUISMARZELLA*

*Departments of Pathology at Huddinge Hospital, Karolinska Institute, Huddinge, Sweden and ?University Hospital, Uppsala University, Uppsala, Sweden I . Introduction 11. Autophagy

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

A . Autophagy during Physiological Cell Function . . . . . . B. Interaction of Autophagy with Synthesis . . . . . . . . C. Autophagy during Physiological Remodeling of Cells . . . D. Autophagy in Pathologically Altered Cells . . . . . . . E. Autophagic Sequestration of Cytoplasm . . . . . . . . F. Autophagic Degradation . . . . . . . . . . . . . . 111. Heterophagy . . . . . . . . . . . . . . . . . . . . . A. Attachment Phase . . . . . . . . . . . . . . . . B. Engulfment Phase . . . . . . . . . . . . . . . . . C. Degradation and Residual Body Stages . . . . . . . . . D. Isotope Labeling . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

149 151 151 153 155 155 155 162 171 173 173 174 175 179

I. Introduction The concept of the degradation of biological membranes and cytosolic components has evolved considerably in the past two decades. The only locus of intracellular degradation that is well characterized to date is the lysosome (Fig. 1). Pathways that permit access to the lysosomal compartment both from the intracellular and the extracellular space have been shown (de Duve, 1969; Ericsson, 1969c; Hamberg et al., 1977; Heifer, 1976). The term autophagy as proposed by de Duve (1969) designates the process of sequestration of intracellular components and their subsequent degradation by the lysosomes. Biological components from the extracellular space can also enter the lysosomal compartment and become degraded through a process designated heterophagy . Besides the lysosomal pathway for protein degradation, Ballard (1977) and Amenta et al. (1977) have discussed the evidence for the existence of an extralysosomal pathway. This alternative pathway probably has different specificities in the sense that it preferentially degrades short-lived proteins, and in particular abnormal proteins. The mechanism of degradation of cell organelles 149 Copyright Q lY8l by Academic Press. Inc All rights of reproduction in any form reserved

ISBN 0-12-364413-9

FIG. I . Schematic representation of formation and function of the lysosomal apparatus. Five different pathways (A-E) for the origin of the isolation membrane are indicated. In the case of de now synthesis, two variants are conceivable: direct formation of a closed vacuole (v. pathway A ‘ ) or formation of a wrapping sheet of unit membrane that ultimately closes to form an isolation membrane (pathway A”, line of development 1V). Segregation via a wrapping, flattened sac (stage wfs) can occur along three different lines (I. 11. and 1111. Lysosomal enzyme (indicated by dots) may or may

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and cytosol during physiological conditions has long been a very controversial subject. At present our knowledge of the existence and properties of nonlysosomal hydrolytic enzymes is limited (Goldberg and St. John, 1976). As applied to the lysosome, the term degradation implies breakdown of biological substances to the level of their constituent molecules, such as monosaccharides and amino acids in the case of glycoproteins, or fatty acids and glycerol as components of lipids. The purpose of this contribution is to summarize some of the current knowledge on the autophagic and heterophagic pathways for degradation of cellular components.

11. Autophagy

A. AUTOPHAGY DURING PHYSIOLOGICAL CELL FUNCTION The process of degradation is of fundamental importance in cell function, because under steady-state conditions subcellular components are broken down and resynthesized (turnover) many times during the life span of the cells. As is apparent from Table I, the rate of turnover of various cell organelles is heterogeneous (Glass and Doyle, 197 1). Furthermore, membrane lipids and proteins have different half-lives; in addition, the turnover of their specific constituents is heterogeneous as well (Omura et al., 1967; Glaumann and Dallner, 1968). This suggests that the synthesis and degradation of membrane lipids are not tightly coupled with those of membrane proteins. With the use of isotope techniques it can be shown that in vivo the loss of label from a specific membrane protein is a first-order kinetic process (Garlick er al., 1976). This implies that both old and newly synthesized membrane components turn over randomly. This is, however, not necessarily the case during induced protein synthesis. Since the first descriptions of intracellular vacuoles containing cytoplasmic material and believed to be engaged in autophagy (Ashford and Porter, 1962; Novikoff, 1959; Essner and Novikoff, 1962), electron microscopic studies have shown the presence of similar structures in a wide variety of cell types ranging from plant cells and unicellular protozoa to highly differentiated cells in vertebrate tissues. The finding of apparent autophagy in normal cells under physioiog-

not be present in the cavity of the wrapping sac. The drawing shows the possible ways by which enzyme preexisting within the sac or in lysosomes (ly) can gain access to the endogenous substrate. Pathways Ib and 111 involve “compaction” of the two limiting membranes, pathways Ia and I1 dissolution of the inner membrane. The final stage is represented by the single unit membrane-limited “autolysosome” (aly). Lysosomes are indicated by ly; er, endoplasmic reticulum; g, Golgi apparatus; pm, plasma membrane. (From Hamberg et al.. 1977.)

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TABLE I ESTIMATES OF TURNOVER RATESOF TOTAL PROTEINS FROM VARIOUS C E L L ORGANELLES" Cell organelle ~

Half-lives (days)

~

Total liver protein Nuclear fraction Mitochondria1 fraction Microsomal fraction Cytosolic fraction Plasma membrane

3.3 5.1 6.8 3.0

5. I 1 .8

~

Data from Arias c'r d.( 1969) and Omura (1980)

ical conditions indicates that intralysosomal breakdown of cytoplasmic components in bulk is an ongoing event (Ericsson and Trump, 1964). This finding suggests that autophagy contributes to the turnover of cell constituents during physiological cell conditions. Autophagy at steady state has been shown to vary among different cell types. The highest activity encountered appears to occur in the proximal tubule cells of rat kidney (Ericsson, 1969~).Diurnal variations in the number of autophagic vacuoles have also been described (Heifer and Scheller, 1975). In addition, both steady-state and induced autophagy can be partly suppressed by insulin (Pfeifer, 1978). and this suppression is related to inhibition of degradation rates as measured in isolated lysosomal fractions (Neely el d., 1977). Studies of regression rates of autophagic vacuoles in liver cells have yielded average half-lives of approximately 9 minutes (Heifer, 1978; Mortimore and Schworer, 1977). From this it can be estimated that the extent of the contribution of autophagy in the physiological degradation of cell organelles is in the order of 40%. Comparable values (30%)have been reached by studying the suppression of proteolysis caused by insulin in isolated cells (Knowles and Ballard, 1976). Additional inhibitors that affect autophagy in various ways caused quantitatively similar decreases in proteolysis. Their effects were not, however, additive (Knowles and Ballard, 1976; Hopgood et af., 1977). From control rat liver one can prepare a mitochondrial-lysosomal fraction in which 50% of the cathepsin D and 35% of the proteolysis present in the total homogenate can be recovered (Marzella and Glaumann, 1980a). This suggests that a substantial amount of proteolytic activity is localized in the lysosomal compartment, even at steady state. Relatively little proteolytic activity is present in the cytosol and in the microsomes. By experimentally altering the density of the lysosomes through injections of an iron compound (Arborgh et al., 1973; Glaumann et al., 1975d), i t is possible to isolate a lysosomal population corresponding to residual bodies that typically contains femtin-like granules. In such lysosomes, the lack of

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morphologically recognizable degradable material correlates with little or no release of degradation products during incubation (Marzella et al., 1980b). This points to the autophagic vacuoles as the main source of degradation products originating from mitochondrial-lysosomal fractions. Another means of testing the participation of autophagy in steady-state degradation involves the use of lysosomal enzyme inhibitors. When inhibitors of cathepsins are introduced into the lysosomal compartment (Seglen, 1977; Seglen et al., 1979), a reduction in degradation in the order of 50% is obtained (Dean, 1975a; Wibo and Pool, 1974). Furthermore, Ieupeptin was found to inhibit protein degradation in normal as well as diseased rat muscles (Libby and Goldberg, 1978). The inhibitory effect varied between 20% for myocardial muscle and 40 to 60% for limb muscles. This effect correlated with inhibition of cathepsin B activity (Goldman and Rothenberg, 1973). The parallel effects of insulin on autophagy and on degradation are of particular interest because they can be demonstrated both in normal and pathologically altered cells. Diabetes mellitus causes an increase in autophagy and enhanced degradation (Fulks et al., 1975). The addition of insulin results in a regression of the lysosomal compartment to normal (Amherdt el at., 1974). In rat livers, a fourfold decrease in the rates of degradation occurs after ingestion of a meal. The decreased catabolism correlates inversely with the amino acid levels and with glucagodinsulin molar ratios in the blood, and is accompanied by decreases in the size and number of autophagic vacuoles (Khairallah, 1978). Glucagon and insulin therefore seem to play an important role in the regulation of proteolysis in the liver by stimulating (glucagon) and inhibiting (insulin) the formation of autophagic vacuoles. The evidence obtained in a variety of experimental systems thus indicates that autophagy plays an important role in the degradation of intracellular components during physiological cell functioning. B. INTERACTION OF AUTOPHAGY WITH SYNTHESIS It is not known why newly synthesized biological components, such as enzymes, are continuously degraded. Studies on the interaction between the autophagic pathway and synthetic processes appear to be of particular importance in this respect. The rates of turnover can be modified by changes in the synthesis and degradation of cell components as has been demonstrated for example by studies with phenobarbital, which causes a proliferation of the endoplasmic reticulum (ER) and induces drug-metabolizing enzymes (Orrenius and Ericsson, 1966; Kuriyama et al., 1969). In this model the involution of the ER following cessation of treatment correlates with an eightfold increase in volume, a twofold increase in number of autophagic vacuoles containing portions of ER (Bolender and Weibel, 1973), and a concomitant increase in hydrolytic lysosomal enzymes

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(Glaumann, et al., 1975a). This seems to suggest that the degradation of ER is due to a rather selective mechanism by means of autophagy. Such a concept is supported by the observation (Kuriyama et al., 1969) that NADPH-cytochrome P-450 reductase turned over significantly faster than normal when the administration of phenobarbital was interrupted after full induction. In the regenerating liver, cytoplasmic growth occurs primarily through a decrease in the rate of degradation (Scornik and Botbol, 1976). The inhibition in degradation is in turn related to a decrease in autophagy. Some inhibitors of protein synthesis-as for example cycloheximide but not puromycin-suppress autophagy (Kovacs and Kovacs, 1980; Marzella and Glaumann, 1980b). This effect seems to be due to inhibition of the formation of autophagic vacuoles, and correlates with inhibition of degradation rates. Both in the case of the isolated perfused liver model and in the case of isolated cells (Mortimore and Schworer, 1977; Knowles and Ballard, 1976), amino acid deprivation induces autophagy. When the amino acids are added, autophagy is suppressed (Neely er al., 1974). When studied, changes in degradation rates have been shown to correlate with changes in autophagy (Neely et al., 1977). Based on these findings, it seems likely that the intracellular levels of amino acids may have regulatory effects on both protein degradation (autophagy) and protein synthesis (Woodside and Mortimore, 1972). It was proposed that the amount of charged cRNA present in the cell could serve this function. However, the levels of charged tRNA seem to remain constant (Goldberg and St. John, 1976). Of obvious importance for the mechanism of autophagic degradation is the manner in which new membranes are synthesized. It seems probable that membrane biogenesis does not occur de now; instead, the existence of a multistep assembly mechanism has been proposed. According to this concept, newly synthesized components are inserted directly into preexisting membranes (“the skeleton”), as is the case for example with cytochrome P-450 in the ER (Omura et a / . , 1967; Dallner er al., 1966). In fact, both lipids and proteins can be incorporated into or removed from the membrane without disrupting the membrane structure thus allowing selective increase or decrease of a certain membrane enzyme. Alternatively, enzymes are transferred from the site of synthesis on the ribosomes to preexisting membranes, for example in mitochondria, where they are assembled (Chua and Schmidt, 1979). The composition, and hence the age, of membranes is also influenced by the bulk, as well as the specific transfer, of components between different intracellular domains, which occurs during secretion (Palade, 1975), endocytosis (Schneider et al., 1979), phagocytosis (Willinger et al., 1979), and “membrane flow.” This migration of proteins, glycoproteins, and lipids may involve ER, peroxisomes, lysosomes, Golgi apparatus, and plasma membranes (Franke et al., 1971). Such a dynamic state can be expected to lead to heterogeneity in “age” of membrane components (DePierre and Dallner, 1975). Accordingly, proteins and

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I55

lipids do have different half-lives even if present in the same membrane. Such a dynamic structure is in accord with the general fluid mosaic model as proposed by Singer and Nicolson (1972). It is therefore not surprising that during autophagy newly synthesized membrane components are as likely to be degraded as old ones. In spite of the fact that available experimental data indicate that old and new membrane constituents are attacked by hydrolytic enzymes equally well, the heterogeneous turnover rates of functionally coupled microsomal enzyme pairs, for example, have been put forward as an argument against autophagic digestion as a principal mechanism for the degradation of endoplasmic reticulum (Omura et al., 1967; Omura, 1980). As will become evident in subsequent sections, we believe that available information on the exact locus for membrane degradation is still limited and more experimental data are necessary before this problem can be settled. C. AUTOPHAGY DURING PHYSIOLOGICAL REMODELING OF CELLS

Increased autophagy in embryonic cells undergoing differentiation has been found in a great variety of tissues (Ericsson, 1969b; Moe and Behnke, 1962). Increased histochemically and biochemically demonstrable activity of acid phosphatase is seen during metamorphosis in tissues from a number of species (Weber, 1964). Both autophagic and heterophagic degradation increase in tissue undergoing involution; examples of this are the mammary gland in the postlactational period and the prostate gland after castration (Helminen and Ericsson, 1968a-c).

D. AUTOPHAGY I N PATHOLOGICALLY ALTERED CELLS Autophagy is induced by numerous treatments, conditions, or agents that cause cell dysfunction. Examples of this are hypoxia (Glinsmann and Ericsson, 1966), ischemia (Marzella and Glaumann, 1981), endotoxin shock (Boler and Bibighaus, 1967), bile obstruction (Novikoff and Essner, 1960), metabolic inhibitors (Ericsson, 1969a; Biempica et al., 1967), irradiation (Hamberg et al., 1977; Lane and Novikoff, 1965), and dietary deficiencies (Howles et al., 1964). E. AUTOPHAGIC SEQUESTRATION OF CYTOPLASM 1. “Classical” Autophagy Morphologically, autophagy is manifested by the demonstration of a membrane-bound vacuole containing cytoplasmic constituents such as mitochondria and ER. Whether this organelle represents a lysosome can only be determined by the demonstration of lysosomal enzyme (usually acid phosphatase) within it. Lack of demonstrable enzyme in the vacuole would suggest

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that the structure corresponded to an "autophagosome. In the absence of cytochemical evidence. vacuoles containing various cytoplasmic organelles are best categorized as autophagic vacuoles (AV) or cytosegresomes (Ericsson, 1 9 6 9 ~since ) their functional significance is unknown. Autophagosomes are created when cytoplasmic components are sequestered into membrane-bound vacuoles (Fig. I ) . De now synthesis of the limiting membranes of forming autophagosomes has been proposed by some investigators (Ashford and Porter, 1962; Napolitano, 1963; Winborn and Bockman, 1968). However, other electron microscopic observations make it more likely that the limiting membranes of the nascent autophagosomes are provided by preformed cytoplasmic membranes. The majority of reports favor the ER (Ericsson and Trump, 1965; Glinsmann and Ericsson, 1966; Novikoff and Shin, 1964, 1978; Trump and Bulger, 1967) and Golgi apparatus membranes (Brandes and Bertini, 1964: Ericsson pt ( I / . , 1965; Frank and Christensen. 1068; Ericsson and Trump, 1966) as the origin of autophagosomal membranes. "

Fic,. 3. Formation of autophagosomes. Newly formed autophagosome ( A V , ) is surrounded by a kwnvoluted cisterna-like double membrane (long arrows). Two mitochondria1 profiles (AV,) are pmially surrnitnded hq' an endoplasmic reticulum cisterna (short arrows). The ER appears to be undergoing dilatation with loss of intracisternal electron density. and degranulation with loss of rihosomes presumably giving rise to a nascent autophagosome. Figure shows portion of a hepatocyte taken from rat treated with vinblastine (Marrella and Glaumann. 1980) to induce autophagy.

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In many instances, the formation of an autophagosome may be initiated by single (Fig. 2) or multiple (Figs. 3 and 4) ER cisternae that appear to wrap around areas of cytoplasm and fuse at their ends. In this manner, vacuoles surrounded by one or several membrane envelopes are formed. The outermost limiting membranes characteristically lack ribosomes and display an electron-lucent cisternal space. However, ribosomes can occasionally be seen on the membranes of nascent AV (Fig. 2). Luminal continuity between rough ER and lysosomes has been shown by serial sectioning (Novikoff and Shin, 1978). The conformational changes of ER cisternae required to induce sequestration of portions of cytoplasm are presumably energy dependent because no autophagy occurs if tissues are incubated anoxically in vitro (Ericsson et al., 1967; Trump et al., 1962, 1965). Interestingly, in the rat prostate, where the rough ER shows extensive convolutions, we find that mechanical disruption of the tissue with a Potter Elvehjem or a Polytron homogenizer causes some ER cisternae to cleave along the cisternal space rather than vectorially across the membranes. The membrane sheets then fuse with the cytoplasmic face (bearing ribosomes) toward the inside of the vacuole, thereby enclosing cell organelles and giving rise to an “autophagosome” in which cytoplasmic components’are found trapped (Fig. 5 ) . In this case autophagosomes seem to be formed in vitro by ER membranes. Studies of autophagy in in vitro cultivated human glia cells (Hamberg et al., 1977) are consistent with the view that the bordering membranes of autophagosomes may be of dual origin. In the case of the glia cells there is some indication that preexisting lysosomes may be able to change their shape so that they are able to flatten out and curve around cytoplasmic organelles, and in this way segregate the organelles. With this mode of formation, AVs would achieve their lysosomal enzymes by leaks or ruptures of the inner membrane of the newly formed double membrane that limits the autophagosome. If the membranes bordering the autophagosome are not of lysosomal origin, injection of enzyme is believed to occur through fusion with a lysosome as suggested by studies using labeled (secondary) lysosomes (Ericsson, 1969a,b). A summary of the possible origins of autophagic membranes, modes of formation of AVs, and distribution of lysosomal enzyme activity during the development of AVs is given in Fig. 1.

2 . Crinophagy A special variant of autophagy is seen in the case of Golgi apparatus-derived components. Secretory vesicles have been found to gain access to the lysosomal compartment through direct fusion. Such a mechanism has been described in the pituitary gland (Smith and Farquhar, 1966) during involution, and in liver following block of export of very low density lipoprotein particles (Figs. 6 and 7). Secretory vesicles in the liver can be seen to enter the lysosomes both via sequestration (autophagy) and via direct fusion. This fusion between a secretory vesicle and a lysosome has been designated crinophagy and may serve to break

FIG.3. Formation of an autophagosorne. Consecutive rings of ER cisternae surround a mitochondrion. An autophagosome appears to be forming by the widening and cleavage of the outer cisternal envelope. Figure shows portion of a hepatocyte taken from rat treated with vinblastine (Marzella and Glaurnann, 1980) to induce autophagy. FIG.4. Peroxisome undergoing segregation into a pocket formed by two ER cistemae. Figure shows portion of a hepatocyte taken from rat treated with vinblastine (Marzella and Glaumann, 1980) to induce autophagy. FIG. 5 . Formation of an apparent autophagosome during homogenization. Homogenization of

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down excess secretory material. Biochemically the isolated Golgi apparatus showed increased degradation of secretory lipoproteins when their export was blocked (Sandberg et al., 1981). 3 . Microautophagy

Another variant of autophagy is thought to occur when soluble cytoplasmic components enter the lysosomal compartment directly by a process analogous to endocytosis. Lysosomal profiles showing membrane invaginations, as well as lysosomes in ring shape (Saito and Ogawa, 1974) or containing single or multiple membrane-bound vesicles (Figs. 8 and 9) suggest the occurrence of this process in vivu. These lysosomal profiles are difficult to catch by EM, presumably because of the rapidity with which these membrane events occur. However, multivesicular bodies may in some instances by interpreted to be the result of multiple microautophagy. Uptake of proteins in vitru into lysosomes has been claimed to occur (Haiashi et al., 1973; Yumoto and Mori, 1979); however, negative reports have also appeared (Huisman et al., 1974). Proteins turning over rapidly (Dean, 1975b) were reported to bind preferentially to lysosomes in vitru. We have recently noted (Marzella et al., 1980a) that isolated lysosomes exposed to polyvinylpymolidone-coated silica particles (Percoll) appear to take up these particles into membrane-bound vesicles. Apparent progressive phases of this process are reconstructed in Figs. 10-1 1. The membranes of these intralysosomal vesicles seem at times to break up (Fig. 11) and allow the particles they contain to gain access to the lysosomal matrix. This is the first indication that lysosomal membranes also in vitru can undergo endocytic-like conformational changes that lead to the internalizationof exogenous material. The applicability of this in vitru physicochemical phenomenon to lysosomal membrane function in vivo remains to be proved. It correlates well, however, with the microautophagy seen in sections of liver and other tissues. It is interesting to note that electron micrographs of lysosomes with membrane continuities (Fig. 12) suggest the possibility that fusion-fission of membranes may be taking place in vitru as well. ~

the.prostatic gland causes cleavage along the lateral plane of the ER cistemae trapping rnitochondna inside derivatives of ER. Note ribosomes attached to the inner surface of the vacuole. FIGS.6 and 7. Fusion between autophagosomes and secretory vesicles. Secretory vesicles (SV) containing very-low-density lipoprotein particles appear to have fused (arrow)with AVs. Note that an SV has also been sequestered in the autophagosome in Fig. 7. Both figures show portions of hepatocytes taken from rats treated with vinblastine (Marzella and Glaumann, 1980) to induce autophagy . FIGS.8 and 9. Secondary lysosomes showing apparent rnicroautophagy. Portions of rat liver. lnvaginations and apparently also enclosed vesicles that might represent later stages of microautophagy are seen.

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4. Trcrrisrnemhnine Permeation of Particles into Lysosomes Both autophagy and microautophagy are associated with membrane movements and fusion during uptake of cytoplasm. Smaller molecules may enter the lysosomal compartment without membrane perturbation, by means of phase distribution. Uncharged weak bases are permeant and become several times concentrated within lysosomes due to the acidic milieu. Examples of such molecules are chloroquine and ammonia (Reijngoud and Tager, 1977).

FIGS. 10-12. Apparent uptake of Percoll particles by isolated lysosomes during incubation in virro (microautophagy). Lysosomes were isolated from rat livers following injections of Jectofer (Arborgh et al., 1973; Glaumann ef at., 1975d).

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5 . Sequestration as Regulatory Mechanism in Catabolism The sequestration of cytoplasmic components into forming autophagic vacuoles seems ideally suited to function as a regulatory mechanism of catabolism. The demonstration that the mode, rates, and specificity of sequestration can vary in response to physiological and pathological alterations supports this notion. As previously discussed, both the mechanism of membrane biogenesis and the constant nondegradative remodeling of membranes (e.g., membrane flow that occurs during normal cell function) may explain the different half-lives of closely associated membrane components such as enzymes of a common electrontransport chain. For this reason, the notion that only old or damaged organelles are sequestered into lysosomes is incorrect. So far as can be judged from the morphology, the cytoplasmic material sequestered into nascent autophagosomes in general appears normal. Moreover, at least in the case of autophagy induced by vinblastine, degradation of both slow- and fast-turning-over proteins increases (Marzella et al., 1980b). Thus, in this sense, autophagy is a random process. It is not known whether there are specific binding sites on the cytoplasmic face of the lysosmal membrane comparable to those known to be active in surface phagocytosis on the plasma membrane. Such binding sites, if present, would provide a mechanism for a selective uptake and turnover of cytoplasmic components or organelles into the lysosomal compartment. As to soluble cytoplasmic components, the rate-limiting step in degradation may be located prior to intralysosomal sequestration. This proposal is based on the demonstration that in the case of proteins, for example, structural characteristics, size, net charge, and chemical structure are important determinants of degradation rates (Dehlinger and Schimke, 1972; Hopgood and Ballard, 1974; Dice et al., 1973; Segal et al., 1974; Ballard, 1977). In vitro studies by Bond (1971) and Segal et al. (1974) are in favor of the concept that the susceptibility of proteins to protease attack correlates roughly to their turnover rates in the cell. This implies that the structure of the protein through different susceptibility to the action of extralysosomal neutral proteases determines the heterogeneous degradation rates of various soluble proteins. The positive correlation between the susceptibility to inactivation in vitro and the turnover rates of some soluble proteins seem to support this proposal. However, many exceptions exist (Dice and Goldberg, 1975). For example, no such correlation has been demonstrated for membrane-bound microsomal proteins (Marzella and Glaumann, 1976). Finally, FIG. 10. Isolated femtin-laden lysosomes incubated with Percoll particles, 5 minutes at 37°C. Note flaplike extension of the lysosomal membrane and trapping of particles. Particles are also seen free in the medium. FIG. 1 1 . A lysosome containing Percoll particles apparently free in the lysosomal matrix (arrow) and in intralysosomal vesicles. FIG. 12. Five lysosomes in the process of apparent fusion.

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the importance of the hydrophobicity of the protein molecules for their turnover rates has also been stressed for cytosolic proteins (Segal et al., 1976). The two most serious objections against the possibility that protein structure and the action of extralysosomal hydrolases suffice to account for degradation of proteins are the following. 1 . The creation of a new proteolytic machinery is required when turnover rates change. There is no evidence that this occurs. 2. No extralysosomal locus capable of degrading most proteins to their constituent amino acids has been defined thus far.

These considerations suggest a possible mode of interaction between lysosomal and nonlysosomal degradation pathways. The autophagic degradation of soluble proteins could in fact begin by the extralysosomal modification of these proteins by means of an initial proteolytic attack. This could in turn result in increased hydrophobic property of the proteins, or denaturation by the proteolytic scission of one or few peptide bonds, binding to membranes of lysosomes and internalization by means of microautophagy as outlined above. It has been established that modifications of proteins through the interaction with various substrates, ligands, and cofactors, and the incorporation of abnormal amino acids into the polypeptide, all influence the degradation of turnover rates of the proteins (Knowles and Ballard, 1976; Weider et d., 1975). It is interesting to mention in this context that some microsomal enzymes are readily detached from their membrane by protease digestion (Takesue and Omura, 1970). Limited proteolysis of microsomal enzymes results in a catalytic portion that is detached from the membrane and a hydrophobic portion remaining in the membrane (Ito and Sato, 1968). Apparently this type of proteolysis will affect the turnover rates. In conclusion, the sequestration of cytoplasmic membrane and soluble components into the lysosomes may play an important role in the regulation of cell catabolism. More work is needed to learn how sequestration interacts with extralysosomal degradation pathways.

F. AUTOPHAGIC DEGRADATION 1. Formation of Residual Bodies from Autophagosomes

In Fig. 1, lysosomal degradation pathways are outlined as we understand them from ultrastructural analyses. After the autophagic vacuoles are formed, the sequestered contents begin to show morphological evidence of degradation. At this stage, the digestive vacuole can be referred to as an auto(phago)lysosome. Histochemical studies suggest, but cannot prove, that nascent AVs lack acid phosphatase (Arstila and Trump, 1969; Ericsson, 1968a) (Fig. 13) and that they

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acquire hydrolytic enzymes after fusion with either primary or secondary lysosomes (Fig. 14). This finding is in line with in vitro studies, since nascent autophagosomes show little proteolytic activity whereas mature autophagosomes are enriched in proteolysis (Marzella et al., 1981). Since it has been shown that microsomes contain certain lysosomal enzymes (Goldstone and Koening , 1973), an alternative mechanism would be that the ER membranes, which at least in

FIGS.13 and 14. Histochemical demonstration of acid phosphatase in different lysosomal populations of rat liver after vinblastine treatment. A nascent autophagosome (AV) lacking reaction product (Fig. 13). An early autophagosome (AV) is about to fuse with a secondary lysosome (SL) that is positive for acid phosphatase (Fig. 14). FIGS. 15 and 16. Fusion of femtin-laden secretory lysosomes with autophagic vacuoles. Secondary lysosomes of rat liver were labeled with femtin-like particles by repetitive injections of Jectofer (Ericsson, 1968 and 1969). In Fig. 15 a secondary lysosome containing femtin-like particles has recently fused with an autophagic vacuole containing a mitochondrion. In Fig. 16 a newly formed autophagosome is undergoing apparent fusion with a femtin-laden secondary lysosome (arrow).

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many instances appear to be part of the forming autophagosomes, also contribute some hydrolytic enzymes. Primary lysosomes are thought to bud off at the Golgi complex or at a specialized portion of the smooth ER located close to the concave face of the Golgi apparatus (Ericsson and Trump, 1966; Ericsson and Glinsmann, 1971). Secondary lysosomes, on the other hand, are 1966; Novikoff et d., lysosomes with signs of digestive events. Lysosomes that show ferritin deposits and/or amorphous electron-dense droplets and lack morphologically recognizable degradable material are defined as residual bodies. Fusion between AVs and secondary lysosomes can be convincingly demonstrated after labeling the lysosomes with electron-dense markers, such as Thorotrast (thorium dioxide particles) or ferritin (Ericsson, 1969a; Trump er a[., 1975) (Figs. IS and 16). Fusion between autophagolysosomes occurs as well. In both cases. the membranes of the two vacuoles merge to form a single continuous membrane and thc contents are mixed. As degradation proceeds, products of small molccular weight diffuse out of the autophagolysosomes into the cytosol. No specific carrier mechanisms for this transport have yet been identified (Reijngoud and Tager, 1977). But there is good evidence that only very small water-soluble molecules (molecular weight 200-300) can diffuse freely across the lysosomal membrane (Reijngoud and Tager, 1977). Larger degradation products will accumulate in lysosomes giving them the characteristic appearance of residual bodies or lipofuscin-containing granules (Brunk and Ericsson, 1972). During later stages of degradation, the autophagolysosomes decrease in size 1979; de Duve and Wattiaux, 1966). (Glaumann rr d.. In forming 4 V s surrounded by multiple limiting membranes, parallel degradation of sequestered content and *‘redundant” membranes may occur (Ericsson, 1969b: Hamberg ef d., 1977). It is not clear how the reduction in the volume of the AVs takes place. Fission into smaller vesicles is a theoretical possibility, as is invagination of the lysosomal membrane to form intralysosomal vesicles, morphologically designated muitivesicular bodies. Lysosomes can also fuse with the plasma membrane and discharge their contents extracellularly (Fig. 17). This has been called exocytosis or cellular defecation and is morphologically equivalent to discharge of secretory products. Exocytosis of intralysosomal content is a physiological event in many protozoa but is seen only in certain cells in higher animals or under pathological conditions. By contrast with the AV, the typical lysosome is always bounded by a single membrane and has an opaque amorphous matrix with dense deposits of varying size; its bordering membrane is 100 A thick. Characteristically, an electron-lucent halo separates the limiting membrane from the matrix. By histochemistry, the presence of acid hydrolases can be demonstrated in these organelles. Despite the obvious heterogeneity in shape, size, and contents the autophagosomes. autophagolysosomes. secondary lysosomes, and residual bodies make up a vacuolar system that is functionally continuous (Brunk, 1973) and within which

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FIG. 17. Exocytosis (defecation) of contents from secondary lysosome. Several AVs are seen, some deep in the cytoplasm of a hepatocyte and one close to the cell membrane only surrounded by a thin rim of cytoplasm. One of the lysosomes has presumably fused with the plasma membrane (arrow) and communicates with the space of Disse.

processes of acid digestion take place. To designate this vacuolar space as a whole, the term “lysosomal apparatus” may be used. The lysosomal space is in addition functionally continuous with endocytic vacuoles (Schneider et al., 1979) and temporarily continuous with secretory vesicles (Smith and Farquhar, 1966; Marzella et al., 1980b) and the ER (Novikoff and Shin, 1978). Our knowledge as to the factors that control the traffic of these lysosomal forms and the manner in which they recognize each other and fuse is still very restricted. The role of microtubules, for instance, in the fusion between AVs and lysosomes is not fully understood. It is interesting in this connection to note that induced occurrence of autophagy and stimulation of degradation occur in the presence of microtubular inhibitors (Marzella and Glaumann, 1980a,b). The multiplicity of lysosomal enzymes has been exhaustively documented (Barrett and Heath, 1977), and their capacity for the degradation of a great variety of biological substrates has been shown (Aronson and de Duve, 1968; Arsenis et al., 1970; Coffey and de Duve, 1968; Fowler and de Duve, 1969; Maharlevan and Tappel, 1968). The classic experiments of de Duve demon-

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strated the latency of lysosomal enzymes, indicating that lysosomes are membrane-bound organelles. The observation that the lysosomal hydrolases in general have acid pH optima suggests a low intralysosomal pH. Estimations both in vitro and in viva have yielded values ranging between 4 and 6 (Reijngoud and Tager, 1975; Jensen and Bainton, 1973; Goldman and Rothenberg, 1973; Reijngoud et al . , 1976). It has been postulated that an ATP-driven pump in the lysosomal membrane functions to maintain this low pH (Mego, 1973; Dell’Antone, 1979; Schneider, 1979). Conflicting views have also been expressed. For example, acidic intralysosomal lipoproteins and glycolipids may suffice to maintain the acid pH by producing a Donnan potential across the lysosomal membrane (Reijngoud and Tager, 1977).

2 . Chamcteristics of Induced Autophagic Degradation Vinblastine sulfate can be used to induce autophagy in rat liver for the study of the degradation of subcellular components (Marzella and Glaumann, 1980a,b). Within 30 minutes after the administration of vinblastine, increases in degradation rate, as measured in vitro, are already measurable and rise two- to threefold by 3 hours. The stimulation of degradation is dose related and is associated with increased lysosomal fragility as determined by the loss of sedimentability of lysosomal hydrolases on incubation. By subcellular fractionation it can be shown that the increase in proteolysis is mainly localized in a mitochondrial-lysosomal (ML) fraction (Fig. 18). Isolated microsomes show no increased catabolism. In the cytosol some increase in proteolysis is seen but only during the first few minutes of incubation. Similar increase in lipolysis is seen in the ML fraction (Fig. 19). In contrast to proteolysis, lipolysis also increases significantly in the cytosol. The pH optimum for the proteolysis is around 4 (Fig. 20). All subcellular components, with the exception of morphologically recognizable nuclear and plasma membranes, are found sequestered into the induced autophagosomes in the ML fraction (Figs. 21-23). No alterations in the activity or distribution of lysosomal enzymes in different subcellular fractions occur. Both the absolute amount and the recovery of proteolytic activity in the ML fractions of vinblastine-treated livers increase approximately threefold compared to control values. Since the recovery of lysosomal enzymes does not increase, this suggests that the ML fractions have become enriched in autophagolysosomes. In fact, morphological analyses show that the relative number of autophagic vacuoles in the ML fractions increases. Subfractionation of the ML fraction from the vinblastine-treated rat in a Metrizamide gradient yields an autophagic vacuole fraction (Fig. 24) that is pure and intact by morphological criteria and shows a high proteolytic activity. This is the first time autophagic vacuoles of such a purity have been isolated (Marzella et al.. 1981). Previous investigations of lysosomal degradation in our and other laboratories

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have been performed with ML fractions that actually consist of only 10% lysosomes. Many of these experiments could with advantage be performed with purified autophagolysosomes. Cycloheximide, an inhibitor of protein synthesis, has been shown to block the autophagy induced by vinblastine and glucagon (Kovacs et al., 1975; Kovacs and Kovacs, 1980; Rumpelt and Weisback, 1978). Both high and low doses of this drug in vivo completely suppress the enhancement in proteolysis caused by vinblastine. In vitro, a cathepsin D inhibitor (iodoacetamide) and an agent known to alkalinize the intralysosomal milieu (NH, C1) inhibit proteolysis in a dose-dependent fashion. Finally, the pH-dependent effects of the degradation process also support the intralysosomal localization of the increased degradation induced by vinblastine (Fig. 20). During in vitro incubation the alterations in autophagic vacuole contents can

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be followed as degradation proceeds (Marzella and Glaumann, 1980b). However, even after 2 hours of incubation abundant sequestered material remains in the AVs, indicating inefficient degradation in vitro and perhaps also overloading of the lysosomal compartment. Morphological analysis of the same livers used to assay degradation rates shows that induced increases or decreases in degradation are paralleled respectively by expansion or regression of the lysosomal compartment.

FIGS.21-23. Examples of sequestered organelles into autophagosomes in the ML fraction of vinblastine-treated rat livers. FIG.21. Sequestered mitochondria and portions of ER. FIG.22. Sequestration of a peroxisome. FIG.23. Sequestered glycogen and ER surrounded by four membranes. FIG.24. Ultrastructural appearance of isolated autophagosomes from vinblastine-treated rat livers. The AVs are often surrounded by two or more membranes. Note the heterogeneous content of the autophagosomes.

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It is interesting to note that the increased intralysosomal degradation induced by vinblastine can be partially (-35%) blocked by insulin (Fig. 18). In cultured cells and in the perfused liver (Knowles and Ballard, 1976; Mortimore and Mondon, 1970), insulin also causes a partial inhibition of proteolysis (30-40%). This effect correlates with a suppression of autophagy. The inhibition of degradation by insulin does not necessarily indicate that intralysosomal degradation has been completely suppressed. Following vinblastine treatment insulin suppresses autophagy only partially. Yet this inhibition is sufficient to lower the degradation rates by one-third (Marzella et al., 1980b). As to the distribution of proteolytic activity among various types of lysosomes, subfractionation experiments have demonstrated that residual bodies lacking morphologically recognizable sequestered membranes show no proteolysis in v i m (Marzella et a l . , 1980b) whereas an isolated AV fraction is highly enriched in proteolytic activity (Marzella et a l . , 1981). 3 . Autophagic Degradation as a Regulatory Mechanism in Catabolism In a previous paragraph we have discussed several ways in which sequestration of cytoplasm by autophagy could contribute to regulate catabolism. Another regulatory site in autophagy could exist if degradation after sequestration were the rate-limiting step, or if only partial hydrolysis of some substrates or membranes were to occur in the lysosomal apparatus. As will be discussed in the section on heterophagy, intralysosomal degradation rates are dependent on the substrate to be hydrolyzed (Glaumann and Marzella, 1981), and at least in the case of ferritin, resistance to lysosomal hydrolases could suffice to explain the slow turnover of this protein in the liver (Coffey and de Duve, 1968; Glaumann and Marzella, 1981). Some evidence for the termination of the degradation of phospholipids extralysosomally has been presented (Fowler and de Duve, 1969). However, in the models of induced autophagy studied, when the volume of the lysosomal compartment expands, parallel increases rather than decreases in sequestration and in degradation rates occur. The concept of membrane vesicles that shuttle between the lysosomal apparatus and other cellular domains (e.g., plasma membranes) suggests a possible mechanism for partial degradation of membrane by the lysosomal hydrolases. The bulk of the membrane is, however, thought to cycle back to its domain of origin (Schneider et al., 1979). In conclusion, although different substrates are degraded at different rates inside lysosomes, lysosomal degradation does not in general appear to be rate limiting under normal conditions. On the contrary, the ability of lysosomes to degrade membranes seems to be sufficient even during induced autophagocytosis. It is therefore unlikely that degradation is regulated by the hydrolytic capacity of lysosomal enzymes. However, exceptions in the case of some proteins and lipids may exist. Additionally, in the case of experiments of nature

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such as lysosomal enzyme deficiencies, diseases develop from the accumulation of substrate in the lysosomes due to slow or no degradation.

111. Heterophagy

As is obvious from the foregoing presentation, the various models on induced or retarded autophagy have contributed to the understanding of the participation of lysosomes in ‘‘bulk” degradation of organelles. However, these experimental models do not permit the study of sequestration into lysosomes of only a single organelle, for example mitochondria or ribosomes. To circumvent this difficulty, a model system would be helpful in which only one specific cell organelle could be sequestered into the lysosomes. In searching for such a model we were attracted by the Kupffer cell due to its great capacity to phagocytose foreign particles (Ericsson er al., 1971). In a series of studies an experimental model was presented in which intracellular organelles were introduced into the lysosomal apparatus of Kupffer cells by means of heterophagy (Glaumann ef al., 1975b,c; Glaumann and Trump, 1975, 1976). In this part of the article some of the experiments along this line will be summarized. This design was set up as a means to mimic autophagocytosis, and more specifically as an attempt to evaluate the capacity of the lysosomes to degrade intracellular membranes. To this end, isotopically prelabeled subcellular organelles were isolated from livers of inbred rats and subsequently injected intravenously into a series of rats. The uptake and degradation of organelles within heterophagic vacuoles could then be followed. By this approach we have studied the manner in which different cellular membranes are digested inside lysosomes and have calculated the degradation rates of various membrane constituents such as proteins, glycoproteins, and various lipids. In this sense, the study of an heterophagic model may have some advantages over induced autophagy ,because the degradation of each membrane constituent or cell organelle can be separately studied. Our in vivo model can obviously be applied to isolated phagocytosing cells (Collins er al., 1980), which might provide easily controlled conditions, especially when studying the biochemical nature and release of degradation products from the lysosomes. A further possibility to study intralysosomal degradation of specific membrane components is to use reconstructed artificial membranes or liposomes. As with biological membranes, injected liposomes are rapidly taken up and degraded by Kupffer cell lysosomes. Liposomes therefore seem to prove useful for the study of intralysosomal degradation, since this approach makes it possible to study a uniform membrane, the size, charge, chemical, and physical composition of which can be easily altered. The process of phagocytosis and intralysosomal

FIG. 25. One minute after intravenous injection of mitochondria. Mitochondria are present in the liver sinusoidal lumen and attached to the surface of Kupffer cells. Note the presence of flaplike cytoplasmic projections embracing the attached mitochondria. Indentation of cell surface is also illustrated. Some mitochondria are apparently already present in phagosomes deeper in the cytoplasm. FIG. 26. One hour after intravenous injection of rough-surfaced microsomes. Most microsomes have lost their rounded shape and appear elongated and ruptured. The ribosomes have detached from the membranes and form clusters and marginate along the inside of the digestive phagosome. Note also fusion between the three digestive vacuoles in the Kupffer cell.

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digestion can be divided into four phases: attachment, phagocytosis, intralysosomal digestion, and residual body phase.

A. ATTACHMENT PHASE Only a few minutes after administration, the injected organelles are present in the liver sinusoids and are attached almost exclusively to the surface of Kupffer cells, and rarely to other sinusoidal cells (Fig. 25). A gap of 2OO-300 A is always present between the adsorbed organelle and the surface of the Kupffer cells as has been described for other types of particles before their engulfment (Wisse, 1970; Vaughan and Boydes, 1964). It seems likely that this gap corresponds to the glycocalyx covering the outer surface of the plasma membrane (Pfeifer, 1970). An interesting finding is that injected isolated plasma membranes do not promptly attach to the surface of Kupffer cells but form clusters in the sinusoidal lumen and remain there for several hours. Thus, in this case the ingestion process proceeds much more slowly than with whole cytoplasmic organelles (Glaumann and Trump, 1976). The mechanisms underlying the attachment are not clear at present. It seems likely that receptors on the membranes of the Kupffer cell surface may play some role in this process. If the injected organelles do not come into intimate contact with the plasma membranes, no ingestion occurs. The necessity of plasma membrane binding for endocytosis is demonstrated conspicuously when glycogen particles are injected (Glaumann et al., 1979). These particles do not bind, presumably because they are physicochemically inert, being composed of branching chains of glucose. This lack of attachment seems to be the main reason only a small proportion of glycogen was recovered in the liver following injection. B. ENGULFMENT PHASE The engulfment phase is completed within a few minutes. In this process the Kupffer cell plasma membrane invaginates into deep folds and extends into flaplike processes containing only cytosolic ground substance and a network of microfilaments. These flaps cup around the cell organelles and eventually fuse to form a vacuole into which the attached organelles are trapped. This engulfment process has been designated the zipper mechanism (Griffin et al., 1976). During FIG.27. Thirty minutes after intravenous injection of mitochondria of a Kupffer cell. The phagocytosed mitochondria are now present in large phagosomes often containing up to 10 or more mitochondria1 profiles in a single section. Note signs of initiated degradation with floccuient material probably representing conglomerates of denatured protein and calcium. The outer mitochondrial membranes are partially ruptured and the cristae form vesicular profiles.

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this process the wormlike invaginations typical of Kupffer cells disappear. The wormlike structures seem to represent an excess of plasma membrane needed in situations of intensive phagocytosis (Wisse, 1977). As the vacuoles containing engulfed cell organelles are translocated into the cells, increasing numbers of organelles are seen within them. This phenomenon may be explained by fusion of incoming phagosomes or emptying of several phagocytic invaginations into one large phagosome. Indeed, fusion took place during all stages of degradation (Fig. 26).

C. DEGRADATION A N D RESIDUALBODYSTAGES As early as 10 to 30 minutes after injection, the phagocytosed organelles show morphological evidence of degradation (Fig. 27). By 8 to 24 hours, depending on the amount and organelle injected, degradation is morphologically completed and organelles are converted to nonrecognizable debris. The sequence of alterations seen during the degradation of mitochondria are intramatrical swelling, rupture of outer membranes, appearance of flocculent densities, and fragmentation of cristae. A gradual accumulation of round electron-dense bodies inside the phagolysosome occurs in the final stage of degradation (Fig. 28). Even after the mitochondria and microsomes were apparently extensively degraded, the lysosomes remained swollen. It is likely that during degradation low-molecular-weight products accumulate within the lysosomes. The intralysosomal osmolarity thus increases and enhanced hydration ensues. Eventually, the degradation products are presumed to diffuse out of the lysosomes into the cell sap. Morphologically, this loss corresponds to shrinkage of the lysosomes (Glaumann et a / . , 1979). Degradation of mitochondria, microsomes, and plasma membranes gives rise to essentially similar types of residual bodies, containing electron-opaque droplets. These densities are therefore not remnants of an organelle-specific membrane component. That these droplets are derivatives of membrane lipids is most likely, since when lipid-depleted microsomes were injected the lipid droplets were much less frequently seen (Fig. 29). Taking available literature data (Fowler and de Duve, 1969) together with our findings, the evidence indicates that these lipid-like residues partially consist of phosphodiesters that are not further hydrolyzed by lysosomes. The esters may either permeate the lysosomal membrane slowly to be finally degraded in the cell sap by neutral lipases or to form intralysosomal lipofuscin complexes. Other known degradation products deriving from lipolysis, such as glycerol and fatty acids, are less attractive candidates to form these droplets, since they should be easily permeable to the lysosomal membranes and escape to the cell sap. In summary, it is obvious that cell organelles are rapidiy degraded in lysosomes and that residual bodies arise from partially digested cell organelles.

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FIG.28. Forty-eight hours after intravenous injection of mitochondria. The Kupffer cell has returned to its normal appearance with projections extending out into the sinusoid. The digestive vacuoles have decreased in size and show the appearance of residual bodies. The vacuoles contain several electron-dense lipid-like droplets. FIG. 29. Injection of lipid-depleted microsomes. Microsomes were isolated from rat livers, the lipids were extracted with chloroform-ethanol, and the lipid-depleted membranes were injected into the femoral vein. The animal was sacrificed 2 hours later. Very few lipid droplets are seen in the Kupffer cell lysosomes. Compare to Fig. 28.

D. ISOTOPELABELING The results from isotope-labeling experiments have substantiated the morphological findings. Vascular clearance of injected isotopically prelabeled organelles is rapid and 50-60% of the injected material is recovered in the liver, and of this 75% was recovered in the ML fraction (Glaumann and Marzella, 1981). When the liver is excluded from the circulation (by applying ligatures

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around the portal vein and the hepatic artery), vascular clearance is considerably longer, demonstrating that the Kupffer cells are the main eliminators in the body of endogenous cell debris. Experiments have been performed to evaluate whether energy is necessary for the degradation process following heterophagy . To this end, cell organelles were injected as described previously and the phagocytic process was allowed to proceed for 15 minutes. Partial or total ischemia was induced either by ligation of the common liver artery in i,ivo or by anaerobic incubation of liver in v i m . With this approach degradation is prolonged from a control half-life ( 2 ; ) of about 1 hour for ['%]leucine-labeled proteins to 3 hours for partial ischemia and to 15 hours for complete ischemia. These results together with work by others seem to indicate that the entire process of lysosomal degradation of protein requires energy, although the hydrolytic catabolism of proteins to amino acids does not seem to require energy (Brostrom and Jeffay, 1970). Estimation of half-lives of cytosolic and membrane proteins varies depending on the technique used. In general, turnover rates are determined by labeling proteins in vivo and measuring the loss of label. Figure 30 demonstrates a phenomenon that influences measurements of intralysosomal degradation, namely that the slope of the decay curve is dependent on the amount of substrate present in the lysosomes. When 6 mg /lo0 gram

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HOURS AFTER ADMINISTRATION FIG. 30. Effect of injected amount of mitochondrial protein on the degradation rate ( t t ) . Different dosages ( 3 , 6. and 9 mg of mitochondrial protein/100 gm of rat weight) were administered intravenously. For each dose, three animals were injected and decapitated 30, 120, and 180 minutes after the administration. The livers were perfused and homogenized and a crude lysosomal fraction was isolated (Glaumann and Trump, 1975). The radioactivity was measured in the TCA-precipitated material.

177

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TABLE I1 SUMMARY OF MEASURED HALF-LIVES OF PHAWCYTOSED MICROSOMAL MEMBRANE COMPONENTS IN KUPFFERCELLLYSOSOMES

Component Proteins Microsomes Microsomes Microsomes Mitochondria Ribosomes Hemeproteins Phospholipids Phospholipids Phospholipids Phospholipids Cholesterol Cholesterol esters Glycoproteins Glycoproteins Ribosomes a Cycloheximide

Isotope

Disappearance curve during interval 0-48 hours

[ I4C]Leucine [14C]Leucine + leucine load [ I4C]Leucine + cycloheximidea [ 14C]Leucine [ 14C]Leucine 55 Fe P [ ‘‘C]Glycerol [ 14C]Glycerol+ glycerol load [ l 4 CIPhosphatidy Icholine [ 14C]Mevalonate/[1 4 C ] ~ h o l e ~ t e r ~ I c Cholesterol-[ l 4 Clpalmitate [ l 4 C]Glucosamine [ l 4 C]Galactosamine [‘*C]Orotic acid

Shortest t (hours)

Triphasic Triphasic Triphasic Biphasic Biphasic Biphasic Biphasic Biphasic Biphasic Monophasic Monophasic Biphasic Biphasic Biphasic Triphasic

0.8-1.5 0.7-1.3 0.8-1.2 1.5 1.5 20 4 6 5-6 4 9 3 2 1.5 0.5

was given 10 minutes before injection of microsomes.

’Data from reconstituted microsomal membrcues (Glaumann and Marzella, 1981). These experiments were performed with microsomes labeled in v i m by incubation.

the lysosomes are “overloaded, ” the proteolysis becomes rate limiting, resulting in apparent prolongation of degradation rates. The heterophagy model proved to be a useful one to evaluate the capacity of lysosomes to degrade various membrane components in vivo. Isotopically prelabeled subcellular organelles were injected into a series of rats and the livers removed for measurements of TCA-nonsoluble (sedimentable)activity in a crude lysosomal fraction. Table I1 gives the results for microsomal proteins labeled with [‘Tlleucine and 55Fe. When experiments are performed with prelabeled phospholipids (PLP), the degradation rate varies depending on which isotope is used. For 32P-labeledPLP the t+ is 4 hours and for [lT]glycerol labeling the corresponding value is 6 hours, as far as can be calculated from the decay curves. Labeling the membranes with [14C]mevalonategives a t+ for cholesterol of 9 hours, whereas when cholesterol esters are labeled with [‘%]palmitate, the decay curve corresponds to a tt for the fatty acid of approximately 3 hours. When microsomes are prelabeled with glucosamine and introduced to the lysosomes of Kupffer cells, the calculated half-life is around 2 hours. A striking feature of the decay curves is a marked heterogeneity. In addition,

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the curves are not strictly exponential but show multiphasic slopes, the first of which is always faster than the following ones. This is in contrast to the decay curves described for the degradation of a single protein in macrophages, namely denatured albumin (Edelson and Cohn, 1974). As can be seen in Table 11, 55Felabeled proteins as well as femtin showed extremely long half-lives. One likely explanation for this heterogeneity is dissimilar rates of degradation of different membrane proteins. Garlick et al. (1976) have also reported complex curves for decay of label in a mixture of liver proteins. Unlike decay curves for homogenous proteins, the curve did not fit a single exponential, but a good fit was obtained with three exponentials. Reutilization of leucine is another possible explanation, although experiments with cycloheximide and dilution with cold leucine do not alter the decay curves significantly as could be expected if reutilization was extensive. However, estimates of catabolism from the decay curves of label in mixed proteins can only be approximations because of the many uncertainties inherent in the techniques used. The analysis and interpretation of the decay curves should therefore be based on the initial slope when reutilization can be expected to be less than at later time intervals. A similar conclusion can be drawn for the degradation of phospholipids. When one specific phosphatide was followed, the decay curve was monophasic, in contrast to the finding with the total microsomal phospholipids. This supports the notion that individual phospholipids are degrated at different rates. The shorter half-life for 32P-labeledPLP compared to glycerol suggests that all the ester linkages of the phospholipid molecule are not split simultaneously. Since glycerol is the “backbone” of the PLP molecule, it can be expected that [‘4C]glycerol-prelabeeled PLP should show the longest half-life, as was also the case. Other possible explanations for the nonexponential appearance of the decay curves, for example isotope reutilization (Poole, 1971) and exchange reactions (Wirtz, 1974), have been discussed in detail elsewhere (Glaumann and Marzella, 1981). It has been postulated that some glycoproteins are only degraded after the carbohydrate moiety has been split off from the apoprotein. If this was so in general, one would expect that the r4of [‘4C]leucine-labeled apoprotein would be longer than that of the carbohydrate moiety. To explore this, we used radioactive galactosamine and glucosamine to label microsomal membranes. Glucosamine is preferentially covalently bound directly to the polypeptide (to asparagine) by an N-glycosidic bond, whereas galactose constitutes one of the end sugars of the chain. Since the rt for leucine, galactosamine, and glucosamine all were very similar (1-2 hours), it seems as if in our model we lack support for the notion that the carbohydrate moieties must necessarily be split off prior to hydrolytic attack on the polypeptide (Goldstone and Koening, 1974). Taking the ultrastructural and biochemical data together, it seems justified to conclude the following:

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1. Lysosomes are able to degrade any cell organelle although at somewhat different rates. 2. Residual bodies originate from lysosomal digestion of cell organelles. 3. Proteins are in general degraded faster than lipids. 4. Different proteins and phospholipids seem to be degraded at different rates. 5. The electron-opaque granules seen in the lysosomes after degradation of membranes originate from membrane lipid derivatives. 6. Autophagolysosomes and heterophagolysosomes constitute the only welldefined and morphologically identifiable locus for the degradation, be it membrane-associated or cytosolic proteins.

ACKNOWLEDGMENTS This work was supported by grants from the Swedish Medical Research Council

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INTERNATIONAL REVIEW OF CYTOLOGY,VOL. 73

Membrane Ultrastructure in Urinary Tubules LELIOORCI,FABIENNE HUMBERT, DENNISBROWN,A N D ALAINPERRELET Institute of Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland

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. . . . . . . . . . . . . . . . . . . . . . . . . General Morphology of the Freeze-Fractured Plasma Membrane . Intercellular Junctions . . . . . . . . . . . . . . . . . Glomerulus . . . . . . . . . , . . . . . . . , . . A. Parietal Epithelium . . . . . . . . . . . . . . . . B. Visceral Epithelium (Podocytes) . . . . . . . . . . . C. Capillary Endothelium . , . . . . . , . , . . . . . D. Mesangial Cells . . . . . . . . . . . . . . . . . Proximal Tubule . . . . . . . . . . . . . . . . . . A. Pars Convoluta . . . . . . . . . . . . . . . . . B. ParsRecta . . . . . . . . . . . . . . . . . . . Loop of Henle (Thin Descending and Ascending Limb) . , . , A. Descending Limb . . . . . , . . . . . , . , . , B. Ascending Limb . . . , . . . . . . . , . . . . .

I. Introduction

11. The Freeze-Fracture Technique

Ill. IV.

V.

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VII. Thick Ascending Limb of Henle and Distal Convoluted Tubule VIII. The Collecting Tubule . . . . . . . . . . . . . . . A. Light Cells (Principal Cells) . . . . . . . . . . . . B. Dark Cells (Intercalated Cells) . . . . . . . . . . . C. Papillary Duct . . . . . . . . . . . . . . . . . IX. Discussion . . . . . . . . . . . , . . . . . . . A. Podocytes . . . . . . . . . . . . . . . . . . B. Mesangial and Lacis Cells . . . . . . . . . . , . C. Proximal Tubule . . . . . . . . . . . . . . , . D. Loop of Henle . . . . . . . . . . . . . . . . . E. Distal Tubule and Juxtaglomerular Apparatus . . . . . F. Collecting Tubule . . . . . . . . . . . . . . . G. Tight Junctions of the Kidney Tubules . . . . . . . . X. Concluding Remarks . . . . . . . . . . . . . . , . References . . . . . . . . . . . . . . . . . . .

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183 184 185 185 189 189 192 196 197 199 199 204 207 209 211 212 216 216 219 221 222 223 223 223 224 225 226 233 235 237

I. Introduction Different regions of the nephron and of the collecting tubule have complex and specific functions, many of which have been well defined from the physiological viewpoint. One of the major structures likely to determine the properties of renal tubules is the plasma membrane of the epithelial cells. It is clear, therefore, that a detailed knowledge of the membrane in different segments of the tubules could contribute to our understanding of their diverse functions. One approach to I83 Copyright @ 1981 by Academic Press, Inc. All rights of repduction in any form reserved. ISBN 0-12-364473-9

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membrane study is the use of morphology, and the membranes of kidney tubules were described with the aid of the electron microscope as early as 1950. The contribution of conventional. thin-section electron microscopy was mainly the establishment of a trilaminar “unit” structure for all kidney membranes. The structures of associated membrane components (i.e,, cell coat and cell web) were also established by this technique, together with the existence of regional variations in membrane thickness. The presence of specific intercellular junctions (i.e., tight junctions, gap junctions, and desmosomes) between epithelial cells was also observed in thin sections, as well as variations in their development related to tubular permeability (see below for references). From 1968 on, the widespread use of the freeze-fracture technique, a breakthrough in the morphological study of membranes, led to a flow of contributions dealing with the ultrastructure of membranes at various levels of the nephron and of the collecting tubule. Unlike thin sections of fixed and embedded tissue. which invariably show three layers of fairly constant width irrespective of the membrane content of the two main chemical species-protein and lipid-freezefractured membranes have a much more differentiated pattern (see Orci and Perrelet, 1975). Freeze-fracture provides a face view of the membrane interior and makes it possible to visualize the respective domains occupied by protein and lipid. A distinction can therefore be made between membranes of different protein-lipid composition, and in addition, membrane domains with specific patterns of protein arrangement can be detected (such as those occurring at the level of intercellular junctions), as well as areas in which protein-lipid relationships are altered by various experimental (physiological and pathological) conditions. As will be seen in the following pages, the kidney proved an exceptionally differentiated structure as far as membrane organization is concerned, a finding well in accordance with the high degree of functional specialization known for the various segments of the nephron and collecting tubule. This article is an attempt to summarize the present state of knowledge concerning the freezefracture ultrastructure of the plasma membrane of epithelial cells at the level of the glomerulus, proximal tubule, loop of Henle, distal tubule, and collecting duct. It includes a detailed qualitative description of the membrane faces and junctional differentiations, as well as a quantitative analysis of the number and size of intramernbrane particles which, as discussed later, represent proteincontaining structures in the membrane. In view of the vast literature concerning kidney ultrastructure and function, we have cited only selected references and reviews in which those interested can find other pertinent works.

11. The Freeze-Fracture Technique

-

Freeze-fracturing involves freezing a tissue specimen at low temperature (150°C), fracturing the frozen specimen with a cooled microtome blade, and

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shadow-casting the frozen, fractured surface with platinum and carbon (Steere, 1957; Moor et al., 1961;Moor and Muhlethaler, 1963). Both the fracturing and shadowing processes are carried out at high vacuum. For the present study, glutaraldehyde-fixed kidneys, mostly from the rat, were chopped into smaller pieces (1 -2 mm3) and infiltrated with 30% glycerol in 0.1 M phosphate buffer for 1-3 hours prior to fracturing. Once infiltrated, pieces of kidney were placed on gold supports in a drop of 30% glycerol and quenched in Freon 22, cooled with liquid nitrogen. They were then introduced into a Balzers 301 freeze-etching device (Balzers High Vacuum Corp., Balzers, Liechtenstein) and fractured at Torr. Platinum was first evaporated at 45” -100°C at a vacuum less than onto the fractured surface, followed by a layer of carbon deposited at right angles. The underlying tissue was removed from the platinum/carbon replicas in sodium hypochlorite, and replicas were further cleaned by sequential treatment with chloroform/methanol (2: l), dimethyl formamide, and distilled water. Replicas were picked up on Parlodion-coated copper grids and examined in a Philips EM 300 electron microscope. 111. General Morphology of the Freeze-Fractured Plasma Membrane

During fracturing, membranes are preferentially split along their interior, hydrophobic plane, so that each membrane yields two complementary halves or fracture faces, corresponding to the insides of the two membrane leaflets (Branton, 1966, 1969). The fracture face associated with the cytoplasm of the cell is known as the P- (protoplasmic) face; the one associated with the extracellular space is called the E- (extracellular) face (Branton et al., 1975; Fig. 1). Morphologically, each face appears as a smooth surface in which globular protrusions are studded (Fig. 1). The smooth matrix is believed to represent the lipid domain of the membrane, whereas the protrusions known as intramembrane particles (IMP) probably correspond to protein-containing structures of the membrane (Vail et al., 1974; Yu and Branton, 1976). Intramembrane particles are usually divided unequally between the two fracture faces, the P-face containing more particles per square micron of surface than the E-face. However, this general rule does not apply to all membranes and may also be affected by fixation in some cell types (Dempsey et al., 1973). A ratio that is useful for describing the distribution of particles between the two fracture faces is the “partition coefficient,” Kp (Satir and Satir, 1974), where Kp =

No. particles per p m in P-face No. particles per p m in E-face

INTERCELLULAR JUNCTIONS Whereas intramembrane particles are often distributed unequally between the two fracture faces, they are usually scattered apparently at random on each

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EXTRACELLULAR SPACE

CYTOPLASM

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187

leaflet. There are, however, important exceptions to this rule, represented by the specific arrangements of particles at the level of intercellular junctions. 1 . Tight Junctions

Tight junctions are formed by the fusion of outer leaflets of the plasma membranes of adjoining cells. In cavitary or tubular epithelia, they form continuous belts around cells and serve to separate to various extents the luminal and interstitial environments, for example, the lumen of the urinary tubules and the interstitial fluid (Farquhar and Palade, 1963). In fixed, freeze-fractured membranes, tight junctions appear as elevated ridges or fibrils on the P-face and as complementary grooves or furrows on the E-face (Staehelin er al., 1969; Chalcroft and Bullivant, 1970; Friend and Gilula, 1972; Montesano et al., 1975; Van Deurs and Luft, 1979). The number of longitudinal, parallel ridges and the “continuity” of these ridges may partially reflect the physiological role of tight junctions. Thus, junctions with few strands (one to three), which are discontinuous, have been found in “leaky” epithelia where the total transepithelial electrical resistance is low, whereas multistranded structures, usually with continuous ridges, are found in tight epithelia in which the paracellular pathway has a lower conductance (Claude and Goodenough, 1973; Pricam et a l . , 1974a; Wade and Karnovsky, 1974). Along the various segments of the nephron and of the collecting tubule, tight junctions of variable development are found in the different regions and in many cases their appearance corresponds to the known “leakiness” or “tightness of the epithelium as measured by a combination of electrophysiological and micropuncture techniques or by lanthanum tracer experiments (see Table XI11 and Figs. 34-41). Thus, the examination of tight junctions by freezefracture can be a useful pointer in determining the contribution of the paracellular pathway to transepithelial fluxes. There are some exceptions, however, and the general rule that structural complexity equals physiological tightness is certainly not as straightforward as was at one time believed (Martinez-Palomo and Erlij, 1975; Friederiksen et al., 1979; Van Deurs and Koehler, 1979). ”

FIG. I . Example of the morphology of freeze-fractured kidney membranes (lateral side of proximal tubular cells). The fracture has exposed the inner (cytoplasmic) membrane leaflet, or P-face. of one cell and the outer (external) leaflet, or E-face, of the adjacent cell. P- and E-fracture faces are separated by a white step representing the intercellular space, and both faces appear as flat surfaces containing a variable number of globular protrusions, the intramembrane particles (two of them are encircled on the E-face). The particles appear randomly distributed and are approximately 10 times more numerous on the P-face than on the E-face in this region of membrane (see Table V). The smoother background i n which the globular particles are embedded represents the lipid domain of the membrane. The drawing shows the path of the freeze-fracture plane within the plasma membrane. The circles in the membrane represent protein-containing particles exposed by the fracture. x !13,000. Bar = 0.25 g m .

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- 13

@-

-

LUMINAL

-

LATERAL

BASAL

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189

2 . Gap Junctions Gap junctions also have a characteristic appearance in freeze-fractured membranes. These junctions consist of aggregates of closely packed intramembrane particles on the P-face and complementary arrays of pits on the E-face (Friend and Gilula, 1972; Staehelin, 1974). Gap junctions are involved in the passage of ions and small molecules (electrical and metabolic coupling) between adjacent cells (Payton et a l . , 1969; Loewenstein, 1970; Revel er al., 1971; Gilula et al., 1972; Goodenough, 1976; Sheridan et al., 1978), probably via hydrophilic channels contained in the gap-junction particles (the connexon, see Goodenough, 1976). The particles in gap junctions often have a dense apical spot that may be a morphological counterpart of the proposed hydrophilic channel (McNutt and Weinstein, 1970). Some particles not associated with gap junctions also have such an apical dense spot, or depression (Orci et a l . , 1977). In the normal kidney, gap junctions appear restricted to mesangial cells, lacis cells, and endothelial cells of the glomerulus, and to epithelial cells of the proximal tubule. Desmosomes, which also have a characteristic pattern of particles in the freezefractured membrane (see Staehelin, 1974) and exist on epithelial cells of the kidney tubules, were not considered in detail in the present article. In the following description, each part of the nephron and collecting duct is identified by a corresponding number in Fig. 2.

IV. Glomerulus Plasma membranes of the following parts of the glomerulus were assessed separately: parietal epithelium and podocytes (visceral epithelium) in Bowman’s capsule, capillary endothelium, and mesangial cells.

A. PARIETAL EPITHELIUM [FIG. 2 (l)] 1. Plasma Membrane

The epithelial cells of the parietal layer of Bowman’s capsule form a squamous epithelium that is continuous with the epithelium of the proximal tubule (Mueller et al., 1955; Rhodin, 1955, 1958a; Latta, 1962, 1970, 1973). Such cells can be FIG. 2. Schema of the nephron and the collecting tubule showing the different portions studied. 1, Parietal epithelium (Bowman’s capsule); 2, visceral epithelium (podocytes) of Bowman’s capsule; 3 , capillary endothelium; 4, mesangial cells; 5 , proximal convoluted tubule (pars convoluta); 6, straight proximal tubule (pars recta); 7 , descending thin limb of Henle’s loop; 8 , ascending thin limb of Henle’s loop; 9, ascending thick limb of Henle’s loop (or straight part of distal tubule); 10, distal convoluted tubule; I I , dark cells in collecting duct; 12, light cells in collecting duct; 13, papillary duct. (These numbers are referred to in section headings.)

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TABLE I P4RTICI t S l L t A N D NUMRER IN T H t PLASMA MFMBRANE OF P A R I E T A L CELLS OF

P-face E-face

BOWMAN'SCAPSULE"

Number of particles/pm*

Diameter of particles (nm)

277 5 18 (28)O 61 2 S ( 1 6 ) K p = 4.5'

10.5 2 2.0 (928)

10.6 ? 2.1 (386)

' I For this and all following tables, results are expressed as mean 2 SEM. Statistical analysis was performed with Student's I test. Figures in parentheses show either the number of photographs used for assessing particle density or the number of particles measured to determine their mean diameter. K p = coefficient of particle partition between P- and E-faces (see Section I'

111).

identified in freeze-fractured preparations by their position surrounding the glomerular capillaries and their associated podocytes, as well as by their broad, flattened shape. The apical membrane (i.e., the one facing the urinary space) has numerous, irregularly arranged, rudimentary microvilli, whereas the basal membrane shows areas with many endmytotic invaginations (Webber and Blackbourn, 197 I ) . These often appear aligned in discrete groups, resembling the bands of invaginations found on smooth muscle cell membranes (Devine et ul., I97 1 ). The particle counts from all membrane regions of the parietal epithelial cell were grouped together (Table I). The P-faces of these cells have, together with those of the mesangial cells and podocyte foot processes, the lowest number of IMPS of any part of the nephron. The particle content of the E-face is, similarly, one of the lowest of the entire nephron, and the size of the IMPS does not differ significantly between the P- and E-faces (Table I).

2. Cell Junctions Desmosomes, intermediate junctions (see Staehelin, 1974), and putative tight junctions have been described (Webber and Blackbourn, 1971) between cells of FIG. 3 . Thin section of a part of a glomerulus showing the structural elements of the filtration barrier. Foot processes (FP)of podocytes are aligned on the urinary side (US) of the basal lamina (asterisks) while the fenestrated endothelium (E) surrounding the capillary lumen (CL) is found on the opposite side. The foot processes are separated from one another by a narrow band of extracellular space. the slit pore. x 15,500. Bar = 1 pm. (From hicam et a / . , 1975-Courtesy of Laboratory Investigation .) FIG. 4. Freeze-fracture replica of a similar region to that shown in Fig. 3. The plane of fracture has exposed successively the capillary lumen (CL),the membrane of the fenestrated endothelium (E), the basal lamina (asterisks), and the tips of podocyte foot processes (IT) Both . the cytoplasmic core . = 0.5 and the plasma membrane of the foot processes can be seen. US,urinary space. ~ 4 2 , 0 0 0 Bar p m . (From Pricam el a l . . 197S--Courtesy of Laboratory Investigation.)

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LELIO ORCl ET AL

the parietal epithelium of Bowman’s capsule. In freeze-fracture replicas of the human kidney (Kiihn et al., 1973, the tight junctions consist of a network composed of two to four anastomosing strands with many discontinuities. This “leaky” morphology is complemented by the fact that these junctions do not prevent the passage of horseradish peroxidase across the epithelium (Webber and Blackbourn, 1971). However, following freeze-fracture of rat, hamster, and Tupaia parietal epithelium, Taugner et al. (1976) reported that the tight junctions between these cells “may not be considered as very leaky.” In addition, gap junctions were found in one species, Tupaia (Taugner et a f . , 1976). B. VISCERALEPITHELIUM (PODOCYTES) [FIG.2 ( 2 ) ] I . Piusma Membrane

These cells are characterized by their interdigitating foot processes, which envelop the glomerular capillaries (Fig. 3; Dalton, 1951; Hall, 1953; Reid, 1954; Pease, 1955; Rinehart, 1955; Sakaguchi, 1955; Yamada, 1955; Bergstrand and Bucht, 1958). The plasma membrane of these cells belongs to the thick symmetrical group (see Farquhar and Palade, 1963) and has an overall thickness of about 11 nm (Farquhar and Palade, 1963; J~rgensen,1967; Latta, 1970). Podocytes can be easily recognized in replicas of both rat (Karnovsky and Ryan, 1975; Kuhn and Reale, 1975a; Pricam et al., 1975; Coleman et al., 1976) and human kidney (Kuhn and Reale, 1975b; Humbert et al., 1976b) because of the characteristic pattern of the foot processes and their position in the glomerulus (Figs. 4 and 5). In both the cell body and the foot processes (Fig. 6 ) ,endocytotic figures were found, as described in thin sections (Policard et al., 1955; Rinehart, 1955; Yamada, 1955: Farquhar and Palade, 1960; Elias et a l . , 1965). For the quantification of intramembrane particles, these two regions were assessed separately, and the main point to emerge was that the P- and E-faces of the cell body have both more and larger particles than the foot processes (Table 11), although an unusual feature of both membrane regions is the low Kp, only the light cells of the collecting duct having a lower value (see Table IX). 2 . Cell Junctions

In the adult glomerulus, only short elements of incomplete tight junctions can be seen between interdigitating foot processes and these only rarely. In contrast, FIG. 5 . Low magnification of podocyte foot processes (FP). Such a fracture plane allows an appreciation of the characteristic complex interdigitations of foot processes. x 13,000. Bar = 1 pm. (From Pricam et a / . . 1975-Courtesy of Laboratory Investigation.) F ~ G6. . Higher power view than Fig. 5 of the P-face of adjacent foot processes. The region of the split pore is seen as a narrow band of regular width (asterisks) separating the foot processes. The membrane of one foot process contains a circular depression corresponding to an endocytotic event (En). See Table I1 for quantitative data on particle number and size at this level. X84,OOO. Bar = 0.5

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PARTICLE

Cell body P-face E-face

Foot process P-face E-face

SIZE A N D

TABLE I1 NUMBER I N PODOCYTE PLASMA MEMBRANES"

Number of particles/pm2h

Diameter of particles (nm)(

( a ) 342 f lO(25) (b) 220 -+ 7 (21) K p = 1.6

13.1 r 2.3 (973) 13.4 t 2.3 (658)

(c) 252 t 15 (18) (d) 131 t 9 (25)

10.4 f 2.1 (788) 11.5 t 2.0 (651)

"See Figs. 4-6. Particle number-a:c and b:d, p < 0.001. ' Particle size-a:c, d:bc, p < 0.001; a:b, p

i 0.005

in the fetal glomerulus tight junctions occurred at both the level of the cell bodies and that of the foot processes, and they appeared to undergo changes in position and shape during differentiation of the podocytes (Humbert et ul., 1976b; Reeves rt (11.. 1978). A development of intercellular junctions also occurs between the podocytes of the nephrotic renal corpuscle (Pricam et ul., 1974c, 1975; Ryan el d., 1975: Caulfield er ul., 1976) and following perfusion of kidneys with polycations, which induces a nephrotic-like state (Seiler et ul., 1977; Kerjaschki, 1978). These junctions consist typically (on the P-face) of rows of individual particles. some short ridges, and small aggregates of closely packed particles that resemble gap junctions (Figs. 7 and 8). On the E-face, linear grooves often containing many adhering particles are found. In both the fetal and nephrotic glomerulus. the presence of junctional specializations seems to be associated with the loss or poor development of podocyte foot processes (Fig. 7) and the obliteration of the slit pore. In the normal adult kidney, the narrow spaces between adjacent foot processes FIG. 7. Nephrotic glomerulus after 14 days of aminonucleoside treatment. The padocyte surface appears as a continuous sheet that lacks the regularly spaced foot processes found on normal cells (compare with Fig. 4). The furrow indicated by arrows represents the region of contact between altered podocytes (obliterated slit pore) in which atypical junctions often develop. US. urinary space; CL, capillary lumen; BL, basal lamina. X43.000. Bar = 1 p m . (From Pricam et al., 1975Courtesy of Laboratory Investigation.) FIG. 8. Nephrotic glomerulus after 10 days of aminonucleoside treatment. The plasma membranes of two podocytes are closely apposed along the obliterated slit pore (arrows). Part of an altered foot process from the upper cell has been fractured away, revealing short rows and small aggregates (arrowhead) of intramembrane particles on the P-face of the adjacent cell, indicating the presence of an intercellular junction at this level. X62.500. Bar = 0.5 p m . (From Pricam et a/., 1975--Courtesy of Laboratory Investigation.)

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are bridged by a thin diaphragm that is visible in thin sections, but is difficult to resolve in freeze-fractured tissue. In deep-etched specimens and in thin sections of tannic acid-fixed kidneys, however, the diaphragm shows a highly ordered, zipper-like structure (Rodewald and Karnovsky, 1974; Karnovsky and Ryan, 1975).

C. CAPILLARY ENDOTHELIUM [FIG.2 (3)] 1 . Plasma Membrane

Endothelial cells can be readily distinguished by their proximity to the basal lamina and by their numerous fenestrae of variable size, but usually between

FIG. 9 . Plasma membrane P-face of the glomerular capillary endothelium. The membrane face is pierced by many characteristic, circular fenestrae (Fe) measuring from 0.1 to 0.2 p m in diamcter. See Table 111 for quantitative data on this membrane. ~67.000.Bar = 0.5 pm.

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TABLE 111 PARTICLE SIZEA N D NUMBERIN PLASMAMEMBRANES OF THE GLOMERULAR CAPILLARY ENLKITHELIUM"

P-face E-face

Number of particles/pm*

Diameter of particles (nm)

808 t 39 (20) 321 2 12(21) K p = 2.5

13.1 k 1.8 (950) 12.9 2.0 (771)

*

" S e e Fig. 9.

0.1 and 0.2 p m in diameter (Fig. 9; Kuhn et al., 1975; Kuhn and Reale, 1975a,b; Pricam et a l . , 1975; Ryan et a l . , 1975; Coleman et a l . , 1976), which were previously described in thin sections (Gautier et al., 1950; Dalton, 1951; Hall, 1953; Farquhar and Palade, 1962; Rhodin, 1962). Compared with other glomerular cell types, P- and E-faces of the endothelial cell membrane have the highest number of particles (Table 111). The particles found on the two fracture faces do not differ significantly in size.

2. Cell Junctions Both gap and tight junctions have been described between adjacent endothelial cells of humans and rat (Kuhn et a l . , 1975). The tight junctions are composed of one or two incomplete strands and probably represent leaky junctions. Regions of close contact between endothelial cells had previously been noticed in thin sections (Yamada, 1955; Farquhar et al., 1961; Latta, 1970). D. MESANGIAL CELLS[FIG. 2 (4)] 1. Plasma Membrane Mesangial cells are stellate cells located in an intercapillary position (Latta et a l . , 1960; Robertson and More, 1961; Farquhar and Palade, 1962; Huhn et a l . , 1962; Suzuki et a l . , 1963), which allows them to be easily identified in freezefractured glomeruli (Fig. 10). Their plasma membrane contains a low number of IMPS both on the E-face and the P-face (Table IV). 2. Cell Junctions The most outstanding feature of the plasma membrane of mesangial cells is the presence of numerous gap junctions (Fig. 11; Pricam et a l . , 1974b; Kuhn et a l . , 1975; Boll et a l . , 1975), which are sometimes associated with tight-junctional fibrils. Extraglomerular mesangium, or lacis cells, also have gap junctions, and on both mesangial and lacis cells, gap junctions were found between processes of the same cell (so-called auto or reflexive gap junctions).

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TABLE IV PARTICLE SIZEAND NUMBER ON PLASMA MEMBRANES OF MESANGIAL CELLS~ _ _ _ _ _ _ _ _ _ _ _

P-face E-face

~~

~

~~

Number of partic1es/pmZ

Diameter of particles (nm)

250 t 9 (24) 83 t 3 (20) K p = 3.0

11.5 2 2.0b (795) 12.4 t 1.8 (372)

aSee Figs. 10 and 11. Significantly different from E-face, p < 0.001.

V. Proximal Tubule The proximal tubule consists of two distinct regions (but see Maunsbach, 1973): the pars convoluta (convoluted portion) and the pars recta (straight portion). The epithelial cell types in these two regions can be distinguished owing to different degrees of development of the brush border (Figs. 12 and 13) and the basolateral invaginations, and also according to the height of the cells and their organelle content (Sjostrand and Rhodin, 1953; Pease, 1955; Caulfield and Trump, 1962; Maunsbach 1966a, 1973; Ericsson et a l . , 1965; Tisher et a!., 1966). The quantitative assessment of membrane organization was performed on convoluted and straight segments, identified on the basis of the parameters described above. In both segments of the proximal tubule, four regions of the epithelial cell membrane were quantified separately: (1) the microvilli, (2) the nonmicrovillar luminal membrane, (3) the lateral plasma membrane, and (4)the basal plasma membrane. [FIG.2 ( 5 ) ] A. PARSCONVOLUTA Plasma Membrane The prismatic epithelium of this region has numerous deep invaginations of the basal membrane, and the microvilli are shorter than in the pars recta. Although the freeze-fracture architecture of these cells was examined by Leak in 1968, the FIG. 10. Replica of the intraglomerular mesangium showing the typical stellate shape of mesangial cells (MC). Mesangial cells are separated from the endothelium (En) by a sheet of basal lamina-like material (mesangial matrix) that appears interrupted at several places (arrows), suggesting contact zones between mesangial and endothelial cells. The encircled areas on the plasma . =1 membrane of the mesangial cell in the center of the field contain gap junctions. ~ 4 3 , 0 0 0Bar Pm.

FIG. 11. High magnification of a mesangial cell plasma membrane showing the characteristic particle aggregates indicating the presence of gap junctions (GJ). These vary from linear to macular in shape and are associated with tight-junctional fibrils (arrows). X63,OOO. Bar = 0.4 jm.

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FIG. 12. Freeze-fracture appearance of the apical pole of a proximal tubule epithelial cell with its brush border. Microvilli fractured at various levels characterize this region, which is separated from the lateral face of the cell by a light junction (arrowheads). The difference in particle content between P- and E-faces can already be appreciated at this magnification (see also Fig. 1 and Table V). ~ 2 3 , 0 0 0 Bar . = 1 urn.

FIG. 13. Luminal and lateral plasma membranes of an epithelial cell from the proximal tubule. The two membrane domains are separated by a tight junction (TJ) whose forming fibrils are arranged in a parallel or a honeycomb pattern. On the lateral plasma membrane, gap junctions (GJ) are present. As detailed in the insets, gap junctions are formed of closely packed particles on the membrane P-face (upper inset) and of closely spaced pits on the E-face (lower inset). x41.500. Insets X92.000. Bar = 05pn.

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TABLE V PARTICLE SIZEA N D NUMBER ON PLASMA MEMBRANES OF PROXIMAL TUBULE EPITHELIAL CELLS"

A. Pars convoluta Microvilli P-face (a) E-face (b) Luminal P-face (c) E-face (d) Lateral P-face (e) (0 E-face (g) (h) Basal P-face (j) E-face (k) B. Pars recta Microvilli P-face (m, E-face (n ) Luminal P-face (p) E-face (q) Lateral P-face (r) (S)

E-face ( t ) (U)

Basal P-face ( v ) E-face (w)

Number of particleslpm"*

KP

Diameter of particles (nm)"

2426 t 61 (16) 2 5 8 t 21 (13)

9.4

10.6 + 1.3 (399) 11.0 t 1.5 (201)

1940 f 34 (15) 1 6 9 2 18(13)

11.4

12.8 t 1.8 (855) 12.0 t 1.8 (207)

2045 t 49(19) 1016f 64 (8)d 127 t 8(11) 275 5 25 (6)"

16.1

3.7*

11.2 5 1.9(3246)

11.2

f 1.9

(622)

11.4

11.2 5 1.9 (704) 1 1 . 1 f 1.7 (285)

2268 t 54(21) 428 rt 35(13)

5.3

11.4 f 1.8 (741) 12.0 2 1.9 (446)

2184 t 68 (26) 346 t 43 (22)

6.3

12.3 t 1.9 (774) 12.4 2 1.8 (332)

2123 f 1497 t 298 t 284 t

79(24) 193 (9)d 17 (25) 26 (9)"

1389 t 92 (9) 286 2 26 (14)

7.I

5.34

4.9

12.0 2 1.9 (774)

12.2 t 2.0 (620)

12.1 t 1.9 (341) 12.7 rt 1.9 (319)

"See Figs. 12-16. *Particle number: (convolutedj a : c e f j , c:fj, e:fj, b:gk, p < 0.001; b:d, p < 0.005;d:k, p < p:sv, r:sv, n:uw. p < 0.001;(convoluted vs straight)-am, c:p, e:r, f s , j:v, not significant: b:n. d:q, f s , k:w, p < 0.001. ' Particle size: (within same segmentja:bcej, d:bcgk, c:ej, m:nprv, w:vn, all p < 0.001: r:t, p < 0.005;n:p, p < 0.01; (convoluted vs straight)-a:m, b:n, c:p, ex. g:t, j:v, k:w, all p < 0.001. "Represents the panicle density of the band of plasma membrane (about 0.1 p m in depth) lying immediately beneath the tight junction. 0.01; (straight)-msv.

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203

highly particulate nature of microvillar and basolateral P-faces in the proximal tubule epithelium was first pointed out by Friederici in 1969. However, the position of the fracture plane was, at that time, a controversial subject and Friederici interpreted the particles as being present on the true membrane surface. Our quantitative data show the highest number of particles on the P-face of the microvilli and the lowest on the basal region of the plasma membrane. The particle contents of the luminal and lateral membranes do not differ significantly (Table V), with the exception of a band of membrane approximately 0.1 p m in depth on the lateral membrane, immediately beneath the tight junction, which contains fewer particles than the rest of the lateral membrane. Similar regions are also found below tight junctions in other tissues (Friend and Gilula, 1972).

FIG. 14. High magnification of the microvillar [Mv(P)J and nonmicrovillar luminal [Lu(P)J plasma membrane of the convoluted proximal tubule. The P-face particles on the luminal membrane are clearly larger than those on the microvilli (see Table V for the quantiative evaluation of the difference). X 102,000. Bar = 0.25 g m .

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B. PARSRECTA[FIG.2 (611 1. Plasma Membrane

The epithelial cells of the straight segment are more cubic than prismatic and have few basal invaginations. However, the brush border is generally longer than in the pars convoluta. In this region, the P-face of the microvilli has the same particle content as the luminal and lateral plasma membranes, but the basal membrane has fewer particles (Table V). The microvilli have the most highly particulate E-face. A comparison of the two segments of the proximal tubule shows that the mean particle contents of corresponding P-faces are not significantly different, whereas the E-faces from all four regions differ. In addition, the coefficient of partition of particles is lower for the convoluted than for the straight tubule in all four membrane regions. When the mean particle size of fracture faces from all four membrane regions of the straight and convoluted tubules were compared, all the particles, except for those on the two luminal E-faces, were significantly different. One of the most striking observations here was the large size difference between particles on the luminal P-face of the convoluted tubule, compared with the other membranes in this segment (Figs. 14 and 15; Table V). In contrast, the particles in the luminal membrane of the straight segment were not significantly larger than those of the basal and lateral membranes.

2 . Cell Junctions (Pars Convoluta and Pars Recta) In the proximal tubule, tight junctions, gap junctions, and desmosomes have all been identified in thin sections (Farquhar and Palade, 1963; Maunsbach, 1966b;Tisher et a l . , 1966, 1969; Ross and Reith, 1970; Silverblatt and Bulger, 1970). and these results have been confirmed in freeze-fracture (Pricam er al., 1974a; Kuhn and Reale, 1975). The tight junctions of the proximal tubule consist of few strands, which have many discontinuities (Figs. 13, 15, 34, 38; Table XIII). In some regions of the tubule, the discontinuity is so marked as to result in the apparent absence of tight-junctional differentiations for distances of up to 1 g m . In other cases, however, three to five ridges or grooves are present (Figs. 13 and 16). On the membrane of the same cell it is possible to find junctions showing sharp transitions from a single to several ridges, or grooves that are arranged in either a parallel or a complex honeycomb pattern (Fig. 13). It has been demonstrated for several species that the tight junctions of the pars recta are more developed than those of the pars convoluta, although the degree of difference varies among species (Roesinger et al., 1978; Schiller et al., 1978; Figs. 15 and 16). In fetal kidney (rat and human), the tight junctions of the proximal tubule

MEMBRANE ULTRASTRUCTURE IN URINARY TUBULES

205

FIGS. 15 and 16. Composite plate showing the difference in tight-junction complexity found in the convoluted part (Fig. 15) and in the straight part (Fig. 16) of the proximal tubule. As seen from the two junctions illustrated (these represent extremes), the overall complexity of tight junctions (i.e., junctional depth, number of strands, and cross-linking strands) is low in the convoluted part and high in the straight part of the proximal tubule. In Fig. 15, note in addition the marked difference in particle size between luminal (above tight junction) and lateral (below tight junction) plasma membrane. See Table V. Fig. 15 (X91,500); Fig. 16 (X72,oOO). Bars = 0.25 p n .

MEMBRANE ULTRASTRUCTURE IN URINARY TUBULES

207

TABLE VI ON THE COMPLEXITY OF TIGHT JUNCTIONS FROM Necturus EFFECTOF SALINE DIURESIS PROXIMAL TUBULES"-*.".~.~ Animal number

Control

Animal number

Saline diuresis

1 2 3 4

4.94 2 0.65 (18; 2359) 4.78 -I- 0.39 (33; 4574) 3.80 ? 0.25 (15; 1579) 4.96 2 0.37 (17; 2141)

5 6 7 8 9

Mean t SEM

4.62 f 0.27

Mean 2 SEM

2.94 5 0.51 (42; 3257) 2.79 0.46 (32; 2505) 2.98 0.21 (33; 2714) 2.93 t 0.30 (22; 1705) 3.45 t 0.79 (30; 2801) 3.01 f 0.11

*

-

I p < 0.005

"From Humbert el al. (1976). *See Figs. 17-19. Values are mean per animal 2 SEM. dThe extension of tight junctions is expressed as the density of boundary length of junctional elements per membrane area. 'The first number in parentheses indicates the number of micrographs examined per animal, the second the total number of intersections measured in these micrographs.

consist of only one or two parallel strands, which are often discontinuous. Gap junctions are also found between epithelial cells of the proximal tubule of the fetus (Humbert et al., 1976b).

VI. Loop of Henle (Thin Descending and Ascending Limb) The freeze-fracture analysis of the loop of Henle was performed on long loops only (Humbert et al., 1975b). It is possible to distinguish between descending and ascending loops by comparing thin sections with freeze-fracture replicas: in thin section, the initial descending part of the loop is characterized by the presence of numerous short microvilli at the luminal surface of the constituent epithelial cells, which have complex cell processes. In contrast, the ascending part of long loops appears to be formed by cells with regular interdigitating cytoplasmic

FIGS. 17-19. This set of figures illustrates the modulation of tight-junction complexity in the proximal tubule induced by a saline load (Necturus maculosus). Bars = 0.25 pm. (From Humbert et al., 1976-Courtesy of the Journal of Cell Biology.) Fig. 17. Control junction. X 104,000.Fig. 18. Saline-loaded animal. The junction shows a decrease in the complexity of strands, which have also many discontinuities (asterisks). X 124,000. Fig. 19. E-face of a tight junction during saline load. The presence of discontinuities in tight-junctional grooves (asterisks) is also apparent (see Table VI). X 123,000.

208

LELlO ORCl ET AL.

processes (Lapp and Nolte, 1962; Osvaldo and Latta, 1966; Bulger and Trump, 1966; Schwartz and Venkatachalam, 1974; Dieterich et a l ., 1975) which are almost devoid of microvilli except along regions of contact with adjacent processes (Fig. 21).

FIG.20. Freeze-fracture appearance of the descending thin limb of Henle. This segment of the nephron yields mostly cross-fractures of the cells with only small areas of the plasma membrane exposed in face view. Such an area of the P-face of the lateral membrane is outlined in the box and shown at high magnification in the inset. Note the extremely high density of intramembrane particles, which are equally numerous on the other (luminal and basal) regions of the cell membrane (see Table VII). TL. tubular lumen; N. nucleus. X 17.500. Bar = 1 jm. Inset. X76.000. Bar = 0.25 p m .

MEMBRANE ULTRASTRUCTURE IN URINARY TUBULES

209

A. DESCENDING LIMB [FIG. 2 (7)] 1. Plasma Membrane In the initial descending limb, the fracture plane mainly exposes the cell cytoplasm and produces relatively few large areas of plasma membrane (Fig. 20). The most striking feature of this membrane is the extremely dense packing of the intramembrane particles on both luminal and basolateral P-faces (Humbert et al., 1975b; Table VII; Fig. 2Linset). The particle content of these membranes is the highest (3500 IMP/pmZ)of all the membranes in the urinary tubule. The E-face has, in comparison, few particles, and that of the luminal membrane contains more particles than the basolateral E-face. Particles on both luminal and basolateral E-faces are characterized by a larger size than those on the respective P-faces. 2. Cell Junctions In the thin limb of Henle, tight junctions consist of only one (rarely two or three) thick fibril(s) (Pricam et al., 1974a; Kiihn and Reale, 1975b; Table XIII). TABLE VII PARTICLE SIZE AND NUMBERIN PLASMA MEMBRANES OF EPITHELIAL CELLS FROM THE Loop OF H E N L E ~ Number of particles/pmZb

Kp

Diameter of particles (nm)c

3444 f 50 (11) 729 +. 33 (20)

4.7

11.8 2 2.0 (250) 12.9 f 2.1 (529)

3507 f 34 (20) 573 f 12 (23)

6.1

11.9 f 2.1 (259) 13.3 f 2.0 (359)

1013 k 25 (24) 64 +- lO(10)

15.8

11.2 t 2.2 (562) 11.9 2 1.7 (226)

1004 t 47 (10) 80 f 9 (12)

12.6

12.6 f 2.1 (295) 11.4 f 1.8 (223)

A . Descending Limb

Luminal P-face (a) E-face (b) Basolateral P-face (c) E-face (d) B. Ascending Limb Luminal P-face (e) E-face ( f ) Basolateral P-face (g) E-face (h) (I

See Figs. 20-22.

* Particle number: (descending)-b:d,

p < 0.005; (descending vs ascending)-a:eg, c:eg, b:fh, d:fh, all p < 0.001. Particle size: (within same segment)-a:b, c:d, e:fg, g:h, all p < 0.001; b:d, e:f, p < 0.005; (descending vs ascending)-b:f, c:g, d:h, p < 0.001; a:e, p < 0.005.

210

LELIO ORCl ET AL

The thick appearance of the fibril on the P-face seems to be a result of the close apposition of two parallel elements covered by adhering E-face from the adjacent cell. This is suggested by examination of the junctional E-face, where two parallel. continuous grooves are found (Figs. 22, 35, and 39).

FIG.21. Freeze-fracture appearance of the ascending thin limb of Henle. This region is characterized by numerous interdigitating cell processes (see also Fig. 2 2 ) . dotted with plump microvilli (arrows). As seen in the inset. the plasma membrane of these cells has a rather low content of particles (compare with Fig. 20, descending limb, and Table VII). TL, tubular lumen: N. nucleus. x 14.OOO. Bar = 1 pm. Inset, ~76.000. Bar = 0.25 pm.

MEMBRANE ULTRASTRUCTURE IN URINARY TUBULES

21 1

FIG.22. Ascending thin limb of Henle, showing the poorly particulated luminal P-face and the lateral E-face separated by a tight junction (arrowheads). In Henle's loop. the tight junction appears often as a single thick fibril (see also Figs. 35 and 39). X70.000. Bar = 0.5 pm.

B. ASCENDING LIMB[FIG.2 (8)] I , Plasma Membrane In the ascending limb, large areas of plasma membrane were often exposed (Fig. 21). The luminal and basolateral membrane P-faces contain many fewer particles than their counterparts in the early descending limb, as do the corresponding E-faces (Table VII; Fig. 21-inset). The largest particles of the ascend-

212

LELIO ORCl ET AL.

ing limb are found on the basolateral P-face, in contrast to the descending limb where the largest particles belong to the basolateral E-face.

2. Cell Junctions The tight junctions found in the ascending long limbs appear similar to those observed in the descending limb (Fig. 22) in both the human and rat nephron (Pricam ez ul., 1974a: Humbert et al., 1975b; Kuhn and Reale, 1975b). However, recent studies on thin limbs of the rat and the rabbit suggest that the freeze-fracture appearance of plasma membranes and tight junctions varies between different regions of long loops, as well as between short loops and long loops (Schwartz et ul., 1979, 1980; Abramow and Orci, 1980; Schiller el al.. 1980). Thus, the highly particulate membranes of descending branches are found principally in the outer medulla, and the particle content gradually decreases as the loop descends further into the inner medulla. In addition, the ascending thin limbs of rabbit have shallower tight junctions than descending limbs, whereas in the rat, the highly particulate regions of the descending limbs also have shallow junctions. In thin sections, differences have also been described in tight-junctional development from various regions of the thin loop of Henle both in rat (Schwartz and Venkatachalam, 1974) and rabbit (Darnton, 1969). However, in the rat, the junctions of both ascending and descending branches are impermeable to lanthanum tracer (Bulger, 1971; Pncam, 1974).

VII. Thick Ascending Limb of Henle and Distal Convoluted Tubule [Fig. 2 (9, lo)] For the quantitative assessment of particle content of the plasma membrane, the thick ascending limb of Henle (or straight distal tubule) was compared to the distal convoluted tubule. Thick ascending limbs could be readily identified in the outer medulla, where distal convoluted tubules are not found. However, both types of tubules are present in the cortex (if the transition is considered to occur at the level of the macula densa), so other criteria are needed. The straight distal part has an epithelium which is thinner than that of the convoluted part, and the basal plasma membrane has many deep invaginations that often reach almost to the luminal surface (Fig. 23). The convoluted distal tubule, on the other hand, has a low prismatic epithelium and less-developed basal invaginations, particularly in the region underlying the nucleus (Rhodin, 1958a,b; Sakaguchi and Suzuki, 1958: Thoenes, 1961;Osvaldo-Decima, 1973; Allen and Tisher, 1976). Particle counts on each segment thus identified were performed separately for luminal. lateral, and basal plasma membrane.

MEMBRANE ULTRASTRUCTURE IN URINARY TUBULES

213

1. Plasma Membrane

In both segments of the distal tubule, the lateral P-face contains more particles than the luminal and basal P-faces. This difference is most evident in the straight part, where the lateral membrane has almost twice as many particles as the luminal P-face and is the most highly particulate membrane face of the entire distal tubule (Fig. 24; Table VIII). The particle sizes on the P- and E-faces of corresponding membranes in the two regions are all significantly different except for the luminal P- and E-faces, where particle sizes do not differ between the straight and convoluted parts. An unusual feature of the luminal membrane of some epithelial cells in the convoluted tubule is the presence of a population of elongated particles on their P-face. The cells with elongated particles probably correspond to the intercalated cells described by Griffith et al. (1968) in the rat distal tubule, a cell type that is also prominent in the collecting duct (see following paragraphs). 2. Cell Junctions In the distal tubule, the tight-junctional fibrils are more numerous than in the proximal tubule or Henle’s thin limb (Pricam er al., 1974a; Kuhn and Reale, 1975b; Table XIII). The strands are usually parallel and closely spaced so that the depth of the junction in the apico-basal direction is relatively shallow (Figs. 24, 36, and 40). The fibrils have few branchings and there are also sharp transitions in the number and packing of the ridges within the same junctional domain. In humans, the tight junctions of the convoluted tubule seem to be rather more complex, appearing as a four- to six-ridge-deep network of anastomosing strands (Kuhn and Reale, 1975b). Nevertheless, it has been reported that, in common with junctions of the proximal tubule, those of the distal tubule are permeable to lanthanum tracer (Martinez-Palomo and Erlij, 1973; Tisher and Yarger, 1973) but not to horseradish peroxidase (Miller, 1960; Venkatachalam and Karnovsky , 1972). 3. The Macula Densa The macula densa is a specialized region of the distal tubule that may be involved in monitoring the urinary load arriving at this level of the nephron (Thurau, 1966; Barajas and Latta, 1967; Thurau et al., 1967; Latta, 1973). Data concerning particle number, size, and distribution are not yet available for this tubule segment. However, the intercellular junctions at this level have been the subject of several studies. It was suggested that the tight junctions of this region were more permeable to lanthanum than those in other parts of the distal tubule (Giacomelli and Wiener, 1976), but not to horseradish peroxidase (Schiller and Taugner, 1979). In a variety of species, the freeze-fracture appearance of tight junctions in the macula densa resembles that described between the other epithe-

MEMBRANE ULTRASTRUCTURE IN URINARY TUBULES

215

lial cells of the distal tubule (Boll et al., 1975; Schiller and Taugner, 1979). Gap junctions are not found between cells of the macula densa (nor between any epithelial cells in the distal tubule), but have been reported to couple adjacent granular juxtaglomerular cells with cells of the extraglomerular mesangium (the lacis dells) (Boll er al., 1975; Taugner et al., 1978). TABLE VIII PARTICLE SIZEA N D NUMBER I N PLASMA MEMBRANES OF THE DISTAL TUBULE EPITHELIUM" ~

A. Pars recta Luminal P-face (a) E-face (b) Lateral P-face (c) E-face (d) Basal P-face ( e ) E-face (f)

B. Pars convoluta Luminal P-face (9) E-face (h) Lateral P-face (j) E-face (k) Basal P-face (m) E-face (n)

~

Number of particledpmZb

Kp

Diameter of particles (nm)"

1052 t 44(11) 130 2 12(17)

8. I

11.5 f 3.1 (518) 12.1 f 1.9 (349)

1866 f 45 (16) 100 f 12(14)

18.7

10.8 t 2.0 (829) 11.3 2 1.9 (284)

1374 2 160 (5) 155 f 17 (8)

8.9

11.3 t 1.7 (145) 12.2 2 1.9 (224)

1236f 56(27) 131 t 14(19)

9.4

11.2 4 2.2 (656) 12.0 f 1.8 (426)

1545 f 45 (28) 133 '. 15(24)

11.6

11.8 f 2.2 (985) 11.8 f 2.0 (333)

1395 t 48 (23) 150 5 9 (24)

9.3

11.8 t 2.2 (747) 11.7 2 1.8 (519)

See Figs. 23 and 24. p < 0.001; bParticle number: (straight)--c:ae, p < 0.001; d:f, p < 0.01; (convoluted)-g:j, (straight vs convoluted)-c:j. p < 0.001. Particle size: (straight)--c:a, fde, b:d, p < 0.001; a:b, p < 0.005; c : e , p < 0.01; (convoluted)-g:hjm, p < 0.001; (straight vs convoluted)+:j, p < 0.001; d:k, f n , p < 0.005; e m , (I

p

< 0.01.

FIG. 23. Replica from the pars recta of the distal tubule showing part of the luminal membrane separated from the lateral plasma membrane by a tight junction (arrowheads). The extensive infoldings of the basal plasma membrane are indicated by asterisks. X20,OOO Bar = 1 pm. FIG. 24. Higher magnification of the junctional area seen in Fig. 23. The tight junctions of the distal tubule are composed of mainly parallel elements that are rather indistinct owing to the adherence of plasma membrane E-face from the adjacent cell (see also Figs. 36 and 40). The lateral plasma membrane P-face contains almost twice as many particles as the luminal P-face in the pars recta (see Table VIII). ~ 5 7 , 0 0 0Bar . = 0.5 pm.

L E U 0 ORCl ET AL

216

VIII. The Collecting Tubule The collecting tubule has two distinct populations of epithelial cells that can be recognized in both semithin and thin sections, as well as in freeze-fracture: the light or principal cell and the dark or intercalated cell (Fig. 25). Dark cells are most frequent in the initial part of the collecting tubule in the cortex and outer medulla, whereas there are few dark cells in the inner medulla and virtually none in the papillary duct (Rhodin, 1958b; Young and Wissig, 1964; Bulger and Trump, 1966: Myers et al., 1966; Osvaldo-Decima, 1973). These two cell types show a remarkable degree of membrane specialization when examined in freeze-fracture (Humbert et al., 1974, 1975~). A . LIGHTCELLS(PRINCIPAL CELLS)[FIG.2 (12)j As for other cell types described so far, the basis of freeze-fracture identification was a careful comparison of thin sections with freeze-fracture replicas. The specific morphological character of light cells is the scarcity of microvilli on their apical membrane. together with a cytoplasm containing relatively few organelles.

1 . Piusma Membrane

The counting and measurement of intramembrane particles in the luminal plasma membrane of light cells was done in three separate parts of the collecting tubule: ( I ) that localized in the cortex and the outer stripe of the external medulla, ( 2 ) that situated in the inner stripe of the external medulla, and (3) that lying in the inner medulla (Table IX). This approach revealed a progressive decrease in the particle density of luminal P-faces from the cortex to the inner medulla (Table IX). This trend continued into the papillary duct, where the corresponding membrane has even fewer panicles than in the inner medulla (Table XII). On the other hand, there is no ~

~

~~

FIG. 2 5 . Thin section of the epithelium lining the cortical collecting tubule. In this region, two distinct cell types are present: the light or principal cell and the dark, intercalated, or mitochondriarich f MR) cell. The dark cells often have a more electron-dense cytoplasm, contain large numbers of mitochondria, and have more microvilli on their luminal membrane. X4500. Bar = 5 pm. (From Humben er ul., 1975-Counesy of Journal of Ultrustrucrure Research.) FIG. 16. In freeze-fracture replicas. dark cells show an unusual membrane specialization: the presence of rod-shaped or elongated intrarnernbrane particles. These particles are present in the luminal. lateral, and basal plasma membrane P-faces of dark cells. As seen in this picture, elongated particles often farm clusters (dashed lines) among globular particles (see also Fig. 28). X60.000. Bar = 0.40 p m . (From Humbert el al.. 1975c-Courtesy of the Journal of Ulirasrrucrure Research.) FIG. 27. Higher magnification of the luminal P-face of a dark cell in which the majority of particles are rod-shaped. ~ 9 8 , 0 0 0 .Bar = 0.25 Frn.

218

LELIO ORCI ET AL.

P A R T I C L E SIZE A N D

NUMBER" IN

TABLE IX MEMBRANES FROM LIGHT COLLECTING Ducrb."

PLASMA

OF THE

Number of panicledpm2n Luminal P-face ( I ) : ( a ) ( 2 ) : th) ( 3 , : IC) Lurninal E-face ( 1 ) : ( d ) ( 2 ) : ie) 13, : t f l Lateral P-lace rg) E-face (h) Basa I P-face (j) E-face (k)

KP

(PRINCIPAL) C E L L S

Diameter of particles (nm)"

11.9

?

1.8 (793)*

12.9

2

2.2 (9521*

2.2 ' 778 t 94 ( 6 ) 723 t 65 (17) 685 ? 70 ( 9 )

1.5: I .2 :{

? 84(26) 450 t 23 (34)

3.15

781 283

2.8

1416

2

62(21)

I 22(19)

11.4 2 I . 8 (688) 13.3 ? 2.1 (756) 1 I .6 ? I .8 (490)

13.0

2

2.3 (428)

" The size and number of particles shown in this table do not include those present in the square arrays that are present on lateral and basal membranes of light cells (see Table X). [' For the luminal membrane only, particle numbers were counted separately in the outer stripe of the external medulla ( I ) : the inner stripe of the external medulla (2): and the inner lnedulla (3). For the measurement of panicle diameter, however. results from these three regions were not separated (*). 'See Figs. 29-33. 'Particle number-xbcj, gxj. def:hk. h:k. p < 0.001. ' Particle size-a:dg. g:h, j:k, p < 0.001: a:j, d:h. p < 0.005.

difference between the particle content of the luminal E-faces (Table IX). One other feature of all collecting duct membranes, but the luminal membrane in particular. is the very low K p value-the lowest of all of the epithelial cell membranes of the urinary tubules in the case of the inner medulla. In addition to the usual set of particles just described, the plasma membrane of the light cell is characterized by two additional distinct features: orthogonal arrays of particles and particle aggregates. The orthogonal arrays are characteristic of the basolatera1 membrane (Humbert et NI.. 1974, 1 9 7 5 ~Fig. ; 29). These arrays are formed of flat-topped particles on the P-face and of pits on the E-face, which appear dispersed among the usual globular particles (Figs. 30 and 3 I ) . They are particularly numerous on the basal membrane where they may occupy up to 15% of the total membrane surface exposed: on the lateral membrane, they cover approximately 3% of the exposed surface (Table X). The close packing of the particles in the orthogonal arrays, as well as their flattened shape, make the precise measurement of individual particle size on the P-face difficult. However. their size

MEMBRANE ULTRASTRUCTURE IN URINARY TUBULES

219

TABLE X PERCENTAGE OF BASOLATERAL PLASMA MEMBRANE (PM) BY ORTHOGONAL PARTICLE ARRAYS IN SURFACE OCCUPIED THE COLLECTING TUBULEA N D PAPILLARY DUCT” Collecting tubule Lateral PM Basal PM

2.8 ? 0.6b 15.0 2.3

(a)

Papillary duct ( C ) 0.6 & 0.2b 11.6 2 1.4

“See Figs. 29-3 1. Significantly different from corresponding basal PM, p < 0.001.

can be estimated by measuring the center-to-center spacing of the corresponding pits on the E-face (Fig. 3 1). This distance is about 7 nm, so a similar diameter is assumed for the particles forming the arrays. The remaining, nonaggregated globular particles on the basolateral membrane are larger than those involved in the arrays. Orthogonal arrays are not observed on the luminal membrane of the light cell, but at this level, loose clusters of globular particles are occasionally found (Figs. 32 and 33). They appear particularly evident on the luminal E-face. Similar clusters have been described in the collecting tubule (light cell) of vasopressin-treated Brattleboro and normal rats (Harmanci ef al., 19781, and they have been associated with membrane-permeability changes. 2 . Cell functions

Tight junctions in the collecting duct are found both between adjacent light cells, and between light and dark cells. The junctions are the most developed found in the entire urinary tubule (Figs. 37 and 41; Table XIII) and consist of several anastomosing strands, usually arranged in a complex honeycomb pattern (Pricam er al., 1974a; Kuhn and Reale, 1975b). There are few or no major discontinuities in the ridges. These junctions belong to the morphological class of “tight” junctions and they are effective barriers to the diffusion of lanthanum tracer (Martinez-Palomo and Erlij, 1973; Tisher and Yarger, 1973). B. DARKCELLS(INTERCALATED CELLS)[FIG.2 ( 1 l ) ] Compared to light cells, dark cells have numerous apical microvilli and a relative abundance of cytoplasmic organelles (Rhodin, 1958b), differences that allow dark cells to be identified in freeze-fracture. Similar ceils have also been found in the terminal part of the distal convoluted tubule (Griffith et d . , 1968; see Section VII).

220

LELlO ORCl ET AL.

FIG. 18. Part of the plasma membrane P-face (luminal) of a dark cell. In this field. rod-shaped particles are not distributed homogeneously throughout the membrane. but appear in discrete clusters (some of which are enclosed in dashed lines). Outside the clusters globular particles are predominant although rod-shaped particles are also present. ~75.000.Bar = 0.25 p m .

I . Plasm~IMembrane

The characteristic feature of the plasma membrane of dark cells is the presence of a population of elongated, or rod-shaped particles, in addition to the more usual globular type (Humbert er ul., 1 9 7 5 ~Figs. ; 26-28). Elongated particles are rare on the lateral and basal P-faces exposed, but on the luminal P-face, they may represent a large percentage of the total particle number (Fig. 27). The number of P-face particles (globular plus elongated) is not significantly different between the three membrane regions (luminal. lateral, basal) of the dark cell. On the other hand. the size of globular particles on the P-face is significantly smaller than that

22 1

MEMBRANE ULTRASTRUCTURE IN URINARY TUBULES

of E-face particles of luminal, lateral, and basal plasma membranes (Table XI). The elongated particles, which are also present in fracture faces of intracellular membranes, are approximately twice the length of individual globular particles found on the P-face. 2 . Cell Junctions

See Section VIII,A,2.

C. PAPILLARY DUCT[FIG.2 (13)] Apart from its location in the papilla, the duct epithelium can be recognized by the shape of the cells. Whereas the cells of the inner medullary collecting tubule epithelium are cubic, those of the papillary duct are prismatic (Bulger and Trump, 1966). In papillary duct cells, the number of particles on the P-face of the lateral membrane was significantly higher than on the luminal and basal P-faces (Table XII). In addition, the number of particles on the luminal P-face of these cells is significantly different from that on light cell P-faces in the inner medulla. The globular particles on the basal and lateral P-faces were larger than those on the luminal P-face, whereas E-face particles from all regions of the cell did not differ

PARTICLE SIZE A N D

NUMBER IN FROM

TABLE XI PLASMA MEMBRANES FOR THE THE COLLECTING DUCT".^.'

Number of particles/pm Z d Luminal P-face (a) E-face (b) Lateral P-face ( c ) E-face (d) Basal P-face (e) E-face (0

2288

?

KP

54 (23)

519 2 35 (22)

4.4

2085 2 300 (3) 271

?

49 (7)

7.7

2314 2 92 (16) 338

?

89 (5)

6.8

INTERCALATED

CELLS

Diameter of particles (nm)'

1 1 . 1 ? 1.6 23.2 2 1.6 (532) 12.9 2 1.6 (385)

11.3 2 1.8 22.7 2 1.7 (85) 12.4 2 1.7 (159) 10.9 ? 2.1 20.2 2 1.7 (446)

On the plasma membrane P-face of these cells, both globular and elongated particles were found. The larger of the two values given for each P-face is the length of the elongated particles. Similar cells were also found in the terminal region of the distal convoluted tubule. (.See Figs. 26, 27, and 28. Particle number-b:d, p < 0.005. Particle size (globular particles only)-a:b, d:c, feb, p < 0.005.

222

PARTICLE

LELIO ORCl ET AL

SIZE AND

NUMBER IN

THE

TABLE XI1 PLASMA MEMBRANE OF PAPILLARY DUCTEPITHELIUM"

Number of panicles/pm2h

Luminal P-face ( a ) E-face ( b ) Lateral P-face ( c ) E-face ( d ) Basal P-face ( e ) E-face (fl

KP

Diameter of particles (nm)"

I .3

8.7 ? 2.0 (561) 10.8 I 2.0 (538)

32 (24) (25)

.<.2

10.4 I 2.0 (675) 10.7 t 2.2 (614)

603 f 20(21) 263 I14 ( 2 5 )

2.3

10.7 2 1.9 (476) 10.6 -t 2.1 (573)

522 t 24 (19) 410 2 28 (24) 1074 334

f

I 20

~~

In the papillary duct. the luminal membrane "lipid" domain has a rough appearance in freezefracture. Some of the resulting rugosities may appear as small particles, but these were not included in the quantification. Particle number-ax, b:f, p < 0.001: d:f, p < 0.005. "Particle size-a:bce, p < 0.001; c:e. p < 0.01. If

significantly in size. Papillary duct cells also have orthogonal arrays of particles or pits on their basolateral membranes, which occupy approximately 12% of the total basal membrane exposed as compared to less than 1 % on the lateral membrane. The comparison of particle sizes in membranes of the papillary duct cells and in the light cells of the inner medulla collecting duct shows that for all membrane regions, the particles are significantly larger in both the P-face and the E-face of the light cells from the inner medulla.

IX. Discussion The application of the freeze-fracture technique to the kidney has revealed that the plasma membranes of its constituent epithelial cells, which have similar trilaminar structures in thin sections, show a marked heterogeneity of structural organization. Thus, in different regions of the nephron and of the collecting tubule, the number, size, shape, and arrangement of intramembrane particles, which probably represent protein-containing structures, as well as the degree of development of junctional specializations, vary greatly in the plasma membranes of different cell types and even between different domains of the membrane from the same cell. Although most of the observed membrane differences cannot, at present, be interpreted in functional terms, with the exception of the variation in tight-junction development, many interesting questions can be raised that are discussed in more detail in the following paragraphs.

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A. PODOCYTES (FIGS.3-8; TABLE11) Podocytes have significantly more particles on their cell body than on the foot processes, and those on the cell body are larger. This raises the possibility that these cells do not act simply as a mechanical support and/or a hydrodynamic regulator of the glomerular filter, but that different regions of their plasma membrane are specialized to serve as yet unknown functions (see also Jones, 1962; and Groniowski et al., 1969). Although tight and gap junctions are not normally found in the adult, they develop in nephrotic glomeruli (Pricam et al., 1975; Ryan el al., 1975; Caulfield et al., 1976) and are common in fetal kidney (Humbert et al., 1976b; Reeves et al., 1978). In both cases, the junctions are macular and are not likely to act as important barriers to intercellular diffusion. The role of these structures is not known but appears to be linked to the absence or alteration of the slit diaphragm and the resulting close proximity of plasma membranes from adjacent cells in fetal and nephrotic glomeruli. B. MESANGIAL A N D LACIS CELLS(FIGS.10 A N D 11; TABLEIV)

Gap junctions occur frequently between mesangial as well as lacis (extraglomerular mesangium) cells of the glomerulus. The coupling properties of such junctions would enable intra- and extraglomerular mesangium to act as a coordinated functional syncytium that may include granular cells of the juxtaglomerular apparatus (Boll et al., 1975; Taugner et al., 1978). These junctions were often found between different processes of the same cell (so-called reflexive gap junctions, for discussion of their possible role, see Majack and Larsen, 1980). Cells having reflexive gap junctions can be divided into two main groups (see Majack and Larsen, 1980): (1) secretory cells such as human uterine decidual cells (Lawn et al., 1971), rabbit luteal cells (Enders, 1973), human ovarian decidual cells (Herr, 1976), dog Leydig cells (Connell and Connell, 1977), and renal medullary interstitial cells (Majack and Larsen, 1980); and (2) smooth muscle cells such as are present in the guinea pig vas deferens (Campbell et al., 1971), in human arteries (Iwayama, 1971), in guinea pig iris (Gabella, 1974), and in hypertrophied rat and guinea pig intestine (Gabella, 1979). Since mesangial cells are rich in microfilaments and contain an actin-like antigen (Becker, 1972), the presence of reflexive gap junctions is another feature suggesting their similarity with smooth muscle cells. In fact, recent studies on cultured rat glomerular cells of probable mesangial origin have shown that these cells contract in response to arginine-vasopressin and angiotensin I1 (Ausiello et al., 1980). C. PROXIMAL TUBULE (FIGS.12-19; TABLES V,VI) In view of the functional differences between the pars recta and pars convoluta, such as the predominance of active secretory processes in the straight part

224

LELlO ORCl ET AL.

and the reabsorption of sugars and amino acids in the convoluted region (Burg, 1969: Tune et al., 1969; Tune and Burg, 197I ; Grantham et a/., 1974; Kawamura et cil., 1975: Jacobsen and Kokko, 1976; Jamison et al. , 1976; Jacobsen and Seldin. 1977), it is interesting that significant differences in the intramembrane particle size and distribution were found between these two regions. In the pars recta, all particles on the basolateral and apical plasma membrane belong to a rather homogeneous population, while in the pars convoluta, the particles present in the apical (luminal) membrane are larger than those found in the basolateral membranes. I n retrospect. it can be said that the larger size of the luminal P-face particles, rather than their increased number. gave the impression of a difference in particle density between luminal and basolateral membranes in the cortical proximal tubules illustrated by Miller and Revel (1975; see Fig. 9 of their paper). The tight junctions of the proximal tubule are morphologically “leaky,” which corresponds well with the low transepithelial resistance measured in this segment of the nephron (Hegel ct a / . , 1967: Boulpaep, 1971) and with the role of the proximal tubule in isotonic reabsorption of the filtered load (Huss and Marsh, 1975: Sackin and Boulpaep, 1975). However, the pars recta has, in several species, junctions that look “tighter” than those of the pars convoluta (Roesinger et ul.. 1978: Schiller et a/., 1978). a feature that may again reflect physiological differences between these two regions of the proximal tubule. In support of a physiological role of tight junctions in the proximal tubule is the demonstration that their development in an amphibian, Necturus maculosus, varies with a change in saline load (Hurnbert et nl., 1975a, 1976a). In control conditions, tight junctions were more developed than in tubules undergoing saline diuresis, a condition that increases the transepithelial electrical conductance (Boulpaep, 1972-see Section G ) . One further important point concerning the proximal tubule is the frequent occurrence of gap junctions between the epithelial cells. Apart from mesangial. lacis, and endotheiial cells in the glomerulus, no other cells of the nephron or of the collecting tubule have been found to possess gap junctions. In view of the role of these structures in ionic and metabolic coupling (Loewenstein. 1970; Gilula er al., 1972), it is reasonable to propose that the proximal tubule epithelium may represent a functional syncytium (provided the junctions are permeable).

D. LOOPOF HENLE(FIGS.20-22; TABLE VII) Our observations on the membrane of epithelial cells of long loops of Henle revealed a marked difference in membrane particle content between the descending (highly particulate) and ascending (sparsely particulate) limbs, that may be related to the differences in transepithelial permeability described in these two regions (De Rouffignac, 1972; Imai. 1977; Kokko, 1974). Since the exact mechanism of tubular fluid concentration in Henle’s loop is unknown (Jamison

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225

and Robertson, 1979), the high particle content in the initial part of the descending limb (see Schwartz et al., 1979) could reflect a high permeability to water (hydrophilic channels in membranes may appear as intramembrane particles in freeze-fracture) or a high ion-transporting activity (membrane-associated enzymes can also appear as intramembrane particles). In contrast, the lower particle content occurs in the ascending thin limb (Humbert et al., 1975b), a segment believed to be almost impermeable to water (Imai, 1977). A similar difference in particle content has been described for the vasa recta of the inner medulla, where the endothelial cell membranes of descending vasa are highly particulate compared to the ascending vasa (Schwartz et al., 1976). In addition, the apical membrane of the stratum corneum from amphibian epidermis, a tissue often used as a model transporting epithelium (Ussing and Zehran, 1951; Ussing and Leaf, 1978) also resembles that of the initial part of the descending loop of Henle (Brown and Ilk, 1979). Another structural characteristic of Henle’s thin loop membranes is the thick appearance of their tight-junctional elements. Morphologically similar junctions have been described between secretory cells in the avian salt gland, a tissue in which a large osmotic gradient is maintained between the lumen and the interstitial space (Riddle and Ernst, 1979). Although these junctions could be classified as ‘‘leaky on the basis of their low number of constituent ridges, this classification does not take into account the unusual, thick aspect of these ridges and their apparent continuity. Indeed, these junctions do not permit the passage of lanthanum tracer (Bulger, 1971; Pricam, 1974). Moreover, recent studies revealing species differences in membrane and tight-junction structure in different regions of Henle’s thin loop, as well as differences between long and short loops (Schwartz er al., 1979, 1980; Abramow and Orci, 1980; Schiller et al., 1980), emphasize the need for a detailed evaluation of structure-function relationships in this part of the nephron. ”

E. DISTAL TUBULE AND JUXTAGLOMERULAR APPARATUS (FIGS.23 and 24; TABLEVIII) As for the proximal tubule, differences in the freeze-fracture appearance of epithelial cell membranes were found between the pars recta (or thick ascending limb) and the pars convoluta of the distal tubule. The most obvious difference was the higher particle content of the lateral cell membranes in the pars recta. A further important distinction between the two regions was the presence, in the pars convoluta, of cells having elongated as well as globular intramembrane particles on their plasma membrane. These cells may be analogous to the dark cells of the collecting tubule (Griffith et al., 1968). The contribution of these structural differences to the known functional heterogeneity of straight and convoluted parts of the distal tubule (Burg and Green, 1973; Burg and Orloff, 1973;

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Giebisch and Windhager, 1973; Rocha and Kokko, 1973) remains to be established. The macula densa is a specialized region of the distal tubule that is associated with afferent and efferent arterioles of the glomerulus and with the cells of the extraglomerular mesangium. Together with modified (renin-containing) smooth muscle cells in the wall of the afferent and (to a lesser extent) efferent arterioles, this collection of cells is known as the juxtaglomerular apparatus (JGA) (Rouiller and Orci. 1969). Although it is assumed that the cells of the macula densa monitor the tubular fluid load, it is not known how this information is transmitted to other cells (in particular the renin-containing cells) of the JGA. Two possibilities of communication can be considered: direct coupling via gap junctions or indirect coupling via substances diffusing in the extracellular space (see Schiller and Taugner, 1979). At present. no gap junctions have yet been found (by freeze-fracture) either between constituent cells of the macula densa or between these cells and other cells of the JGA, which therefore renders the direct-coupling hypothesis unlikely. On the other hand, granular cells of the JGA are coupled by gap junctions to cells of the extraglomerular mesangium (Boll et al., 1975), and since these cells are, in turn. coupled to the intraglomerular mesangium (Pricam ct d., 1974b). the possibility exists that these cells form a functional syncytium that may be involved in the regulation of some aspects of glomerular function. Studies aimed at determining whether the tight junctions between cells of the macula densa are more “leaky” than those in the remainder of the distal tubule (thus supposedly facilitating equilibrium of distal tubular fluid at the level of the macula densa with the interstitial space in this region) are not conclusive. Giacomelli and Wiener (1976) reported that these junctions were, indeed, more permeable to lanthanum than junctions from the rest of the distal tubule (but see Tisher and Yargcr. 1971). whereas Schiller and Taugner (1979) found that all distal tubular junctions. including those at the level of the macula densa. were impermeable to horseradish peroxidase (at least in mouse and Tupaia, the only species examined with this tracer). Furthermore, the freeze-fracture appearance of tight junctions from the macula densa was no different from that of junctions between the remaining distal tubular cells (Boll et ul.. 1975: Schiller and Taugner. 1979).

F. COLLECTING T ~ I B U L E I . Light ( Priticiptil f Cells ( F i g s . 2-5 mid 3 - 3 3 ; Table3 I X . X , and X l l )

The luminal plasma membrane of the light cell shows great regional differences i n particle content from the outer stripe to the papillary ducts, cells from the latter region having the fewest particles and those from the outer stripe and cortex the most. This suggests that the term “light cell” includes a heterogene-

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227

ous population of cells that differ along the length of the collecting tubule. The characteristic specialization of basolateral membranes of light cells, the orthogonal arrays of particles, are also less numerous in the papillary duct. The fact that similar arrays exist on a variety of cell types including astrocytes (Dermietzel, 1973; Landis and Reese, 1974), intestinal epithelial cells (Staehelin, 1972), gastric parietal cells (Bordi and Perrelet, 1978), toad urinary bladder mesothelial cells (Wade, 1978), and basal cells from larval salamander epidermis (Robenek and Greven, 1980)-has not helped to uncover the role of these arrays, which is still entirely unknown. The only progress made since their original description has

FIG.29. Detail of the basal membrane of a light cell from the collecting duct. The complex infolding of the basal membrane produce irregularly shaped regions of P-fracture face surrounded by a wavy narrow band of extracellular space. The circles delimit some areas of the membrane containing orthogonal arrays of particles (see also Figs. 30 and 31). x71.OOO Bar = 0.5 pm.

228

LELIO ORCl tT AL.

FIG>.30 and 31. High magnification of the basal P- and E-faces of a light cell showing the subunit structure of the orthogonal arrays. On the P-face, the constituting particles appear square and flat-topped. On the E-face. the regular geomefq of the orthogonal arrays can be appreciated more readily: the pits complementary to the P-face particles form a bidimensional lattice with a periodicity of about 70 A . Larger globular particles are also visible. Fig. 30. x I16.000. Bar = 0.25 p.m. Fig. 31. x143.ooO. Bar = 0.1 p m .

been to exclude their gap-junctional nature, and a currently held hypothesis is that they are somehow involved in membrane permeability (Dermietzel, 1974; Rash et (11.. 1974; Anders and Brightman. 1979). The other characteristic membrane specialization of the light cell is found on the luminal plasma membrane: loose clusters of intramembrane particles that often adhere to the E-face. Previously described in the collecting duct (light cell) of vasopressin-treated Brattleboro and normal rats (Harmanci et ( i f . , 1978). these were later reported to be present in cells from the inner medulla but not the papilla (Lacy. 1980). Clusters of particles also appear on the luminal membrane of the amphibian urinary bladder granular cell following an ADH-induced increase in water permeability of the epithelium (Chevalier et al., 1974; Kachadorian el ul.. 1975). and they have also been found following isoprenaline or vasopressin treatment of toad epidermis, which causes a hydroosmotic response in this tissue (Brown er al., 1980). Although the particle aggregates found in the

Frm. 32 and 33. Fig. 32. Lurninal plasma membrane of light cells of the collecting tubule in the inner medulla. Some cells such as illustrated here have loose clusters of particles (circles). Clusters are present here in the E-face. but they can also he found on P-faces. The outpocketings indicated by arrows correspond to E-face views of the bases of rnicrovilli. x4I.OOO. Bar = 0.5 jm, Fig. 33. Higher magnification of the particle clusters (asterisks) in an otherwise poorly particulatcd mernbrane uYR.000. Bar = 0.25 @ni.

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LELIO ORCl ET AL

bladder and epidermis do not resemble morphologically the loose particle clusters found in the kidney, the observation nevertheless suggests that a rearrangement of luminal membrane components occurs in association with permeability changes in certain cell types from vasopressin-sensitive epithelia. 2. The Durk (Intercalated) Cell ( F i g s . 25-28; Table X l ) Dark (intercalated) cells are abundant in the initial part of the collecting tubule and become progressively scarcer toward the more terminal region. They seem to be completely absent from the papillary duct. The elongated intramembrane particles that characterize the plasma membrane of this cell type have also been found on dark cells from Chinese hamster, Acomys, and rabbit (Humbert et al., 1975~.1977, and unpublished observations), as well as on cells from the collecting duct of the amphibians Xenopus laevis, Ram ridibunda. and Necturus muc~it/osus(Brown, 1978, and unpublished observations). In addition, similar elongated particles have been described on a specific population of cells in several other transporting epithelia: the toad urinary bladder (Orci et al., 1974, 1975; Wade, 1976; Bourguet et al., 1976), amphibian epidermis (Brown e f al., 1978; Brown, 1979), rat epididymis (Brown and Montesano, 19801, hen lower intestine (Eldrup et a l . , 1980). primate colon (Neutra, 1979), and supporting cells of mouse olfactory mucosa (Kerjaschki and Horandner, 1976; Menco, 1980). In the kidney, bladder, epidermis, and epididymis, these cells have been called “mitochondria-rich (MR)” cells, since they contain more mitochondria than the surrounding epithelial cells. MR cells have a further common feature: they contain large amounts of the enzyme, carbonic anhydrase (Lonnerholm, 1971: Rosen, 1972: Rosen and Friedley, 1973; Lonnerholm and Ridderstrale, 1974; Cohen et a / . , 1976; Brown, 1980). So far, in those cells examined, the presence of elongated membrane particles is correlated with a high content of this enzyme, although the reverse is not necessarily true; that is, not all cells that contain carbonic anhydrase have elongated intramembrane particles (e .g., erythrocytes). Although the exact function of MR cells is unknown, evidence has FIGS.34-37. Composite plate showing the representative morphology of tight junctions on the P-faces of four regions of the urinary tubules (see Table XI11 and Figs. 38-41 for the E-face appearance of similar junctions). The tight junction of the pars convoluta of the proximal tubule (Fig. 34) i s characterized by junctional fibrils that are often ill-defined and discontinuous, indicative of a ”leaky“ junction. In the ascending thin limbof Henle (Fig. 35). the tight junction appears usually as one continuous strand. In the distal tubule (Fig. 36). the tight junctions are composed of several parallel elements to which portions of the E-face of h e adjacent cell often adhere (this also applies to the fibril of Henle’s thin loop). The tight junction is most developed at the level of the collecting tubule (Fig. 37). where it is formed by numerous branching fibrils. Fig. 34, x 104,oOO; Fig. 35, ~80,000; Fig. 36. ~ 9 5 , 0 0 0 Fig. ; 37, X50.000. Figs. 34-36: Bar = 0.25 pm. Fig. 37: Bar = 0.5 wm. (From Pricam,M.D.Thesis, University of Geneva, 1974.)

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been presented suggesting a role in acidification (Rosen et al., 1974; Frazier, 1978), which would be compatible with their high carbonic anhydrase content. Other work, however, has implicated them in sodium transport (Saladino et al., 1969; Scott el al., 1974; Ehrenfeld er al., 1976), chloride fluxes (Whitear, 1972; Voute and Meier, 1978), secretory processes (Ehrenfeld er al., 19761, and potassium exchange (Oliver ef al., 1957; Richet and Hagege, 1975). Freeze-fracture has revealed an increase in the number of cells with elongated particles in the rat collecting tubule in animals given a potassium-deficient diet-a treatment enhancing ammonia excretion and potassium reabsorption in this tubular segment (Stetson et al., 1980)-although the number of MR cells in various epithelia can also be modified under other experimental conditions (Hancox and Komender, 1963; Hagege et al., 1968; Frazier, 1978; Ilic and Brown, 1980). In addition, MR cells were believed to be the target cells for aldosterone (Voute er al., 1972; Sapirstein and Scott, 1975) and/or vasopressin (Scott er al., 1974; Goodman ef al., 1975) in the epithelia in which they occur, although more recent work has not confirmed these results (Handler and Preston, 1976; Rossier ef al., 1979). In fact, the response of amphibian skin to ADH is independent of the MR cell population of the epidermis (Brown er al., 1981). At present, therefore, there is much confusion surrounding their exact role, although some of the proposed functions just mentioned are not mutually exclusive. It is, however, probably safe to conclude that MR cells from different epithelia share a common transport function at the level of the cell membrane, in which the elongated intramembrane particles are implicated. G. TIGHTJUNCTIONSOF

THE

KIDNEYTUBULES

Figures 34-41 allow a direct comparison among junctions from different regions of the urinary tubule. When their structure is compared with the electrical resistance of the various segments, it can be seen that, with the exception of Henle’s loop, resistance increases along with junctional complexity (Table XIII). Since tight junctions are a major component in the control of the paracellular pathway, these results are in accordance with data relating well-developed tight junctions with low epithelial conductance, and vice versa, in other epithelia (Claude and Goodenough, 1973). However, in some cases, such a correlation is less straightforward (MartinezPalomo and Erlij, 1975; Frederiksen et al., 1979; Van Deurs and Koehler, 1979) FIGS.38-41. Composite plate showing the representative appearance of tight junctions as seen on the E-fracture face of the plasma membrane. On the E-face, the junctional element appears as a groove and the overall description of the junctional elements given in Figs. 34-37 also applies here. Fig. 38. Pars convoluta of the proximal tubule. ~ 7 1 , 5 0 0 .Fig. 39. Thin limb of Henle’s loop. ~80,500. Fig. 40. Distal tubule. X82,OOO. Fig. 41. Collecting tubule. X60,OOO. Bar in all figures = 0.25 pm, except Fig. 41 = 0.5 pm. (From F’ricam, M.D.Thesis, University of Geneva, 1974.)

234

LELIO ORCl ET AL. TABLE XI11 MORPHOLOGY OF THE TIGHT JUNCTIONS COMPARED WITH TRANSEPITHELIAL RESISIANCEIN EACH SEGMENT OF T H E URINARY TUBULE“**

Tubular segment

Transepithelial resistance‘ (fl/cm 2,

Proximal Henle Distal Collecting

5-7 ( 1 ) 700 (2)” 350 (3) 867 (4)‘

Number of junctional strands (minimal to maximal range)

0-6‘ 1-4

2-11? 6-14

Junction depth (nm) in apico-basal direction (minimal to maximal range)

0-130 15-190 30-210 140-400

~

~~

“Table adapted from Pncam er a/ [ 1974a) “See Figs 34-41 Number\ in parentheses are references from which the physiological data were taken ( 1 ) Hepel cr ol (1967). ( 2 )Abramow and Orci (1980). ( 3 ) Malnic and Giebisch (1972). (4)Helman er a/ (I97 I ) Values from the rabbit ’ Values from pars recta and pars convoluta 1

and in the kidney, the structure of the junctions found in the loop of Henle, which have an apparently “leaky” morphology, should be interpreted with caution. Thesejunctions are shallow and often consist of only one or two parallel strands. However. these strands are continuous and may indeed reflect a relatively ”tight” paracellular pathway. In the rabbit, where a correlated freeze-fractureelectrophysiological analysis has been performed for the first time on isolated thin descending limbs of the inner medulla, a high resistance (700 R/crn? was measured across the epithelium, but the tight junctions consist of only one to four strands (Abramow and Orci, 1980). This study shows, therefore, that junctions with relatively few parallel elements can nevertheless be an effective barrier to paracellular ionic diffusion, a fact that could not have been suspected on morphological grounds alone. Since ultrastructural appearance is not, therefore, a totally reliable means of determining the “tightness“ of a given junction, electrical and ultrastructural parameters should ideally be compared in the same tissue or tubule segment. Such an approach would, for example, help to determine the functional significance of the modulation of tight-junction structure that can be induced under a variety of experimental conditions (see Montesano er (11.. 1976; Orci ct ul., 1973: Wade and Karnovsky, 1974). This has been successfully achieved in the proximal tubule of the amphibian Nucturus muculosus, undergoing saline diuresis. It has previously been shown that in this animal, saline loading results in an increased paracellular conductance in proximal tubules (Boulpaep, 1972).

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When a quantitative analysis of the tight junctions from saline-loaded animals is performed, they are found to be significantly less developed than junctions from control animals (Table VI; Figs. 17.-19). In particular, individual ridges or grooves appear interrupted more often and the number of superposed elements is reduced (Humbert et af., 1975a, 1976a). These results complement the work of Bentzel (1972), who provided thin-section evidence for a reduction in size of tight junctions of Necturus proximal tubules during volume expansion. Thus, in this experimental model, a clear correlation exists between paracellular electrical conductance and the complexity of tight junctions, which emphasizes the importance of the paracellular pathway in determining overall permeability in this leaky epithelium (Windhager el al., 1967; Boulpaep, 1971; Anagnostopoulos and Velu, 1974; Bulger et al., 1974).

X. Concluding Remarks The systematic application of the freeze-fracture technique to the kidney has revealed an impressive mosaic of membrane structure among the epithelial cells lining the urinary tubules: there are considerable variations in the density, size, and shape of intramembrane particles between membrane domains of the same cell and among different cells, as well as in the degree of development of intercellular junctions between cells. Since intramembrane particles are considered to represent protein-containing structures, this morphological demonstration of particle heterogeneity can be interpreted to reflect different properties of the plasma membranes. In the same way, the difference in the number, continuity, and complexity of tight-junctional fibrils can be interpreted in terms of differences in the permeability of the paracellular pathway of the various tubular segments. In addition to providing important clues about membrane heterogeneity as far as proteins are concerned, the scope of the freeze-fracture technique has been recently widened by a new method involving pretreatment of tissue with the polyene antibiotic filipin. In this way, one of the major membrane lipids, cholesterol, can be detected morphologically (Elias et al., 1979; Montesano et al., 1979; Orci et a l . , 1980; Robinson and Karnovsky, 1980). Preliminary data obtained with this technique applied to kidney membranes are presented in Figs.. 42-44. Thus, a map of cholesterol distribution among kidney tubular membranes will soon be added to the map of morphological detectable proteins detailed in the present work. Clearly, morphology alone will not answer the basic questions concerning tubular functions, but it must be considered an essential part of future multidisciplinary studies using not only entire kidney but also isolated tubule segments (see Burg and Orloff, 1973; Humben et al., 1977; Morel et al., 1976, 1977; Abramow and Orci, 1980), isolated cell types and purified membrane fractions.

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ACKNOWLEDGMENTS The work (published and unpublished) summarized in this article has received continuous support from the Swiss National Science Foundation since 1973. The authors acknowledge the invaluable assistance of Miss Jacqueline Rial in collecting the numerical data, of Miss Isabelle Bernard in the typing of several versions of the manuscript, of Mrs. Marthe Sidler for the photographic work and of Mr. Max Baumann, Michel Bernard, Patrice Fruleux. and Patrick Sors for freeze-fracture and electron microscopy. We also thank Dr. R. C. De Sousa for helpful suggestions during the writing of the manuscript.

REFERENCES Abramow, M., and Orci, L. (1980). In!. J . Eiochem. 12,23-27. Allen, F., and Tisher, C. C. (1976). Kidney Inr. 9, 8-22. Anagnostopoulos, T . , and Velu, E. (1974). Pjlugers Arch. 346, 327-339. Anders, J. J.. and Brightman, M. W. (1979). J. Neurocyrol. 8, 777-795. Ausiello, D. A , , Kreisberg, J. I., Roy, C., and Kamovsky, M. J. (1980). J. Clin. Invest. 65, 754-760. Barajas, L., and Latta, H. (1967). Circ. Res. Suppl. 2, 20, 2 1 15-28. Becker, C. G . (1972). Am. J . Parhol. 66, 97-110. Bentzel. C. J. (1972). Kidney In?. 2, 324-335. Bergstrand, A,, and Bucht, H. (1958). 2. Zeluorsch. 48, 51-73. Boll, H. U., Forssmann, W. G., and Taugner, R. (1975). Cell Tissue Res. 161, 459-469. Bordi. C., and Perrelet, A. (1978). Anar. Rec. 192, 297-304. Boulpaep, E. L. (1971). In “Electrophysiology of Epithelial Cells” (G. Giebisch, ed.), pp. 98-1 12. Schattauer-Verlag, Stuttgart. Boulpaep, E. L. (1972). Am. J. Physiol. 222, 517-531. Bourguet, J., Chevalier, J., and Hugon. J . S. (1976). Eiophys. J. 16, 627-639. Branton, D. (1966). Proc. Natl. Acad. Sci. U.S.A. 55, 1048-1056. Branton, D. (1969). Annu. Rev. Plant Physiol. 20, 209-238. Branton, D., Bullivant, S., Gilula, N. B., Karnovsky, M. J . , Moor, H., Miihlethaler, K., Northcote,

FIGS.42-44. Freeze-fracture of kidneys fixed in the presence of 300 p m filipin. Filipin binds to membrane cholesterol and related 3-P-hydroxysterols and forms multimolecular filipin-sterol (FS) complexes which are visible as 25-30 nrn diameter studlike protuberances (or pits) on the fracture face. FS complexes are easily distinguished from the much smaller (8-10 nm) intramembrane particles. FIG.42. Fracture of the glomerular filtration barrier (compare with Fig. 4 ) showing a paucity (about 60/pmZ)of FS complexes (circles) on that part of the foot process membrane (FP) which lies against the basal lamina (BL). In contrast, the foot process membrane is heavily (about 500 complexes/pmz) labeled (asterisks) above the level of the slit diaphragm (dotted line), and the capillary endothelial membrane (En) adjacent to the basal lamina also contains many FS complexes (data from Orci et a/.. submitted for publication). US, urinary space. X52.000. Bar = 0.5 pm. FIG. 43. Part of the luminal P-face from 3 interdigitating cell processes in the ascending thin limb of Henle. The membranes of cells 1 and 3 contain many FS complexes while that of cell 2 has only very few. Some complexes are encircled (from Orci et al., in preparation). X30.000. Bar = 1 pm. FIG.44. Part of the basal plasma membrane of a collecting tubule light cell showing FS complexes (circles) sparsely distributed among characteristic orthogonal arrays (see Fig. 29). X82,000. Bar = 0.25 p m .

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A . Arthropod Tight Junctions in Thin Sections . . . . . . . B . Freeze-Fracture Appearance . . . . . . . . . . . . . C . Localization and Coexistence with Other Junctional Types . . Functional Significance . . . . . . . . . . . . . . . . A . Permeability Barriers . . . . . . . . . . . . . . . B . Compartmentalization of Plasmalemma . . . . . . . . . C . “Leakiness” Correlated with Ridge Number and Discontinuities D . Tissue Rigidity Affected by Extent of Junctional Reticulum . E. Adhesion . . . . . . . . . . . . . . . . . . . Arthropod Groups in Which Tight Junctional Structures Have Been Reported . . . . . . . . . . . . . . . . . . . . . A . Primitive Arachnids . . . . . . . . . . . . . . . . B . Crustaceans . . . . . . . . . . . . . . . . . . . C . Insects . . . . . . . . . . . . . . . . . . . . D . Arachnids . . . . . . . . . . . . . . . . . . . E . Myriapods . . . . . . . . . . . . . . . . . . . Junctions Exhibiting Certain Features in Common with Tight Junctions . . . . . . . . . . . . . . . . . . . . . A . Pleated Septate Junctions . . . . . . . . . . . . . . B . Continuous (Smooth Septate) Junctions . . . . . . . . . C . Reticular “Septate” Junctions . . . . . . . . . . . . D . Retinular Junctions . . . . . . . . . . . . . . . . E . GIial Cell Junctions in Primitive Arachnids . . . . . . . Comparison of Arthropod Zonulae Occludentes with Those of Vertebrates and Lower Chordates . . . . . . . . . . . . A . Differences in Localization . . . . . . . . . . . . . B . Differences in Fracturing Characteristics . . . . . . . . C . Differences in Types of Leakiness Observed . . . . . . . D . Differences in Developmental Stages . . . . . . . . . E . Differences in Insect versus Vertebrate Physiology . . . . F . Disruption of Junctions . . . . . . . . . . . . . . Stages in the Assembly and Breakdown of Arthropod Tight Junctions A . Intrainembranous Particle (IMP) Migration . . . . . . . B . Arthropods Yield Evidence for Distinction between Tight and Gap Iunctional Precursors . . . . . . . . . . . . . Models Constructed to Clarify the Features of Arthropod Tight Junctions . . . . . . . . . . . . . . . . . . . . . Possible Explanations for the Existence of Both Simple and Complex Tight Junctions in Arthropods-Evolutionary Implications . . . Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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243 Copynght 0 1981 by Academic Press. loc. All righia of reproduction In any form reserved. ISBN 0-12-364.173.’)

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I. Introduction Whether tight junctions exist in the tissues of invertebrates is an issue that has vexed at least some cell biologists for a considerable time. Many investigators have reiterated Satir and Gilula’s (1973) statement that ionulae nccludentes, as originally described in 1963 in vertebrate epithelia by Farquhar and Palade, are absent in tissues from nonchordate organisms. Those species possessing these sealing junctions include ones from all the conventional vertebrate phyla (see Claude and Goodenough, 1973; Staehelin, 1974; Gilula, 1978) together with such lowly chordates as the tunicates (Lorber and Rayns, 1972; Georges, 19791, included and so categorized because of their larval notochord. Until recently the remaining invertebrate groups appeared to lack any structurally comparable junctions and the physiologically analogous structures responsible for producing permeability barriers were thought to be the septate junctions (Satir and Fong, 1973; Satir and Gilula, 1973; Noirot and Quennedy, 1974; Lord and diBona, 1976; Filshie and Flower, 1977; Noirot-Timothee er a / . , 1978; Green et a / . , 1979; Noirot-Timothee and Noirot, 1980). Another aspect of the problem which has led to confusion concerning the existence of invertebrate tight junctions is that relating to the interpretation of thin-sectioned material. The advent of the technique of freeze-fracturing enabled evidence to be produced that demonstrated the three-dimensional nature of tight junctions in vertebrate tissues (Kreutziger, 1968; Staehelin et a / . , 1969; Chalcroft and Bullivant. 1970). Before this time, tight junctions were identified in thin sections merely as being close appositions of adjacent cell membranes (Farquhar and Palade, 1963). Moreover, before the advent of en bloc staining with uranyl acetate (Farquhar and Palade, 1965), it was not possible to distinguish accurately between the close appositions of tight junctions and those of gap junctions in that both structures appeared to be pentalaminar or five-layered. Given this ambiguity and the related problem that many investigators did not begin to use err bloc staining for a considerable time, it is perhaps not too surprising that the vertebrate literature in the 1960s, and even the early 1970s, is peppered with references to “tight” junctions which are, with the knowledge of hindsight, clearly gap junctions. They can readily be so identified since the close membrane apposition referred to earlier is maintained over some length of cellto-cell contact, which is characteristic of the gap junction in thin section, but not of the punctate tight junction. Such erroneous references to “tight” junctions also occur in the literature pertaining to the invertebrates, particularly the arthropods, relating to many insect tissues (Locke 1965; Osborne, 1966; Berridge and Gupta, 1967; Grimstone er al., 1967; Maddrell and Treherne, 1967; Smith 1968: Stuart and Satir, 1968; Oschman and Wall, 1969; Smith et al.. 1969; Schiirmann and Wechsler, 1969; Lane and Treherne, 1969, 1970; Treherne et

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al., 1970; Zacharuk t’t al., 1971; Leslie and Robertson, 1973; McLaughlin, 1974a; Peacock and Anstee, 1977). Examination of such published micrographs reveals that most, if not all of them, are very likely to be gap junctions. It must be borne in mind however, that although there were earlier references to the structure of the “nexus” (Dewey and Barr, 1963; Robertson, 1963) the septilaminar, en face subunit-packed gap junction was not described as such until 1967 when Revel and Karnovsky showed, by lanthanum staining, how two cells could be thus associated. So until the late 1960s the mistaken label of “tight”junction was frequently applied to gap junctions in a whole range of tissues. In addition, some five-layered profiles, resembling tight junctions, are labile, depending on the method of preparation (Johnston and Roots, 1967; Brightman and Reese, 1969). Here, unlike true tight junctions where it is less, the width of the middle dense line equals the sum of the thicknesses of the two outer leaflets of the apposed membranes (Johnston and Roots, 1972). These artifactual, labile, pentalaminar configurations are also distinguished from true tight junctions in that they do not exhibit the mirror image undulations in the two participating membranes, but are flatly apposed. The distinction between these tight and gap junctional types and the recognition that the two can and do coexist in insect tissues first came from thin sections (Lane and Treherne, 1972) in the central nervous system (CNS) of cockroaches. Earlier tracer studies had revealed that a permeabiity barrier existed to the entry of exogenous molecules (Lane and Treherne, 1970, 1971) while physical disruptions of the supposed cellular basis of the barrier (Lane and Treherne, 1969, 1970), the outer glial cell layer round the CNS, called the perineurium, destroyed this ‘‘barrier” effect. Moreover, electrophysiological studies showed that this cellular layer presented a restriction to the free entry of cations such as Na+ and K+ (Hoyle, 1953; Twarog and Roeder, 1956; Treherne et al., 1970; Treherne and Pichon, 1972). For example, incubation of the intact CNS in Na’-free saline did not lead to any diminution of the conventionally propagated nervous impulses within the CNS (Treherne et al., 1970); were there no barrier, the system should have ceased functioning in the absence of Na+. These experiments all suggested that a true permeability barrier existed here similar to those observed in a variety of vertebrate epithelia (Erlij and Martinez-Palomo, 1978). However when the fine structure of the insect perineurium is examined carefully, punctate cell appositions are not in evidence near the apical surface. Instead, the cell borders are found to interdigitate in a highly complex way, exhibiting many gap and septate junctions between them (Maddrell and Treherne, 1967; Lane et al., 1977a) with rather basally positioned punctate appositions (Lane and Treherne, 1972; Lane, 1972; Lane et al., 1977a). There seems little doubt that the presence of extensive septate junctions here in the perineurium, as well as between the lateral cell borders of a range of other

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invertebrate epithelial types, such as the gut, salivary gland, and Malpighian tubules (see references in Lane and Skaer, 1980) led to the assumption that they were physiologically Comparable (Noirot and Noirot-Timothee. 1976)to the tight junctions found in similar positions in vertebrate epithelia. However, two main points have to be considered here. First, such studies as have been made with tracers on invertebrate epitheiia have been rather erratically done. Frequently, studies on colloidal lanthanum staining (where lanthanum is added to the fixative) are erroneously said to be comparable to the more physiologically meaningful technique of incubating in ionic lanthanum (where low concentrations of lanthanum are added to physiological saline) before fixation. No really thorough, consistent, and rigorous study has been made of the freedom of access of tracers into these invertebrate tissues, although there is the occasional suggestion that the data indicate that the septate junctions are not occluding to the entry of certain ions and molecules (Maddrell and Gardiner, 1974; Lane, 1978b, 1979a). Second, the physiology of invertebrates is markedly different from the vertebrates in a variety of cases. This is especially well documented in the case of the insects, with which a great many studies have been particularly concerned. It seems that most substances taken into the insect digestive system can readily penetrate through the gut wall into the hemolymph (Maddrell, 1980). Important systems such as the CNS, testis, and eye are then protected from the fluctuating ionic composition of the hernolymph by having evolved barrier systems. Of the gut tissues through which must pass the ingested substances via an intracellular or intercellular route, preliminary studies show that most do not seem to restrict completely the free extracellular passage of materials (Skaer et al., 1980); the adjacent cells are associated in nearly every case, not by tight junctions, but by extensive septate junctions. The eye, testis, and CNS of insects which possess a blood-eye (Shaw, 1977), blood-testis (Szollosi and Marcaillou. 1977; Jones, 1978), and blood-brain barrier (Lane et a / . , 1977a), possess both punctate tight junctions and septate junctions. However, it appears that it is the tight junctions which restrict the entry of exogenous substances, although the septate junctions may play some part in modulating the entry of certain molecules. For example, a highly efficient blood-brain bamer operates in the tobacco hornworm, the CNS of which lacks septate junctions entirely (Lane and Swales, 1979; Tolbert and Hildebrand, 1981). There seems then to be evidence from both physiological experiments and tracer incubations that occluding tight junctions exist in the tissues of arthropods as entities distinct from the septate junctions. Before considering further details of this evidence in various systems, the structural features of the Zonulae occludentes found in such organisms, as visualized by both thin section and freeze-fracture techniques, should be analyzed.

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11. Fine Structure

A. ARTHROPODTIGHTJUNCTIONSIN TH INSECTIONS 1 . Historical Background

Tight junctions proper, in contrast with gap junctions incorrectly thought to be tight junctions in non-en bloc stained tissues (see Section I), were originally found in tissues of the cockroach in 1972 by Lane and Treheme in the glial layer, or perineurium, that ensheathes the insect central nervous system. They were also seen in the perineurial layer in the CNS and large peripheral nerves of other insects as well, such as the adult moth, Manduca sexta (Lane, 1972) and the locust (Schistocerca gregaria) (Lane and Treheme, 1973). In all cases their presence could be correlated with the presence of a permeability barrier measured both electrophysiologically (Treheme e t a / . , 1970; Pichon et al., 1972; Treheme and Pichon, 1972) and by impedance to the free entry of tracer molecules (Lane and Treherne, 1971, 1972; Lane, 1972). Establishing the unequivocal existence of punctate tight junctional appositions proved to be difficult because the location of the junctions was found to be basal, in a tissue where extreme interdigitation along the lateral border was a prominent feature (Fig. I); in such a complex system, it was not easy to discern punctate appositions which were not only abbreviated, but also relatively infrequent. Subsequent studies corroborated the existence of punctate tight junctions in the insect CNS in thin sections in that they were reported in the larval and pupal perineurium of Manduca sexta (McLaughlin, 1974a), between the perineurial cells of hatchling and later instars of Carausius rnorosus (Leslie, 1973), and in the CNS of a range of insects (Lane et al., 1977a), including both the larval and adult blow fly Calliphora erythrocephala (Lane and Swales, 1978a,b) and the embryonic and hatchling tobacco hornworm moth Manduca sexta (Lane and Swales, 1979). Further investigations on other tissues, such as glial cells (Lane and Swales, 1978a,b; Lane and Skaer, 1980), the testis, where a blood-testis barrier is operative (Szollosi and Marcaillou, 1977), parts of the gut (Lane, 1978b, 1979a; Skaer et al., 1980), and the eye (Lane, 1981b) where a bloodretinal barrier exists, have all revealed in thin sections, a number of punctate appositions between cells which could represent tight junctions. The perineurial layer in the CNS of other arthropods has also yielded evidence to support the existence of tight junctions in thin sections, such as in the spiders (Lane and Chandler, 1980; Lane, 1981a), scorpions (Lane and Harrison, 1980; Lane et al., 1981), and centipedes (Lane, unpublished data). In all cases the thin-sectioned appearance of the tight junctions was very much the same, as described in the following section.

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2. Features in en Bloc Stained Preparations

The characteristic appearance of tight junctions in arthropod tissues is essentially similar to that reported for zonulae occludentes in vertebrate material as described originally in the now classic papers by Farquhar and Palade (1 963, 1965) and substantiated by subsequent investigations in a range of tissues (see Staehelin, 1974; Gilula, 1978). The membranes of the adjacent cells that are associated by tight junctions are fused together in a number of punctate appositions so that the intercellular cleft, normally 10-20 nm in width, may be totally obliterated at these points (Fig. 2). The effect of a number of these occurring close together is to produce an undulating, mirror-image scalloped appearance along the lateral border of the two participating cells (Fig. 3). At these points of cell-to-cell apposition the total width of the two membranes in thin sections is less than the sum of two measured separately, as originally calculated in vertebrate tissue by Reese and Karnovsky (1967), indicating a degree of real fusion between the membranes. This will obviously affect the movement of ions and molecules through the intercellular space since such substances will be unable to diffuse freely past cell-to-cell fusions; in this respect the overall arrangement of the structures composing the junctions is important since their three-dimensional conformation as seen by freeze-fracture will clearly have an effect on the completeness of the seal produced. The impact of these morphological restrictions and the changes in permeability imposed by their structural variability on the FIG. 1 . Outer perineurial layer of an insect CNS, in this case the moth Mariduca sexra. illustrating the manner in which the lateral borders of adjacent cells are interdigitated with one another in a highly complex way. Note that the result of this is that the cell borders lie parallel to the neural lamella or periphery of the tissue, not at right angles to it. X36,700. FIG. 2. Intercellular junctions from the perineurium of the spider Tegenaria. Gap junctions (GJ) are intercalated between punctate tight junctions (arrows) which produce a scalloped appearance. En bloc uranyl-calcium staining enhances the density of the outermost membrane leaflet. (From Lane and Chandler, 1980.) X81,500. Inset shows a tight junction without uranyl-calcium treatment. ~83,500. FIG. 3. Multiple punctate tight junctions (arrows) along the interdigitating intercellular cleft between adjacent perineurial cells (PN) of a late embryonic moth. X 104,400. FIG. 4. CNS of a garden spider incubated with ionic lanthanum; the tracer has penetrated the perineurial clefts partway, leaking past discontinuous tight junctional ridges (arrows) which appear as stain-excluding fibrils. X46.500. FIG.5. Tight junction from a blood vessel of an adult garden spider. Aranaeus, showing that the opaque ionic lanthanum is unable to penetrate beyond a punctate junctional fusion (arrow). X 100,600. FIG. 6. Thin section of CNS and peripheral nerve of the cockroach, P e r i p h e t a , after incubation in ionic lanthanum; the tracer penetrates the neural lamella (NL) and in the CNS moves down into the perineurial (PN) clefts, but does not move beyond into the underlying region of glia (G) and axons (A). In the peripheral nerve, the perineurium is absent and no tight junctions are present: here the tracer reaches the surface of the axons (arrows). X 13,900.

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physiology of the tissues possessing these junctions will be considered later (see Section 1II.A). The structural features of tight junctions are readily demonstrated in thin sections by en bloc staining procedures (Fig. 2). In some cases, for example when the tissue is treated with both calcium and uranyl salts, or when lanthanum is added to the fixative, the stains are unable to gain access to the lines of fusion between the two cell membranes held together by these junctions. Hence these are left unstained as nonopaque fibrils lying against the background stain (Fig. 4) which adheres to the external half of the membrane leaflets (Fig. 2). The tight junctions revealed in thin sections in arthropod tissues are, in nearly every case, homocellular, that is, they occur between homologous cells. This is the case for those between adjacent perineurial cells, glial cells, and the epithelial cells of the gut (see Section 1II.A). To date, there are few reports of heterocellular tight junctions in the arthropods: one such report relates to those between the perineurial sheath cells and the underlying glial cells of the embryonic moth nervous system (Lane and Swales, 1979). In this case. the two kinds of cells appear to be heterologous but as they are both thought to be glial in origin, they are rather an equivocal example of a heterocellular interaction. Another report is that between glial cells and nerve cells, that is, axoglial tight junctions and those between glial cells and tracheae, called tracheoglial junctions (Lane and Skaer, 1980), both of which are ill-defined because they are probably fasciar rather than zonular, and unpredictable in terms of location and precise physiological significance.

3. Frcirrtrrs (IJkrIncubation nith Tracers: Total E.rclusion or Partiul Lecrkagc When arthropod tissues are incubated in saline to which tracers, such as ferritin. horseradish peroxidase, microperoxidase, ferrous gluconate, or lanthanum. have been added, the inward intercellular penetration of these exogenous substances is restricted. Since the lateral borders between adjacent cells in many arthropod epithelia are enormously interdigitated (see Fig. I ) , it was initially less than easy to discern the precise point at which the tracers were denied entry and by what structures. Further. in the nervous system, one of the most frequently studied arthropod tissues, the tight junctions may be at the basal surface of the perineurial cell layer. as in insects (see Lane and Skaer, 1980) or near the outer surface, as in spiders (Lane and Chandler, 1980). The entry of the tracer in tissues from the latter can be seen to be relatively promptly stopped (Fig. 5 ) near the periphery of the ganglion (Lane and Chandler. 1980); in the insects the perineurial occlusion occurs nearer the base of the intercellular clefts (Fig. 6). In both cases. as in the scorpion CNS, the tracers may be seen to move past what appear to be close membrane appositions (Fig. 8) before being stopped by subsequent fused membrane appositions. The explanation of this is evident once their structure is studied in freeze-fracture replicas (see Section 11,B42):discon-

/-Cytoplasm-/-

E Face -

Lanthanum in

-\+- intercellular -+P

Face'-

,

FIG. 7. Model of the leaky tight junctions found in such arthropods as the scorpion. The junctional ridges, on the EF in fixed tissue, may be discontinuous. in which case lanthanum can leak around them, but they serve to pull the adjacent membranes a bit closer together (as at D and E). Hence the intercellular cleft is reduced, but not obliterated as it is at A, B, and C where the ridges are continuous and the adjacent membranes are, therefore, fused together all along the length of the ridges. The tracer can move through the reduced clefts (at D and E). but not beyond the points of fusion where the extracellular space is obliterated (at A, B, and C). The unstained fibrils Seen in the lanthanum-filled intercellular space would be where the ridges in the two faces are fused together to exclude the tracer. The P face exhibits grooves complementary to the EF ridges. (From Lane eral.. 1981.)

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tinuous tight junctional ridges, when present, may allow leakage of tracers through the interstices created by these very discontinuities (see model in Fig. 7). This partial leakage, slight in the spider CNS, is really extraordinarily extensive i n the scorpion nervous system (Lane ef a / . , 1981). The entire lateral perineurial borders seem peppered with close appositions through which the tracers move (Fig. 8). This unimpeded entry occurs near the peripheral border of the nervous tissue, by the neural lamella, but with inward penetration, the tracers finally are stopped (Fig. 8) before they reach the base of the perineurium. These partial pinchings together of the adjacent perineurial membranes in the periphery may also be explained by the freeze-fracture data (Section II,B,2) as discontinuous intramembranous ridges between which the tracers are free to percolate but only up to the point where discontinuities disappear (Lane and Harrison, 1980). Such a partial leakage between discontinuities in the tight junctional ridges has also been observed in the internal blood sinuses, or channels, within the arachnid central nervous system (Lane et a / . . 1981). These seem to be more leaky electrophysiologically than the outer perineuriurn, and also admit tracers between close intercellular appositions for some distance inward from the injected, tracer-filled, intraganglionic sinuses (Fig. 9) (Lane el d.,1981). Although less striking visually. the crustacean nervous system bears some resemblance to this,

FIG 8 Thin-section from the perineurium of the scorpion, Paritroctonus. after incubation with ionic lanthanum. The tracer has moved through the outer neural lamella (NL) and down into the penneurial (PN) clefts. These exhibit a number of nonopaque fihrils (arrows) which are assumed to he discontinuous stain-excluding ridges or areas where the two cell memhranes are fused together in a fasciiu way. As a result, the lanthanum can leak around the discontinuities and does so over quite romr distance but is ultimately stoppcd at the base of the perineurium (B) in the CNS. ~ 3 4 , 8 0 0 . F K . 9 . lntraganglionic blood channel (BC)from the C N S of the scorpion showing the accessibiiits of these channels to tracer injected into the hcan. The perineurial-like cells lining the channels restnct the entry of tracer along their intercellular clefts such that it never gets along their full extent: this restriction is effected by the intercellular tight junctions. The arrows indicate the areas wherc discontinuous junctional strands occur. similar to those found in the perineurium proper (as in Fig. 8). x I I.2(!0. FIL. 10. Freeze-fracture replica of a simple insect tight junction with the typical single intramembranous PF ridges (arrows) lying parallel to the outer CNS periphery. x60.?00. FIG;. I 1 Simple tight junction from an insect showing the relatively parallel alignment of two or more ridges in the perineurium. x 74.100. FK,. 12. Simple insect tight junctions revealing that they are composed of intramemhranous particles aligned side by side and fused into ridge-like structures. X 71.600. i-~ti. I ? . Complex tight junctions from an adult spider which show some degree of latcral fusion betwecn thc aligned intramembranous particles that comprise the junctions. X40,lSO. FIG. 14. Embryonic insect perineurium with intramembranous particles (arrows) becoming aligned into ridges in the presumptive junction-bearing region tci form simple tight junctions. v 35.200. FIG. 15. Insect glial cells demonstrating the existence of short discontinuous EF grooves. ..57.xOo.

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in that the macular tight junctional perineurial appositions are circumvented by tracers (Lane et a l . . 1977b); however, in both the crayfish and the crab, the perineurium is ultimately relatively leaky and, unlike the scorpions or spiders, the underlying nervous tissue is accessible to circulating ions and molecules (Abbott, 1970, 1972; Lane et a l . . 1977b; Abbott et al., 1978). B. FREEZE-FRACTURE APPEARANCE

Freeze-cleaved replicas have the advantage over thin sections for visualizing junctional structures in that en face views of membranes are revealed and the intramembranous patterns assumed by junctional particles may then be studied. The tight junctional particles are aligned in ridges which are shared, by fusion of the plasma membranes of adjacent cells, thereby leading to closure of the intercellular space. With this additional dimension it becomes possible to distinguish two chief types of tight junctions in arthropods which cannot be discerned to be different in thin sections. These two types exhibit the same basic structural features, but display variations in the degree of complexity of the arrangements of their component intramembranous ridges; they are referred to here as simple and complex tight junctions. The terminology used to describe the plasma membrane in freeze-fracture replicas refers to fracture face P (P face or PF), the cytoplasmic or inner membrane half, and fracture face E (E face or EF), the extracellular or outer membrane half. 1 . Simple Tight Junctions

Simple tight junctions are not entirely unique to the arthropods since some vertebrate tissues also possess very simply organized tight junctional structures. These latter are often referred to as “leaky” junctions (Claude and Goodenough, 1973), since they have fewer linear ridges indicative of cell-to-cell fusion with which to occlude the extracellular space (see Section 111,A). Details of these in comparison with those of insects will be considered later (Section Vi). These junctions are found around the circumference of the cells forming diffusion barriers, and are characterized by single intramembranous P face ridges or E face grooves which are often discontinuous but variable in length (Fig. 10). In some cases two or three may lie in parallel with one another, occasionally in fairly close proximity (Fig. 1 1). They are clearly moniliform and hence appear to derive from linear arrays of intramembranous particles, 8-10 nm in diameter, which are fused laterally. The degree of this fusion is variable and ranges from the distinctly bead-like (Fig. 12) to ridges displaying partial fusion (as in Fig. 13). to smooth strands (Fig. 20); this aspect of course may be affected to a large degree by the angle of shadow, but may also be a developmental feature, the ridges possibly deriving from the moniliform alignments by the addition of “covering material” (Montesano er al., 1975; Elias and Friend, 1976; Luciano

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et a l . , 1979) as occurs in some vertebrate tight junctions with maturation. This feature is considered at greater length elsewhere (see Section VII) but it might be noted in passing that bead-like arrays, as distinct from ridges, are often particularly prominent in embryonic tissues (Fig. 14). The E face grooves (Fig. 15) are considered to be coincident with the PF ridges or fibrils, but complementary replicas have thus far not been successfully made of simple arthropod tight junctions. Moreover, fracture face transitions from PF to EF are rare in insect junctional areas. Since insect tight junctions are primarily simple ones, which occur in regions of complex interdigitating cell processes in most tissues, the ridges or grooves themselves are only rarely encountered. The result of these phenomena is that unequivocal face transitions to show (a) the obliterated intercellular cleft and (b) the coincidence of PF ridges with EF furrows across such transitions, are as yet unavailable. To this extent the evidence for the simple tight junctions in insect tissues has been unsatisfactory and has not convinced some sceptics that they could form an effective seal (Green et al., 1979). The discontinuous nature of the strands has also raised doubts (NoirotTimothee and Noirot, 1980) as to the validity of the theory that the simple ridges and grooves in insect tissues could be the morphological basis of the electrophysiologically observed permeability barriers. That this might in fact be so is considered at length elsewhere (Lane et a l . , 1977a; Section III,A,6).

2 . Complex Tight Junctions-Range of “Tightness” The distinctions between these and the simple arthropod tight junctions are made entirely on the basis of their freeze-fracture morphology because the electrophysiological evidence for the barrier properties of the complex tight junctions is too slight as yet to be able to judge differences in their relative permeabilities. Complex tight junctions in arthropods are characterized by a distinct circumferential reticulum around the cells forming permeability barriers; they are composed of a network of moniliform ridges on one fracture face, usually the PF (Fig. 16), and a complementary interconnecting array of grooves, usually in the EF (Fig. 16). These preferential fracturing faces are found consistently in insect and some arachnid tissues independent of the pretreatment, that is, whether the material is frozen without fixing or after chemical fixation. However, in other arachnid tissues, notably the scorpions, the fracture face varies depending on whether or not the tissue has been fixed; in such cases, the unfixed cryoprotected material reveals the conventional P face ridges and EF grooves, while fixed, cryoprotected tissue possesses EF ridges. In possessing EF ridges they resemble the tight junctional system in some vertebrate tissues, notably that in the bloodtestis barrier (Nagano and Suzuki, 1976) and endothelium (Simionescu et al., 1978) (see Section V1,B). These results are at variance with the theory proposed by van Deurs and Luft (1979) which suggests that chemical fixation causes the particulate ridges to cleave onto the PF, while in unfixed tissues, the ridges shift

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over to the E face. Speculations as to the reasons for these unexpected results in the arthropods are considered later (see Section VIII). The complex tight junctions found in arthropod tissues display the obliterated intercellular cleft at the point where punctate membrane fusion occurs (Fig. 17). As a result of these lines of fusions, a “quilted” appearance is found in the E face where the furrows (valleys) occur (Fig. 18) while the opposite effect, that of a series of raised ridges (hills) occurs on the P face (Fig. 18). This imagery of valleys and hills was first described in vertebrate tight junctions (Chalcroft and Bulliant, 1970). These membrane undulations result in a scalloped look when the intercellular cleft is cross-fractured (Fig. 19), with both membranes pinching in at the points of fusion. When the fracture plane cleaves across a face transition, the PF ridges, although offset, are coincident with the EF grooves (Figs. 19 and 20), thereby indicating their complementary nature. The component intramembranous particles which compose these fibrils or ridges are about 8-10 nm in diameter, the same size as those that make up simple insect tight junctions (Lane and Skaer, 1980) and vertebrate zonulue occludentes (Gilula, 1978). In some cases, due, it seems, to the discontinuous nature of some of the monilifortn fibrils, a PF ridge will terminate at a face transition, and no complementary furrow will therefore be found in the EF (asterisk in Fig. 16). This could be misleading insofar as it could suggest a lack of complementarity between the ridges and grooves, but it seems very likely that the numerous ridge discontinuities (Fig. 21) are responsible; ridges may suddenly terminate just as the plane of fracture shifts from the PF up to the EF so that no groove will be found. Although the angle of shadowing may well affect this feature, the ridges in some cases seem offset with respect to the furrows [Figs. I9 (large arrows) and 201. In other instances, it is difficult to determine if this is so. Due to lipid collapse (Bullivant, 1977), it is impossible to be sure whether the relatively considerable height of the PF fibrils is so because they have fractured off as two fibrils together or because they have cleaved as a single fibril, the height of which is enhanced by the collapse of the surrounding lipid. In either case, the fibrils in some regions appear as solid smooth strands (Fig. 20) and in others as beadlike linear arrays (Fig. 16). In mature arachnid tissues the latter appear to be as FIG. 16. Complex tight junctions from the spider Tegemria showing the networks, existing as PF ridges and EF grooves. that compose them. X43.700. FIG. 17. lnterdigitating perineurial borders from a spider exhibiting the punctate fusions (arrows) where adjacent cells are sealed together. The P face (PF) ridges are coincident across face transitions with E face (EF) grooves. X43.700. FIG. 18. Complex tight junctions from a spider with the P face (PF) ridges sitting up on membrane elevations, and the E face (EF) grooves resting in furrows or troughs. C, cytoplasm. x25,100. FIG.19. lnterdigitating perineurial processes in a spider with numerous punctate tight junctional appositions (as at arrows) along the lateral borders. Thick arrows indicate EF furrows coincident, but offset, to the PF ridges. X30.700.

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common as the former, so it seems that there can be no consistent developmental change involving a “covering up” of the particles with some other substance (cf. Lucian0 et al., 1979); in any case, it is difficult to visualize how this could happen within the membrane. The range of tightness exhibited by’these complex occluding junctions may be considerable, depending on the extent or depth of the junctional reticulum, the intricacy of its network, and the number of discontinuities that are present. This is reflected in the extent to which such a junctional complex allows tracers like ionic lanthanum to permeate across its “territory.” If the junction has many breaks in the ridges, tracers are able to leak at least partly through, although not usually, in those tissues thus far studied, completely so, presumably due to the topography of the cellular interdigitations. It may be that some ionic leakage does occur, as happens in the case of the simple insect tight junctions where, for example, there is a finite leak to Kf in the CNS (Pichon et al., 1971). Such evidence as is currently available for the complex junctions relates only to Ca2+ and Mg2+ and it seems that they can leak in (or out) more rapidly between the junctions in the blood channels within ganglia than they can through those located in the peripheral perineurial layer (Lane et al., 1981). This may be related to the overall surface area occupied by each of the junctions (see Section 111,C). C. LOCALIZATION A N D COEXISTENCE W I T H OTHER JUNCTIONALTYPES 1 . Localization and Distribution

The tight junctions thus far observed in arthropods have been found between cells in those tissues where it has been established that a permeability barrier exists (see Section 111,A). These include the central nervous system where there FIG. 20. Complex arachnid tight junctions with the P face (PF) ridges coincident. but slightly offset, with E face (EF) grooves over face transitions. Unfixed tissue. ~ 7 3 , 6 0 0 . FIG. 21. Perineurial tight junctions from the spider with typical PF ridges and EF grooves showing the discontinuities that exist. especially toward the apical or basal part of the junctional belt. Gap junctions (*) are intercalated between them as the two junctional types frequently coexist in intimate spatial association with one another. x23.700. FIG. 22. Tight junctions (arrows). gap junctions (CJ), and desmosomes (D)between adjacent perineurial cell borders in an insect. Septate junctions also coexist with tight junctions, as shown in the inset. X 116,000. Inset, X 169,100. FIG. 23. Replica of glial cells from the CNS of the horseshoe crab, Limulus. Gap junctions (GJ), characterized by clusters of E face (EF) particles and P face (PF) pits lie in close proximity to PF particle alignments and complementary (at arrows) EF grooves. X 53,400. FIG.24. Extensive E face (EF) grooves in Limulus glial cells, fracturing down (at arrows) into coincident PF ridges: in some cases there appears to be an obliteration of the intercellular cleft at these junctures. X26,600.

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is a blood-brain barrier (Treherne and Pichon, 1972; Lane, 1972; Lane er af., 1977a), the compound eye, where a blood-retinal bamer exists (Boschek, 1971; Shaw, 1977, 1978), and the germ cells, where a blood-testis barrier (Szollosi and Marcaillou, 1977; Jones, 1978) is to be found. There are other sites where diffusion bamers or concentration gradients have been observed, for example, in such tissues as epidermal sensory organs (Keil and Thurm, 1979), or others where occluding structures have been found, for example, in certain parts of the digestive tract (Skaer et a l . . 1980); here confirmatory structural or physiological data are as yet not forthcoming to corroborate these as unequivocal barrier systems. Within a given tissue the position of the tight junctions ha5 tended to be rather different from that in vertebrates, where in epithelia, for example, they are always located near the apical or luminal surface (Farquhar and Palade, 1963, 1965). In insect tissues, they usually are basal rather than apical, if the tissue is epithelial or epithelial-like whereas in arachnids they are usually apical (but see Section VI,A for a consideration of the terminology of “basal” and “apical” in this context). They always occur between the borders of cells which are highly interdigitated (see Sections IV,A-E), rather than those with straight lateral borders as occurs in vertebrate epithelia. The tight junctions appear to have a circumferential distribution around the lateral borders of each cell in the layer of tissue in which they are found. In ganglia, for example, they occur around the cells that in turn ensheath the nervous system; hence their beltlike distribution can serve as the basis for an effective permeability barrier. The distribution of the complex tight junctions is more readily discerned (e.g., Lane and Treherne, 1980. Fig. ID) than that of the simple tight junctions because the former are more apically oriented, cover a broader area of membrane, and hence are easier to “spot.”

2. Coexistence Tight junctions coexist with a vanety of other junctional types. The most frequent and striking is the gap or communicating junction and this association has been found to be so in nearly every case thus far examined (Lane and Skaer, 1980). With simple tight junctions both junctional types are found on the same cell membranes. In the case of the complex tight junctions, the gap junctional intramembranous particle clusters occur in the interstices of, or near, the network of ridges which comprise the zonulne occludentes (Fig. 21) (Lane and Chandler. 1980: Lane Pt a/.. 1981). Since the gap junctional particles fracture onto the E face in arthropod tissues (Flower, 1972) and since they are larger than the tight junctional particles (Lane 1978a; Lane and Skaer, 1980). they are readily distinguished as a separate class of intramembranous particle. This feature has been extremely useful in developmental studies where stages in the formation of the two categories of junction can clearly be separated (see Section VI1,B); since this

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distinction is not always possible in vertebrate tissues, it is a convenient arthropod characteristic as it clearly demonstrates that each junctional type originates from a different class of precursor particle (Lane, 1980, 1981a,c). Tight junctions also, in certain cases, coexist with septate junctions. This is always so, thus far. in insect tissues with the notable exception of the moth Manduca (Lane and Skaer, 1980) but never so, as far as is yet established, with Arachnids. In the insect nervous system, the simple tight junctions lie beneath or between extensive septate junctions, as well as gap junctions and desmosomes in the perineurium (Fig. 22) (Lane et a l . , 1977a). Similarly, in the rectal pads of cockroaches the simple tight junctions lie along the same lateral border as both septate and scalariform junctions in addition to gap junctions and desmosomes (Lane, 1979a,b); in this case the tight junctional elements appear to be restricted to the basal part of the cells, near the basement membrane (Lane, 1979a). In esophageal tissues they also coexist with gap and septate junctions as well as desmosomes and tend to occur near the luminal, cuticle-covered surface (see Figs. 28 and 29). 111. Functional Significance

A. PERMEABILITY BARRIERS Tight junctions or zonulae occludentes (Farquhar and Palade, 1963) are considered to provide a seal between epithelial cells, thereby producing a diffusion barrier to the intercellular movement of ions and molecules. A measurable electrical resistance develops if the concentration gradient across the cells thus associated is steep and the two compartments so formed are kept distinct. In arthropods, the tight junctions that have been observed are not always found between epithelial cells that form a layer with circulating body fluids on either side. Frequently tissue lies on one side with only a very restricted extracellular space between it and the epithelial-like cell layer which possesses the tight junctions. This poses certain problems in terms of measuring the transepithelial resistance and in analyzing the movement of tracers in both directions across the tight junction-bearing cell layers of arthropods in comparison with the situation in vertebrate tissues possessing tight junctions, such as kidney, gall bladder, .intestine, endothelium, and so on. That the tight junctions are the structures involved in permeability control has been demonstrated in vertebrates by correlating experimentally induced changes in permeability with morphological changes, such as blistering, in the junctions (Wade et al., 1973; Wade and Karnovsky, 1974b). More direct evidence for vertebrate tissues derives from the exclusion of tracer molecules at the level of the focal tight junctional occlusions (Reese and Kamovsky, 1967; Goodenough and Revel, 1970). In the arthropods, the evidence is often less precise as will be apparent in the following sections.

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I . Blood -Bruit1 Burrier-Perineurial

and Glial Effects

The central nervous system in insects is avascular, hence ions and molecules are only able to gain access to the nerve cells and their associated glial cells by dint of moving across the sheath of cells that surround them. Oxygen gains entrance via the tracheoles, of which there is an abundant supply. The sheath cells are modified glial cells, called perineurial cells (Wigglesworth, 1959), and the basal lamina which separates their outer surface from the hemolymph is commonly termed the neural lamella (see Ashhurst, 1968; Lane, 1974). There is also a perineurium and neural lamella around the central nervous system in the crustacea and in such Arachnids as spiders and scorpions, but in these organisms it appears that the ganglia are vascularized (see Lane and Treheme, 1980). Blood sinuses and intracerebral channels have been reported in crustacea (Sandeman, 1967; Abbott, 1971, 1972), in the scorpion (Lane e f al., 1981) and in the spider (Chandler, personal communication) so that here the perineurium would seem at first to play a lesser role in regulating the neural environment. However, in the crustacea, no effective blood-brain barrier is present (Abbott. 1970; 1972: Lane et d.. 1977b). Moreover, it has been shown in the scorpion (Lane et d.,1981) that the cellular linings of the intracerebral blood channels possess intercellular tight junctions, and resemble inpushings of the perineurim rather than a separate variety of vascular endothelium; effectively therefore the organism has simply increased the surface area of its perineurium by invagination. Since there are seemingly no tracheoles in the CNS of the terrestrial scorpion with which to supply oxygen, this “vascularization” represents the same reasonable solution to a potentially critical problem as has evolved in the vertebrates. Eiectrophysiological data and experiments with tracers were first responsible for the demonstration of the insect blood-brain permeability barrier. Changes in the chemical composition of experimental solutions bathing intact preparations of the central nervous system and large peripheral nerves of insects were shown to result in delayed neuronal responses (Hoyle, 1953; Twarog and Roeder, 1956) as compared with the more rapid responses obtained with desheathed preparations or with injection of solutions beneath the perineurial sheath (Treherne et al., 1970; Treherne and Pichon, 1972). This peripheral diffusion barrier was thought at first to be associated with the outer fat body layer or the connective tissue sheath (Weidler and Diecke, 1970); however, the former in some cases is a discontinuous layer or may be absent in early instars (Leslie 1973), while tracer studies show that molecules as large as horseradish peroxidase (40,000 MW) as well as other tracers penetrate both the fat body sheath (Lane and Treherne, 1971) and the underlying neural lamella (Lane and Treherne, 1970, 1971, 1972; Lane 1972). Eiectrophysiological evidence also supports such observations in that the neural lamella does not represent an appreciable barrier to the inward diffusion of inorganic cations (see references in Treherne, 1974). Hence it be-

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came clear that the perineurial cell layer must represent the permeability barrier in insects; further tracer studies confirmed this since the entry of exogenous substances was restricted to the perineurial clefts and never invaded the underlying nervous tissue (Lane and Treherne, 1970, 1971, 1972; Lane, 1972; McLaughlin, 1974b; Leslie, 1975; Houk and Beck, 1975). Microprobe studies corroborated the absence of such tracers as lanthanum in areas beneath the perineurium (Lane et al., 1977a). However, when the perineurial layer was removed or disrupted, exogenous tracers moved into the axonal and glial clefts as well as into the cytoplasm of any damaged glial cells (Lane and Treherne, 1969, 1970, 1972). Ionic lanthanum was found to penetrate both septate and gap junctions (Treheme er al., 1973) and so the remaining punctate appositions or tight junctions found between adjacent perineurial cells (Lane and Treherne, 1972; Lane, 1972) were presumed to be the morphological basis for the observed physiological barrier. Moreover, in those peripheral nerves of insects which are so small as to lack a proper perineurium (Lane and Treherne, 1973), the interaxonal and interglial extracellular spaces are accessible to externally applied lanthanum (Lane and Treherne, 1972) since no tight junctions are present to restrict tracer entry. Subsequent freeze-fracture studies on a range of insects revealed that their basal perineurium possesses a simple tight junctional system of ridges and grooves (Lane et al., 1975a, 1977a; Lane and Swales, 1978a,b, 1979). Hence these tight junctions are ideally positioned to be the structural correlate of the existing diffusion barrier. However, they are not so ideally structured as they are positioned, in that they are composed frequently of discontinuous ridges. This would appear to be a problem in topography since the perineurial cells interdigitate very extensively; an effective barrier in insects, in comparison with vertebrates and their relatively straight lateral borders, could be a much less extensive structure spread over a larger membrane area. It appears, in any case, to be the number of parallel ridges rather than the extent to which they are interwoven which determines their effectiveness in occluding substances (Claude and Goodenough, 1973); moreover, more interdigitation produces more junction per unit area of luminal surface (Claude and Goodenough, 1973) while at the same time contributing to the overall transepithelial resistance by virtue of the narrowness of the intercellular clefts (Wright er af., 1972). Such factors support the contention that the ridges, albeit discontinuous, which lie in parallel in the insect perineurium, are the restrictive elements. The morphological barrier produced by tight junctions may not totally occlude all ions (DiBona, 1972; Machen et al., 1972) and indeed the insect perineurium may exhibit a finite leak to K+ (Pichon el al., 1971). The “tightness” of the seal physiologically may be maintained by devices such as reverse perineurial pumping which would counteract any such intercellular leakage; perineurial pumps have been demonstrated electrophysiologically in insects (Schofield and Tre-

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heme, 1975) as have high densities of intramembranous PF particles in the outward facing perineurial membranes (Lane er al., 1977a). Another device to compensate for any inward leakage through the perineurium could be a reduction in the underlying interglial diffusion. Although no unequivocal physiological evidence for a glial-based back-up is available for insects, glial cells do possess discontinuous intramembranous PF ridges and EF grooves (Fig. 15) (Lane and Swales, 1978b). In insect peripheral nerves, where neurohemal areas consist of neurosecretory axons releasing products into the hemolymph by interposing themselves between perineurial cells to terminate in the neural lamella, incubation with tracers may lead to leakage around such axons back into the subperineurial area (Lane et u l . , 197Sb). Equally, tracheoles penetrate the perineurium and ramify through the insect ganglia: leakage can also occur around their peripheral cell borders. In both instances, any leakage of tracer seems to be relatively limited (Lane et al.. 1975b) and possibly interglial tight junctions, albeit focal or fasciar. are involved in preventing extensive movement of exogenous materials through the nervous system. The perineurial layer in such arachnids as spiders and scorpions is believed to provide a diffusion or blood-brain barrier in the same way as it does in the insect central nervous system, but the evidence is much more slender. The primary basis for this contention is that tracers may penetrate partway into the clefts of the perineurial sheath and the blood channels, within the ganglia, but have never been seen to move beyond the region of the tight junctions (Lane and Chandler, 1980: Lane and Harrison, 1980; Lane et al., 1981). Preliminary electrophysiological results with the scorpion suggest that the outer perineurial sheath forms a bamer to the inward diffusion of some cations and that the less extensive tight junctions lining the network of intraganglionic blood channels and the peripheral nerves are rather more leaky to ions (Lane et a l . , 1981). Further physiological studies are clearly required to uncover more details of these systems.

2 . Blood-Rrtiricil Barrier There have been a number of recent reports which contend that there is a blood-retinal barrier in insect compound eyes. The locust has been found to possess a blood-eye diffusion barrier both by electrophysiological criteria (Shaw. 1977) and by tracer studies (Shaw, 1978); the zone of restriction seems to be in the area of receptor axons and pigmented glial cells where narrowing intercellular clefts have been reported (Shaw, 1978). Earlier claims of tight junctions in thin sections of the locust eye (Eley and Shelton, 1976) were not entirely convincing but freeze-fracturing the eye has produced evidence for simple PF ridges and EF grooves in both the locust (Skaer, unpublished data; Lane and Skaer, 1980) as well as in the honeybee (Nickel and Scheck, 1978). The situation in the locust eye is rendered more complex by the presence within them

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of extracellular channels containing hemolymph, lined by perineurial-like cells (Northrop and Guignon, 1970; Shaw, 1978), rather similar to the blood channels found in the ganglion of the crustacea and scorpions. The precise morphological basis of the locust barrier, however, remains obscure at present. The compound eyes of dipterans have also been investigated but they differ from those of the locust in a variety of ways, including lacking any internal, perineurial-lined channels. The existence of glial cell barriers was suggested by Boschek (1971) but electrophysiological evidence of high resistance in the eye, together with tracer studies have led to the speculation (Zimmerman, 1978) that tight glial wrappings may reduce the extracellular space to such an extent that a continuous complex of tight junctions need not be necessary. However, other data support the proposition that tracers such as ionic lanthanum are restricted from moving through the glial clefts below the basement membrane near the distal region of the eye where the retinular axons leave the pigmented retinal area (Lane, 198lb). In addition, freeze-fracture evidence reveals that in this same region of both Cailipora and Musca, complex, vertebrate-like tight junctions are to be found (Lane, 1981b) (see Figs. 30-33 and Section 1V,C,2). Earlier reports of “close” junctions (Chi and Carlson, 1976) in Musca eye in fact referred to gap junctions (Ribi, 1978) and striking ridge-groove systems seen near retinular axons in Musca and Calliphora appear to have unreduced clefts (Carlson and Chi, 1979; Chi etal., 1979; Lane, 1979c), so that, unlike the glial tight junctions mentioned above (Lane, 1981b), they cannot be a form of tight junction proper. Further studies to map out the exact distribution of the tight junction-bearing glial cells will be very helpful and in this regard a splendid series of reports is currently forthcoming from the laboratory of Carlson, Chi, and Ste. Marie which, it seems likely, will soon elucidate many of these recalcitrant points. The eyes of scorpions have also recently been found to possess tight junctions; these appear identical in thin sections, after tracer incubation and in freezefracture replicas, to those in the perineurium of the scorpion CNS (Lane et al., 1981 ). No information on their physiological role is currently available, nor has their precise localization been analyzed with respect to the details of the fine structure of the scorpion eye as reported elsewhere (for example, Fleissner and Siegler, 1978; Schliwa and Fleissner, 1980). In the lateral eyes of Limulus, the close junctions observed (Lasansky, 1967) between glial cells or between glial and visual cells, are quintuple-layered, but nevertheless appear to be electrotonic gap junctions, not occluding ones, 3. Blood-Testis Barrier A permeability barrier has been found in the testis of insects; here most of the evidence relates to the germ cells of the locust. By using tracers such as horseradish peroxidase, iron saccharate, lanthanum chloride, and iron gluconate, a basal compartment containing the differentiating spermatids has been shown to

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be tightly closed (Marcaillou and Szollosi, 1975; Szollosi and Marcaillou, 1977; Jones. 1978: Toshimori e t 01.. 1979). Although one report showed that tracers did not cross the totality of the septate junctions which appeared, therefore, to be the main structures restricting the entry of the tracers (Szollosi and Marcaillou, 1977). others indicated that simple tight junctions were also to be found between the cell layers ensheathing the developing germ cells (Skaer, Szollosi, and Connell, unpublished data; Toshimori et a / . , 1979; Lane and Skaer, 1980). In freezefracture replicas these are nonanastomosing, frequently discontinuous, PF ridges that are moniliform in structure (see Lane and Skaer, 1980, Fig. 47). Unequivocal tight junctional ridges of the simple variety have been found lying he/ow the septate junctions in the cyst envelope of the silkworm and this same area i s there where tracer restriction has been observed (Toshimosi et ul., 1979). N o information is as yet available on the sequence of events occurring during junctional development, but the barrier does not arise as such until the second instar and some "barrier-stimulating" factor is thought to induce its formation (Jones 1978). Other investigators believe that the isolation of the gonial compartment is not based upon any morphological structure and is due rather to a metabolic "inertia." the mechanism of which remains to be clarified (Marcaillou et ul.. 1978). The barrier is believed to be present owing to the requirement by the germ cells for a specialized environment for spermatid maturation and development (Jones, 1978), but clearly more work must be done on this system in a range of species to clarify the precise role of the different junctional types which could conceivably vary in different genera. 4. Di8iision Btrrrirrs in Epidermal Senson Organs

In insect sensilla, there also appear to be intercellular diffusion barriers, for after incubation with such tracers as ionic lanthanum, no tracer appears in the receptor lymph cavity of either Colliphoro or Achetn (Keil and Thurm, 1979; Kuppers and Thurm, 1980). The main diffusion barriers have been thought to be provided by septate junctions since the lanthanum penetrates only partly through these structures: hence they are considered to retard at least to some extent the inward diffusion of tracers. This may well be so, but these sensilla have not been examined hy freeze-fracture and so, in addition, simple tight junctions, which would only be properly revealed by freeze-cleaving, may yet be found to be present. Moreover, in thin sections, occasional punctate appositions have been observed (Keil, personal communication).

5 . Et*rdencefor Occlusion in the Digestive Truct There is some evidence from studies on the digestive tract of a range of arthropods that tight junctions exist both in the esophagus and in the rectal tissues. Punctate cell-to-cell fusions occur, albeit relatively infrequently. in thin sections (Fig. 28) but have been observed in such diverse arthropods as the

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horseshoe crab Limulus, the bug, Rhodnius, and the moth, Manduca (Lane, 1978b, 1979a,b; Skaer et al., 1980; Lane, unpublished data). After freezefracturing, intramembranous PF ridges and EF grooves have also been found in Limulus, Periplaneta, and Manduca (Figs. 27 and 29). These structures appear beadlike and tend to be nonanastomosing; they either occur as single strands (Fig. 27) or may lie in parallel (Fig. 29). Where the fracture cleaves across a face transition, the typical tight junctional pinching together of the PF and EF may be seen (Fig. 29). In the esophagus the observed tight junctions occur in the lateral borders near to the cuticular lining. In the rectum they are found near the basement membrane in the intercellular clefts between the adjacent rectal pad cells (Lane, 1978b, 1979a,b). Incubation in tracers shows that ionic lanthanum, applied at the basal surface of the rectal pads, enters the clefts but traverses them only for a short distance, stopping in the regions of the tight junctions (Lane, 1979a,b). In the esophagus, such substances applied from the basal surface penetrated for some distance along the intercellular cleft but do not pervade beyond the tight junctions (Skaer et af., 1980). Tracers applied at the luminal surface of the foregut do not enter due to the thick and relatively impermeable cuticle (Maddrell and Gardiner, 1980), but the physiological significance of these tight junctions, apart from possibly serving as a back-up to the cuticle, is unclear. In the rectal pads the occluding junctions may act to prevent back-flow of substances into the tissue from the hemolymph which would otherwise interfere with the unidirectional solute flow from rectal lumen through the intercellular clefts to the hemolymph circulating around the basal regions (Lane, 1979a,b). 6 . Relationship to Septate Junctions

For some time the septate junctions have been considered to be the basis for the permeability barriers observed in the invertebrates (for example, Wood, 1959, 1977; Satir and Gilula, 1973; Newel1 and Skelding, 1973; Flower and Filshie, 1977; Noirot-Timothee et al., 1978; Noirot-Timothee and Noirot, 1980; Wood and Kuda, 1980). There are reports in the literature,-however, which show that tracers are able to penetrate these junctions (Leik and Kelley, 1970; Treherne et a l . , 1973; Leslie, 1975; Lord and DiBona, 1976; Ryder and Bowen, 1977; Lane, 1979b; Toshimora et a l . , 1979) although they may not always move completely through them. Moreover, there are recent reports of septate junctions between the two-cell stage in embryos (Vernet and Gontcheroff, 1980) where they could not possibly be forming a barrier. The fact that tight and septate junctions frequently coexist (Connell, 1978; Toshimori et a l . , 1979; Lane, 1979a) might be taken to indicate that they are unlikely both to form barriers unless they provide alternative barriers to one another, differing in the subtleties of their interference with the intercellular passage of ions and molecules. When the electrical resistance of a vertebrate tissue is changed by bathing in

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hypertonic solutions, the tight junctions become altered and intercellular “blisters” form (di Bona, 1972; Wade er ul., 1973). Similarly in planarians (Lord and Di Bona, 1976) and arthropods (Mills et al., 1976; Green et uf., 1980) blisters form between or in the middle of septate junctions. Results of this sort have persuaded some investigators that the vertebrate tight junctions and the invertebrate septate junctions are functionally alike and that the latter may have even given rise to the former (Green, 1978). However, in the blistering experiments the elevation of the osmolarity of the fluid environment to unphysiological levels has been so extreme as to undermine the validity of these results. There also seems little doubt that the effectiveness of both these junctions may be correlated with the steepness of the gradient that may be supported; the efficiency with which a steep concentration gradient can be maintained across a cellular layer will obviously depend in part on the relative resistance of the occluding junctions to ionic movement. With the active transport of ions across the cellular route into the intercellular space, a potential back leak may result. In “leaky” vertebrate systems, it has been suggested that tight junctional permeability may be regulated by cellular mechanisms which could involve dynamic changes in the tight junctions themselves (Bentzel and Hainau, 1979). However, differences in the gross arrangement or number of the tight junctional fibrils do not fully explain the differences observed in transepithelial resistance (Martinez-Palomo and Erlij, 1975: Mdlgard et ul.. 1976; Erlij and Martinez-Palomo, 1978). Hence other parameters such as chemical or electrical charge differences in the junctional proteins themselves may determine the junctional permeability (Claude, 1978; van Deurs, 1980). This would be readily explained in the tight junctions (see van Deurs. 1980, Fig. 3) where as a result we have fairly clear models to work with, and speculations could also be made for the septate junctions. For these, although there is still some question as to whether the intercellular septa and the intramembranous particle rows can be correlated one with another (NoirotTimothee et u l . . 1978), tentative models can be constructed (Flower and Filshie, 1975: Lane and Skaer, 1980). Without unequivocal evidence we cannot be sure whether the septa are attached on both sides of the cleft or, if only on one side, how easily they may be deflected, as might a curtain, by the passage of ions or molecules. Alternatively, they could be fenestrated so as to produce a permeable, sieve-like structure or may be distributed with so many septa1 discontinuities as to produce a maze-like structure through which molecules may ultimately make their way by a circuitous but accessible route. Binding of ions to a charged intercellular matrix could also occur, which would interfere with diffusion. More information is required on the effects of septate and tight junctions on ionic and molecular accessibility in different tissues and in diverse arthropods before any definitive conclusions can be reached as to the precise differences in their function.

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B . COMPARTMENTALIZATION OF PLASAMALEMMA

The plasma membrane of vertebrate cells is divided up by any circumferential junctions which serve to establish and maintain cellular polarity (Staehelin and Hull, 1978). They achieve this by preventing the translateral migration of intramembranous particles (IMPs) from the outer or luminal surfaces to the lateral or basal ones, by providing a physical barrier; this is in the form of junctional fusions in the case of the tight junctions. As a result, differences in the IMP populations of the luminal membrane, compared with those of the lateral and basal membrane, have been found (de Camilli et af., 1974) in vertebrate epithelia. Although the spiders and scorpions with their luminal tight junctions have the potential to slot into this category, the intercellular tight junctions in the insects tend to be near the basal, not the luminal surface, and so they would seem not to be analogous. However, it may be that the insect membrane polarity is comprised of luminal surface and lateral borders separated from the basal surface; both the former are packed with IMPs, more so than the other membranes (Lane et al., 1977a) and high levels of ion transport have been recorded across these outwardly facing perineurial surfaces (Treherne and Schofield, 1978). Such data may indicate that the outer and lateral membranes in the insect perineurium are active in a way quite different from those of the basal surface. Specific information of this kind is not yet available for those arachnids, such as the spiders and scorpions, which bear their perineurial junctions closer to the outward-facing membrane and so the possibility of their providing membrane compartmentalization must remain speculative for the time being. Another possibility which cannot be disregarded, is that, in insects, the septate junctions, which girdle the apex of many cells, could maintain polarity (Noirot-TimothCe and Noirot, 1980). C. “LEAKINESS” CORRELATED WITH RIDGENUMBERA N D DISCONTINUITIES It was originally suggested that a positive correlation should exist between the number of sealing strands or ridges in a tight junction, and its transepithelial resistance or permeability (Claude and Goodenough, 1973), the more strands, the higher the resistance and greater the impermeability. “Tight” tight junctions seemed to have a complex reticulum of tight junctional ridges, while “leaky” tight junctions were considered to have fewer strands and a less complex network. Arthropod tight junctions at first seemed not to exhibit variations like this since the insect perineurial, rectal, and esophageal junctions were all simple. However, with the discovery of the more complex tight junctions in the insect eye (Lane, 1981b), a range is seen to exist within the Insecta, and with the observations on the spider perineurium (Lane and Chandler, 1980) and the cells of the scorpion perineurium and those linking their vascular channels

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(Lane and Harrison, 1980; Lane et ul.. 1981), a range also exists within the whole of the Arthropoda. Unfortunately these can only be ascribed “leakiness” in so far as such criteria as permeability seen by tracer penetration or freezefracture features are concerned, not as regards resistance measurements. However. in the case of the insects, the perineurial cells have been shown by electrophysiological techniques to possess a finite leak to K’ (Pichon et ul., 1971) which indicates that the simple tight junctions must permit some degree of ionic leakage. Addition of urea enhances this leak of K’ and also Na’ but the entry of higher molecular weight molecules is still inhibited (Treherne et a[., 1973). Here it is thought that modifications induced in the tight junctions by urea could be responsible for the increased leakage, so that the zonulue occludentes might be involved in regulating intercellular permeability by differential junctional sensitivity i n response to various agents. This could be correlated with the situation in vertebrates where recently there have been claims that differences in the chemistry of the junctional molecules (Claude, I978), their molecular arrangement, or electrical charge (van Deurs, 1980) would explain the observed differences in transepithelial resistance rather than the number of tight junctional strands (Martinez-Palomo and Erlij, 1975; Mdlgard et ul., 1976; Erlij and Martnez-Palomo. 1978). Changes in electrical resistance induced by treating with hypertonic solutions leads to “blistering” of the tight junctions (di Bona, 1972): this has been taken to mean that the junctions are accessible to water and provide a “limiting” effect rather than totally preventing the paracellular passage of ions and water. In certain resorptive epithelia which are “leaky,” it has been suggested that tight junctional permeability is subject to regulation by cellular mechanisms (Bentzel and Hainau, 1979). Tight junctional units are envisaged as being assembled and reversibly disassembled in a dynamic feedback loop, regulated by the cytoskeletal system and the state of active cation transport; this rapid turnover of the ultrastructural components of the tight junctions would represent the response of the epithelial layer to physiological stimuli (Bentzel and Hainau. 1979). Since cytoskeletal elements have been shown to be associated with tight junctions (Meza e f ul., 1978; Cereijido et al., 1980) and to be involved in displacement and subsequent resealing of tight junctions (Meza et ul., 1980), it is thought that they could act to anchor or stabilize the tight junctional precursor particles (Bentzel and Hainau, 1979) during the retardation of ridge assembly, the act of assembly itself being stimulated by inhibition of active Na’ transport. Any such dynamic interpretation of the events occurring in the tight junctionbearing tissues of arthropods is not yet feasible due to lack of data; for example, further information is required on the cytoskeletal system since the apparent lack of microfilaments associated with the tight junctions has not yet been corroborated by sufficient observations.

TIGHT JUNCTIONS IN ARTHROPOD TISSUES

27 1

If ridge discontinuities in the simple tight junctions found in the insects are responsible for the finite ionic leakage observed electrophysiologically, then the discontinuous ridges in the scorpion tissues would also be likely to lead to ionic, if not molecular, leakage via the disruptions in their intramembranous ridge structures (see Section IV,D, 1). What few electrophysiological experiments have been carried out would tend to support this (Lane et al., 1981), since the junctions in the very extensive, rather attenuated, and hence low-junction-bearing area of the perineurial-like layer lining the blood channels of scorpion ganglia are leakier to cations than those in the deep, high-junction-bearing, outer perineurial sheath. However, in the arthopods, there is at present not enough evidence to determine whether the degree of permeability is proportional to the observed complexity of the tight junctional intramembranous ridge network or whether some other unresoivable chemical feature of the fibril network is the determining factor; further experiments are required to elucidate this point.

D. TISSUERIGIDITYAFFECTED BY EXTENT OF JUNCTIONAL RETICULUM There have been several reports on vertebrate tissues in which it has been suggested that the fibril morphology of the tight junctions may be related to passive shape changes in the cells. For example, cells with loosely interconnected arrays of ridges, in contrast to highly anastomosing arrays, may be better able to stretch or expand under such strains as the accumulation of secretory products (Pitelka ef al., 1973; Hull and Staehelin, 1975, 1976; Staehelin and Hull, 1978). In the Sertoli cells of the testis which have no network of tight junctions but only parallel arrays of ridges, it is thought (Suzuki and Nagano, 1978a) that this particular geometry of the ridges may be associated with the capacity to handle the increases jn tension that develop due to the pressure of the contents of the lumen. In this context, the insect tight junctions may be required to be simple in order that the tissues respond appropriately to external stress or motion and to allow for variability in cell shape (Lane and Skaer, 1980). This suggests that these junctional configurations are not permanent structures but dynamically reconstructable ones. The existence of the other more complex arthropod junctions may signify that tissues such as the dipteran compound eye, and spider and scorpion ganglia, all of which possess the complex reticular form of anastomosing tight junctions are subjected to less external movement or pressure or, although subject to such stress, are able to resist changing their shape which may be in the best interests of the organism. Whether this difference between simple and complex tight junctions could be related to such distinctions as the relative number of interweaving tracheoies (low or nonexistent in the Arachnids) or the degree of vascularization (nonexistent in the Insecta) is not clear. It may be possible that the pressure which might be expected from a

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ramifying system of intraganglionic blood channels could be better withstood by the more anastomosing patterns taken up by the Arachnid tight junctions. Such remarks are of course highly speculative since at present we do not know which of the functions of the tight junction is most important to the animal; shape changes in cells responsible for ensheathing the CNS are not so likely to occur as those in secretory tissues or storage organs.

E. ADHESION In many instances, junctions between cells appear to serve in at least a dual capacity. In the case of tight junctions indubitably this appears to be so in that in the very act of creating a diffusion bamer by punctate cell-to-cell fusions, the cells are held firmly together and, in so doing, maintain the integrity of the tissue in which they occur. In arthropods, adhesion as a primary role would appear to be the property of the desmosomes, both rnacula and zonula adhaerens (Farquhar and Palade, 1963), which seem not to fulfill any obvious alternative function. Septate junctions on the other hand are altogether more difficult to characterize in this respect. They have an unequivocal role in adhesion in arthropod tissues as many investigators, too innumerable to cite, have mentioned, but they may also play a part in maintaining permeability barriers (for example, Wood, 1959; 1977; Satir and Gilula, 1973; Noirot-Timothee and Noirot, 1980; see references in III.A.6). The fact that they do not appear to occur in vertebrates (although reports of septate-like junctions have been published-Friend and Gilula, 1972b; Schnapp and Mugnaini, 1975; O’Connell, 1978; other references in Lane and Skaer, 1980) makes the question of their physiological significance the more intriguing. There seems little doubt that they may play some part in creating diffuson barriers; probably this is their primary role in some lower invertebrates (Wood, 1959, 1977; Wood and Kuda, 1980) and perhaps a role secondary to the tight junctions in many arthropods (Lane and Skaer, 1980). Certainly the septate junctions also act as adhering devices to maintain complex cellular interdigitations in epithelia which may be very flattened and upon which great pressure may be imposed (see Noirot-Timothee and Noirot, 1980; Lane and Skaer, 1980). Although in Arachnids, the tight junctions also form a broad apical girdle which could be fairly effective as an adhesive device, in the insects they are not nearly so extensively distributed and hence seem less likely to be as important in adhesion as would the septate junctions. On the other hand, when the insect tight junctions are disrupted in early pupation (Lane and Swales, 1978b). the perineurial cells and glia become dissociated from one another. The precise role and relative importance, as adhesive devices, played by septate and tight junctions, remain therefore at present a matter for conjecture.

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IV. Arthropod Groups in Which Tight Junctional Structures Have Been Reported Reports indicating the existence of structures which could be considered to be tight junctions have been forthcoming for each of these arthropod groups, the Xiphosura, Crustacea, Insecta, Arachnida, including scorpions and spiders as well as the Myriapoda, as summarized in Table I. The structures observed are not precisely the same in all cases, and indeed this diversity is a feature of arthropod zonulae occludentes. The evidence in some cases derives from the lines of study which would normally be required to prove the existence of this kind of sealing structure. As indicated earlier, these include (1) the occurrence in thin sections of punctate appositions between adjacent cell membranes which obliterate the intercellular space, (2) the existence in freeze-fracture replicas of a junctional belt composed of intramembranous ridges with complementary grooves, and (3) the presence of a permeability barrier where these junctions are found, demonstrated either by the impedance of tracer entry or electrophysiologically. In certain instances in the various arthropod classes, as will emerge later in this section, only one or two of the requjsite criteria have been shown to be fulfilled. In the arthropods, which exhibit many physiological differences from vertebrate species (Maddrell, 1980) (see Section III,A), there has been a search for tight junctions which has usually been, in the first instance, in tissues known to possess permeability barriers. Since, until recently, the amount of information available for the arthropods on such phenomena has been scanty, there have been relatively fewer physiological ‘‘leads’’ to follow up in comparison with vertebrate tissues. The ones which have been most fruitful, are those deriving from electrophysiological studies on the CNS. Hence, in many cases, those arthropod tissues in which tight junctions were first “sighted” were those that form part of the nervous system. This will emerge from the subsequent discussion of such junctions in the various classes of arthropods.

A. PRIMITIVE ARACHNIDS 1 . Xiphosuran Chelicerates

The primitive arthropod Limulus polyphemus, the horseshoe crab, is a marine organism which attains a considerable size at maturity. Hence its organs tend to be large by arthropod standards and its CNS consists of a relatively massive ganglion. This is composed of fused supra- and subesophageal ganglia (Fahrenbach, 1976) and by virtue of its size requires a mechanism other than simple

Arrangement of ridges and grooves

Class Ardchnidr

Common name Hou\e \pider Garden \pider Scorpion

Genus namc

Tissue

Centipede

Lirhohius

lnsecta

Blowfly. house fly

Culliphoru rryrhrowphulu, Muscu dornesricv

Perineurium o f CNS

Glial cells in compound eye, between outer retina and inner neuropile Perineuriurn of CNS and Tobacco horn worm moth Munducu sexru large peripheral nerves Calliphoru rryhr(~c.ephir/u Perineurium of CNS and Blowfly large peripheral nerves Periplunetu iirnericunu Perineurium of CNS and Cockroach large peripheral nerves Schisrocercir greguriu Perineurium of CNS; Locust testis Botnbw mori

Cyst envelope of testis

Complex and reticular t

Outer glial cell\ (perineurium) of CNS 4 riiti(ii*us Perineurium ot CNS P t r r ~ ~ r ~ ~ r ome~iic~n\r~ riio Perineurium of CNS rnd peripheral nerve\ Trqcwuriu

Myriapoda

Silkworm

Simplc and discontinuous

+ +

+

+ + + +

+

Lane and Chandler ( 1980); Chandler and Lane ( 1980) Lane (1980, 1981a) Lane and Hamson ( 1980). Lane er irl. ( 198 I ) Lane (unpublished data)

+

+

Keterence

Lane and Treherne ( 1980); Lane and Skaer ( 1980); Lane (1981b) Lane el ul. ( 1977a); Lane and Swales (1979) Lane er ul. (1977a); Lane and Swales (1978a.b) Lane er ul. (1977a) Lane erul. (1977a); H. Skaer, A. Szollosi. andC. Connell (personal communication) Toshimori PI al. (1979)

TIGHT JUNCTIONS IN ARTHROPOD TISSUES

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diffusion in from the hemolymph at the periphery to ensure that oxygen and nutrients are available throughout its mass. This problem appears to be solved by the presence of a ramifying network of blood vascular spaces all through the CNS; these are lined by basal lamina and glial cells which are not associated with one another by any special junctional complexes (Harrison and Lane, 1981). Hence free penetration of ions and molecules may occur from the hemolymph into the nervous system as observed by tracer penetration (Harrison and Lane, 1981 ) and by electrophysiological recordings (Willmer and Harrison, 1979). However, in some parts of the glial system in the fused ganglia, linear junctions occur in freeze-fracture replicas which bear many of the features of tight junctions. These are composed of rows of aligned P face particles, which may sometimes be fused laterally into ridges (Fig. 23); these appear to be coincident with EF grooves across face transitions and hence seem to be complementary. In some cases there are also suggestions that at these junctures the intercellular cleft is obliterated (Fig. 24). However, unequivocal obliterations have not yet been shown to be present in thin sections, and their barrier properties are suspect because it is not clear where or whether they form a circumferential junctional belt; possibly they might occlude around one small area of the CNS. Experiments are currently underway to elucidate the further features of these structures (Lane and Harrison, unpublished data). Although other tissues in Limulus have been examined after tracer incubation and by freeze-fracture (Lane and Harrison, unpublished data), no other comparable occluding structures have been found except possibly in parts of the digestive tract. For example, the midgut and hepatopancreas possess smooth septate junctions (Herman and Preus, 1972; Johnson ef al., 1973; Lane and Harrison, 1978) and the rectum, pleated septate junctions (Lane and Harrison, unpublished data); these, as will be discussed later (see Sections V,A and B), are quite different from tight junctions in that they are characterized by a considerable intercellular cleft of 15-20 nm (Flower and Filshie, 1975; Noirot-Timothee and Noirot, 1980; Lane and Skaer, 1980). However, in thin sections the esophagus contains apparent tight junctions, as does the rectum. Moreover, in the latter, although rarely, intramembranous ridges have been found (Fig. 27) which would correlate with the punctate membrane appositions seen in thin sections (Lane, unpublished data). In the ommatidia of Limulus,the associations observed appear to be gap junctions (Lasansky, 1967). Apart from these, other possible sites of tight junctions, such as the testis, have not thus far been investigated. 2. Ticks Like Limulus,only some of the organs of the tick have been investigated for an analysis of junctional structures. The salivary gland is known to possess pleated septate junctions (Beadle and Megaw, 1979) and the mid-gut contains

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typical smooth septate junctions (Lane and Binnington, unpublished data). The CNS possesses no septate junctions, nor occluding tight junctions, and is accessible to tracers added to the hemolymph (Binnington and Lane, 1980; Hart et al., 1980). However, in the glial cells of the CNS, occasional intramembranous ridges have been found in freeze-fractured replicas (Fig. 29 in Binnington and Lane, 1980) which bear some resemblance to those forming part of conventional right junctions. These may be analogous with the ridges found in insect glial elements (Lane and Skaer, 1980); if so, whether they could be some primitive structure, representing an early nonfunctional precursor to junctions that subsequently developed, or alternatively, a residual structure of some kind cannot be ascertained.

B. CRUSTACEANS Within the crustacea, a good many of the studies an intercellular junctions have been concerned with gap junctions (Peracchia, 1973a,b; Lane and Abbott, 1975; Peracchia and Dulhunty, 1976; Graf, 1978b), desmosomes (Shivers and Brightman, 1977; Shiver and Chauvin, 1977), or continuous (smooth septate) junctions (Hudspeth and Revel, 1971; Graf 1978a). There have been some studies that suggested the occasional presence of junctions of a fascia or maculu occiudens variety both in thin sections (Lane and Abbott, 1975) and in freezefracture replicas (Shivers, 1977). In the latter, the junctional structures were reported to be rows of intramembranous EF particles, running into clusters of similar EF particles, referred to as desmosomes. The rather unexpected spatial association of tight junctions and desmosomes was remarked upon (Shivers, 1977), but what was unstated, but clearly a distinct possibility, is that the observed intramembranous arrangements may be interpreted as gap junctions in the process of assembling. In such an event, linear arrays of EF particles would be predicted and such a process would be expected in regenerating tissues, which is what was being examined (Shivers, 1977). The fact that the intramembranous particles fractured on the EF also makes them somewhat atypical, although there are a few other EF tight junctional particle rows (see Section IV,D). Other preliminary observations of EF ridges in crustacean nervous tissue (MartinezPalomo, personal communication), also occurring near EF clusters of gap junctional particles, make it clear that here is an area worthy of further exploration. Hence, the presence of true tight junctions in the crustacea has yet to be proved unequivocally. In the CNS of crayfish, where permeability and electrophysiological studies have been made, there is no blood-brain barrier, in that, although the entry of ions and moecules is slowed down and to some extent impeded (Abbott ~t a/., 1977), there is no ultimate restriction to entry (Abbott, 1970). The tight junctions observed in thin sections of crayfish central ganglia are relatively rare

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and seem not to form a belt-like zone around the neuropile (Lane and Abbott, 1975) as would occur if the junctions were truly of the zonulae occludentes type.

C . INSECTS 1 . Simple Tight Junctions

The “simple” tight junctions characterized as such by their thin section or freeze-fracture appearance described earlier (Sections II,A and B) are relatively infrequent in occurrence in insects, or at least are rarely observed; this may perhaps be due to the difficulty in finding single ridges in a replica in contrast with the ease of recognizing an extensive and ramifying network of fibrils such as chordates tend to possess. Moreover, simple insect tight junctions are not to be found in a distinctive and readily monitored intercellular region in tissues, as, for example, occurs between the relatively straight lateral borders of vertebrate epithelia, where the tight junctions are positioned fairly consistently by the luminal surface (Farquhar and Palade, 1963, 1965; Staehelin and Hull, 1978). This orderly arrangement is in complete contrast to the simple variety of insect tight junctions which occur toward the basal surface of epithelia; as described earlier, these epithelial cells exhibit an unusually high degree of intercellular infolding and interdigitation, such that the lateral borders are often running parallel to the outer surface of the epithelial layer (Fig. 1). a. Perineurial Tight Junctions in the CNS. The historical background relating to the original observations on the existence of arthropod tight junctions in the perineurial layers ensheathing the CNS of insects has already been considered (see Section II,A, 1). The reader may recall that electrophysiological data (Hoyle, 1953; Twarog and Roeder, 1956; Treherne et a l . , 1970) first suggested the presence of a permeability barrier for which, it was supposed, must exist some morphological basis. This was first visualized as points of punctate membrane fusion in the basal region of the perineurial layer, the area wherein the penetration of tracers was prevented (Fig. 6) (Lane and Treherne, 1970, 1972; Lane, 1972). Subsequent freeze-fracture studies revealed intramembranous ridges and grooves in this same region (Lane et a / . , 1975a, 1977a) which appeared to represent a simple form of tight junction. Although these are often discontinuous elements, they appear nevertheless to be able to effect a restriction to the entry of molecules and most ions, albeit a finite leak to potassium may be present (Pichon et a l . , 1971). These simple ridges have been shown to be present in the perineurium of the CNS of the cockroach Periplaneta, the locust Schistocerca (Lane et a/., 1977a), the blow fly Calliphora (Lane and Swales, 1978a,b), and the moth Manduca (Lane and Swales, 1979) among others. These are in sharp contrast to the more complex ridges

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and grooves (Figs. 16-19) which have been found between the perineurial cells in the spider Tegenaria (Lane and Chandler, 1980) and Aranaeus (Lane, 1980. 1981a) as well as in the compound eye of Muscu and Calliphoru (Lane, 1981h) and the eye and CNS of the scorpion Parurocronus (Lane and Harrison, 1980: Lane P t ui., 1981) (see Sections IV,C,2 and 1V.D). These are usually found. however. in the more apical part of the perineurium and, although discontinuities occur. especially toward the basal side (Fig. 25), the junctional “belt” is usually fairly broad in arachnids. This may also be the case for insects, for, although tracers may leak partway through the more basal insect belt because of the breaks in the membrane-to-membrane fusions, they are ultimately stopped. Urea treatment of these insect perineurial junctions has the effect of destroying the barrier to Na‘ entry but not that to the entrance of larger molecular weight tracer substances (Treherne et a/.,1973). b. Giinl cind Truc-heal Junctions. The glial cells that encompass the neurons in insects have occasionally been found to exhibit punctate appositions in thin sections (Fig. 26) and short intramembranous PF ridges or EF grooves in freezefracture replicas within their plasma membrane (Fig. 15) (Lane er al., 1977a; Lane and Swales, 1978a.b); these are most commonly encountered in the interganglionic connectives where they are enveloping axons only. In the replicas it is not always clear whether these are glial-glial tight junctions or whether they are axoglial membrane modifications, or merely nonjunctional (Lane, 1979d). If they are junctional they clearly do not have a circumferential distribution, but might be expected to produce focal areas of restriction to the entry of ions and molecules. This seems to be corroborated by the fact that when there is backleakage of tracer from neurosecretory terminals exiting through the perineurium to the neural lamella, it is severly limited in its interglial passage, presumably by these short tight junctional contacts (Lane et al., 1975b). Rather similar short intramembranous ridges occur along the longitudinal axis of tracheae and tracheoles where they lie alongside glial cells, as well as within the mestracheon region (Lane et ul., 1977a; Lane, 1979d; Lane and Skaer, 1980). Punctate appositions are also found in these areas as tracheoglial junctions or mestracheal junctions. Their role is unclear but the former may be related to stopping any leakage inward from the hemolymph that could occur when tracheoles penetrate tissues with permeability barriers such as the CNS or the compound eye, and the latter may restrict any leakage beyond the cuticular lining of the tracheal lumen. ~~

~~

FIG. 25. Freeze-fracture replica from the prineurium of spider CNS revealing extensive, but relatively discontinuous, tight junctional ridges on the P face (PF). These are alignments of discrete 8-10 nm intramernbranous particles which appear as grooves (arrows) on the membranous E face IEF): here too occur the arrays, often loosely clustered, of gap junctional (GJ) particles. x44.600. FIG. 26. Thin section through glial cells ( G )surrounding and ensheathing axons (A). The glial processes are often associated with one another by punctate tight junctions (arrows). X 129,200.

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NANCY J. LANE

TIGHT JUNCTIONS IN ARTHROPOD TISSUES

28 1

c. Rectal Pads. These junctions were originally detected by freeze-fracture criteria as simple PF ridges and EF grooves in the basal region of cockroach rectal pads (Lane, 1978b, 1979a,b). Similar intramembranous ridges have also been observed in the rectum of Limulus (Fig. 27). Incubation with tracers such as lanthanum showed restriction to the entry of such molecules in the areas where the simple tight junctions were found in insects (Lane, 1979a). It seems possible therefore that sealing junctions are present to prevent back flow of substances during the pumping of solutes across the cockroach rectal pad cells from the lumen to the basal area during water and ion reabsorption (see Section 111,AS). d. Esophagus. The cuticle-lined epithelium of the arthropod esophagus is generally a highly flattened cell layer and much interdigitated along its lateral borders. Here desmosomes and pleated septate junctions abound (Lane and Skaer, 1980), and in a number of organisms, including the horseshoe crab Limulus, the blood-sucking bug Rhodnius, and the moth Manduca sexta punctate appositions also occur between the adjacent cell borders (Skaer et al., 1980; Lane, unpublished data). These look like conventional tight junctions in thin sections (Fig. 28) and after freeze-fracturing, intramembranous ridges and grooves (Fig. 29) are to be found in the same areas. Tracer studies are currently underway (Skaer et al., 1980) but since the permeability of the cuticular lining of the foregut of a variety of insects has been shown to be very low (Maddrell and Gardiner, 1980), the tight junctions in the esophagus may act in a back-up capacity for any ions and molecules that filter through the cuticle. e. Testis. The testis of insects such as the locust has been shown to possess a diffusion barrier to ions and molecules in that it exhibits a restriction to the entry of exogenous tracers (Szollosi and Marcaillou, 1977; Jones, 1978; Marcaillou et al., 1978; Toshimori et a l . , 1979). This is found along the inner margin of the cell layer ensheathing the developing germ cells. There is some evidence to suggest that the morphological basis of this barrier is simple nonanastomosing intramembranous ridges and grooves in the locust (Skaer, Connell, and Szollosi, personal communication; Lane and Skaer, 1980) and in the silkworm (Toshimori FIG.27. Rectal tissue from Limulus showing the intramembranous PF particle alignments that exist therein. X97,OOO. FIG.28. Esophagus of Rhodnius which possesses both gap junctions (GJ)and punctate tight junctional appositions (arrows) between adjacent epithelial cells. X 8 6 . 6 0 . FIG.29. Replica from esophagus of the moth Manduca showing the parallel alignments of PF particle rows (at arrow) and EF grooves (longer arrows) that lie in membrane troughs or depressions. . Occasionally the junctional particles fracture onto the EF. X42,lOO. FIGS.30-33. Complex tight junctions from the compound eye of dipteran insects showing the coincidence of PF ridges and EF grooves (Fig. 30), the positioning of the ridges on PF elevations (Fig. 31). and the grooves in EF troughs (Fig. 32); the interdigitating glial borders show punctate points of membrane fusion (at arrows in Fig. 33) when there is a cross-fracture. Fig. 30, X54,300; Fig. 31, X30.600; Fig. 32, x40,100; Fig. 33, ~ 2 5 , 7 0 0 .

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NANCY 1. LANE

et af.. 1979). However, there may be species differences in these structures since studies by Szollosi on the lepidopteran testis (personal communication) indicate that rather more extensive arrays of ridges occur there. Septate junctions are also present in the testis and may provide additional restriction to the entry of molecules (Szollosi and Marcaillou, 1977). This blood-testis barrier may exist due to a requirement by the germ cells for a specialized environment for meiosis (see Section III.A.3).

2 . Insecrs: Complex Tight Junctions The complex insect tight junctions as described earlier (Section II,B,2) have thus far been found only in the dipteran compound eye (Lane, 198I b; Lane and Treherne, 1980). Here they have been found in the lamina ganglionaris of both the house fly Musca domestica and the blow fly Cailiphora erythrocephala between the glial cells that form the blood-eye barrier (Fig. 30). It is just possible that similar, more complex, junctions may also be found in the regions of the blood-testis barrier (Szollosi, personal communication). The existence of an electrical resistance in the insect compound eye has been demonstrated by Shaw (1977, 1978) in the locust eye, and also in the fly eye (Zimmerman, 1978). Morphological investigations show that the first optic neuropile is shielded from the outside world by a number of layers of glial cells that include marginal glia. epithelial cell, and satellite glial cells (TrujillozCenoz, 1965; Boschek, 1971). These glial cells are associated by a variety of junctions, including gap junctions and septate junctions (Carlson and Chi, 1979; Lane. 1981bl as well as retinulajunctions (Chi eral., 1979; Lane, 1979~); these last bear a superficial resemblance to the freeze-fracture appearance of tight junctions but are not in fact the same as they exhibit a considerable intercellular cleft (see Section V,D and Fig. 47). The interglial tight junctions of the Diptera are referred to here as “complex” because they are much more extensive than the simple ones found in the perineurium and elsewhere in arthropods. They form a reticulum or network of PF ridges which sit on raised membrane protuberances to produce a quilted appearance (Fig. 31) much in the same way as the vertebrate tight junctions. The corresponding EF grooves lie in troughs (Fig. 32) as would be expected from the pinching together of the adjacent cell membranes in cross-fractured preparations (Fig. 33) or thin sections. Moreover, the PF ridges and EF grooves are coincident across face transitions (Fig. 30) where the intercellular cleft is obliterated. In all these respects, therefore, the observed tight junctions are much like those of vertebrates, and are complex. rather than simple in configuration. D. ARACHNIDS

Although hitherto they have been virtually neglected, the junctions in the tissues of arachnids exhibit a range of morphology which is in many ways very

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similar to that found in other arthropod groups. For example, they display characteristic smooth septate junctions (continuous junctions; see Section V,B) between the lateral borders of such tissues as the midgut and hepatopancreas, and typical pleated septate junctions (see Section V,A) between the adjacent cells of esophagus, salivary gland, and compound eye (Lane and Harrison, unpublished data). The existence of these unequivocally septate junctional structures is interesting in view of the presence in Arachnid tissues of equally unequivocal vertebrate-like tight junctions (Lane and Chandler, 1980; Lane and Harrison, 1980). The coexistence of these two types of intercellular cell association in the same organism, albeit in different organs, tends to argue for a different function for each kind of junctional structure (this will be considered at greater length later in Section V). The tight junctions found in the arachnids appear to be of two varieties which I have called the “leaky” and the “tight,” to borrow the terminology applied in subdividing tight junctions in vertebrate epithelia (Claude and Goodenough, 1973) with reference to variabilities in the degree of intercellular permeability. The differences observed in the arachnids thus far investigated appear to be due to both a varying number of ridges or strands which comprise the tight junctional structures as well as a varying number of discontinuities which occur in the composite strands. Unlike the vertebrate tissues, extensive permeability studies have not thus far been carried out in the arachnids so that the degree of leakiness for the junctional types to be described is based purely on structural considerations and on the inability of various tracers and dyes to penetrate beyond the regions where the tight junctions are found. 1. “Leaky” Tight Junctions-Scorpions The scorpions, as a group, have not been extensively studied, except for the features of their visual system and peripheral nerves (Foelix, 1975; Foelix and Troyer, 1980; Schliwa and Fleissner, 1980), but recent investigations reveal that certain of their tissues possess structures (Lane and Harrison, 1980) very like the zonulue occludentes found in vertebrate epithelia and in the vascular endothelium of the CNS; these tissues of the scorpion include the ganglia, eye, interganglionic connectives, and peripheral nerves (Lane et al., 198 1). Not only are they characterized by tight junctions (Fig. 34) but also by many gap junctions as well as by other “close membrane appositions”; these last refer to regions seen in thin sections where the adjacent plasma membranes come to lie very close together in a punctate manner, but, unlike tight junctions, are not quite fused. These various kinds of cell appositions are particularly striking after lanthanum infiltration (Fig. 35) since the tracer meanders through the gxtracellular cleft, negatively staining the plasma membrane and the intercellular structures (Fig. 36) if any such exist. It manages to move past the close appositions (Fig. 35) so that it is clear that they cannot be sealing off the intercellular space completely. As is the case with the gap junctions, the tracer must be moving through these narrowed intercellular

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clefts. However, in instances where the tracer appears on either side of a real occlusion (Fig. 37), it cannot be moving through, but must be moving around a punctate or discontinuous point of cell-to-cell fusion such as a macular tight junction. Freeze-fracture replicas yield an apparent explanation for these phenomena observed in thin sections for such cleaved preparations exhibit a number of intramembranousEF ridges in the junctional belt region. These ridges, however, nearly always possess a number of discontinuities in their structure (Fig. 38) which provide ideal sites for leakage of tracers between the apposed cell membranes, since the plasmalemma cannot be fused together at these points via ridges if none are present. It seems unlikely that the missing bits of ridge have all fractured onto the PF, since the PF tends to possess mainly grooves. However, complementary replicas are required to clarify this point. A diagrammatic illustration of how this system could operate has been constructed (Fig. 7) and here it can be seen how the discontinuousjunctional fibrils or ridges could be involved in holding the cells together, and even pinching the adjacent membranes closer together to produce close appositions. The ridge from one cell’s fracture face does not fuse with that in the opposite membrane’s fracture face at such points but only alongside them. The regions of real membrane fusion are considered to be where the ridges do fuse together in a continuous way, thereby excluding tracers and producing classical punctate tight junctional appositions. In scorpion CNS, these do seem to exist, but more frequently much deeper into the tissue, since tracers tend to migrate into the perineurial clefts for quite some distance before being restricted (Fig. 8). These clefts reveal, in enfuce views, an array of unstained, discontinuous strands (Fig. 36) which represent the regions of partial fusion of the adjacent cells’ membranes via the intramembranous fibrils. Ultimately, however, the tracer penetrates no deeper and never attains the whole depth of the perineurium and beyond into the central nervous system (Fig. 8).

FIGS.34-38. Discontinuous tight junctions from the scorpion CNS. FIG. 34. Conventional preparation showing gap junction (GJ) and tight junctional apposition (arrow) along the perineurial lateral borders. X 134,400. FIG.35. Section after incubation with ionic lathanum; the tracer has penetrated the neural lamella (NL) and moves through the perineurial (PN) clefts past close membrane appositions (arrows). ‘x 135,700. FIG.36. En face view of section such as that shown in Fig. 35. The stain-excluding fibrils represent the discontinuous lines of membrane fusion of these leaky junctions. X81.400. FIG. 37. Incubation in ionic lanthanum leads to the tracer being found in beadlike perineurial (PN) clefts which indicate that the lanthanum must have migrated around the discontinuous, fasciar lines of membrane fusion (at arrows). NL, neural lamella. X54,200. FIG. 38. interdigitating perineurial clefts showing cross-fractured fusions (large arrow), P face (PF) grooves. and E face (EF) ridges. The latter are frequently discontinuous although in some areas the intramembranous particles are fused laterally into solid ridges (at arrows). X44,650.

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The central nervous ganglia of the scorpion are penetrated by a ramifying system of blood channels which are lined by cells which possess the characteristics o f the outer perineurial cell layer. That is, they interdigitate extensively with one another on their lateral borders and are associated with one another by comparable "leaky" tight junctions (Fig. 9) which allow tracer to permeate past many of their discontinuous intramembranous ridges (Lane ef a / . , 1981). They appear to represent invaginations of the outer perineurial sheath which branch throughout the ganglionic mass.

2. ' 'Tight" Ocduding JunctionsSpiders In the spiders. as with the scorpions, tissues such as the esophagus and salivary gland exhibit pleated septate junctions while the mid-gut possesses smooth septate junctions (see Sections V,A and B). The nervous system, however, lacks septate junctions but has vertebrate-like tight junctions (Lane and Chandler, 1980) between the adjacent lateral borders of the modified glial cells that ensheath the central nervous system. These junctions appear to be tight in contrast to the scorpion junctions in that tracers are not usually found moving past close appositions or around the punctate tight junctions (as in Fig. 5); moreover, in freeze-fracture replicas, although there may be some discontinuities in the intramembranous ridges forming the junctions, they are far fewer than those found in the "leaky'' junctions of scorpions. In thin sections, then, the interdigitating lateral borders are associated by a string of punctate cell-to-cell appositions may of which exhibit fusion of the adjacent membranes (Fig. 2) so that at these points the intercellular space is clearly occluded. That these regions of fusion are between the linear intramernhranous ridges of two adjacent plasmalemma, as seen in freeze-fracture, is suggested by en f u c ~sections of uranyl-calcium treated tissues. Here the dense uranyl stain binds to the external leaflet of the exposed membrane but is unable to associate with the regions where adjacent cell membranes are fused to one

FIG, 39. Freeze-cleaved replica from the spider CNS revealing tight junctional ridges on the P face (PF) and grooves on the E face (EF). Note that these are coincident (at arrows) over fracture face transitions, thereby indicating their complementary nature. X44,lOO. FIG.40. Low power view of a replica from a freeze-fractured specimen of house spider perineurium to indicate the belt-like arrangement of the tight junctions (mows) around the outer circumference of the CNS. x 15,700. FIG.31. Replica of garden spider perineurium with tight junctional structures fracturing either onto the P face (PF) or partly onto the E face (EF) where the particles lie separately or in short ridges fat arrows) In EF grooves. Gap junctional (GJ) plaques occur on the same face. The inset shows the continuous EF ridges in arachnid tight junctions fracturing down into complementary PF grooves. In some cases, the EF ridges have been fractured away ( m o w ) and here the coincidence of the ridge-free EF with PF grooves suggests that the ridges may be offset with respect to the grooves, C. cytoplasm. x 39,700, insert. x 53,900.

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another, as that portion of the membrane is inaccessible to the stain (Figs. 2 and 4; see Lane and Chandler, 1980, Fig. 2). Moreover, the total width of the two membranes at the point of fusion is usually less than that of two separate membranes. After incubation of nervous tissue possessing tight junctions with tracers such as lanthanum (Lane and Chandler, 1980), India ink, or vital dyes such as rnethylene blue (Chandler, personal communication), the junctions are found to form permeability barriers since they exclude these substances which never penetrate beyond the level of the peripheral tight junctions. Apart from these and gap junctions, no other junctional type is to be found here. In the spider, then, the tight junctions appear to form the morphological basis of a blood-brain barrier but as yet they have not been observed in other tissues in these animals apart from the blood vascular system. The blood vessels that are associated with the CNS of adult spiders, both in Tegenaria (Chandler, personal communication) and Aranurus (Lane, unpublished data), apparently possess intercellular junctions that exclude tracers (Fig. 5) and are not unlike those found in the perineurial FIG.42. Thin section through outer perineurial layer in the CNS of the millipede, lulus. Note the prevelance of both gap junctions (GJ) and close membrane appositions (arrows) where the adjacent cell membranes are pinched together to varying extents. X78.800. FK.. 43. Perineurial cell layer from the centipede, Lirhobius. revealing extensive linear arrays of gap junctional EF particles. These frequently are associated with discontinuous grooves (arrows) which appear to form the tight junctional system, visihle in thin sections as punctate cell-to-cell appositions or fusions (arrows in inset). ~38.600.inset. ~75,300. Flti. 44. Freeze-fracture replica of a pleated septate junction from the rectum of a centipede, showing the extensive P face (PF) rows of aligned, yet separate intramembranous particles and complementary EF grooves. These structures, possibly by serving as insertion sites for intercellular ribbons or septa. have been considered by some investigators to be occluding in nature. in spite of the 15-20 nm intercellular cleft ( C ) . ~38.600. FIG. 45. Replica of smooth septate or continuous junctions from the mid-gut of the moth, Munducu. These are characterized by extensive arrays of intramembranous particles which are aligned so closely as to be fused laterally with one another into ridges. These ridges lie in closepacked parallel rows near the luminal microvilli ( M V ) and are rather more loosely arrayed beneath as undulating rows of P face (PF) ridges or complementary E face (EF)grooves into which. in this fixed tissue, only a few particles fracture. The continuous ridges were the structure that originally led investigators to believe, in spite of the 15-20 nm intercellular cleft, that these junctions resembled vertebrate tight junctions. X32.500. FIG. 46. Freeze-cleaved replica from the rectal papillae of the blowfly, Caf[iphorn.showing the reticular "septate" junctions that occur between the stacked scalarifom junctions. These display extensive ridges composed of intramembrananous particles in the membrane P face (PF) and complementary grooves in the E face (EF). The intercellular space ( S ) between adjacent membranes is distendible and may attain a considerable size. C , cytoplasm. ~30.200. FIG. 47. Replica featuring the retinular junctions found between rhabdomeres or retinular axons in the photoreceptor region of the compound eyes of CuUiphoru. These junctions are composed of rows o f intramembranous PF particles, fused into ridges. which are scattered over the membrane face. x38.ooO.

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layer. Since very few junctional studies have yet been made on arachnids, however, it seems a reasonable prediction that tight junctions may also occur in both eye and testis where blood-retinal (Shaw, 1978) and blood-testis (Szollosi and Marcaillou, 1977). bamers have been found in other arthropod systems, in particular the insects. Freeze-fracture data on the spider tight junctions thus far studied (Lane and Chandler, 1980) reveal that they exhibit all the classical features of vertebrate tight junctions, namely ( 1) in cross-fractures, the adjacent membranes make punctate appositions or fusions into one another (Fig. 17); (2) at these junctures the PF ridges are coincident across the face transitions with EF grooves on the other membrane face (Fig. 39); (3) there is an occlusion of the extracellular space at these points so that it is totally obliterated; (4)there is a circumferential region of intramembranous PF ridges and their complementary EF grooves (Fig. 40) which lie around the region that is sealed off; (5) these ridges and grooves are arranged as a network or reticulum exhibiting anastomosing ridges with only a few ridge discontinuities (Fig. 16); and (6) the ridges are composed of intramembranous partices fused laterally into ridges with intercalated gap junctional particle lying within the reticular framework (Figs. 21 and 41). Such characteristics differ from vertebrates only with respect to ridge discontinuities and in that the gap junctions in arthropods possess EF. not PF intramembranous junctional particles; these are loosely, not tightly, packed and measure about 13 nm in diameter in contrast with the 8-10 nm particles of the tight junctional arrays. Here then, in this group of the Arachnids, are true complex zonulae occludentes which form unequivocal permeability barriers. In other groups of spiders (Lane, unpublished data). the perineurial tight junctions are characterized by discontinuous ridges which fracture on both PF and EF (Fig. 41) and which may, within a single replica, exhibit both PF and EF ridges. In some cases there are also regions with a mixed ridge population, usually a few short ridges or particles sitting within EF grooves (Fig. 41). Clearly here complementary replicas are required in order to determine precisely how discontinuous the ridges actually are.

E. MYRIAPODS Both millipedes and centipedes are found in the class of arthropods known as the My-iapoda but relatively little is understood about their junctional complexes. Their neuromuscular junctions have been examined (Reger and Fitzgerald, 1981) and their midgut has been found to possess smooth septate junctions (Juperthie-Jupeau, 1979; Lane and Lee, unpublished data). However, the ensheathing glial cell layer round their CNS exhibits quite atypical junctional complexes in comparison with those found i n insects and spiders. In millipedes, thin sections of the CNS reveal curious glial cell-to-cell associations. possibly ones which operate via intercellular septa although the intercellu-

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lar space is much reduced (Fig. 42), together with many gap junctions; the latter, in replicas, can be seen to be very loosely clustered, but no features characteristic of any kind of tight or septate junctional structures have thus far been found in the freeze-fractured material (Lane, unpublished data). In freeze-cleaved replicas of centipede CNS, the same outer glial region exhibits an unusual mixture of linear gap junctions and discontinuous tight junctional arrays (Fig. 43). The latter are also identifiable as punctate appositions in thin sections (Fig. 43, inset) and appear as lengthy, yet interrupted EF grooves running into the linear gap junctions (Fig. 43) or PF particle ridges in the freeze-cleaved material (Lane and Lee, unpublished data). As yet, however, no definitive statement as to their precise nature and distribution can be made although they appear to be in some degree leaky to ionic lanthanum and ferritin (Lane and Swales, unpublished data) which would correlate with the observed discontinuities in their component ridge/ groove system (as in Fig. 43). It is clearly of enormous importance to be able to distinguish true occluding junctions from other junctions which may also bear a superficial resemblance to the tight junctions in replicas. For this reason, and because of the controversial history of the invertebrate occluding junctions, the sections that follows (Sections IV,A-E) deal briefly with the kinds of junction found in arthropods that are nor occluding, but which either have been, or may be, misinterpreted as such. These either have been thought to have a sealing action, or are structurally sufficiently similar to tight junctions in one way or another, to have been considered by some investigators as indistinguishable from them.

V. Junctions Exhibiting Certain Features in Common with Tight Junctions Table 11 summarizes the features that these various junctions share with tight junctions, together with the respects in which they differ. A . PLEATED SEPTATE JUNCTIONS

Pleated septate junctions occur in a wide diversity of arthropod tissues (Wood, 1959; Noirot-TimothCe and Noirot, 1980; Lane and Skaer, 1980), in particular epithelial cells where they may frequently occur near the luminal border. This positioning resembles that of the tight junctions in vertebrate epithelia and although little was known of the septate junctional permeability characteristics for a very considerable period, the concensus of opinion was that invertebrates lacked tight junctions per se, and formed their permeability barriers by means of these septate junctions (Satir and Gilula, 1973; Noirot-Timothee et al., 1978; Green, 1978; Green et al., 1979, 1980). In fact, many places where septate junctions are found lack any obvious barrier function, and although some inves-

Comparison with tight junctions Tissue

Name of junction

Ewphagus. rectum. salivary glands, testis. ovary. CNS. epidermis, tracheae

Pleated septate junctions

Extensive arrays of PF particles and EF grooves which are complementary

15-20 nm clefts Particles always separate

Insects. Cells of mid-gut. hepatoArachnids. pancreas, cecum, and Myriapods. Malpighian tubules and Crustaceans

Smooth septate, or ronultr conrinuu (continuous junctions)

Rows of PF particles often fuaed into ridges + EF grooves in fixed tissue

15-20 nm intercellular cleft If unfixed. ridges fracture onto EF

Inhects: Diptera

Retinular axons pound eyes

Retinular junctions

Insects: Diptera

Rectal papillae

Primitive Xiphosuran horseshoe crab,

Glial cells of CNS

Organism Insects, Arachnids. Myriapods. Crustaccans

Limuluu

in

com-

Common features

Distinctive featurex

Reference Wood ( 1959, 1977): Flower and Filshie (1975): Noirot-Timothee and Noirot (1973. 1980); Lane and Skaer ( 1980) Noirot and Noirot-Timothee (1967); Noirot-Timothde and Noirot ( 1974, 1980); Flower and Filshie ( 1975); Lane (1978a); Lane and Skaer (1980); Skaer et (11.. (1979) Lane ( 197%): Carlson and and Chi (1979); Chi et d. ( 1979): Lane and Skaer (1980) Lane (IY7Yb.c); Lane and Skaer ( 1980); Flower and Walker (1979)

Rows of PFparticles fused 15-20 nm intercellular into often discontinuous clefts ridges + EF grooves Ridges and grooves often are noncomplementary Reticular septate junctions Rows of PF particles fused Variable cleft 15-20 nm or into PF ridges, often greater discontinuous. + EF Ridges and grooves often grooves noncomplementary Junction not in circumferential bands Tight-like junctions Rows of PF particles fused Cleft not (always) obliter- Harrison and Lane ( 198 1 ) into ridges + EF ated grooves Not in circumferential bands

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tigators claim that septate junctions retard the intercellular passage of substances to a variable extent (see references in Section III,A,6), others found that they presented no occlusion to the intercellular passage of ions or molecules. These junctions then have been confused with tight junctions from the physiological point of view, due to their localization rather than ( 1 ) to their physiological similarity to vertebrate tissues (indeed, see Maddrell, 1980, for details of the ways in which they differ), (2) to any structural likeness, or (3) to any clear-cut support for their forming an effective permeability barrier. Many reports which claim to show that they inhibit tracer entry deal with preparations in which tracers are added to the fixative (see Lane and Skaer, 1980) and so are not meaningful physiologically. The structural features of septate junctions differ dramatically from those of the Zonulae occludentes, for they consistently exhibit a regular 15-20 nm cleft wherein exist undulating septal ribbons which in thin sections cut transversely appear as ladder-like structures (Fig. 22, inset) (see Noirot-Timothee and Noirot, 1980). After freeze-fracturing they possess intramembranous PF particles which are separated from one another by fairly consistent distances, arranged in distinct rows with complementary EF grooves or rows of pits; these lie in undulating tracts (Fig. 44) over the membrane fracture faces and do not form networks. Structurally, then, they do not resemble tight junctions in thin sections or in freeze-fracture replicas in spite of the similarity of the two in their cytological localization.

B . CONTINUOUS (SMOOTH SEFTATE)JUNCTIONS When these continuous junctions were first described (Noirot and NoirotTimothee, 1967) they were called zonula continua and attention was drawn to the observation that in freeze-fracture, they looked structurally similar to vertebrate tight junctions. This was due to the fact that their intramembranous composition is that of particles fused laterally into extensive fibrils or moniliform ridges, on the one membrane face, with complementary furrows on the other membrane face (Fig. 45). The face to which the fibrils adhere depends on whether the tissue is fixed or unfixed (Flower and Filshie, 1975). However, they are restricted to certain tissues, for the most part (see Skaer et al., 1979) of endodermal origin, such as the midgut and hepatopancreas, and they tend to run in parallel tracts rather than to branch into a network like the vertebrate tight junctions; there is, moreover, a constant 15-20 cm cleft between the adjacent membranes so that no intercellular occlusion occurs. This intercellular cleft contains both septal ribbons and columns (Flower and Filshie, 1975; Noirot-Timothee and Noirot, 1980; Lane and Skaer, 1980) and under some circumstances it has been suggested that these may restrict the passive intercellular diffusion of ions and molecules (Mills er al., 1976); the evidence for this, however, is not yet convincing (Maddrell and

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Gardiner. 1974; Lane and Skaer, 1980, p. 70). Although there is some disagreement on the following point (Noirot-Timothee et d.,1978). it is possible that the intercellular septa1 ribbons attach to the membrane at the sites of the intramembranous ridges (see Flower and Filshie, 1975; Lane and Skaer, 1980, Fig. 23). Whether or not this is so, the junctions are clearly distinct from tight junctions proper.

C. RETICULAR“SEPTATE” JUNCTIONS The reason that these junctions may be confused with tight junctions is their freeze-fracture appearance; they are composed of rows of fused intramembranous particles which appear as ridges arrayed in a semireticular fashion (Fig. 46); these are found over the face of the membranes lying on either side of the stacked scalariform junctional arrays in the Dipteran rectal papillae (Lane, 1979b; Flower and Walker, 1979). The intercellular space which is 15-20 nm or greater seems to contain not septa, but faint striations which may be columns in some regions (Lane, 1979b). For this reason it has been argued that they ought not to be called reticular septiite junctions (Noirot-Timothee and Noirot, 1980). However, the term was originally coined in an attempt to incorporate both the features of the regular 15-20 nm width of the cleft with its striated appearance and the reticular arrays of intramembranous ridges (Lane, 1979b). The physiological role of these junctions is still fairly speculative but it is not an occluding one; it has been suggested (Lane. 1979b). however, that they may serve to regulate the speed and extent of dilation that occurs in the interstack regions of rectal papillae; by spreading the pressure applied to these membranes by solute flow, an even pushing apart of the membranes would be achieved by the presence of the ridges. This would ensure that the hydrostatic pressure was adequate to support the rapid solute flow which is a feature of these rectal tissues. D. RETINULAR JUNCTIONS The retinular junctions (Lane, 1979c; Lane and Skaer, 1980), also called R cell junctions (Carlson and Chi, 1979; Chi et al., 1979), are found between retinular (and glial) cells in the regions of the photoreceptor cell axons or the rhabdomere region of dipteran compound eyes. Again, as with continuous and reticular “septate” junctions, they look to be tight junctions proper by virtue of their freeze-fracture appearance, which is that of PF ridges or EF grooves arrayed at a whole galaxy of angles to one another (Fig. 47); however, these are frequently discontinuous and the intercellular cleft is not obliterated but measures about 15-20 nm. Moreover, the ridges and grooves are often noncomplementary. These features are obviously not those of true zonulae occludentes although it

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has been suggested that they may maintain localized concentration gradients between retinular cells (Chi et al., 1979).

E. GLIALCELLJUNCTIONS I N PRIMITIVE ARACHNIDS Primitive Xiphosuran chelicerates, such as is typified by the horseshoe crab, Limulus polyphemus, appear to have no clear-cut permeability bamer in the CNS (Harrison and Lane, 1981) nor have the acarine arachnids, the ticks (Binnington and Lane, 1980; Hart er al., 1980). In both cases tracers are able to penetrate between the perineurial clefts and beyond into the underlying glial cells and axons. As might be expected, the perineurial glial cells are associated by gap junctions but not tight junctions or septate junctions. The latter clearly exist in these tissues for the pleated variety can be found in the tick tracheoles (Binnington and Lane, 1980), hypodermis and salivary gland (Beadle and Megaw, 1979) and in Limulus rectum, testis, and esophagus (Lane and Harrison, unpublished data), while the midgut of both tick and horseshoe crab possesses smooth septate junctions (Lane and Harrison, 1978; unpublished data). Although no tight junctions per se exist in the tick, the basal perineurial cells, or the immediately underlying glial cells, are sometimes found to possess PF ridges (Binnington and Lane, 1980, Fig. 29). In Limulus, regions of glial cells, as yet uncharted precisely within the ganglia, exhibit extensive PF ridges with complementary EF grooves (Fig. 23) (Harrison and Lane, 1981). The evidence is as yet equivocal, but in some cases the face transitions from PF ridges to EF furrows appear reduced or obliterated (Fig. 24) as might be expected for occluding junctions. Whether this means that there are some focal regions within the brain of Limulus from which ions and molecules are excluded is not yet clear nor is there any immediate explanation for such a structure, save for the remote possibility that rudimentary tight junctions had appeared early on, but that no effective barrier function subsequently evolved.

VI. Comparison of Arthropod Zonulae Occludentes with Those of Vertebrates and Lower Chordates A. DIFFERENCES IN LOCALIZATION The tight junctions of arthropods may be divided into two categories in terms of the position they assume with respect to the basal lamina (termed, in the case of the nervous system, the neural lamella). In insects they tend to lie toward the inner (basal) border of the peripheral cells between which they occur; however, in arachnids such as the spiders and scorpions, as well as in the myriopods such

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as the centipedes, they are found toward the outer (apical or luminal) border, as they are in vertebrates (Claude and Goodenough, 1973) and lower chordates (Georges, 1979). This terminology relates to the rather unusual arrangement and position of the "epithelial-like" perineurial cells wherein the outer border is referred to as apical. However, since this is where the neural lamella or basal lamina-like layer is found, this face could be construed to be basal. There is no distinctive luminal border, because this layer, like the tight junction-containing myelin sheath of vertebrates (Schnapp and Mugniani, 1975; Shinowara et al., 1980), is not interposed between two extensive fluid compartments as are vertebrate epithelial layers. It is, in fact, lacking in morphological criteria such as microvilli, and is arranged in the reverse way to that of vertebrate epithelia. Hence the terminology cannot be precisely comparable. The distribution of the simple tight junctions in insects may be related in some cases to the bracelet cell phenomenon of the CNS where the morphological basis of the diffusion barrier may be between overlapping bracelet cell processes that are very attenuated (Fig. 53) (McLaughlin, 1974a; Swales et al., 1981): the junctions between these may only occur in the basal regions of the interdigitating perineurial cells. The topographical location of the complex tight junctions in the insect compound eye is not clear either, since here the junctions are not linking adjacent epithelial cells but are associations between complex glial arrays (Lane, 1981b) which abut onto other cell types rather than extracellular space. In all cases the arthropod tissues exhibit junction-bearing lateral borders with a high degree of interdigitation in contrast with the more straight-sided lateral borders of vertebrate tissues (Lane and Skaer, 1980, p. 54). The physiological significance of this, apart from the maintenance of structural integrity, is not clear, especially in those arachnids which possess vertebrate-like tight junctions near the outer border of the cellular sheath.

B. DIFFERENCES I N FRACTURING CHARACTERISTICS The zonulae occludentes of arthropod tissues tend to possess rows of aligned or fused intramembranous particles which fracture onto the P face. This is the case whether the tissues have been fixed before cryoprotection or not, in insect tissues, as well as in most arachnid and myriapod material. The exceptions to this generalization include the scorpion and at least one genera of spider; in such systems the ridges fracture onto the E face in fixed tissue (Fig. 38) and onto the P face if unfixed (as in Fig. 20). In some cases, intermediate situations prevail and i n fixed tissues, only part of the EF particle population may adhere to the E face after fixation (Fig. 41); this is a variable phenomenon, and in the same area of membrane face solid EF ridges may be found together with fragments of EF ridges or EF particles associated with EF grooves, with concurrent PF particle

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rows (Fig. 41). The forces binding the intramembranous particles to the inner half of the membrane or to each other (see Section VIII) can therefore presumably not be completely due to cross-linking induced by glutaraldehyde fixation as has been suggested for vertebrate tissue (van Deurs and Luft, 1979). Indeed, in this respect the arthropods are quite distinct from vertebrates in that in the latter they are adherent to the EF only if unfixed according to van Deurs and Luft (1979). However, the situation in some other vertebrate or lower chordate tissues, which have been chemically fixed, may be rather different. The tight junctions there may exhibit E face particle rows, such as in the Sertoli cells of the testis (Gilula er al., 1976; Nagano and Suzuki, 1976; Connell, 1978), in the endothelial cells of small blood vessels (Simionescu et al., 1978) and in two out of the three species of tunicates studied (Georges, 1979). It has been suggested, since these intramembranous particles fracture onto the EF and tend to lie in rows, rather than becoming fused into ridges, that they do not respond to glutaraldehyde fixation in the conventional way (Van Deurs and Luft, 1979). A rationale for this idea or support for the theory has yet to be produced, however. In the tunicates, tight junctions have been observed in adult tissues, such as differentiated pharyngeal and epidermal epithelia; they are here arranged in some 6 to 13 strands of distinct particles forming a circumferential network at the apical part of cells (Georges, 1979). The component particles range from 6.5 to 25 nm in diameter for some species or are 12 +2 nm in others and in some cases fracture onto the EF. No septate junctions were observed in larval tadpoles or adult tissues (Cloney, 1972; Georges, 1979), although gap junctions and spot desmosomes are present (Georges, 1979). The form or pattern assumed by tight junctions in tissues of arthropods is possibly more variable than that observed in vertebrates. In the former, the simple junctions exhibit only simple, discontinuous linear ridges; the complex junctions may be fairly continuous with branching to form a network but this is often disrupted when the ridges or particles rows abruptly terminate (Fig. 25). Moreover, although the simple insect ridges tend to lie parallel to the outer surface of the CNS, the more complex arachnid ones are more open meshworks of ridges rather than parallel, interconnected alignments such as characterize many vertebrate tight junctions (Claude and Goodenough, 1973). Tunicate tight junctions consist of a number of particle rows arranged in a loose network in a beltlike position (Lorber and Rayns, 1972; Georges, 1979); in some cases, their apical parallel strands are underlain by a network of ridges, which increase in looseness with the distance from the cell apex (Georges, 1979). Precisely how steep a concentration gradient these, and the various forms of the arachnid tight junctions, produce in the relatively impermeable barrier systems observed, is as yet unclear, since relatively few physiological studies have been made on them in comparison with the vertebrates.

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C. DIFFERENCES I N TYPES O F LEAKINESS OBSERVED As just stated, in the arthropods, there have been fewer physiological studies made to date on the permeability barriers present, than is the case for vertebrate tissues. In some of the earlier studies on the latter, it was possible to try to correlate the strand morphology of the tight junctions with the recorded physiological degree of “leakiness“ or “tightness” (Claude and Goodenough, 1973); here the case was put forward that the more complex the junctional network and the greater the number of parallel ridges, the “tighter” it would be in terms of restricted intercellular permeability. However, since then, more extensive studies have revealed (Martinez-Palomo and Erlij, 1975; Mollgard er al., 1976; Erlij and Martinez-Palomo. 1978) that the degree of leakiness cannot be correlated simply with strand morphology. For example, tissues where identical tight junctional ridges are found may exhibit very different degrees of leakiness (Martinez-Palomo and Erlij, 1975). The chemistry or degree of charge of the ridge elements or some other, as yet unresolvable, aspect of the tight junctions, is held to be responsible (van Deurs and Luft, 1979). This may well also be the case for the arthropods; for example, urea treatment leads to a leakiness to Na+ but not to lanthanum ions (Treherne et a/.. 1973). However, in some “leaky” vertebrate epithelia, there is no doubt that grossly discontinuous junctional elements permit extracellular tracers to leak partway along the intercellular cleft (Friend and Gilula. 1972: Reale er al., 1975). This situation is paralleled in the scorpion blood channels and perineurium where many of the tight junctional ridges are discontinuous and tracers are observed to leak around the incomplete restrictions imposed by these elements (Figs. 35 and 36) (Lane et d., 1981), particularly in the peripheral nervous system.

D. DIFFERENCES I N DEVELOPMENTAL STAGES During the assembly of tight junctions in both vertebrate and arthropod tissues, individual intramembranous particles become inserted into the presumptive junctional region. In vertebrates, this area is relatively clear of other intramembranous particles (Suzuki and Nagano, 1978b) and the precursor particles may be rather larger (Porvaznik er id., 1979; Luciano er al., 1979; Yee and Karnovsky, 1979); studies with filipin labeling indicate that it is also relatively cholesterolfree (Montesano, 1981). Comparable data are not yet available for arthropods. The individual intramembranous particles gradually become aligned into rows, presumably by translateral migration in the fluid lipid bilayer. After this part of the assembly process, the particulate elements of vertebrate junctions frequently fuse laterally to form moniliform rows which are then “coated” to produce smooth ridges (Luciano er a/., 1979; Montesano er d.,1975). This does not occur in tunicates where the particles remain separate (Georges, 1979) nor is it

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clear whether it happens in arthropods (Lane and Swales, 1978a,b, 1979). In some cases the ridges remain particulate while in others they appear smooth. This need not necessarily be due to the addition of a “coating” but may reflect the angle of shadowing during replication., The capacity of tight junctions to coexist with other intercellular junctions seems a universal quality. In both vertebrate and invertebrate systems, the tight junctions are to be found along the same cell border as gap junctions and desmosomes, and, in some special insect examples, along with septate junctions. The first of these junctional types is the most common partner of the tight junctions. Indeed, their persistent coexistence has led to the possibility of making studies of their concurrent developmental changes. In young spider hatchlings, the different preferential fracture planes to which the particles of tight (PF) and gap (EF) junctions adhere enable one to follow their distinct patterns of insertion into the presumptive junctional membrane and their subsequent differentiation via translateral intramembranous migration. This combined with .their distinctive sizes, the former (8-10 nm), compared with the latter (12-14 nm), makes it possible to distinguish them unequivocally during development (Lane, 1980; 1981a). This has made more possible the assertion that there is no one precursor particle from which both tight and gap junctional particles have evolved (Lane, 1981a) (see Section VII,B).

E. DIFFERENCES I N INSECT VERSUS VERTEBRATE PHYSIOLOGY Insects are remarkable organisms in that they appear to absorb through the wall of their gut almost anything they consume, which tends to lead to fluctuating and unpredictable hemolymph ionic proportions and concentrations (Maddrell, 1980). Seemingly in response to this, the particularly important and potentially vulnerable systems have become sealed off from the circulating hemolymph by permeability barriers; these include the blood-brain barrier, blood-testis barrier, and blood-retinal barrier. Malpighian tubules and rectal tissues are then active in regulating the hemolymph composition. In vertebrate gut there is a strong dependence on active absorption, whereas in insects the system seems to depend to a greater extent on passive, and possibly less selective, uptake mechanisms (Maddrell, personal communication). The situation in arthropods other than insects is not yet so clear-cut, although permeability barriers may exist in CNS, eye, and blood vessels. In Comparison with insects, vertebrates exhibit many more diffusion barriers, possessing tight junctions in a great many of their epitheliaintestine, kidney, gall bladder, etc. (see Claude and Goodenough, 1973); lower chordates also possess the morphological basis of a barrier in the pharynx as well as between epidermal cells which in this case is considered to represent protection against water loss (Georges, 1979).

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F. DISRUPTION O F JUNCTIONS A number of treatments have been found to disrupt tight junctions in vertebrate systems, particularly the application of hypertonic solutions such as urea (Rapoport et al.. 1971), sucrose, or disaccharides (Goodenough and Gilula, 1974: Van Deurs, 1980), and trypsinization (Dermietzel et al., 1977) or mechanical shear (Metz et 01.. 1978). Under such conditions, the junctions became leaky, due, it has been thought, to separation of the junctional elements (Wade and Karnovsky, 1974b). Other treatments, involving osmotic stress, lead to blistering of the adjacent membranes which are held together by tight junctions (DiBona, 1972). Changes in permeability have been induced by lowering the Ca' levels, from treatment with Ca2+-freesolutions to those with EGTA (Meldolesi et ul., 1978; Martinez-Palomo er al., 1980). The molecular basis for this is thought to be charged interactions between adjacent tight junctional particles (van Deurs and Luft, 1979; Martinez-Palomo and Erlij, 1978). There is evidence that microfilaments may play a part in positioning the tight junctional strands and may influence the degree of sealing of the junctions in cultured mammalian cells (Meza et al.. 1980). Such an association of components of the plasmalemma with microfilaments has not yet been observed in arthropod tissues although it has been reported for tunicate tight junctions (Georges, 1979). In the arthropods, relatively few experiments have been carried out along the lines of junctional disruption. Urea treatment leads to inward leakage of physiologically important ions but no change in the restriction to entry for tracer molecules (Treherne et al., 1973). Blistering experiments with hyperosmotic solutions have thus far been carried out only on tissues which possess septate, but not tight, junctions (Lord and diBona, 1976; Mills el a l ., 1976; Green et u1.. 1980).

VII. Stages in the Assembly and Breakdown of Arthropod Tight Junctions A. INTRAMEMBRANOUS PARTICLE (IMP) MIGRATION Analysis of the development of tight junctions can normally occur in vhio only during embryonic and very early hatchling stages in arthropods or possibly during the regeneration of injured tissues. In some groups, however, such as the holometabolous insects, metamorphosis presents another possible occasion for the formation of junctions during the reassembly of cells from disrupted larval tissues into organized adult systems, which tissues may differ markedly in certain respects one from the other. The first studies to be made on developing tight junctions in the arthropods were in the holometabolous dipteran blow fly, Calliphoru. Here the tight junctions of the developing perineurial layer, the cellular basis of the blood-brain

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permeability barrier, were studied during the stages of their assembly in embryonic and early life (Lane and Swales, 1978a) as well as during pupil metamorphosis (Lane and Swales, 1978b). It emerged that the barrier and hence the tight junctions do not develop until after hatching in Calliphora during the first few hours of postembryonic life. In this situation, the advent of a barrier to the entry of tracer through the perineurial clefts was correlated with the appearance of simple nonanastomosing tight junctional ridges (Lane and Swales, 1978a). Observations of the CNS during the various stages of pupal metamorphosis revealed that in the first few days of pupal life the blood-brain barrier broke down. Tracers leaked in around the nerve cells (Fig. 48) and only fragments of ridges were to be found in the fractured perineurial membrane faces (Lane and Swales, 1978b). Toward the middle of pupal life, ridges reformed, possibly by reassembly of the fragments into tight junctions again; tracers were once more excluded from the CNS by the end of pupal life. The precise details of assembly and reassembly were rendered indecipherable because of the extreme difficulty involved in actually finding these very simple and elusive tight junctions; such rarity made the following of a step-by-step sequence of the events leading to intramembranous ridge assembly rather unlikely. It was only possible to know when the effective events had occurred by watching carefully for the lanthanum exclusion. One feature that did emerge was the possibility of reutilization of the IMPS from the old junctions for assembling the new ones (Lane and Swales, 1978b), a possibility which has also recently been set forth for the formation of vertebrate tight junctions (Polak-Charcon and Ben Shaul, 1979) when they reassemble after in vitro degradation. Preexisting tight junctional components have also been reutilized in resealing cultured mammalian cells it seems; the process was found not to require protein synthesis (Martinez-Palomo er a f . , 1980). Evidence to suggest comparable IMP dispersal and subsequent reaggregation of the same junctional particles has also been put forward to explain uncoupling and recoupling of gap junctions between migrating glial cells in insect pupae (Lane and Swales, 1980; Lane el al., 1980). Here, as in the case for tight junctional breakdown and reassembly, hormonal stimuli are probably the cues to promote these events. As in insect pupae, tight junctions undergoing disassembly or degradation in vertebrate tissues also possess broken strands with decreasing lengths of ridges and more dissociated particles (Revel et al., 1973; Revel 1974; Decker and Friend, 1974; Dermietzel et al., 1977; Metz et al., 1977, 1978; Polak-Charcon and Ben Shaul, 1979; Decker, 1980). Although internalization of these remnants has been suggested (Staehelin, 1973, 1974; Porvaznik, 1979) for some tissues, in the insect pupae there is no evidence for this. Breakdown of junctional ridges, perhaps by sequential weakening of interactions between the IMP subunits (Decker, 1980, 198I), and subsequent reutilization of the junctional elements, does, however, seem possible, as stated above. This reassembly could be a “hemicon-

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servative” process (Dermietzel et al., 1977) whereby junctional remnants serve as condensation points from which the assembly proceeds as it does when they first form in embryonic development. A subsequent developmental study in insects, on the holometabolous moth Manducu sextu was made, where the tight junctions were rather less infrequent (Lane and Swales, 1979) than in Calliphoru; various stages in apparent junctional assembly were followed in the late embryonic life of the larval form. These stages involved IMPs becoming aligned into short parallel ridges on the P face (Fig. 14) which ultimately became fused into more lengthy ridges lying in parallel with other simple unbranching ridges (Lane and Swales, 1979). No formation plaque areas, nor larger IMPs seemed to be involved nor did the ridges appear to become covered with a “coating material” as has been reported in vertebrate material (Porvaznik et al., 1979; Yee and Karnovsky, 1979; Luciano et al., 1 979). In the locust, Schistocercu, tracer and freeze-fracture studies on the perineurium permit analysis of tight junctional differentiation which occurs during embryonic development. Glial cells migrate to the surfaces of the nerve cell mass and begin to form a layer that excludes tracers (Fig. 49). The perineurial cells are extremely attenuated and few in number; they come to overlap with one FIG.48. Thin section of CNS of the blowfly, Calliphoru, during early stages in metamorphosis, after incubation in ionic lanthanum. The perineurial tight junctions have been disrupted and the electron opaque tracer has leaked in around neurons and glial cells. x 1 1,100. FIG. 49. Perineurial cells (PN) of the embryonic locust CNS, in the early stages of forming a sheath around the nerve and glial cells. The outer neural lamella (NL) is very attenuated and lanthanum can be seen in it and in some of the underlying cellular clefts where the junctions are beginning to form. The axons (A) beneath this are as yet disorganized and have not yet assumed their final positions. X 16,300. FIG.50. Early stage in the development of tight junctions in spider perineurium. lntramembranous 8-10 nm PF particles appear to migrate translaterally to take up linear arrangements, first in short rows of 2 to 3 particles, later, in more extensive alignments. X63,600. FIG. 5 1. Slightly later stage in the development of spider perineurial tight junctions where the P face (PF) ridges have become quite extensive (large arrow), although still clearly moniliform, and the complementary E face (EF) grooves are fairly widespread. Between these are scattered 13 nm EF particles which, in certain regions (arrows), are beginning to become aggregated into macular arrays as is typical of mature gap junctions. The precursor EF particles of these are clearly distinct from those that give rise to the PF tight junctional structures. x33,600. FIG.52. Later stage in the differentiation of perineurial tight junctions in hatchling spiders: the aligned intramembranous particles are fused into ridges which are frequently quite smooth in appearance. This could be due to the angle of shadowing or could result from the addition of a “covering” material of some sort. ~ 5 7 , 4 0 0 . FIG.53. Perineurium (PN) around interganglionic connectives of the cockroach ventral nerve cord. One of these peripheral sheath cells can be seen to possess various cytoplasmic processes projecting inward between axons (A) and glial cells. It forms a bracelet cell, lying around a large proportion of the nerve cord’s circumference and overlapping (along from*j with another perineurial cell. x 1 1.600.

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another very extensively, in bracelet-like configuration (as in Fig. 53). The tight junctions, therefore, need only form in the crucial areas of overlap of one bracelet cell process with another (Swales et al.. 1981). This they do in the conventional way via PF particles aligning into ridges; these are more basally oriented than the gap junctions some of which seem also to be fully formed by the same stage as the tight junctions are forming, about two-thirds through embryonic development. This stage can be correlated with the advent of an electrophysiologically demonstrable blood-brain barrier in another orthopteran, the grasshopper, Melanoplus, where the cells also form an ensheathing and effective barrier at the same relative time in the embryonic life cycle (Goodman and Spitzer, 1979). A rather more striking example of arthropod junctional assembly has been found in the arachnids. This is in the spider nervous system where the perineurial layer around the CNS is associated on its lateral borders by gap junctions, intimately associated with rather more complex tight junctions (Lane and Chandler, 1980) than are encountered in the insect CNS. Stages in the assembly of these junctions have been followed in very young hatchling spiders wherein the blood-brain barrier is not yet completely formed (Lane, 1980, 1981a). In this system the 8- 10 nm tight junctional particles are found first as free IMPS on the P face; eventually they begin to become aligned alongside one another into short (Fig. 50) and then increasingly long (Fig. 51) PF ridges. In some cases these bead-like ridges later become very smooth in appearance (Fig. 52). It is not clear whether this is due to the angle of shadowing or whether there is an addition of “covering material” to mark the final stages in ridge maturity as is thought to occur in vertebrate tight junctions (Montesano et a / . , 1975; Elias and Friend, 1976; Luciano er al.. 1979). If the phenomenon does occur, it is inconsistent, however, since even in mature animals one may find moniliform ridges; this could be a reflection of the variability of the interactions between the component, facing particles that comprise the junctional strands (see Luciano et al., 1979). Ultimately the fibrils form a reticular network over much of the outer portion of the lateral perineurial borders (Fig. 40) in the spider CNS (Lane and Chandler, 1980; Lane, 1980, 198la). The early stages in arachnid tight junctional development are not marked, as has been reported for a variety of vertebrate tissues (Porvaznik et al., 1970; Luciano et a ( . , 1979; Yee and Karnovsky, 1979). by the presence of larger precursor particles, nor have formation zone ‘ridges’ been observed (Porvaznik er a / . ,1979).

B . ARTHROPODS YIELD EVIDENCE FOR DISTINCTION BETWEEN TIGHTA N D GAP JUNCTIONAL PRECURSORS An interesting aspect of this developmental process in the spider perineurium is that, as might be expected, the gap junctions tend to develop from dispersed particles into macular aggregates at the same time or just slightly after the events

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described above for tight junctions (Fig. 51). Since they are intimately associated with the ridges forming the tight junctions, their developmental sequence occurs around the same time as that for the tight junctions and the two can be followed simultaneously (Lane, 1980, 1981a). The two processes are, however, quite distinct, because the gap junctional particles fracture onto the EF and measure about 13 nm in diameter, while the tight junctional ones fracture onto the PF, and measure only 8-10 nm in diameter (Fig. 51). During this period of concomitant assembly it is evident, from their component particle size and plane of preferential fracturing, that the two junctional types are composed of intramembranous particles that are quite distinct from one another (Lane, 1980, 1981a). They are therefore unlikely to arise from a common precursor particle as has been suggested in some vertebrate tissues (Montesano et al., 1975; Elias and Friend, 1976; Larsen, 1977; Porvaznik et al., 1979); in the latter, the junctional particles of both tight and gap junctions are P face and of comparable diameter and so in many cases it is not possible to distinguish them during their concurrent formation (see Revel and Brown, 1976; Decker, 1976). However, the arachnid evidence would appear to put an end to this hypothetical common precursor particle; this theory in any case was intellectually unsatisfying since it suggested that two structures with very different physiological significance, in particular the gap junctional particles which possess a central pore for ionic exchange, could be similar in origin. The proteins comprising these junctional structures might be expected to be quite distinct purely on physiological grounds.

VIII. Models Constructed to Clarify the Features of Arthropod Tight Junctions There have been a number of models proposed to account for the features of vertebrate tight junctions (for example, Chalcroft and Bullivant, 1970; Staehelin, 1974; Wade and Karnovsky, 1974a; Bullivant, 1978; van Deurs and Luft, 1979) but hitherto there has been only one (Lane and Skaer, 1980) for the invertebrates, notably that for the insect tight junctions (see Fig. 54) and a modification of it (Fig. 7) for the scorpion occluding junctions (Lane et af., 1981). The reasons for this are obvious; tight junctions in invertebrates have only recently been described. Moreover, those that have been sighted had until recently not been seen in great quantities; hence there were too few results for the requisite rigorous analysis. However, now, by virtue of the concentration of tight junctions found between glial cells in dipteran compound eyes (Lane, 1981b), such models can be reliably put forward for insect tight junctions. Moreover, sufficient data have not accumulated from the arachnids to make it clear that their zonulae occludentes, as seen between the outer glial cells in the spider CNS, are rather different in certain respects from those seen between adjacent perineurial cells in

-

CYTOPLASM

*

-

E FACE

-,-LANTHANUM

IN INTERCELLULAR SPACE --',,

-

P FACE

-

FIG. 54. Model of an arthropod tight junction showing the appearance of the two fracture faces, with the P face exhibiting the junctional ridges and the E face the grooves. The intercellularspace is shown as it would appear with lanthanum leaking past a ridge that is discontinuous, had been disrupted. or that was in the process of forming. When continuous, or once completed, the tracer would not move past the junctional structure, as indicated in the left-hand side of the model where the punctate apposition between adjacent membranes is illustrated. A double fibri1 structure is depicted, and the two are thought to be offset with respect to each other.

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the “leaky” tight junctions of the scorpion CNS (Fig. 7). The models of these junctions incorporate information both from thin sections, conventionally treated and lanthanum incubated, and from the freeze-fracture data. It is clear that although there is a difference in complexity between insect and spider tight junctions, they both have comparable fracturing characteristics, that is, PF ridges and EF grooves in both fixed and unfixed tissues (Lane et ai., 1977a; Lane and Chandler, 1980; Lane, 1981a). In scorpions, however, the ridges fracture onto the EF in fixed tissue and onto the PF only if the material is unfixed, although occasionally in fixed tissues they may fracture partly onto the EF and partly onto the PF (Lane er al., 1981). The original report on spider tight junctions dealt with Tegenaria, the common house spider (Lane and Chandler, 1980) (see Fig. 40); subsequent observations have been made on the garden spiders such as Aranaeus (Lane, 1980, 1981a) (Fig. 51). Here the junctions were also comprised of PF ridges and EF grooves. However, other species of garden spider may possess zonulae occludenfes in the perineurium with ridges which fracture either entireIy or partially (Fig. 41) onto the E face (Lane, unpublished data). Such phenomena occur in fixed tissue and may coexist in the same replica. This suggests that there may be local variations in the way that the tight junctional particles are associated with either side of the membranes or laterally with each other, since clearly the one piece of the tissue was treated in exactly the same way throughout, unless there were differences due to speed of fixative penetration. In vertebrate tissues it has been suggested that the P face images in glutaraldehyde-fixed tissue are due to the junctional particles crosslinking first with each other to produce continuous ridges or fibrils and second with the protein-rich cytoplasmic surface of the membrane to produce the ridge-enriched PF images; brief or weak fixation would lead to intermediate particle distribution on both PF and EF, while fresh-frozen tissues would have rows of E face particles (van Deurs and Luft, 1979). However, examples of EF ridges in vertebrate tight junctions exist, for example, in Sertoli cells (Gilula et al., 1976; Nagano and Suzuki , 1976) or vascular endothelial cells (Simionescu et al., Palade, 1978) where the ridges are particulate, not smooth, even after conventional glutaraldehyde fixation. It has been suggested, therefore, that these images are the result of the fact that the tight junctional proteins in these systems lack glutaraldehyde-reactive sites, which would normally cause them to bind to the cytoplasmic membrane surface and laterally to each other to form smooth ridges (Van Deurs and Luft, 1979). This theory supposes that in uivo the junctional particles are quite separate entities which have a greater affinity for the external half of the membrane. Since ridges are sometimes found in cryoprotected but unfixed tissue (Bullivant, 1978) it is thought that the glycerol may also contain crosslinkers which cause formation of fibrils from separate particles (Van Deurs and Luft, 1979). Rather than the number of interconnecting strands determining tightness to ions (Claude and Goodenough, 1973), some other properties of the tight junctions may be respon-

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sible for the permeability observed at times (Martinez-Palomo and Erlij, 1975) whereby the tight junctional strands could be effectively “open” (Claude, 1978; Lucian0 et u l . . 1979). These suggestions of the presence of spaces between the tight junctional particles in vivo which themselves could possess different charges and side chains depending on their source and state, would permit varying and possibly selective permeability of ions between them (van Deurs and Luft, 1979). Bullivant (1978) points out that this is readily explained in the offset two fibril model where slight lateral separation of the two fibrils where they overlap would permit such leakage (see Fig. 54). The model favored by Van Deurs and Luft (1979) is not the single fibril model (Wade and Karnovsky. 1974a) but the offset double fibril model (Bullivant, 1978) which has also been used in a modified way in the insect tight junctional model (Lane and Skaer, 1980, Fig. 53; Fig. 54). Although four protein particles (Fig. 7) rather than two (Fig. 54) are suggested in the earlier model (Lane and Skaer, 1980), it seems unlikely that there is more than one transmembrane junctional protein particle in each participating membrane (Fig. 54). The EF ridges seen in fixed scorpion tissue would not be predicted by the hypothesis of van Deurs and Luft (1979); therefore, scorpions either lack glutaraldehyde-reactive sites in their tight junctional proteins, or some other explanation must be found. Further, in the tight junctions in some scorpion and garden spider replicas, there is a mixture of PF and EF binding (see Fig. 41). Unexpectedly, the EF particles tend to fuse laterally into fibrils in some areas (Fig. 38). while the PF ridges appear more separated and beadlike (Fig. 41). Moreover, in the same tissue, the EF may possess grooves with only a few EF particles fracturing onto that face (Fig. 41). The forces between these proteins, therefore, seem to be erratic; the reasons for this are at present unknown, as is their interaction, if any, with the lipid in the membrane. Removal of membrane lipid allows tracers to move past the tight junctional fibrils of some vertebrate junctions into the extracellular compartments (Goodenough and Revel, 1970) and so the state of the lipid probably affects the junctional protein (see Costello and Gulik-Krzywicki, 1976). There are some areas of replica in arthropod tissues where the fibrils appear to be offset with respect to one another (see Figs. 19 and 20) but the ridges nevertheless appear to be very prominent. The former indicates a double fibril but the latter suggests a single fused fibril model (Wade and Karnovsky, 1974a). However, lipid collapse (Bullivant, 1977) has been suggested to account for the enhanced height of some intramembrane proteins; therefore, quite apart from shadowing differences, the ridges could be more pronounced for this reason, although they must indeed be double not single fibrils if they appear offset (see Fig. 20). Further studies including complementary replicas are required to clarify points such as whether the arthropod tight junctions are composed of single or double

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fibrils as well as what the effects of the surrounding lipid may be on their structure. Since the data suggest that they are offset with respect to one another, this supports the double fibril model of Bullivant (1978) and this feature is incorporated in the current model (Fig. 54) of the arthropod zonulae occludentes.

IX. Possible Explanations for the Existence of Both Simple and Complex Tight Junctions in Arthropods-Evolutionary Implications Various questions arise upon considering the distribution of tight junctions in arthropods; for example, why should insects possess both simple and complex tight junctions while arachnids appear to have only the latter and some other groups of arthropods, no tight junctions at all? It must be presumed that in this last case, there is no physiological need for these organisms to have tight permeability barriers: such protection as is required may be provided by septate junctions. The varying complexity of the tight junctions that do occur in arthropods may be supposed to be a function of both their environment-aquatic organisms, for example, not encountering the rigors of maintaining a stable hemolymph that terrestrial forms must contend with-and possibly the sophistication of their nervous system in terms of their behavior. The arachnids exhibit very complex behavior patterns, and it may be that they must therefore protect their nervous system particularly carefully; this would be important so that the nervous tissues do not suffer from problems resulting from direct contact with the extracellular fluids, given the liklihood that their hemolymph could be very variable in its concentration and ionic composition depending on factors such as the animal’s diet. This, however, remains speculative in view of our ignorance of many of the parameters involved. In this respect it is worth drawing attention again to the arthropods’ unusual physiological mechanisms in comparison with vertebrates. It has been shown, as mentioned earlier (Section VI,E), that insects are less selective in what is absorbed through the gut wall, so that food substances are rapidly taken up into the hemolymph, tending to give rise to great variation in its composition (Maddrell, 1980). Mechanisms to ensure reabsorption of water and physiologically important ions then occur in the Malpighian tubules and rectal tissues leaving important organs, such as brain, eye, and testis sealed off from the vagaries of a changing hemolymph by means of efficient permeability barriers (Lane and Skaer, 1980; Maddrell, 1980). Although we do not have much information as yet on the physiology of arachnid systems, there is no reason to suppose that they would be radically different from the insects in this regard: certainly their brain and eye possess extensive diffusion barriers (Lane et al., 1981). Within the Insecta it is striking that the ganglia, connectives, and larger peripheral nerves possess simple tight junctions (Lane and Skaer, 1980) while the

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compound eye possesses complex ones (Lane, 198I b; Lane and Treherne, 1980). One possible explanation for this lies in the topography of the two systems. In the central and peripheral nervous system, it is clear that the perineurial layer that ensheathes the nervous tissue consists of cells that not only interdigitate extensively (Lane and Skaer, 1980) but also send their attenuated processes around large parts of the ganglionic circumference in the form of “bracelets” (McLaughlin, 1974a). This is particularly clear in embryonic systems where the separation of the CNS from the circulating body fluids by the developing perineurial processes occurs by long cell projections “reaching around” and overlapping with another cell process over some distance (Swales er al., 1981, unpublished data). This arrangement is often obscured in adult systems where the increased perineurial bulk produces an indecipherable meshwork of interdigitating processes. In certain situations. particularly in interganglionic connectives or nerves, where the perineurium is much thinner than in the ganglia, enhanced electron density of a single perineurial cell makes it possible to visualize this “bracelet” arrangement (Fig. 53). It is then clear that there is only one small area of overlap that requires the presence of tight junctions to effect a sealed system, and it is here that freeze-fracture studies of embryonic locust CNS reveal the appropriate ridges and grooves (Swales er al., 1981, unpublished data). Simple short ridges found in the adult insect CNS, could, as in the embryo, also be occurring in just the requisite position of cell overlap, but it would be impossible to judge this with any certainty due to the increased perineurial volume and complexity. The compound eye of insects, would, however. represent a rather different situation. Here, glial cell tight junctions must seal off the distal from the proximal portion of the same photoreceptor axon by intertwining around these axons as they run through at right angles to the barrier region (see Section VI,C,2). Simple overlapping cells with a discontinuous or noninterconnected ridgelgroove system would not be sufficient to provide an effective permeability barrier in this situation, so the more complex, vertebrate-like system appears to have evolved (Lane. 1981b). This question of the evolution of these junctions remains, perforce, an enigma. However. it is tempting to speculate on the events of the past. Attempts have been made (Green, 1978) to place the septate junctions of invertebrates, by virtue of their intramembranous ridges, in the position of evolutionary forerunners to vertebrate tight junctions: this disregards the obvious differences between septate and tight junctions, not least of which is the size of the intercellular cleft (Lane and Skaer, 1980, pp. 150-151). Such an evolutionary relationship seems unlikely in the light of recent advances. It seems more feasible that arthropod and chordate tight junctions arose independently as two separate lines of evolution, since the phylogenetic tree shows the chordates arising as a quite separate branch from that which gives rise to arthropods and annelids (Roots, 1978). Equally, the

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complex tight junctions of insect compound eye and arachnid nervous system must have arisen separately, since no direct evolutionary link seems likely between arachnids and the diptera; the Xiphosura, arachnids, and crustacea arose, it is thought, from primitive annelid stock, separately from the myriapods and the insects (Roots, 1978). The interesting preliminary data from myriapod systems are fascinating in view of the theory that holds that the six-legged larval form of some early myriapods could be the ancestor of the insects, originating by neoteny , which is the persistence of a larval characteristic. Centipede junctional systems, at least around the nervous system, provide preliminary intimations of both extensive linear gap junctions and rather leaky, discontinuous tight junctions (Lane, 1981, unpublished data) (Fig. 43) (see Section VI,E); they would appear more advanced than the millipedes in that the latter exhibit no evidence of ridges, or other tight junctional structures at all (Lane, unpublished data). Further details of these systems need to be established before such a theory can be seriously advocated. However, it does seem reasonable to suggest that when arthropod cells are required to form permeability barriers, they have the capacity to do so by means of cell-to-cell fusion via intramembranous ridges. This plasticity of the eukaryotic cell membrane may have been brought to fruition more than once during the evolutionary process. So it seems, that, as with the almost universal capacity of eukaryotic cells to couple via intramembranous gap junctional proteins, so too may they seal off two compartments by punctate tight junctional ridge-to-ridge fusions. The further question of why they are absent in invertebate groups other than arthropods can thus far only be countered by enquiring whether this absence is not so much real as apparent, due merely to a dearth of data. Perhaps further more extensive investigations of other invertebrate groups will yield additional examples of tight junctions. Until this occurs, it must be supposed that they may not be forthcoming and that the requirements for permeability barriers in such groups are less rigorous; perhaps septate junctions provide an adequate device to slow down and modulate the entry of exogenous molecules. Certainly there seems no reason why the existence of septate junctions should have precluded the simultaneous development of another type of occluding junction, possibly more effective in forming seals-the tight junction. The evidence thus far available by examining tissues like the CNS of such other groups as molluscs (Lane and Treheme, 1972b; Sattelle and Lane, 1972; Sattelle and Howes, 1975; Pentreath and Cottrell, 1970) or annelids (Skaer et al., 1978) yields no support for the existence of junctions other than desmosomes and gap junctions between the glial cells ensheathing the central ganglia. However, recent preliminary data (Hall et af., 1980; Abbott et al., 1981) suggest that simple tight junctions may occur between some glial cells in molluscs and it may be these which form a partial diffusion barrier, as has been proposed (Reinecke, 1976), on physiological grounds.

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To recapitulate. it seems then that certain kinds of cell associations are widespread between eukaryotic cells. These include desmosomes, gap junctions. and now, it seems. also tight junctions (Lane. 1981b). Septate junctions, and variations on that theme. although not normally encountered, may also be capable of forming between vertebrate cells (for example, Friend and Gilula, 1972; Schnapp and Mugnaini, 1975: Connell, 1978), in comparison with invertebrate tissues where they are extremely prevalent (Noirot-Timothee and Noirot, 1980). Indeed, there seems to be a progressive transformation in the complexity of septate junctions which conforms with phylogenesis (Dallai et al., 1977). In the case of the arthropods, the increase in complexity of tight junctions cannot be directly linked to phylogeny since the complex Arachnida are thought, as stated above. to have no direct evolutionary link with the Insecta (Roots, 1978). The evolutionary relationships, then, between these different kinds of occluding junctions have yet to be elucidated; further data of both a physiological and fine structural nature are clearly required.

X. Concluding Remarks It is clear that there are many facets of arthropod tight junctional structure and function which remain to be clarified. Their existence at least now seems established as does their involvement in regulating transepithelial permeability. However, the nature of the interactions between the adjacent particles that compose the intramembranous ridges, as well as the modifications that the junctional ridges are capable of manifesting with time and different levels of cellular activity, have yet to be elucidated. Equally, the control mechanisms of such dynamic events, the stimuli that lead to the transformations the junctions undergo during developmental assembly or disruption and the mechanisms by which these are expressed. remain unknown: it seems likely, however, that in development, hormones are involved. Clearly, though, the concept of some additional or alternative sort of short-term cellular regulation of permeability is an attractive one and recent studies on membrane turnover and recycling have emphasized the nonstatic nature of membranes and their specializations. Rapid-freezing without cryoprotectants and specific junctional labeling will aid in the clarification of some of these points, but the latter first requires isolation and purification of the junctional proteins which at present has eluded investigators of even the extensive and more wide-spread tight junctions of vertebrate tissues. Moreover, labeling the junctions in replicas, in spite of recent developments (for example, Rash et a/., 1980), remains technically complicated. Advances in our understanding of such parameters must therefore await new procedures and greater technical expertise. The nature of the tight junctions of arthropods specifically, however, also requires much further routine investigation to extend our knowledge of those

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tissues in which they are to be found, as well as those groups of organisms in which such associations are present and to what extent they affect tissue permeability. More electrophysiological experimentation needs to be done to establish the features of the gradients produced by these structures and under what conditions they may be modified. One puzzling aspect of arthropod tight junctions is the complex nature of the interdigitating cellular borders along which they occur in contrast to those where the vertebrate zonula~occludentes are found. Since this is the situation whether the junctions are simple or complex, it cannot be, as first thought, merely a compensatory device evolved to ensure the effectiveness of the permeability barrier imposed by the simpler junctions (Lane ef a f . , 1977a; Lane and Skaer, 1980). Whether the differences between vertebrates and arthropods, rather than having a physiological basis, is a reflection of different trends in evolutionary patterns, cannot be established, but if the two lines evolved separately, as seems likely, this could be one possibility. Undoubtedly much work remains to be done in this field; the next decade should extend the information available to us and thereby perhaps modify our thinking as much as the research of the past 10 years has done in proving that tight junctions i n many diverse forms, and in spite of predictions to the contrary, do exist in the tissues of invertebrates.

ACKNOWLEDGMENTS

I should like to express my gratitude to Mr. William Lee who prepared the photographic montages and to Mr. John Rodford who produced the diagrams. I am deeply indebted to Mrs. Vanessa Rule who painstakingly typed this manuscript and to Dr. Simon H. P. Maddrell and Dr. Helen Skaer for many stimulating discussions. I am also grateful to my colleagues Ms. Lesley Swales and Mr. Banie Harrison who have been involved in a number of the projects mentioned in this article.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 73

Genetics and Aging in Protozoal JOAN SMITH-SONNEBORN Zoology and Physiology Department, University of Wyoming, Laramie, Wyoming I . Introduction . . . . . . . . . . . . . . . . . . . .

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11. Clonal Aging in Ciliates

Ill. IV. V. VI. Vfi.

A. Background B. The Ciliate Genetic Complex . . . C. Breeding Life-styles . . . . . . . D. Paramecium . . . . . . . . . E. Euploies . . . . . . . . . . . . F. Tokophyra . . . . . . . . . . G . Terrahymena . . . . . . . . . Ciliate Fertilization: Marvel or Menace . Environmental Modulation of Longevity . Aging in Multicells and Unicells . . . Mechanisms of Cellular Aging . . . . Summary.. . . . . . . . . . . References . . . . . . . . . . . .

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319 321 321 322 326 321 333 334 334 336 339 341 344 348 349

I. Introduction Protozoa can exhibit a finite growth potential and can escape extinction by fertilization and formation of successive generations. Yet certain protozoa do not require fertilization for survival and appear immortal. This paradox within unicellular organisms can be understood only when the biology and evolutionary relationships of these protozoans are well characterized. At present, we glimpse only a microscopic image of a massive reservoir of untapped information. The present article will present an overview of the genetic complex and life-style among protozoa as they relate to longevity assurance. My premise is to recognize that protozoa are different from each other and from humans, but where similarities are found, there too may be found the fundamental mechanisms that regulate life processes. The genetic system in eukaryotes must be broad enough to include not only DNA but the epigenetic influences that impinge on DNA regulation. The eukaryotic genetic complex that controls heritability in cells and their regulatory 'This article is dedicated to the memory of Professor Tracy M. Sonnebom who leaves with us his legacy of excellence to strive for in our research and teaching. 319 Copyright Q 1981 by Academic &ss. Inc. All rights of reproduction in any form reserved. ISBN 0-12-362473-9

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events must encompass ( a )the interaction of DNA with proteins to form chromatin: ( h ) the subchromosomal organization of DNA with the primary nucleotide sequence. the secondary double-stranded helix, the tertiary superhelical coils, and the quaternary multiple superhelical domains-all of which contribute to the ultimate regulation of gene function (Lipetz, 1981); ( c ) the concept that DNA damage occurs not only with changes in the primary DNA sequence but can also refer to alterations at other levels of DNA organization (Brash and Hart. 1978): (zl) the contribution of long-term modulation of gene activity by cytoplasmic and environmental interactions (Sonneborn, 1966, 1977): and ( e )the role of preexisting cell structure in directing the replication and/or position of the organellar structure. and maintenance of cell morphology (Sonneborn, 1963: Beisson and Sonneborn, 1965). since life maintenance and self-reproducibility depend not only on the basic structure of DNA but also on DNA regulation and integrity of cell structures. It must be stressed that protozoa span enormous periods of evolutionary time and exhibit life-span durations that last from days to immortality (Sonneborn, 1960). In general, protozoa that differ on the basis of their fascinating forms, peculiar appendages, size, and life-style are recognized as organisms long separated in evolutionary time. DNA base composition ranges from 46 to 61% guanine and cytosine ( G + C ) in flagellates, and from 22 to 45% G + C in ciliates (Schildkraut ef a / . . 1962: Sueoka, 1961). The ciliates Paramecium and Blephwismci may be no more closely related than Homo and Salmo (trout) on the basis of DNA hybridizations and competition studies (Gibson, 1966). The protozoa that show morphological similarities and even identity are more deceptive. Morphologically similar species of Terr-ahymena range in G+C composition from 25 to 33% (Suyama, 1966) and scarcely show any molecular homologies (Nanney and McCoy. 1976). The spectrum of finite to infinite life span among these protozoa may well reflect the divergent strategies for survival in organisms widely separated in evolutionary time. It took 3 billion years to proceed from the origin of life to unicellular diploids and only one-sixth as long to evolve the diversity seen in multicellular organisms (Margulis, 1970; Ohno, 1970). Fundamental processes for survival that evolved during the first 3-billion-year period were surely conserved. These fundamental processes can be meshed in unique ways to achieve different biological objectives. It is the challenge of the investigator to ask the proper questions and to discover underlying unity in biological systems of common origin (Nanney, 1 968). The scope of this contribution will be restricted to those ciliates whose life cycle and mating behavior are well characterized both with respect to life span and the genetics of temporal patterns of gene expression. The inheritance and longevity in Amoeba have been thoroughly reviewed (Danielli, 1959; and Muggleton-Harris, 1979) and therefore will not be extensively reviewed here.

GENETICS AND AGING IN PROTOZOA

32 1

Other protozoa that offer potential for elucidation of mechanisms that regulate proliferative capacity are found among the collective Amoeba, colonial flagellates, and some other ciliates (see Grell, 1973; Coleman, 1979). The colonial green flagellates exhibit embryogenesis' including inversion stages with separation of somatic and germ-line cell types with different proliferative capacities. A genetic contribution to the extent of cell-proliferative capacity is suggested from the Mendelian genetic behavior of developmental mutants in these organisms (Sessoms and Huskey , 1973).

.II. Clonal Aging in Ciliates A . BACKGROUND Ciliate aging was described by Maupas (1889), and the early pioneering studies are reviewed from different points of view by Calkins (1926) and Jennings (1929). The discovery of mating types by Sonneborn (1937) provided the basis for breeding analyses. The longevity studies of Jennings (1944a,b,c, 1945) and Sonnebom (1954) established the reality of ciliate aging. Genetic regulation of longevity is implied by the characteristic differences in longevity of different species (Sonnebom, 1960; Siegel, 1967; Smith-Sonnebom, 1981). Aging in ciliates exhibits predictable, progressive, species-specific rates of decline in function and, finally, death. The probability that two viable products will be produced at the next cell division declines as the number of cell divisions (clonal age) increases since the last fertilization. The parental generation dies unless fertilization sets back the age clock. In the absence of fertilization, the cells undergo repeated cell divisions and produce cells that are progressively less vigorous than the progenitor cell. Clones like Paramecium retraureliu. which experience repeated fertilizations at short intervals, yield successive generations without loss of vigor; but when cultivated in the same medium with no fertilization, the clones age and die. Clonal aging is therefore an intrinsic process of these cells, not induced by the culture medium (Sonnebom, 1954; Siegel 1967). Thus clonal aging refers to the aging phenomenon in those species that in order to escape extinction must undergo fertilization to initiate a new sexual generation. The life span of an individual cell or cell type within a clonal aging species also can be studied. For example, Tokophyru exhibits the stalked adult form that produces the swimming buds that then metamorphose to the adult form. The period of bud production during the life of the adult, and the life span of this single nondividing cell type, can be monitored as a function of clonal age. The methodologies used to study clonal aging vary with investigators, but in general, cells after fertilization are placed in growth medium and transferred daily or at regular intervals. The total number of cell divisions or days from the

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origin of the clone at fertilization to death represents the life span. Daily isolations of several sublines (representatives) of a clone allow observation of the number of cells produced by each single cell (log, of that number is the daily fission rate) each day after fertilization. A single cell from a population of cells produced in the previous 24 hours in a subline is reisolated on successive days and the sum of the fissions per day is the age of the cell. When an isolated cell fails to survive on any day, the sum of the number of cell divisions or days since fertilization represents the life span of that subline. The mean life span is the average clonal age for all sublines in a control or experimental group. Maximal life span is the highest number of cell divisions or days attained by any subline within an experimental or control group.

B . THE CILIATE GENETIC COMPLEX Characterization of the DNA structure at different levels of organization is available for the ciliates. Typical eukaryotic chromatin structure of nuclei in Tctruhynena (Gorovsky er al., 1973) and Stylonychiu (Lipps and Morris, 1977) has been elucidated and studies indicate superhelical coiling of DNA in P . retruureliu (Lipetz and Smith-Sonnebom, 1980). In general, the ciliates contain two kinds of nuclei, the micronucleus or germ line and the macronucleus or somatic line. however amicronucleated strains have been observed in both Tetrahymena and P . multimicronucleutum (Nanney, 1974; Sonnebom, 1957). Although the nuclei occupy a common cytoplasm the different nuclei have separate functions. The micronuclei are the repository for genetic information for the progeny after fertilization and show little transcriptional activity during the asexual binary cell division cycle (Sonneborn. 1946, 1954: NobiIi, 1962: Pasternak, 1967; Gorovsky and Woodard, 1969; Murti and Prescott, 1970; Klass, 1974). The macronucleus dictates the metabolic activity of the cell (Sonneborn, 1947, 1954a: Siegel, 1963). The transcriptionally active macronucleus of Trtruhymena has been shown to have acetylated histones, whereas the transcriptionally inactive micronucleus does not (Gorovsky et ul., 1973). In ciliates, fertilization can occur by conjugation (mating), autogamy (selffertilization), or selfing (mating within a clone). During conjugation in Puromeciurn, cells come together side by side and eventually exchange nuclear garr.etes through a temporary transfer organelle (Fig. 1 ). Comparable to nuclei of sex cells of higher organisms, the micronuclei undergo meiosis to generate gametes that function during fertilization. In each mate, meiosis of the two micronuclei produces eight gametes, seven of which disintegrate, and only one haploid gamete is retained. This gamete duplicates into two identical haploid gametes. One remains stationary, the other migrates into the mate and fuses with the mate’s stationary gamete. Thus, in true conjugation, the two members of a

GENETICS AND AGING IN PROTOZOA

323

mating pair become genetically identical, each with one-half from its own duplicated haploid complement and one-half from its partner’s. The details of the developmental events after fertilization differ with species but normally the zygote undergoes differentiation of a new micro- and macronucleus for progeny cells. In autogamy, fertilization occurs in a single cell. After meiosis, one haploid gamete is retained, and duplicates into two identical gametes that fuse to form a homozygous diploid gamete (for review of ciliate genetics see Preer, 1968).

FIG. 1 . Paramecium retruureliu during mating. The intact ancestral macronucleus has started to stretch out or “skein. The micronuclei have undergone meiosis and the cells will exchange gamete nuclei at the juncture between the cells. The exchanged gametes will fuse to form the zygote nucleus, which will differentiate the new micro- and macronuclei for progeny cells. The cells are stained with acridine orange (0.3 mg/ml) and viewed with fluorescence microscopy. X720. ”

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If the genotype of the two gametes is denoted “A” and “A” in one mate and “a“ and “a” in its partner, then after mutual exchange of gametes both members of the pair become Aa. In autogamy, the two identical gametes fuse in a single cell yielding AA or aa depending on the genotype of the parent cell. After conjugation or autogamy ,the zygote nucleus (the synkaryon) divides mitotically twice to form four genotypically identical diploid nuclei. In Tetrtihvmenci, one disintegrates. one stays a micronucleus, and the remaining two develop into macronuclei (for review see Sonnebom, 1977). The position of the synkaryon products within the cell can determine which nuclear differentiation is favored (Nanney. 1953: Sonnebom, 1954a). In the cell division that follows, the micronuclei divide but the macronuclei pass without division to each daughter cell producing two sister caryonides. (A subclone derived from one of the macronuclei ofthe fertilized cell is called a caryonide.) Macronuclear development proceeds by several rounds of DNA replication with half or more occurring in the first cell division and the rest in the second to provide a Paramecium macronucleus with 800 times the DNA content of the haploid micronucleus. The ancestral macronucleus can function during the development of the new filial macronucleus (Berger, 1973, 1974. 1976). The RNA produced by the ancestral macronucleus represents a maternal cytoplasmic environment reminiscent of the stored RNA in oocyte cytoplasm in higher organisms. If the new macronucleus fails to form, the ancestral macronucleus can persist (Tefruhgmena),or fragments of the ancestral macronucleus can regenerate (Paramecium).termed macronuclear retention or regeneration, respectively. The sensitive period of development in Paramecium refers to the interval from synkaryon division until the end of the second postfertilization cell division. In this period. differentiation of nuclei and determination of macronuclear gene functions occur. Some determinations of gene function occur early, others late in the sensitive period, and the determinations are subject to change due to intracellular and extracellular environmental conditions (Sonnebom, 1977; Sonneborn and Schneller, 1979). Paramrcium tetraurelia and certain Tetrahymena retain most of the micronuclear genome in the macronucleus (Yao and Gorovsky, 1974; Doerder and Debault, 1975: Cummings, 1975). Chromosome polytenization and chromosome diminution followed by rapid resynthesis is observed in lower ciliates (Kovaleva and Ravikov, 1978) and in some hypotrichs (Ammermann, 1965, 1970; Ammermann er al., 1974; Bostock and Prescott , 1972; Riewe and Lipps, 1977; Lauth tit al., 1976: Lawn et al., 1977). Parameciiim bursuria show strains in which amplification of certain chromosomal segments and chromosome resorption or condensation were observed in the macronucleus (Schwartz, 1975). Therefore, the strategy for macronuclear development from the synkaryon varies with species, and generalizations from one species to another must be avoided.

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Ciliate genetics has revealed that certain traits are inherited in a typical Mendelian pattern. The F, heterozygous clones show dominance or codominance and the F2 autogamous generation segregates allelic traits in the expected proportions (for text see Beale, 1954). Inheritance in the ciliates has gained prominence, however, by its apparent deviation from Mendelian genetic principles. Caryonidal, cytoplasmic, and nucleocytoplasmic inheritance have all been observed in ciliates. In fact, as studies continued, it became apparent that genotypically identical cells can show phenotypic diversity not unlike differentiated cell types of higher organisms-although there is argument as to whether all cells of higher organisms are in fact genotypically identical (Briggs, 1977). A cross between genotypically identical cells expressing different phenotypes can result in phenotypically different offspring since the development of the new macronucleus is sensitive to the cytoplasm and extracellular environment of its mother cell. No segregation is expected or found in the ex-autogamous F2 generation since there was no change in genotype during the cross. There was no violation of Mendelian inheritance, rather the cross was between two cells of the same genotype with different genotypickytoplasmic differentiations. In addition to nuclear inheritance, self-reproduction of cells includes replication of surface structure and its pattern. Autonomy of the surface pattern of organelles has been established and cortical differentiations are essential for their own selfreproduction; the old structures serve as scaffolds for the synthesis of new structures (Sonneborn, 1963; Beisson and Sonneborn, 1965; for review see Frankel, 1979). The ciliate genetic system has been established as a model for problems of developmental genetics despite the fact that cells that express different loci or alternative alleles may exhibit morphological identity. The system is likewise applicable to studies of aging, since fertilization marks the initiation of a new life cycle and represents a short period of nuclear development that can be synchronized and modulated to discover cytoplasmic and environmental effects on gene determination with respect to longevity. The protozoan model offers an in vitru and in vivo system of cellular senescence, since age-related changes can be followed in individuals or populations of cells and a single cell is a complete organism. The ciliate systems of cellular aging are free of complications introduced by tissue-tissue interaction in multicellular organisms and are not subject to possible artifacts introduced when certain tissues are cultivated outside their natural environment. The genetics of aging in ciliates has focused on the role of fertilization in species longevity. Crosses between cells as a function of clonal age have been used to monitor the contribution of cell parts to progeny life span. Certain cell parts can be exchanged during fertilization and by microsurgical manipulations. The various options for fertilization can involve unique sets of cell parts. For

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example, it is possible to monitor and in some species to control to some degree the amount of cytoplasm exchanged between mating pairs. Cytogamy (selffertilization within one or both members of a mating pair) can occur with cell-cell contact and cytoplasmic transfer without exchange of germ nuclei. Conjugation permits the assessment of nuclear exchange after various amounts of cytoplasmic transfer. The effect of cytogamy versus conjugation on progeny survival and life span can be monitored and the impact of the cytoplasm determined by comparison of cytogamous versus true conjugant progeny vitality. Autogamy. on the other hand, does not involve cell contact or cytoplasmic transfer. Thus progeny vitality from autogamous old (or young) cells can be compared with cells of the same clone after cytogamous or conjugal mating. When other variables are held constant. a difference in progeny survival found between autogamous and cytogamous cells would imply cell membrane/ cytoplasmic influences. If nuclei exert a major life-span effect, progeny from the young-old exconjugants will have the same life span since they are genetically identical. If the conjugants must exchange massive cytoplasm for equivalent life spans of progeny, a nucleocytoplasmic effect is indicated. Immediate death in the F, generation of old-young offspring imply dominant lethals or interference with normal development. Recessive lethal mutations can be easily detected in those ciliates that i’ndergo autogamy (i.e., when all genes become homozygous after autogamy). The ciliates that have been most thoroughly studied with respect to genetics of aging include Parclmecium, Euplotes. Tokophyra, and Tetrahymena, and aspects of their life-style, aging, and genetics are presented and compared in this contribution. Since each species has a characteristic breeding system that impacts on progeny longevity, the options and consequences of breeding systems precede the description of aging in particular species.

C . BREEDINGLIFE-STYLES The various ciliates can be classified with respect to their commitment to inbreeding or outbreeding (for review see Sonnebom, 1957). Outbreeders tend to mate with “strangers” and therefore tend to accumulate genetic variety. To favor outbreeding, these organisms have multiple mating types (increasing the chance that an encountered stranger will be of a mating type different from itself and therefore will be suitable as a sexual partner), long immaturity periods (to allow spatial separation between closely related individuals before mating can occur), and inability to undergo self-fertilization (discouraging homozygosity). Inbreeders can undergo self-fertilization, have only two mating types, and have short immaturity periods. Spontaneous recessive mutations quickly become homozygous after autogamy, and if nonadaptive in that genetic and environmental background, the possessor of that mutation may die or be overpowered by neighbors,

327

GENETICS AND AGING IN PROTOZOA TABLE 1 THELENGTHOF IMMATURITY

VS LIFE SPAN"

Median period fissions (days) Organism Paramecium tetraurelia

Paramecium biaurelia Paramecium primaurelia Paramecium caudatum Euplores woodrufi (marine) Euplotes patella

Haploid Amicronucleate Diploid Paramecium bursaria Paramecium multimicronucleatum

Immaturity 0

13.5 17.5 29 33.5

Life span

(0)

244

(3.5) (4.5) (27) (21)

286 340 528 339

62.5 (59) 63.5 (66.5) 176 (173) (484) 60+ (80')

(50)

(162) (120)

(205) (181)

613 (789) 616 (824) 1332 (1830) (2585) (5400)

References Sonneborn (1954, 1957); Smith-Sonneborn er a/. (1974) Sonneborn (1954, 1957) Sonneborn (1954, 1957) Takagi and Yoshida (1980) Kosaka(1974) Katashima (1971) Katashima (1971) Katashima (1971) Jennings (1944a) Sonneborn (1957)

"The length of immaturity and life span are presented as median values. In those cases where ranges were not available, the value was omitted or represented with a +. Amicronucleate clones in E. parella arose spontaneously in a small fraction of the progeny from a normal cross between diploids. Haploids in E . patella arose when amicronucleateswere crossed with diploids. As the commitment to inbreeding decreases, both the immaturity period and life span increase. Maturity can begin relatively early, however, in both the outbreeders P . bursaria and P. multimicronucleatum.

reducing genetic variety. Inbreeders are less resistant to environmental stress and fluctuation than outbreeders (Nyberg, 1974). Outbreeding organisms may require a lower mutation rate than inbreeders to achieve comparable genetic variety (Nanney, 1974) and their repair systems may be evolutionarily adjusted for their respective genetic strategies (Sonneborn, 1978). Ciliates can be arrayed in a linear series of life spans relative to the degree of commitment to inbreeding or outbreeding (Sonnebom, 1957). In those ciliates whose life duration is well characterized, the length of the immaturity period is highly correlated with the length of life (Table I; Fig. 2), suggesting a common cellular calendar for both life-cycle events. D. Paramecium

1. P . aurelia There are 14 species in the P . aurelia complex. P . primaurelia, biaurelia, and tetraurelia have been studied thoroughly and show maximal life spans of 350,

328

JOAN SMITH-SONNEBORN FISSIONS

r

= 0 986

MEDIAN LIFESPAN IFISSIONSI

FIG. 2. The length of the immaturity period and life spans for Porrirneciirm are given in Sonneborn (1954. 1957); for E . pcrrelln. in Katashima (1971): and for E. rcwodrufli in Kosaka (1974). The life spans as measured in cell divisions (fissions) were not available for the long-lived P. hi4r.wn‘l2 and P. mu/rimic.rcmuclearurn. A high correlation coefficient was found when immaturity and life span were measured in days when P . hirrsriricr was included or excluded. Inclusion of the values resulted in loss of a correlation between immaturity and life available lor P . ~~ticlfimirronitcleciritnz span. It is possible that undetected selfing occurred in the P , multimirmnuclmrum clones, andlor extreme outbreeders should he separated from inbreeders when comparisons of immaturity and life spans are made.

303, and 200-325 asexual cell divisions, respectively (Sonnebom, 1954, 1957; Smith-Sonneborn, 1979). The maximal life-span durations vary due to both intraclonal variations and selection procedures used by different investigators. Age-associated changes in the P . crurelin complex are presented with those of other ciliates in Table 11. The capacity for fertilization to provide fully vigorous rejuvenated offspring declines with increased parental age (Pierson, 1938; Raffel, 1932; Sonneborn and Schneller, 1955: Mitchison, 1955; Smith-Sonneborn et d.,1974). Another lifecycle event that is shortened in the progeny as parental age increases is the timing of maturity (Siegel, 1961). Ultraviolet irridation can shorten both immaturity in P. cuiidrrritrii (Takagi, 1974) and life span in P . tetruureliu (Smith-Sonneborn, 1979), suggesting coordinated or common cellular clocks for immaturity and life span. Increase in micronuclear damage with age has been found (Sonnebom and Schneller, 1960: Rodermel and Smith-Sonnebom, 1977) and could contribute to decreased longevity in the progeny from older parent cells after autogamy (selffertilization). It is not clear whether the “age clock” is not reset to zero or the

GENETICS AND AGING IN PROTOZOA

329

rate of aging is accelerated when the parents are old. The progeny from the older parents can show the youthful fission rate, but they subsequently exhibit a more rapid decline in vitality implying an accelerated aging. Alternatively, offspring may not show the youthful fission rate and live a longer life span than a sister clone with a rejuvenated fission rate. In general, progeny from older parents show greater variations in vitality than offspring from young parents. Fertilization, if delayed too long, does not necessarily rejuvenate the clone (Sonneborn, 1954; Smith-Sonneborn et a / ., 1974). In true conjugation each member of a pair has one-half its own complement and one-half the genetic complement of its partner. Thus members of a hybrid pair produced in young-old crosses are genetically identical, but they differ in cytoplasm, one having predominantly young, the other predominantly old cytoplasm. Cytoplasm mixing by increased duration and intimacy of the union could provide 50% young and 50% old cytoplasm for both mates. The F, autogamous (self-fertilization) progeny survival from the cytoplasmically old and young mates was compared and a chromosomal basis for agerelated damage was indicated by increased frequency of F2 dominant and recessive lethals in progeny from the young member of the mating pair (Sonneborn and Schneller, 1960; Williams, 1980). The’old member of the pair, however, shows greater F, lethality than the young member (Williams, 1980). The difference in F2 mortality from the presumed genetically identical mates may reflect some increased recombinational repair of age-correlated nuclear damage in the young mate and/or interference with normal nuclear development in the aged cytoplasm of the old mate. Abnormalities due to aged cytoplasm were identified by studies using “merogones” (old cells whose nuclei fail to function that receive the young nuclear complement). Although these merogones usually do not survive, indicating detrimental effects of aged cytoplasm, occasional survivors do show restoration of the youthful phenotype (Sonneborn and Schneller, 1960). Therefore, a young nucleus can rejuvenate aged cytoplasm. Evidence that recessive mutations were not the sole cause of age-related death after fertilization was obtained from studies of aged lines in P . primaurelia and P . caudatum. In P . primaurelia most death occurred within zero to three fissions after autogamy in aged lines; recessive lethal mutations show a longer phenotypic lag (Mitchison, 1955). However, dominant micronuclear mutations and interference with normal development of nuclei may reflect germ-line damage and contribute to possible cytoplasmic effects on postautogamous survival. In P . caudatum, micronuclei from aged lines that exhibited death and abnormal macronuclear development after selfing were microsurgically transferred into young amicronucleated recipient cells. The typical youthful increased survival and normal nuclear development were found after selfing of the young recipients containing the aged micronuclei (Karin0 and Hiwatashi, 1981). Rejuvenation of

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JOAN SMITH-SONNEBORN

aged micronuclei by young cytoplasm was not possible when the aged cells progressed further into senescence. These studies indicate that aged cytoplasm is incompatible with normal macronuclear development and/or some repair of age damage is possible in the young cell although germ-line erosion eventually becomes irreversible. TABLE ll AGE-RELATED CHANGES I N Life-cycle changes Immaturity. maturity. and senescence

Organism" P.a. P.b. P.C. Em.

E.p.

THE

CILIATES References

Sonneborn ( 1957): Jennings (1944a): Takagi and Yoshida ( 1980); Heckmann (1967); Katashima (1971); Kosaka ( 1974); Colgin-Bukovsan ( 1979)

E.w. T.I. En~.irnnmenmlmidiclarion of lifie-cycle chongc'.s

UV-induced repair UV-induced changes in immaturity Ion and temperature effects on nuclear development Lffects (IJ old-\ oirng i rossc5 Induced damage to progeny of young mate

P.a. P.C. P.a. T.I.

Smith-Sonneborn (1979); Williams and Smith-Sonnebom (unpublished) Takagi ( 1974) Sonneborn (1977); Nanney (1976); Nanney and Meyer ( 1977)

P.a.

Williams (1980); Sonneborn and Schneller (1960); Katashima ( 19711; Kosaka ( 1974) Sonneborn and Schneller ( 1960)

Micronuclear aberrations

P.a. E.p.

Variability in numberofmicronuclei

P.a.

Sonnebom (1954a); Sonneborn and Schneller (1960a); Dippell (1955); Fukushima (1975); Nyberg (1978); Rodermel and Smith-Sonnebom ( 1977); Sonneborn and Dippell ( 1960); Katashima (197 I ) Sonneborn and Dippell (1960); Mitchison (1955)

Rescue of old cytoplasm by young nucleus Senescent chonges Increase3 wirh ugr Micronuclei

Mac~ronucleirs

lnvagination in macronucleus Fused chromatin Abnormal appearance

P.a. P.a. P.a.

Sundararaman and Cummings (1976) Sundararaman and Cummings (1976) Siege1 ( 1 967); Sundararaman and Cummings (1976); Schwartz and Meister (1973); Takagi and Kanazawa (1980)

33 1

GENETICS AND AGING IN PROTOZOA

The decline in vitality and increase in micronuclear erosion in the P . aurelia complex may (see Sections V and VI) reflect loss of ability to repair DNA since: ( 1 ) sensitivity to UV irradiation increases with clonal age, suggesting loss of excision-repair ability (Smith-Sonnebom, 1971); (2) life span is modulated by amount of UV-induced damage and repair (Smith-Sonnebom, 1979); and (3) UV-induced lethality after autogamy is increased by treating young clones with TABLE I1 (Continued) Life-cycle changes Cytoplasm Toxicity of cytoplasm

Lysosomal activity Sensitivity to ultraviolet Sensitivity to X-rays Decreases with age Progeny viability

Organism"

References

P.a.

Williams and Smith-Sonnebom (1980); Sonnebom and Schneller (1960, 1960b); Katashima (1971) Karino and Hiwatashi (1981) Sundararaman and Cummings (1976, 1976a); Rudzinska (1961) Smith-Sonnebom (1971, 1979) Tixador et al. (1972); Fukushima ( 1974)

E.p. P.a. T.i. P.a. P.a.

P.a. E.p.

Nucleolar volumes DNA amount

P.a. P.a.

Ribosomal clusters Food vacuole number and synthesis Fission rate

P.a. P.a. P.a. P.C. E.p. E.w. P.a. P.a.

Sonnebom and Schneller (1960); Williams and Smith-Sonnebom (1980); Pierson (1938); Smith-Sonnebom et al. (1974); Sonnebom and Schneller (1955, 1955a, 1960); Sonnebom (1954); Katashima (1971); Kosaka ( 1974) Heifetz (1979) Klass and Smith-Sonnebom (1976); Schwartz and Meister (1973, 1975); Takagi and Kanazawa (1980) Sundararaman and Cummings (1977) Smith-Sonnebom and Rodermel (1976) Sonnebom (1954); Smith-Sonnebom et a / . (1974); Takagi and Yoshida (1980); Katashima (1971); Kosaka ( 1972) Smith-Sonnebom and Klass (1974) Klass and Smith-Sonnebom (1976)

P.a. P.a.

Nyberg (1978) Williams and Smith-Sonnebom (1980)

DNA template activity RNA synthesis No change with age Copper tolerance DNA polymerase activity

" P.a. is the Paramecium aurelia complex; P.b. is Paramecium bursaria; P.c. is Paramecium caudatum; E.m. is Euploresminuta; E.p. is Euplorespatella; E.w. is Euplotes woodruffi (marine); T.t. is Terrahymena thermophila; T.I. is Tokophyra lemnarum; T.i. is Tokophyra infusonium.

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JOAN SMITH-SONNEBORN

novobiocin (Lipetz and Smith-Sonneborn, 1980). a drug that can reduce DNAnegative superhelicity, interfere with repair of UV-induced damage, and reduce normal replicative DNA synthesis (see Section VI). Aside from the nuclear damage in Purumecium, organellar damage can contribute to intraclonal variation in vigor. The division products of asexually dividing cells are not equal, since in the formation of organelles to produce two daughters from one cell, the anterior cell (the proter) retains the old, but the posterior cell (the opisthe) receives the new oral apparatus. Studies of successive lineages of proters and opisthes showed that the fission rate was higher for the posterior line with the new oral apparatus (Siegel, 1970). The hypothesis advanced was that the old gullet could not be repaired should accidental damage occur, thereby reducing the viability of these individuals in the clone and contributing to variation in vigor within the clone. Thus nuclear, cytoplasmic, and organellar function contribute to longevity.

2 . Other Species of' Purutnecium Purumecium rcrudrmm has a life span of 600-700 cell divisions lasting about 200 days (Takagi and Yoshida, 1980) and exhibits an inbreeding life-style (Sonneborn. 1957). Although these clones do not undergo autogamy, they can selfcross (mating within the clone) and initiate a new sexual generation. These organisms have an intermediate life span between the short life span of Parameciuin retraureliu and the 15 year life span of Paramecium multimicronur.lrutun1. Paramrc~iitmhursnriu shows a life span of around 3000 days and is an outbreeder. Rejuvenation by conjugation was documented in these organisms, but conjugation produced. in addition to youthful vigorous clones, early death and pathologically deprived cells. Jennings noted 5-14 times as much death when related clones were crossed, compared with crosses between unrelated mature populations (Jennings, 1944a,b,c, 1945). As parental age increased, F, lethality increased until most or all exconjugants died. At least some, but not all, of the nonviable conjugants resulted from unfavorable mutations accumulated with clonal age. However. induced abnormalities in nuclear behavior were noted in young members of an old-young pair even prior to exchange of nuclei, reminiscent of lethality resulting from "foreign" cytoplasm in intraspecies crosses of P . hursaria (Chen. 1946). Fertilization in these organisms, then, is not always a rejuvenating process. P . multimic~rot~uclecltutn,the most long-lived of the Paramecium, have a life-span duration of about 5400 days. They are extreme outbreeders with immaturity periods lasting months to years. The aged P . multimicronuclearum are found to have become amicronucleated without loss of vigor (Sonneborn, 1957).

GENETICS AND AGING IN PROTOZOA

333

E. Euplotes Species of Euplotes are found in marine and fresh-water environments. Marine species in general show an outbreeding behavior, though facultative inbreeding species are found (Katashima, 1971). Autogamous strains of the marine species E . rninuta have been investigated, and there is evidence for genetic control of the immaturity period (Luprino, 1970). Fresh-water Euplores tend toward an inbreeding behavior (Katashima, 1971). The life span of the marine E . woodruffi ranges from 235 to 442 cell divisions (Kosaka, 1974), and E . patella can live 1300 cell divisions (Katashima, 1971). The fresh-water E . woodrufl exhibits a life'span of at least 208 cell divisions (Kosaka, 1970, 1972). In marine E . woodrufl, crosses between old and young stocks resulted in high mortality for both the young and old mates (Kosaka, 1974). Only 12% survivors were found among the 50 pairs studied. The relative contribution of nucleus and cytoplasm to lethality could not be assessed in these studies. In old-young (mature) crosses of E . parella, progeny viability declined in both members of the pair (Katashima, 1971). The mature nucleus could function in the mature, but not in the old cytoplasm. That aged cytoplasm was toxic could be ascertained as well from the observation that the mature mate was seriously damaged by contact with the old mate even before nuclear exchange occurred (Katashima, 1971). Stomatogenesis could not be completed in the old mate. The toxic influence or incompatibility of aged cytoplasm with normal development of nuclei or organelles was established in these crosses and correlates with the aged cytoplasmic effects detected in the P . aurelia complex and P . caudaturn (Mitchison, 1955; Sonneborn and Schneller, 1960a; Williams, 1980; Karino and Hiwatashi, 1981). In E . crassus, mating type is determined directly and uniquely by the genotype. Five different alleles of a single mating type locus were revealed by cross-breeding analyses. The five alleles show serial dominance each over all those below in the hierarchy. In any combination of two alleles, the higher index dominates (Heckmann, 1964). Heterozygous clones that had been phenotypically stable for long periods suddenly showed selfing conjugation after 500 cell divisions. The appearance of selfing in old age implies a loss of rigid dominance replaced by oscillation of expression between mutually exclusive types, which allows cells within a clone to express complementary mating types. Exceptional heterozygous clones were found that did not self even after 800 consecutive cell divisions. A genetic block against mating-type instability explains the exceptional nonselfing heterozygous old clones and implies that mating-type instability in old age is not an accidental event but an actively triggered process under genic control (Heckmann, 1967). Old-age selfing is seen also in P . bursaria (Jennings,

334

JOAN SMITH-SONNEBORN

1941) and, like autogamy in the Paramecium uureliu complex, provides a mechanism for fertilization if the members of the aging clone have failed to find a mate. Selfing, like autogamy, initiates a new life cycle (Heckmann, 1967).

F. Tokophyra The study of clonal aging in Tokophya Iemnarum (Colgin-Bukovsan, 1979) was possible after the discovery of mating types in this species (ColginBukovsan, 1976). Senescence is noted by decreased survival after conjugation and by complete fusion of some pairs. The immaturity period is 0-60 cell divisions and maturity lasts at least 800 fissions. The fate of individuals within a clone can be assessed since the parent cell (of a given clonal age) is sessile, and its buds (offspring) swim away and later metamorphose into the stalked sessile adult. The adult form passes through a period of immaturity when buds are not produced, a mature period of bud production, and a senescent period when no more buds are produced. The adult lives a maximum of 10 days in T . infusionurn (Rudzinska, 1961) and 16 days in T. lemnurum (Colgin-Bukovsan, 1976). The sessile adult Tokophyru is adapted primarily for forming buds and is not even provided with an organelle for excretion of solid wastes, and thus its life maintenance is sensitive to the amount of food ingested (Rudzinska, 1962). if given an opportunity to overfeed by supply of excess prey (Tetruhymena), the adult will gorge itself. Overfeeding shortens adult life span and intermittent starvation extends life span (Rudzinska, 1951, 1961, 1962). Since these parent cells have no organelle for solid-waste excretion, it is not surprising that feeding conditions modulate life span. The malfunction of excretory organelles in any cell would lead to buildup of toxic products and cellular injury and death. Morphological changes that accompany overfeeding are seen also in aged cells. In both overfed and aged adults. tentacle loss occurs and feeding ceases. Death follows by starvation. The adult Tokophyra may represent a model of toxic effects of intracellular excess solid wastes in a nondividing cell type. Studies of adult life span as a function of clonal age should be performed. G . Tetrahymena The ciliate Tetruhymena can exhibit an infinite or finite life span, and different species appear widely separated in evolutionary time (for review see Nanney, 1974). Although long-lived Tetrahvmenu do not show a progressive decline in somatic function, dead and defective lineages appear at a constant strain-specific rate. One strain showed an increased rate of abnormality with increased age (Nanney, 1959). When long-lived strains are inbred, detrimental effects include (1) exconjugants that die within the first fission or within 10 fissions postconjugation; (2)

GENETICS AND AGING IN PROTOZOA

335

nonconjugants in which a new macronucleus does not develop (the old macronucleus is retained); or (3) accelerated onset of micronuclear anomalies showing “inbreeding deterioration” (Nanney, 1957). Successive inbreeding and stringent selection eventually produce viable progeny (Nanney, 1957). Senescent-like changes induced by inbreeding include severe disorganization of cell division, asynchrony of division of micronuclei and macronuclei, and abnormalities in micronuclear structure. Anomalous micronuclei may appear as early as 13 fissions but often between 40 and 100 fissions after fertilization (Nanney, 1957; Allen, 1967). The micronuclear spindle is often displaced in organisms with micronuclear anomalies (Nanney , 1957). Age-related micronuclear erosion is found in some but not all species of Tetrahymena and the onset of mutations appears to be random, not programmed to increase with age (Simon and Nanney, 1979). When cells with defective micronuclei are crossed with normal cells, full vigor can be restored to the senescent clone (Weindruck and Doerder, 1975). In the cross, replacement of the abnormal micronucleus occurs but the ancestral macronucleus can be retained, producing heterokaryons. If new macronuclei are formed, the anlage appear to undergo autolysis and eventually the cells die (Doerder and Shabatura, 1980). After a strain-dependent interval, the rejuvenated heterokaryote clones show signs of senescence, implying ancestral macronuclear regulation of onset of premature micronuclear abnormalities (Weindruck and Doerder, 1975). Micronuclei, like the germ-line cells of higher organisms, are shielded from natural selection during somatic life since micronuclear damage will be expressed only when amplified or activated after fertilization. Therefore, it is not surprising that germ-line damage can occur at a rate independent of the somaticline damage. Genetic death precedes somatic death in most ciliates and in Tetrahymena. The genetically conditioned short-life-span variants produced by inbreeding represent exceptional strains. Most clones appear to continue to reproduce indefinitely (for review see Nanney , 1974). Tetrahyrnena, which does not normally manifest age-related somatic decline or timing of onset of germ-line abnormalities, exhibits other well-ordered aspects of its life cycle. The onset of maturity, the timing of expression of antigenic types, and various enzymes can be ordered. Tetrahymena represents an organism that can “differentiate” or show timed changes in gene expression without revealing any senescent changes and represents an exception to the view that “differentiation” and age share a common cellular clock. The expression of different phenotypes postfertilization in Tetrahymena differ significantly from Euplotes and Paramecium. For example, when cell lineages were followed for 100 or more divisions, cells heterozygous for the H immobilization locus expressed one or the other allele as would be expected of homozygous, not heterozygous, lines. Crosses of the lines revealed the germ line was still heterozygous (Nanney and Dubert, 1960). Several other

336

J O A N SMITH-SONNEBORN

loci behave in a similar manner and can be arrayed into a Tetruhymenn calendar of timed events (Bleyman, 1971; Nanney, 1974). Somatic assortment of loci is not universal since one temperature-sensitive mutant exception was found not to assort (McCoy. 1973). The structure of the macronucleus in any ciliate is still an unsolved problem. All macronuclei are compound in that they contain multiple copies of some or all the genetic elements. Since expression of phenotype in Tetruhymena differs from other ciliates, it is reasonable to assume that difference in organization exists between the somatic nuclei of Tetruhymenci and at least some other ciliates like Puirurmc~ciirmand certain Euplotes, and this difference, if understood, may explain its immortality and exceptional pattern of timing of life-cycle events.

111. Ciliate Fertilization: Marvel or Menace

Fertilization in ciliates represents a risk that can result in immediate death, reduced life span. or resetting the age clock to zero. The outcome of the process is dependent on breeding behavior, individuals participating in the cross, and their age. Lethality immediately after fertilization can be due to expression of germline-dominant mutations, developmental anomalies in nuclear and structural processes, and/or chromosomal and cytoplasmic incompatibilities. Reduced life span could be attributed to expression of detrimental mutations and changes in the regulation of timing of gene expression. Complete rejuvenation implies full function of all cell parts. Repair of any damage must be possible at all levels of molecular and cellular organization. If deterioration of any cell part exceeds the capacity of the cell to correct that damagc, reduced life span ensues. In the lab, change from the normal breeding behavior of the species can result in reduced progeny survival. Inbreeding an outbreeding species like T e f r~1l7yncnnor Purritnec~iiitnhirrstrritr allows increased homozygosity of recessive lethals and detrimentals and thus their expression. Outbreeding an inbreeder like Purcrm~cirrmtetrcrirrcliu can bring together unpairable regions of chromosomes o r whole unpaired chromosomes, since different stock show characteristic differences in chromosome numbers (Dippell, 1954). The results o f fertilization must be viewed in the context of the organism’s life-style and the need for species. not individual, survival. The requirement for fertilization in Pnrcimeciwn and Euplotes but not in certain immortal Trrrurhytnenii is perplexing. The best explanation of the facts may lie in their ability to repair or replace defective cell parts. Defective cells could also be eliminated by selection for the more vigorous lines. Immortal Trtrirtz~menucan repair its gullet without undergoing mating while certain obligate breeders i n Ptiratnrc~iumrequire fertilization for gullet replace-

GENETICS AND AGING IN PROTOZOA

337

ment (Frankel, 1970; Sonneborn, 1978). The number of cells eliminated during asexual reproduction or after fertilization must be less than the number of individuals in the population. Thus damage and repair levels must be appropriately regulated to assure species survival. . Sonneborn (1978) has pointed out that aging arose after the advent of and within the diploids. Haploid unicellular species are immortal, and since they possess only one chromosome of each kind, they can accumulate mutations only very slowly and precariously. Deleterious mutations will be expressed and eliminated by death or selection against the less-vigorous cells that bear the mutation. Diploids, on the other hand, can carry recessive mutations of all kinds covered by the normal allele. Genetic adaptation to variable environmental conditions involves accumulation af mutations in the same individual, since some mutations may be favorable only in combination. Diploidy, sex, fertilization, and recombination allow accumulated mutations to be assorted in all possible combinations including some combinations in which both genes of a pair are mutant and can be expressed. The different combinations can be subject to the changed environment for their adaptive advantage and natural selection can preserve the beneficial combinations. Mutations that assure a program for a limited period of fruitful fertilization followed by aging and death were selected (or there was no selection against those combinations) and fertilization ceased to be optional and became mandatory for species survival (Sonneborn, 1978). The immortality of haploid organisms could be considered to be, at least in part, a consequence of selective elimination of individuals with harmful mutations, and the price of adaptation and fertilization in diploids was inadvertently the evolution of aging. The somatic immortality of certain strains of Terrahymena appears to be exceptional to the onset of aging with diploidy. Since the phenotype of the ciliates is regulated by the somatic macronucleus, the organization of macronuclear structure can be assumed to be relevant to the duration of life span. Although the macronuclear structure of any ciliate is still not understood (for review see Nanney and Preparata, 1979), Tetrahymena does exhibit the unique ability to assort loci during somatic reproduction. Thus sublines of the same clone express alternative alleles (i.e., the nucleus behaves as a homozygote or functional haploid). However, if macronuclear damage accumulated with age were to be assorted also, then the phenotype of the mutant would be expressed and possibly eliminated by selective growth of the more vigorous nonmutated sublines. A “phenotypic homozygote” is equivalent to a haploid since mutant genes can be expressed. Tetrahymena may be able to utilize the “haploid” mechanism of elimination of harmful mutations from the population by expression and selection against them during somatic growth to achieve immortality, and “purification” of the macronucleus during vegetative growth could occur by segregation and elimination of cells that carry harmful mutations (Pitts, 1979). On the other hand, Paramecium and Euplotes do not show any similar phenotypic assortment. Old-

338

JOAN SMITH-SONNEBORN

age selfing in Euplotes and in P . bursaria shows an oscillation between expression of alternative alleles, not segregation of the alleles to subline cells of the population. Therefore, those ciliates that cannot segregate and express harmful macronuclear mutations would accumulate damage and age. Although this attractive hypothesis would help explain the difference in longevity between Tetrahpmeno and some other ciliates, experimental approaches have not yet been possible to test its validity. It should be stressed that haploidy per se will not result in immortality or long life since ( 1) the rate of forward and backward mutation rates must be genetically adjusted relative to the reproductive rate to assure survival of some members of the species, and (2) increased longevity in haploids includes the opportunity for continuous selective pressure for the more vigorous cells and therefore is primarily restricted to dividing and not nondividing cells. In Paramecium, both the germ-line and somatic-line nuclei can accumulate mutations. After fertilization with development of macronuclei from the micronuclear synkaryon, some detrimental recessive mutations in the germ line can be expressed and eliminated from the population by death or selection against the cells that bear those detrimental loci in the new macronucleus. Thus, sexual reproduction represents a mechanism for “purification” of the somatic nucleus in Purameciurn, since the ancestral macronucleus is normally discarded and the new macronucleus is derived from the micronuclear genome. The most vigorous survivors of the fertilization process can then be selected. At least one advantage of fertilization. then, is the formation of a new somatic nucleus, though fertilization must take place before age-accumulated genetic erosion of the germ line is so severe that there can be no survivors. Aging in inbreeding Paramecium may be a means to provide greater genetic variety, and lower repair capacity adjusted to favor more variety in the genome may therefore be a major contributor to its aging process and short life span. Conjugation provides the opportunity for assortment of accumulated mutations and natural selection can preserve the adaptive combinations. The option for genetic assortment in outbreeders is greater than in inbreeders, since mutations will become homozygous at a much slower rate after fertilization (and therefore in the outbreeders, detrimental mutations-favorable only in certain genetic backgrounds-will not be “prematurely” eliminated from the gene pool). Thus the opportunity is increased for those mutations that may be favorable only in certain combinations to come together in the same cell and favor longer life by greater adaptation to environmental stress and presumed higher repair capacity. The diverse array of genetic combination after fertilization that induced short life-span variants in Terrahpmma (Nanney, 1974) may reflect ( 1 ) homozygosity of detrimental and lethal mutations, (2) the disruption of the optimal combinations of mutant genes present in immortal strains, andor (3) the rate of assortment of defective alleles. Although scholars disagree about the advantages of assorting

GENETICS AND AGING IN PROTOZOA

339

harmful mutations into favorable combinations, the evolutionary record attests to its value (Sonneborn, 1978). Outbreeders have longer lives and are more tolerant to stress than inbreeders (Sonneborn, 1957; Nyberg, 1974).

IV. Environmental Modulation of Longevity The diverse predictable life spans of different ciliates suggest genetic control of longevity that is subject to the influence of the extranuclear environment. A cytoplasmic initiator can induce macronuclear synthesis in Stentor (deTerra, 1967). An aged cytoplasm can result in increased F, mortality in Paramecium terraurelia, and can interfere with proper morphogenetic functions in a mature Euplotes. An ancestral macronucleus can induce premature germ-line erosion in Tetrahymena. A young nucleus, if it survives the effects of aged cytoplasm, can rejuvenate the cell and an aged nucleus in a youthful cytoplasm can be like a young nucleus (Sonneborn and Schneller, 1979; Karino and Hiwatashi, 1981). The data indicate that aging is not controlled by the nucleus or cytoplasm, but that each is dominated by the other, dependent on the time in the cell and life cycle investigated, and is best described as the result of nucleocytoplasmic interactions. Determination of alternative phenotypes can occur during the sensitive period of development. Maturation of the new macronucleus (Sonneborn and Schneller, 1979) and determinations of gene function that affect life-span duration may also be subject to the nuclear environment during the developmental period. Temperature, nutrition, and ion concentrations during the sensitive period can alter nuclear differentiation and can prejudice the resulting phenotype in both the P . aurelia complex and in Tetrahymena (for reviews see Sonneborn, 1977; Nanney, 1976; Nanney and Meyer, 1977). Cell surface changes with age have been observed in Euplotes (Katashima, 1971; Frankel, 1973). Structural changes in the cell cortex of the ciliate Stentor and invertebrate eggs can modulate intracellular events and interfere with normal nuclear division (for review see deTerra, 1970, 1974). Temporal correlation of cortical morphogenesis with micronuclear and macronuclear events were observed in Euplotes eurystomus (Ruffalo, 1980). Since cell surface changes must .be coordinated with intracellular events for maintenance of self-reproducibility, changes in cell surface could impact on longevity. The decline in gullet function in cell lineages with the old gullet represents organelle-mediated loss of vigor in even young sublines and contributes to intraclonal variations (Siegel, 1970). X-Ray administered by repeated fractionated doses for a total irradiation of 138,000 roentgens during the asexual cycle reduced life span and accelerated the decline in fission rate (Fukushima, 1974). However, irradiation with 80,000 R at the two-anlage stage of autogamy (when induced mutations should be amplified

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by several rounds of DNA replication) did not accelerate aging (Kimball and Gaither, 1954). The difference in results may reflect difference in timing of X-ray administration and the dosage used by the different investigators. Ultraviolet irradiation reduced life span of Puraniecium tetruureliu but not if the damage was enzymatically repaired by subjection to photoreactivation repair (Smith-Sonnebom. 1979). The cumulative effect of two cycles of irradiation followed hy photoreactivation repair resulted in significant mean and maximal life-span extension ( Smith-Sonnebom, 1979). Although clones show increased sensitivity to UV irradiation with age (Smith-Sonnebom, I97 I ) , those cells pretreated with UV light and then photoreactivated responded like younger cells. I t is not known whether the beneficial effect represents a decelerated rate of aging or that the age clock was reset with respect to this age-correlated trait (decreased sensitivity to UV relative to clonal age). If the UV-induced damage exceeds the capacity of the cells to repair the damage, reduced life span results; if the damage does not saturate the repair system, increased repair capacity also might facilitate correction of some age-correlated damage. Should some age damage be repaired the ape clock would be reset to some extent, but if repair of new damage is facilitated the rate of aging may be retarded. Recent studies indicate increased levels of DNA polymerase activity after UV irradiation, which could provide increased levels of repair (Williams and SmithSonnebom, 1980). Since DNA polymerase activity does not decline with age (Williams and Smith-Sonnebom, 1980), other enzymes or variables may contribute to the beneficial effects of the ultraviolet and photoreactivation treatment. For example, UV-altered modulation of gene expression, greater accessibility of DNA to repair. increase in repair, and/or removal of age-accumulated damage contained within patches of UV-induced damage may be responsible for the increased longevity observed after U V irradiation and photoreactivation cycles. Carcinogen-induced life-span extension in P . multimicronucleatum has also been reported (Spencer and Melroy. 1949). but unfortunately selfing in the respective control and treated populations was not monitored, Nutrition-induced alteration in longevity has been noted in adult Tokophyru as already described, and can impose finite life spans on immortal Amoeba. Although Amoehti appear to multiply indefinitely on a growth diet, a limited life span can be induced by exposure to a maintenance diet for more than 2-9 weeks prior to transfer back to the growth diet (Muggleton and Danielli, 1958; Danielli and Muggleton. 1959). The ability to transfer nuclei and cytoplasm using this protozoan provided a means for study of nucleocytoplasmic relations in life-span determination (for review see Danielli, 1959; Muggleton-Harris, 1979). The finite life-span cells were type A or B; type A showed stem-line growth in which one of the two daughters died after binary fission, and the other reproduced again to yield another viable and nonviable cell until death, and type B produced two viable cells at each division until death. Transfer of normal nuclei to limited

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life-span cells did not rescue the cells from a finite growth potential and all showed type B growth irrespective of the type A or B recipient cytoplasm. Transfer of a finite nucleus to normal cytoplasm induced type A cells. In finite life-span cells, type B cytoplasm is dominant, while the nucleus carries type A behavior. Cytoplasmic injections from limited life-span cells to normal cells conferred the limited life-span phenotype (Muggleton and Danielli, 1968). These studies emphasize the importance of environment and nucleocytoplasmic interactions for longevity. In many protozoa there is evidence indicating that the genome is subject to environmental modulation during development, but some plasticity remains at least during the mature stages of the life cycle. Cells that are immortal, like Amoeba, can be regulated by nutritional ‘changes that affect their proliferative capacity, and certain chemical and physical agents may be capable of inducing changes in gene function even in differentiated nuclei.

V. Aging in Multicells and Unicells The uniformity of the structure of the DNA molecule and the universality (or nearly so) of the genetic code imply a common origin for all living forms. The ciliated protozoans can be viewed not only as individual cells but also as clones that after fertilization show predictable timed changes in functionthough unlike multicellular organisms, these unicells remain separated in space and do not necessarily show morphological changes. The unicells, as eukaryotes, are a link between prokaryotes and multicellular organisms. The limited life-span ciliates show a predictable decline and finally death as has been shown for normal human cells in culture (Hayflick and Moorhead, 1961; Hayflick, 1965). Similarities between age-associated changes in protozoa and multicellular organisms are noted (Table 111),and these and other similarities among unicells and multicells are now described. Age-dependent germinal damage is widespread in the protozoans and is also seen in older human parents (for review see Kram and Schneider, 1978). The capacity for repair of some germ-line damage has been documented for chemically induced DNA damage after fertilization in mice. The repair capacity was relative to the stock of mice used and the kind of DNA lesions induced and indicates that repair capacity of specific lesions may be genetically controlled (Generoso et a l . , 1979). The determinative role of the cytoplasm in regulation of development of undifferentiated nuclear function is well documented in multicellular and unicellular organisms. For example, interactions between the nucleus and oocyte cytoplasmic proteins can produce a heritable state of nuclear activation in multicellular organisms (Brothers, 1976), and in Paramecium cytoplasmic states and external

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JOAN SMITH-SONNEBORN TABLE 111 SIMILARITIES IN AGE-RELATED CHANGES IN PROTOZOA MULTICELLULAR ORGANISMS"

AND

Life-cycle changes Immaturity, maturity, and senescence Length of immaturity directly related to length of life Cytoplasmic determinations of gene activity during development Cytoplasmic modulations in differentiated nuclei Senescent changes Parameters that decrease DNA content DNA synthesis RNA synthesis Ribosomal RNA, nucleolar volume DNA template activity Endocytic activity Asexual cell reproduction Age-induced decline in progeny viability DNA repair Parameters that increase Abnormal mitochondria Age pigments, chromosomal aberration Lysosomal activity Sensitivity to X-ray

For protozoan references, see Tables I and 11; for multicellular organism references, see review by Hayflick (1977) as well as Stiffel et a / . (1956) and Wagner et a/. (1966). for endocytic changes with age: Rockstein et a / . (1977) and Cutler (1978) for correlations of immaturity period with longevity; DeRobertis and Gurdon (1977) for cytoplasmic determinationsof gene activity during development; and Gopalakrishnan and French Anderson (1979). Gopalakrishnan era/. (19771, and Lipsich e t a / . (1979) for cytoplasmic modulations of differentiated nuclei.

environment states can determine the heritable expression of alternative alleles (for review see Sonnebom, 1977; Sonneborn and Schneller, 1979). The sensitivity of a differentiated nucleus to cytoplasmic changes has been explored in both multicellular and unicellular organisms. When a somatic cell nucleus is introduced into an oocyte, the oocyte cytoplasm brings about activation and repression of different genes in the newly introduced nucleus (DeRobertis and Gurdon, 1977). The introduction of a differentiated nucleus into another differentiated cytoplasm has been investigated using cybrids (cell composites formed by the nucleus and cytoplasm of one cell type and the enucleated cytoplasm of an alternative type). Cytoplasts from a nonerythroid cell, when introduced into an erythroid cell, can cause the extinction of the cell's ability to make

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hemoglobin (Gopalakrishnan et al., 1977). Cytoplasmic induction of gene activity has also been found (Gopalakrishnan and French Anderson, 1979; Lipsich et a f . , 1979). Liver-specific enzyme function can be induced in fibroblast-derived nuclei by cytoplasm from liver cells, and the induced gene expression can last 75-100 cell generations (Lipsich et al., 1979). Likewise, in paramecia, injection of cytoplasm from an immature paramecium to a mature one can result in temporary reversion to immaturity (Miwa et a f . , 1975). Although these examples are not cytoplasmic effects on senescence per se, they raise the possibility that some determination of nuclear function may be modulated by the cytoplasm not only during development, but even after nuclear differentiation. A detrimental effect of aged cytoplasm has been apparent in different species of both Paramecium and Euplotes. Similar detrimental cytoplasmic effects have been seen with “foreign” cytoplasm in Paramecium (Chen, 1946) and in multicellular organisms (Hennen, 1963). The similarity between aged and foreign cytoplasm may reflect a cytoplasmic state (not necessarily the same state) that is incompatible with normal nuclear development. Loss of ability to maintain specialized states of differentiation of mating type in aged cells have been seen in Euplotes (Heckmann, 1967) and in P . bursaria (Jennings, 1941), and is suggested in multicellular organisms. The number of globin mRNAs and the sequence diversity of murine leukemia virus RNA increases in aging mouse brain and liver (Ono and Cutler, 1978), though loss of regulation need not be responsible for this observation. New macromolecules appear in aging brain cells (Terry and Gershon, 1976). Constitutive and induced levels of enzyme activity are altered with age (Finch, 1972; Adelman and Britton, 1975; Fulder and Tarrant, 1975; Wilson, 1973; Lipetz, 1981). These results imply change of gene regulation for specific cell functions, which could have deleterious effects on organismal function. DNA damage and/or programmed loss of function could contribute to the loss of ability to maintain the differentiated state (Lipetz, 1981). The respective contributions of the nucleus and cytoplasm in human cell hybrids, heterokaryons, or cybrids formed between immortal transformed cells and limited life-span cells and/or young and old cells is under active investigation, but a comprehensive view is still not possible (for review see Nonvood, 1978; Muggleton-Harris and Palumbo, 1979; Bunn and Tarrant, 1980). Environmental conditions such as radiation may also induce changes in gene expression that could contribute to the observed UV-induced life-span extension and modulation of timing of maturity. Evidence for radiation-increased repair is found in paramecia (Smith-Sonnebom, 1979; Williams and Smith-Sonnebom, 1981), insects (Ducoff, 1976), and mammals (Calkins and Greenlaw, 1971). The amount of DNA damage was found to be correlated with longevity (SmithSonnebom, 1979). Ultraviolet treatment in prokaryotes is known to initiate a

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complex induction process culminating in derepression of a group of metabolically diverse but coordinated functions that could promote survival (Witkin, 1976) or long life. DNA damage and repair have been correlated with longevity in mammals (for review see Hart et al., 1978). Age-accumulated DNA damage and loss of repair are found in some aging human cells, though experimental design and cell type influence the results obtained (for review see Tice, 1978; Williams and Dearfield, 1981). L o s s of repair in aged paramecia is suggested by increased sensitivity to UV with increased age (Smith-Sonnebom, 1971 ). The repair capacity for inbreeders may be adjusted (lower) to attain genetic variety comparable to outbreeders, and longevity is greater in outbreeding species. Length of immaturity and life span are also correlated in ciliates (Table I) as in multicellular organisms (Rockstein et al., 1977; Cutler, 1978), though in ciliates immaturity and longevity are measured in cell divisions, not days since fertilization (Kroll and Barnett, 1968; Miwa and Hiwatashi, 1970; Takagi, 1970, 1974; Smith-Sonnebom and Reed, 1976; Takagi and Yoshida, 1980). An apparent difference between many multicellular organisms and some ciliates is the ability to undergo autogamy, a process apparently lost in mammals. Recent studies indicate, however, that the mouse oocyte, under appropriate conditions, can be induced to undergo duplication of the oocyte nucleus and proceed to the blastocyst stage (Markert and Petters, 1977). This amazing result implies that these higher cells have not completely lost the potential for “parthl enogenesis” under special environmental conditions. Sight should not be lost of the fact that the fundamental unit of the multicellular organism is the eukaryote cell, possibly a direct descendent of the original prokaryote cell 3 billion years ago (Margulis, 1970). Patterns of gene expression can contribute to life-span duration of specific cell types determined during development. Different cell types may have a role in the determination of the organismal longevity in different genera, but each may be meshing fundamental regulatory mechanisms that are utilized in protozoa for survival and longevity. Fundamental regulatory mechanisms may be suggested by their demonstration in several evolutionarily diverse organisms and the verification of their fundamental character assessed by the extent of their universality (i.e., maintenance throughout evolutionary time).

VI. Mechanisms of Cellular Aging The immortality of the germ line implies that the necessary longevityassurance loci for infinite cell life span reside in the genome. However, regulation of these loci must be adjusted during development and somatic life span to provide a long enough individual organismal life span for a subsequent fruitful fertilization, and new repository for the germ line. Maintenance of DNA integrity

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and regulation of those loci that are necessary for life maintenance must be genetically determined for survival of the species. Integrity of the germ-line DNA can be preserved if fertilization occurs prior to the onset of any genetic damage. Should germ-line damage accumulate, repair may be possible when homologous chromosomes pair during fertilization (Martin, 1977). The demonstration that chemically induced DNA damage to sperm can be repaired in some recipient female strains of mice (Generoso et al., 1979) implies that age-induced damage, if similar to chemically induced lesions, also may be reparable after fertilization. Regulation of genome function includes both determinations during development and temporal patterns of gene expression. During fertilization in Paramecium tetraurelia, determinations of genome function occur with 48 hours after mating-pair separation. Within minutes after separation of pairs, the differentiation of two nuclei occurs and a metabolically active macronucleus and relatively inactive micronuclei are formed. The ancestral nucleus continues to produce RNA until the filial macronucleus suppresses the ancestral nucleus (Berger, 1974, 1976; Sonneborn, 1954a; Mikami, 1979). Mating types are determined within the first cell division following fertilization, and the ability to discharge arrow-like trichocysts occurs later (see reviews Sonnebom, 1977; Sonneborn and Schneller, 1979). Molecular mechanisms that can direct determinations of gene function are emerging rapidly. The presence of repressors or derepressors in the cytoplasmic environment of the developing nucleus can turn on or off certain genes, and, once activated or repressed, perpetuate that state. The absence of a derepressor can lead to a “permanently” inactive locus but cannot account for the determinations of nuclear function observed in Paramecium described above (Sonneborn and Schneller, 1979). Altered gene function can be mediated by insertion sequences (Starlinger, 1977), structural inversions (Zieg et a l . , 1977; Bukhari and Ambrosia, 1978), base modification (Sager and Kitchin, 1975; Holliday and Pugh, 1975), and heterochromatinizations (Cattanach, 1975). Although determinations of gene functions are made in the relatively short interval after conjugation, expression of the genotype may be delayed by many cell divisions. Temporal patterns of gene expression need not imply sequential gene activations though there is ample evidence for a sequential pattern in mating-type expression in Paramecium bursaria. There is evidence that the mating type is’ determined at clonal initiation but expressed only 30-150 cell divisions later-that is, immature cells lack mating-type substances, adolescent cells form a single substance, and mature cells form two substances. The temporal sequence is under genic control; the B locus not only directs production of the B substance, but expression of the nonallelic A locus (Siegel, 1967). A hypothesis was developed by Nanney (1974) that proposes that random events can be used to measure biological time and thus convert noise into order. The

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attractiveness of this model rests in the demonstration that temporal patterns can be attained without necessarily invoking a stepwise sequential series of gene expression and may provide an explanation for the exceptional ciliate Tetruhymena, which can show timed gene expression without necessarily showing senescence. Although sequential activation of gene expression may represent a model for development and aging, it is prudent to consider the achievement of an orderly sequence of timed events occumng by alternative mechanisms, since more than one means may be employed to acquire the desired biological objectives in different species. A general solution to problems of development may be nonexistent; rather there are many combinations of solutions operative in different organisms (Sonneborn, 1970a). Mechanisms for regulation of eukaryotic chromosomal function that may be critical during development and aging include interactions of DNA with histone and nonhistone proteins into the supramolecular complex chromatin. Postsynthetic modifications of chromosomal proteins such as phosphorylation, acetylation, ADP ribosylation, and methylation alter the structural organization of DNA and template activity (for review see Kanungo, 1980). Cook ( 1973) proposed that DNA superstructure can control differentiation and Lipetz (1981) reviews evidence suggesting a pivotal role for superhelicity (supercoiling) in the aging process. The three-dimensional structure of the doublestranded molecule of DNA may be described by the coiling of the helix into loops or supercoils. Local denaturation can be a natural consequence of DNA supercoiling and thus can alter accessibility of DNA to enzymes, transcription, recombination, and repair (Benham, 1979). Since DNA gyrases can introduce DNA supercoiling, regulation of these enzymes can modulate specific genome expression in bacteria and bacteriophages (Botchan et a / . , 1973; Richardson, 1975; DeWyngaert and Hinkle, 1979; Yang et af., 1979) by alteration of RNA-binding sites (Botchan et a / . , 1973; Benham, 1979). DNA supercoiling in eukaryotes was inferred from the expected sedimentation pattern in sucrose gradients with the intercalation agent ethidium bromide before and after treatment with DNA nicking agents using nucleoids (permeabilized and protein-depleted nuclei) (Cook and Brazell, 1975; Cook el a l . , 1976). Novobiocin, an antibiotic that reduced DNA-negative superhelicity and DNA synthesis in prokaryotes, also shows a sedimentation pattern, after novobiocin treatment. consistent with reduced numbers of DNA supercoils in mammalian cells (Mattern and Painter, 1979). Novobiocin-induced effects on eukaryotes, which are consistent with loss of DNA-negative superhelicity, include both decreased rate of DNA synthesis (Mattern and Painter, 1979; Lipetz el a l . , 1980)-though novobiocin can inhibit some eukaryotic DNA polymerase (Sung, 1974: Lynch et al., 1 9 7 6 h a n d decreased cellular repair of UV but not Xray-induced damage (Collins and Johnson, 1979). Another inhibitor of prokaryo-

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tic gyrase, nalidixic acid, inhibits both replicative and unscheduled (presumably repair) DNA synthesis (Lipetz et al., 1980), and RNA and protein synthesis (Whittaker and Carnevali, 1977). The cumulative effects of novobiocin and nalidixic acid represent indirect though not compelling evidence for enzymatic regulation of DNA superhelicity in eukaryotes. Evidence for decreased DNA-negative superhelicity with increased age has been noted in human cells and in mice (Lipetz et al., 1980; Lipetz, 1981), and novobiocin-treated paramecia show enhanced sensitivity to UV irradiation (increased death after autogamy) when young cells were used (Lipetz and SmithSonnebom, 1980). It is tempting to speculate that decline of DNA-negative superhelicity occurs with age and results in reduced repair capacity, contributing to the observed loss of UV dark-repair capacity (Smith-Sonnebom, 1971) and novobiocin-enhanced UV sensitivity. The DNA-superhelical theory of aging predicts that DNA alterations in superhelicity will result in altered transcriptional control of all genes in a domain. A domain is a large DNA segment whose superhelicity is modified as a single unit (Cook and Brazell, 1976). DNA strand breaks, as well as other forms of DNA damage, may alter DNA superhelicity and may affect the function of many genes (Lipetz, 1981). Base sequences are not altered in this process, so that DNA damage need not imply changes in primary DNA sequence (Brash and Hart, 1978). The impact of DNA damage leading to alterations of gene function may be the major factor that contributes to pathologies of both carcinogenesis and aging (Brash and Hart, 1978; Lipetz, 1981). Enzymes that can regulate the superhelicity of DNA may be responsive to stimulation by hormones at the cell surface (Lipetz et al., 1980b). Spermidine inhibits DNA nicking-closing enzyme, or relaxation of DNA superhelicity, and activates gyrase (Liu and Wang, 1978; Lipetz et al., 1980b). The net result of spermidine would be the maximization of DNA superhelicity. Loss of polyamines with age (Raina et al., 1977; Reinnert and Shukla, 1978) might favor decline in regulation of DNA superhelicity with age. Since polyamines are known to be second messengers of cell membrane stimuli (Tabor and Tabor, 1976), DNA superhelicity could be responsive to cell surface stimulation. DNA-confonnational changes need not be the primary event, but rather an integral part of a cascade of events in aging. Regulation of superhelical coiling may be a critical intermediate step in the expression of several age-related pathologies (Lipetz, 1981). The genetic loci that are candidates for longevity assurance include those genes that are involved with repair of UV damage, free-radical detoxifying enzymes, enzyme systems that activate polycyclic aromatic hydrocarbons to their mutagenic form, and immune function (controlled by the major histocompatibility loci). The first indication of association of DNA repair with longevity was the relationship between maximal life-span potential of mammals and their

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excision-repair capacity (Hart and Setlow, 1974; for reviews see Hart et a f . , 1978; Tice, 1978; Williams and Dearfield, 1980). Modulation of life span in 'Paramecium was found to be correlated with the levels of repair of UV-induced damage (Smith-Sonnebom, 1979; Williams and Smith-Sonnebom, 1981). The life spans of inbreeders are correlated with their commitment to that mode, suggesting that repair may be related to breeding life-style with the function of generation of genetic variation for adaptation to environmental changes. Regulation of superoxide dismutase for detoxification of free radicals in different genetic mutants was directly correlated with their longevity, that is, increased enzyme activity was coupled with increased longevity while decreased enzyme activity was coupled with decreased longevity (Munkres, 1980). The ability of organisms to activate polycyclic aromatic hydrocarbons to their mutagenic form is inversely correlated with organismal longevity (Schwartz, 1975). All of these loci are involved with maintenance of DNA integrity (though not exclusively), either by reducing endogenous or exogenous genotoxic agents or by repair of DNA damage that may occur. DNA repair may even impact on age-related changes in immune functions. Excision repair of UV-induced damage in erythrocytes was found to be higher in longer lived mice, and the possibility of a tricornered relationship between the main histocompatibility complex, which primarily controls age-related abnormalities in immune function (for review see Walford et al., 19781, one form of DNA repair, and life span of different strains of mice was suggested (Hall et ul.. 1980). The number of longevity assurance loci is unknown, but identification of such genetic systems is a prerequisite for some comprehension of an aging process; regulation of their function to improve or extend the duration of quality life may then be possible. Organismal survival depends on the function in concert of all cell parts both at the molecular and organellar level. Loss of function of any vital part that cannot be renewed will lead to cell death. The weakest link in the chain of cell vitality, then, will determine the length of life, and need not be the same "link" in all organisms (Smith-Sonnebom, 1979).

VII. Summary The view through the microscope reveals a panorama of organisms that have evolved mechanisms to achieve preservation of the species, though not of individuals. Each species need not, and in fact does not employ the same mechanisms in the same way to assure survival. The protozoa present an untapped reservoir for an evolutionary approach to identification of fundamental mechanisms that are utilized in the assurance of longevity. The differences in protozoa and humans belie the underlying fundamental similarities. The challenge

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is to identify common longevity-assurance characters, the options for regulation of their function, and the contribution of cell parts andlor their repair to maintenance of vigorous life. The array of regulatory mechanisms need not be large since meshing mechanisms in a variety of ways will generate exponential increases in the number of ways to achieve the desired biological objectives. A common regulator in a variety of functions such as chromatin conformation may represent a target for modulation of some of the pathologies of aging.

ACKNOWLEDGMENTS The constructive comments of Professor T. M. Sonnebom, D. L. Nanney, P. D. Lipetz, G. L. Fisher, and R. Kitchin are gratefully acknowledged.

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Index

A Aging cellular, mechanisms of, 344-348 in multicells and unicells, 34 1-344 Aleurone tissue, 84-92 function of, 92-93 Animal glycoprotein, structure and biosynthesis of carbohydrate units, 105-110 Arachnids primitive glial cell junctions in, 295 tight junctions and, 273-276 tight junctions in, 282-290 Arthropod(s), comparison of zonulue occludenres with those of other organisms developmental stages and, 298-299 disruption of junctions, 300 fracturing characteristics and, 296-297 insect versus vertebrate physiology, 299 localization of, 295-296 types of leakiness observed, 298 Arthropod groups, with tight junctional structures arachnids, 282-290 crustaceans, 276-277 insects, 277-282 myriapods, 290-291 primitive arachnids, 273-276 Arthropod tissues fine structure of tight junctions in freeze-fracture appearance, 254-259 localization and coexistence with other junctional types, 259-261

thin sections and, 247-254 functional significance of tight junctions adhesion, 272 compartmentalization of plasmalemma, 269 leakiness correlated with ridge number and discontinuities, 269-27 1 permeability barriers, 261-268 tissue rigidity and, 27 1-272 junctions exhibiting features in common with tight junctions continuous (smooth septate) junctions, 2%-294 glial cell junctions in primitive arachnids, 295 pleated septate junctions, 291-293 reticular junctions, 294-295 reticular septate junctions, 294 stages in assembly and breakdown of tight junctions evidence for distinction between tight and gap junctional precursors, 304-305 intramembranous particle migration, 300304 tight junctions both simple and complex, 309-312 models of, 305-309 Autophagic degradation characteristics of induced degradation, 166I70 formation of residual bodies from autophagosomes, 162-166 as regulatory mechanism in catabolism, 170171 355

356

INDEX

Autophagy degradation and, 162-171 interaction with synthesis. 153-155 pathologically altered cells and, 155 physiological cell function and, 151- 153 physiological remodeling of cells and, 155 sequestration of cytoplasm and, 155-162

c; Carbohydrate units, of animal glycoproteins biosynthesis, 108-1 10 glycopeptide bond, 105- 106 structure of, 107-108 Cell endosperm, 65-69 pathologically altered, autophagy and, 155 physiological function. autophagy and, 151 153 physiological remodeling of, autophagy and, 155 Cell wall endosperm. formation, 76-82 of eykaryotic algae, 13-14 Cholesterol biosynthetic sequences common to phosphorylated dolichol and, 134- I37 glycoprotein assembly and, 137-142 Ciliate(s) clonal aging in background, 321-322 breeding lifestyles, 326-327 ciliate genetic complex, 322-326 Euplotes, 333-334 Paramecium, 327-332 Tetrahyrnena. 334-336 Tokophnw, 334 fertilization, marvel or menace, 336-339 Clonal aging in ciliates background, 321-322 breeding lifestyles. 326-327 ciliate genetic complex, 322-326 Euplotes, 333-334 Paramecium, 327-332 Tetrahymena. 334-336 Tokophrya. 334 Collecting tubule, membrane ultrastructure, 226-233 dark cells, 219-221 light cells, 216-219

papillary duct, 22 1-222 Crustaceans, tight junctions in, 276-277 Cytoplasm, autophagic sequestration of classical autophagy, 155- 157 crinophagy. 157-159 microautophagy, 159 as regulatory mechanism in catabolism, 161I62 transmembrane penetration of particles into lysosomes, 160

D Dolichylpyrophosphorylsaccharide, formation of intact cell systems, 114-1 18 subcellular systems, 112-1 14 Dolichylsaccharides relationship to asparagine-linkedcarbohydrate units of glycoproteins, 122-123 regulatory role of glucose, 123-125 trimming and maturation of, 125-127 structure of carbohydrate portion, 118-122

E Endocycles, development of polytene chromosomes of plants and, 22-25 Endosperm aleurone tissue, 84-92 function of, 92-93 cell wall formation, 76-82 central cell and, 56-63 functions of, 93-95 haustoria, 82-84 function of, 93-95 primary nucleus, 63 ruminate, 76 types of formation cellular, 71-75 helobial, 75-76 nuclear, 69-7 1 Endosperm cell, 65-69 Euplores, clonal aging in, 333-334

F

Freeze-fracture, technique of, 184- I85

357

INDEX

G Glomerulus, membrane ultrastructure capillary endothelium, 196-1 97 mesangial cells, 197- 199 parietal epithelium, 189-192 visceral epithelium, 192- 196 GIycoprotein bacterial systems, lipid intermediates and, 110-1 12 carbohydrate units biosynthesis of, 108-1 10 glycopeptide bond, 105-106 structure of, 107-108 eukaryotic formation of dolichylpyrophosphorylsaccharides intact cell systems, 114-1 18 subcellular systems, 1 12- 1 14 structure of carbohydrate portion of dolichylsaccharides, 118-122 regulation of biosynthesis, 133-134 biosynthetic sequences common to phosphorylated dolichol and cholesterol, 134-137 interdependence of cholesterol synthesis and glycoprotein assembly, 137-142 relationship of dolichylsaccharides to asparagine-linked carbohydrate units of, 122- 123 regulatory role of glucose, 123-125 trimming and maturation of asparaginelinked saccharides, 125-127 Glycosylation reactions, dolichol-dependent cell surface-associated mannosyltransferases, 131-132 disposition of polyisoprenol derivatives in membrane systems, 130-131 spatial and temporal relationships to ribosomal machinery, 127-130

engulfment phase, 173-174 isotope labeling and, 175-179

I Insects, tight junctions in, 277-282

L Lipid intermediates bacterial glycoproteins and, 1 10-1 12 eukaryotic glycoproteins and formation of dolichylpyrophosphorylsaccharides intact cell systems, 114-1 18 subcellular systems, 112-1 14 structure of carbohydrate portion of dolichylsaccharides, 118-122 Longevity, environmental modulation of, 33934 1 Loop of Henle, membrane ultrastructure, 207208, 224-225 ascending limb, 21 1-212 descending limb, 209-2 11

M Mannosyltransferases, cell surface-associated, dolichol-dependent glycosylation reactions, 131-132 Morphogenesis, of eukaryotic algae, 16 Myriapods, tight junctions in, 290-291

0 Organelles, of eukaryotic algae, 14-16

P H Haustoria, endosperm, 82-84 function of, 93-95 Heterophagy, 171-173 attachment phase, 173 degradation and residual body stages, 174175

Paramecium, clonal aging in, 327-332 Phaseolus, polytene chromosome of, 30-37 Plasma membrane, freeze-fractured, general morphology and intercellular junctions, 185- 189 Podocytes, membrane ultrastructure, 192-196, 223

358

INDEX

Polyisoprenol derivatives, disposition in membrane systems, 130-131 Polytene chromosomes of plants development through endocycles, 22-25 evolutionary aspects of, 4448 functional aspects of, 4 2 4 4 historical background, 22 Occurrence and induction, 25-28 structure general aspects, 28-30 Phuseolus, 30-37 Rhinanrhus. 38-42 Tropeohm, 37-38 Protoplasts of eukaryotic algae experimental work cell walls, 13-14 morphogenesis, 16 organelles, 14- 16 physiology, 16- 17 methodology culture conditions, 3-4 enzymes and other methods, 4-6 organisms, 2-3 osmotica, 6-9 regeneration, I 1- I3 separation, 9- 1 1 viability. I I Proximal tubule, membrane ultrastructure, 223-224 pars convoluta, 199-203 pars recta, 204-207

R Rhinunthus, polytene chromosome of, 38-42 Ribosomes, dolicol-dependent glycosylation reactions and. 127-130

7 Terrahwnena, clonal aging in, 334-336 Thick ascending limb of Henle and distal convoluted tubule, membrane ultrastructure. 212-215

Tight junctions arthropod groups with arachnids, 282-290 CNStaCeanS, 276-277 insects. 277-282 myriapods, 290-29 1 primitive arachnids, 273-276 in arthropod tissue freeze-fracture appearance, 254-259 localization and coexistence with other junctional types, 259-261 thin sections and, 247-254 both simple and complex in arthropod tissues, 309-3 12 functional significance adhesion, 272 compartmentalization of plasma lemma, 269 leakiness correlated with ridge number and discontinuities, 269-271 permeability barriers, 26 1-268 tissue rigidity and, 271-272 junctions with common features and continuous (smooth septate) junctions, 293-294 glial cell junctions in primitive arachnids, 295 pleated septate junctions, 291 -293 reticular junctions, 294-295 reticular septate junctions, 294 models of, 305-309 stages in assembly and breakdown, 300-305 Tokophryu. clonal aging in, 334 Tropeohm, giant chromosomes of, 37-38

U Urinary tubules, membrane ultrastructure, 222 collecting tubule, 226-233 distal tubule and juxtaglomerular apparatus. 225-226 loop of Henle, 224-225 mesangial and lacis cells, 223 podocytes, 223 proximal tubule, 223-224 tight junctions of kidney tubule, 233-235

Contents of Recent Volumes Volume 50

Cytophysiology of the Amphibian Thyroid Gland through Larval Development and Metamorphosis-ELIANE REGARD Cell Surface Enzymes: Effects on Mitotic Activity and Cell Adhesion-H. BRUCEBOSMANN The Macrophage as a Secretory Cell-Roy C. PAGE,PHILIPDAVIES,AND A. C. ALLISON New Aspects of the Ultrastructure of Frog Rod outer Segments-JtiRGEN ROSENKRANZ Biogenesis of the Photochemical Apparatus -TIMOTHYTWFFRY Mechanisms of Morphogenesis in Cell Cultures J . M. VASILIEV AND I. M. GELFAND Extrusive Organelles in P r 0 t i s t s - K ~HAUS~~~ Cell Polyploidy: Its Relation to Tissue Growth MANN AND I. V. L ~ c ~ ~ ~ s - J AC.Y BROWNAND RICHARDC. and Functions-W. YA. BRODSKY URYVAEVA HUNT Action of Testosterone on the Differentiation SUBJECT INDEX and Secretory Activity of a Target Organ: The Submaxillary Gland of the Mouse-MONIQUE Volume 53 CHRBTIEN SUBJECT INDEX

Regular Arrays of Macromolecules on Bacterial Cell Walls: Structure, Chemistry, Assembly, and Function-UwE B. SLEYTR Volume 5.1 Cellular Adhesiveness and Extracellular SubStrata-FREDERICK GRINNELL Circulating Nucleic Acids in Higher Organisms -MAURICE STROUN, PHILIPPEANKER, Chemosensory Responses of Swimming Algae and Protozoa-M. LEVANDOWSKY AND D. C. PIERREMAURICE, AND I%ERB. GAHAN R. HAUSER Recent Advances in the Morphology, Histochemistry, and Biochemistry of the De- Morphology, Biochemistry, and Genetics of veloping Mammalian O V ~ ~ ~ ~ A S. R D UPlastid L Development in Euglena gracilis-V. NIGON AND P. HEIZMANN GURAYA Morphological Modulations in Helical Muscles Plant Embryological Investigations and Fluorescence Microscopy: An Assessment of (Aschelminthes and Annelida)-GIuuo Integration-R. N. KAPILAND S. C. TIWARI LANZAVECCHIA The Cytochemical Approach to Hormone Assay Interrelations of the Proliferation and Dif-J. CHAYEN ferentiation Recesses during Cardiac Myogenesis and Regeneration-PAVEL

P. RUM-

SUBJECT INDEX

YANTSEV

The KWbff Cell-hTER A. REVELL Circadian Rhythms in Unicellular Organisms: An Endeavor to Explain the Molecular Mechanism-HANsGEoRG SCHWEIGER AND MANFRED SCHWEIGER SUBJECT INDEX

Volume 52 Cytophysiology of Thyroid Parafollicular Cells-ELADIo A. NUNEZAND MICHAEL D. GER~HON

Volume 54 Microtubule Assembly and Nucleation-MARC W. KIRSCHNER The Mammalian Sperm Surface: Studies with specific Labeling Techniques-JAMES K. KOEHLER The Glutathione Status of C~IIS-NECHAMA S. KOSOWER AND EDWARD M. KOSOWER Cells and ~aMwX.tX+ROBERT ROSEN Immunocytology of Pituitary Cells from Teleost Fishes-E. FOLLBNIUS,J. DOERR-SCHOTT, AND M. P. DUBOIS

359

360

CONTENTS OF RECENT VOLUMES

Follicular Atresia in the Ovaries of Nonmamma- Cytoplasmic Structure and Contractility in Amoeboid Cells-D. LANSING TAYLOR AND lian VertebratedRINlVAs K. SAIDAPUR JOHNS. CONDEELIS Hypothalamic Neuroanatomy: Steroid Hormone Binding and Patterns of Axonal Projec- Methods of Measuring Intracellular CalciumANTHONY H. CASWELL tiOnS-hNALD w . PFAF'F AND LILY c. A. Electron Microscope Autoradiography of CalCONRAD cified Tissues-ROBERT M. FRANK Ancient Locomotion: Prokaryotic Motility SystemS-LELENG P. TO A N D LYNNMARGULIS Some Aspects of Double-Stranded Hairpin Structures in Heterogeneous Nuclear RNAAn Enzyme Profile of the Nuclear Envelope-I. HIROTONAORA B. ZBARSKY Microchemistryof Microdissected Hypothalamic SUBJECT INDEX Nuclear Areas-M. PALKOVITS SUBJECT INDEX

Volume 55 Chromatin Structure and Gene Transcription: Nucleosomes Permit a New SynthesisTHORU PEDERSON The Isolated Mitotic Apparatus and Chromosame Motion-H. SAKAI Contact Inhibition of Locomotion: A Reappraisal-JOAN E. M. HEAYSMAN Morphological Correlates of Electrical and Other interactions through Low-Resistance Pathways between Neurons of the Vertebrate Central Nervous System-C. SOTELOA N D H. KORN Biological and Biochemical Effects of Phenylalanine Analogs-D. N. WHEATLEY Recent Advances in the Morphology, Histochemistry. Biochemistry. and Physiology of Interstitial Gland Cells of Mammalian O V X ~ - S A R DS. U GURAYA L Correlation of Morphometry and Stereology with Biochemical Analysis of Cell Fractions -R. P. BOLENDER Cytophysiology of the Adrenal Zona FasCiCUhtaAASTONE G . NUSSWRFER.GIUSEPPINA MAZZOCCHI. A N D VIRGILIOMENEGHEI.I.1 SUBJECT I N D E X

Volume 57 The Corpora Allata O f InSeCtS-PIERRE CASSIER Kinetic Analysis of Cellular Populations. by Means of the Quantitative RadioautographyJ.-C. BISCONTE Cellular Mechanisms of Insect PhotoreceptionF. G. GRIBAKIN Oc~CyteMaturation-YosHio MASUIA N D HUGH J. CLARKE The Chromaffin and Chromat'Tin-like Cells in the Autonomic Nervous SyStem-JACQUES TAXI The Synapses of the Nervous System-A.'A. MANINA SUBJECT INDEX

Volume 58 Functional Aspects of Satellite DNA and Heterochromatin-BERNARD JOHN A N D GEORGEL. GABORMIKLOS Determination of Subcellular Elemental Concentration through Ultrahigh Resolution Electron Microprobe Analysis-THOMAS E. HUTCHINSON

The Chromaffin Granule and Possible Mechanisms of ExocytosisHARvEY B. POLLARD, J. PAZOLES, CARLE. CREUTZ, CHRISTOPHER A N D ORENZINDER Volume 56 The Golgi Apparatus, the Plasma Membrane, and Functional Integration-W. G. WHALEY Synapses of Cephalopods-COLLETTEDUCROS A N D MARIANNE DAUWALDER Scanning Electron Microscope Studies on the Development of the Nervous System in V i w Genetic Control of Meiosis-I. N. GOLUBOVSKAYA and in Vitro-K. MELLER

CONTENTS OF RECENT VOLUMES

36 1

Hypothalamic Neurons in Cell Culture-A. Cell Reparation of Non-DNA Injury-V. YA. TIXIER-VIDAL AND F. DE VITRY ALEXANDROV The Subfornical Organ-H. DIETER DELL- Ultrastructure of the Carotid Body in the M A N N AND JOHNB. SIMPSON M ~ ~ ~ ~ s - A L A IVERNA N SUBJECT INDEX The Cytology and Cytochemistry of the Wool Follick-bNALD F. G. ORWN SUBJECT INDEX

Volume 59 The Control of Microtubule Assembly in Volume 61 ViVo-ELIZABETH c. RAFF Membrane-Coating Granules-A. F. HAYWARD The Association of DNA and RNA with Innervation of the Gastrointestinal TractMembranes-MARY PATMOVER GIORGIO GABELLA Electron Cytochemical Stains Based on Metal Effects of Irradiation on Germ Cells and EmChelation-DAVID E. ALLENAND DOUGLAS bryonic Development in Teleosts-Noeuo D. PERRIN EGAMIAND KEN-ICHIIJIRI Cell Electrophoresis-THOMAS G. PRETLOW, I1 Recent Advances in the Morphology, CytoA N D THERESA P. PRETLOW chemistry, and Function of Balbiani's VitelThe Wall of the Growing Plant Cell: Its Threeline Body in Animal OOC~~~S-SARDUL S. Dimensional OrganiZatiOll-JEAN-CLAUDE GURAYA ROLANDAND BRIGIITEVIAN Cultivation of Isolated Protoplasts and HybridiBiochemistry and Metabolism of Basement zation of Somatic Plant CellS-RAISA G. Membranes-NlcHoLas A. KEFALIDES, BUTENKO ROBERT ALPER,AND CHARLES C. CLARK SUBJECT INDEX The Effects of Chemicals and Radiations within the Cell: An Ultrastructural and Micrurgical Study Using Amoeba proreus as a Single-Cell Model-M. J. ORD Volume 60 Growth, Reproduction, and Differentiation in Transfer RNA-like Structure in Viral ~cunthnmoebu-THoMAs J. BYERS SUBJECT INDEX GeIIOmeS-TlMOTHY c. HALL Cytoplasmic and Cell Surface Deoxyribonucleic Acids with Consideration of Their OriginBEVAN L. REID AND ALEXANDER J. CHARLSON Volume 62 Biochemistry of the Mitotic SpindlePETZELT CHRISTIAN Calcification in PlantS-ALLAN PENTECOST Alternatives to Classical Mitosis in Hemopoietic Cellular Microinjection by CeH Fusion: TechE. ENTissues of Vertebrates-VIEEKE nique and Applications in Biology and GELEBERT Medicine-MmuRu FURUSAWA Fluidity of Cell Membranes-Current Concepts Cytology, Physiology, and Biochemistry of and Trends-M. SHINITZKY Germination of Fern Spores-V. RAGHAVAN AND P. HENKART ImmunocytochemicalLocalization of the VerteMacrophage-Lpphocyte Interactions in Imbrate Cyclic Nonapeptide Neurophypophyseal mune Induction-MARC FELDMANN, ALAN Hormones and Neurophysins-K. DIERICKX AND PETER EREI ROSENTHAL, Recent Progress in the Morphology, 'Histochemistry, Biochemistry, and Physiology of Immunohistochemistry of Luteinizing HorDeveloping and Maturing Mammalian mone-Releasing Hormone-ProducingNeurons TestidARDUL s. GURAYA of the Vertebrates-JuLIEN BARRY

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CONTENTS OF RECENT VOLUMES

Transitional Cells of Hemopoietic Tissues: Ori- V d u m 65 gin, Structure, and Development PotentialCell Surface Glycosyltransferase ActivitiesJOSEPHM. YOFFEY MICHAELPIERCE, EVA A. TURLEY,AND Human Chromosomal Heteromorphisms: Nature STEPHEN ROTH and Clinical Significance-Rm S . VERMA The Transport of Steroid Hormones into Animal A N D HARVEY DOSIK Cells-ELEONORA P. GIORGI SUBJECT INDEX Structural Aspects of Brain Barriers, with Special Reference to the Permeability of the Cerebral Endothelium and Choroidal Volume 63 Epithelium-B. V A N DEURS Immunochemistry of Cytoplasmic Contractile Phvsarum polvcephalwn: A Review of a Model PrOteinS-uTE GROSCHEL-STEWART System Using a Structure-Function Ap- The Ultrastructural Visualization of Nucleolar proach-EUGENE M. GOODMAN and Extranucleolar RNA Synthesis and Microtubules in Cultured Cells: Indirect ImA N D E. PUVION Distribution-S. FAKAN munofluorescent Staining with Tubulin Anti- Cytological Mechanisms of Calcium Carbonate body-B. BRINKLEY. s. FISTEL. J. M. Excavation by Boring Sponges-SHIRLEY A. MARCUM. A N D R. L. PARDUE POMWNI Septate and Scalarifom junctions in Ar- Neuromuscular Disorders with Abnormal MusthpodS
CONTENTS OF RECENT VOLUMES Structure in and around the Liver Sinusoids, and Vitamin A-Storing Cells in Extrahepatic Organ+KENJIRO W A K E

363

A. H. WYLLIE, J. F. R. KERR,A N D A. R. CURRIE INDEX

SUBJECT INDEX

Volume 69 Volume 67 Membrane Circulation in Neurons and Photoreceptors: Some Unresolved Issues-Em A N D ARTHUR M. MERCURIO HOLTZMAN Ultrastructure of Invertebrate Chemo-, Thermo-, and Hygroreceptors and Its Functional Significance-HELMUT ALTNERAND LINDE PRILLINGER Calcium Transport System: A Comparative Study in Different CellS-ANNE GODFRAIND AND TH~OPHILE GODFRAIND DE BECKER The Ultrastructure of Skeletogenesis in HerS.NJOHNSTON matypic C O ~ ~ ~ S - I A Protein Turnover in Muscle Cells as Visualized by Autoradiography-J. P. DADOUNE Identified Serotonin Neurons-NEVILLE N. 0sBORNE A N D VOLKER NEUHOFF Nuclear Proteins in Programming Cell RAO Cycles-M. V. NARASIMHA SUBJECT INDEX

The Structuresand Functions of the Mycoplasma Membrane-D. B. ARCHER Metabolic Cooperation between Cells-M. L. A N D J. H. SUBAK-SHARPE HOOPER The Kinetoplast as a Cell Organelle-V. D. KALLINIKOVA Chlorplast DNA Replication in Chlumydomoms reinhardtiiSTEPHEN JAY KELLER AND CHINGHo Nucleus-Associated Organelles in Fungi-I. BRENTHEATH Regulation of the Cell Cycle in Eukaryotic CellS-ROSALIND M. YANISHEVSKY A N D GRETCHEN H. STEIN The Relationship of in V i m Studies to in Vivo Human Aging-EDWARD L. SCHNEIDER AND JAMESR. SMITH Cell Replacement in Epidermis (Keratopoiesis) via Discrete Units of Proliferation-C. S . POTTEN INDEX

Volume 68 Volume 70 Moisture Content as a Controlling Factor in Seed Developmentand Germination-C. A. ADAMS Cycling Noncycling Cell Transitionsin Tissue A N D R. W. RINNE Aging, Immunological Surveillance, TransApplications of Protoplasts to the Study of Plant formation, and Tumor G I O W ~ ~ ~ E Y M O U R Cells-Lamy C. FOWKEAND OLUFL. GAMGELFANT BORG The Differentiated State of Normal and MaControl of Membrane Morphogenesis in Bacterialignant Cells or How to Defm a “Normal” phage-GREooRY J. BREWER Cell in Culture-MINA J. BISSELL Scanning Electron Microscopy of Intracellular On the Nature of Oncogenic Transformation of Structures-KEIICHI TANAKA CellS--GERALDL. CHAN The Relevance of the State of Growth and TransMorphological and Biochemical Aspects of Adhesiveness and Dissociation of Cancer formation of Cells to Their Patterns of MetaCe~s-HlDEO HAYASHI AND YASUlI ISHIMAbolite U p t a k e - R ~ ~ ~ KOREN RU Intracellular Source of BiolumineswnceThe Cells Of the GastIiC MUCOSa-HERBERT F. BEATRICE M. SWEENEY Differentiation of MSH-, ACTH-, Endorphin-, HELANDER and LPH-Containing Cells in the Hypophysis Ultrastructure and Biology of Female N. Gametophyte in Flowering Plants-R. during Embryonic and Fetal DevelopmentKAPILAND A. K. BHATNAGAR JEAN-PAUL Dumuv INDEX Cell Death: The Significance of Apoptosis-

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Volume 71

Transplantation-ROBERT

GILMOREMCKIN-

NELL

Integration of Oncogenic Viruses in Mammalian Ceh-cARLO M. CRoCE Mitochondria1 Genetics of Paramecium a u r e l i u 4 . H. BEALEA N D A. TAIT Histone Gene Expression: Hybrid Cells and Organisms Establish Complex Controls-PHiLip HOHMANN Gene Expression and Cell Cycle RegulationSTEVEN J . HOCHHAUSER. JANETL. STEIN, A N D GARY S. STEIN The Diptera &s a Model System in Cell and Mokcular Biology-ELENA c . ZEGARELLISCHMIDT A N D REBAGOODMAN Comments on the Use of Laser Doppler Techniques in Cell Electrophoresis: Reply to Pretlow and F'retlow's Review-JOEL H. KAPLAN A N D E. E. UZGIRIS Comments on the Use of Laser Doppler Techniques as Represented by Kaplan and Uzgiris: Reply to Kaplan and UZgiris-THOMAS G . PRETLOW I1 A N D THERESA P. PRETLOW

Expression of Nuclear and Cytoplasmic Factors in Ontogenesis of Amphibian Nucleocytoplasmic Hybrids-CLAuDE-bUIS GALLIEN The Separate Roles of Nucleus and Cytoplasm in the Synthesis of DNA-M. J. ORD Reassembly of Cellular Components for the Study of Aging and Finite Life SpanAUDREYL. MUGGLETON-HARRIS SUBJECT INDEX

Supplement 1 0 Differentiated Cells in Aging Research

Do Diploid Fibroblasts in Culture Age?-

EUGENEBELL, Louis MAREK,STEPHANIE CHARLOTTE MERRILL, DONALD LEVINSTONE, A N D IANYOUNG Urinary Tract Epithelial Cells Cultured from S. FELIX A N D J . W. Human Urine-J. LITTLEFIELD The Role of Terminal Differentiation in the FiINDEX nite Culture Lifetime of the Human Epidermal Keratinocyte-JAMES G . RHEINWALD Long-Tern Lymphoid Cell CUkUreS-GEORGE Supplement 9 Nuclear Transplantation F. SMITH,PARVIN JUSTICE, HENRIFRISCHER, Overview-JAMES F. DANtELLl AND MARIEA. LEE KIN CHU,A N D JAMES KROC Type I1 Alveolar Pneumonocytes in V i m DIBERARDINO WILI.IAM H. J. DOUGLAS, JAMES A. Nucleocytoplasmic Interrelationships in MCATEER, JAMESR. SM~TH, A N D WALTER R. Acerubulario and Some Other Dasycladaceae-HANS-GEORG SCHWEIGER BRAUNSCHWEIGER A N D SIGRIDBERGER Cultured Vascular Endothelial Cells as a Model Compatibility among Cell Components in Large System for the Study of Cellular Free-Living Amebas-KwANG w.JEONAND Senescence-ELLIOT M. LEVINE AND STEPHENM. MUELLER I. JOAN~ R C H Vascular Smooth Muscle Cells for Studies of Nuclear Transplantation Studies in Amoeba Cellular Aging in V i m ; an Examination of proteus-A. L. YUDIN Prologue: Nuclear Transplantation in AmChanges in Structural Cell Lipids-OLGA 0. phibia-THOMAS J. KING BLUMENFELD,ELAINE SCHWARTZ, VERONICA M. HEARN, A N D MARIE J. KRANEPOOL Genetics of Cell Type kterIIIinatiOn-ROBERT Chondrocytes in Aging Research-EDWARD J. BRIGCS MILLERA N D STEFFENG A Y Nuclear and Chromosomal Behavior in AmphiA . DIbian Nuclear Transplants-MARIE Growth and Differentiation of Isolated CalBERARDINO varium Cells in a Serum-Free MediumReprogramming of Transplanted Nuclei in A. PECK JAMESK. BURKSA N D WILLIAM Amphibia-J. B. GURDON,R. A. LASKEY, E. Studies of Aging in Cultured Nervous System AND G. A. PARTINGTON M. DE ROBERTIS, Tissue-DONALD H. SILBERBERG A N D SEUNG The Pluriptential Genome of the Frog Renal U. KIM Tumor Cell as Revealed by Nuclear Aging of Adrenocortical Cells in CultureSHER,

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365

PETERJ. HORNSBY, MICHAEL H. SIMONIAN, Protoplast Fusion and Somatic Hybridiz a t i o n 4 m o SCHIEDERA N D INDRAK. AND GORDON N.GILL Thyroid Cells in Cuhe-FruNcEscO s. VASIL AMBESI-IMPIOMBATO AND HAYDEN G. COON Genetic Modification of Plant Cells Through Uptake of Foreign DNA<. 1. KADOAND A. Permanent Teratocarcinoma-Derived Cell Lines KLEINHOFS Stabilized by Transformation with SV40 and Nitrogen Fixation and Piant Tissue CultureSV4OtsA Mutant V~N~~S-WARREN MALTZMAN, DANIEL1. H. LINZER,FLORKENNETH L. GILESA N D INDRAK. VASIL ENCE BROWN, ANGELIKAK. TERESKY, Preservation of Gemp~esm-LYNDSEY A. WITHERS MAURICEROSENSTRAUS, A N D ARNOLDJ. intraovarian and in Vitro Pollination-M. LEVINE ZENKTELER Nonreplicating Cultures of Frog Gastric Tubular Endosperm Culture-B. M. JOHN, P. S. CelkSaERTRUDE H. BLUMENTHAL AND SRIVASTAVA, AND A. P. RASTE DINKAR K. KASBEKAR The Formation of Secondary Metabolites in SUBJECT INDEX Plant Tissue and Cell Cultures-H. WHM Supplement 11A Perspectives in Plant Cell Embryo Culture-V. RAGHAVAN The Future-GEORG MELCHERS and Tissue Culture SUBJECT INDEX

Cell Proliferation and Growth in Callus Cultures-M. M. YEOMAN AND E. FORCHE Cell Proliferation and Growth in Suspnsion Vofume 72 Cultures-P. J. KING Microtubuie-Membrane Interactions in Cilia and CytodifferentiatiOn-R~ CHARD PHILLIPS Fiagella-WrLLIm L. DENTLER Organogenesis in Vitro: Structural, Physiological, and Biochemical A S ~ ~ ~ ~ S - T R E A. V O RThe Chloroplast Endoplasmic Reticulum: Structure, Function, and Evolutionary SigniTHORPE fiCanCe4ARAH P. GIBBS Chromosomal Variation in Plant Tissues in DNA Repair-A. R.LEHHANN A N D P. KARRAN Culture-M. W. BAYLISS Clonal Propagation-INmA K. VASIL A N D Insulin Binding and Glucose TrmsponANDROGER RUSSELLHILF, LAURIEK. SORGE, VIMLAVASIL 3. GAY Control of Morphogenesis by Inherent and Exogenously Applied Factors in Thin Cell Cell Interactionsand the Control of Development in Myxobacteria PopulatiOnS-DAVID WHITE Layers-K. TRANTHANH VAN Ultraswcture, Chemistry, and Function of the Androgenetic Haploids-INDRA K. VASIL Bacterial Wall-T. J. BEVERIDGE Isolation, Characterization, and Utilization of INDEX Mutant Cell Lines in Higher Plants-PAL MALIGA SUBJECT INDEX

Supplement 11B: Perspectives in Plant Cell and Tissue Culture Isolation and Culture of Protoplasts-INDRA K. VASILA N D VIMLAVASIL

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