Ciliary and Flagellar Membranes Ciliary and Flagellar Metnbranes

Edited by Robert A. Bloodgood University qf Virginia School of Medicine Charlottesville, Virginia

PLENUM PRESS • NEW YORK AND LONDON Library of Congress Cataloging-In-Publication Data

Ciliary and flagellar me~branes I edited by Robert A. Bloodgood. p. cm. Includes bibliographical references. ISBN-13: 978-1-4612-7845-0 e-ISBN-13: 978-1-4613-0515-6 DOI: 10.1007/978-1-4613-0515-6 1. membranes. 2. Cilia and ciliary motion. 3. Flagella (Microbiology) I. Bloodgood, Robert A. [DNLM, 1. Cell Me.brane--physiology. 2. Cell Movement. 3. Cllia• -physiology. 4. Flagella--physiology. 5. Protozoa--physiology. CS 532.5.E7 c5721 CHSO 1.C55 1989 574.87·S4--dc20 DNLM/DLC for Library of Congress 89-23227 CIP

© 1990 Plenum Press, New York A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013 Softcover reprint of the hardcover 1st edition 1990 All rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfIlming, recording, or otherwise, without written permission from the Publisher Contributors

Joseph C. Besharse Department of Anatomy and , Emory University School of Medicine, Atlanta, Georgia 30322; present address: Department of Anat• omy and Cell Biology, The University of Kansas Medical Center, Kansas City, Kansas 66103 Robert A. Bloodgood Department of Anatomy and Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia 22908 Emmanuelle Boisvieux-Ulrich Centre de Biologie Cellulaire C.N.R.S., 94205 Ivry sur Seine Cedex, France G. B. Bouck Department of Biological Sciences, University of lllinois, Chicago, lllinois 60680 Richard A. Cardullo Worcester Foundation for Experimental Biology, Shrews- bury, Massachusetts 01545 Bernadette Chailley Centre de Biologie Cellulaire C.N.R.S., 94205 Ivry sur Seine Cedex, France William L. Dentler Department of Physiology and Cell Biology, University of Kansas, Lawrence, Kansas 66045 Cynthia J. Horst Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322 Edna S. Kaneshiro Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221-0006 F. M. Klis Department of Molecular Cell Biology, University of Amsterdam, 1098 SM Amsterdam, The Netherlands P. J. Levasseur Department of Biological Sciences, University of lllinois, Chicago, lllinois 60680 A. Musgrave Department of Molecular Cell Biology, University of Amsterdam, 1098 SM Amsterdam, The Netherlands Robin R. Preston Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706

v Preface

While there have been many recent books on the cell surface and a few on the topic of cilia and flagella, this is the fIrst volume that attempts to bring together the available informa• tion on ciliary and flagellar membranes. This reflects a slow awakening by cell biologists and other scientists to the signifIcance of ciliary and flagellar surfaces. When Michael Sleigh edited an excellent book entitled Cilia and Flagella in 1974, not one of the sixteen chapters was devoted to ciliary or flagellar surfaces. When W. B. Amos and J. G. Duckett edited the very fIne 25th Symposium of the Society for Experimental Biology on Prokaryotic and Eukaryotic Flagella in 1982, only two of the twenty chapters on eu• karyotic cilia and flagella were devoted to ciliary and flagellar surfaces. Only in 1989 has the timing become right to produce a volume entirely devoted to the nonaxonemal struc• tures and functions of eukaryotic cilia and flagella. The fIfteen chapters in this volume cover a wide spectrum of (from protozoa and algae to birds and mammals) and an equally wide spectrum of topics (from sexual interactions in the algae to the binding of pathogens in the lung). It is hoped that almost any biological or medical scientist will fInd at least a few chapters of interest and I will bet that few who peruse this book will fail to discover at least a couple of roles for ciliary and flagellar surfaces that they had never imagined. For those readers who may be relatively new to the subject, Dr. George Witman has prepared an introductory chapter that introduces the reader to the general features of cilia and flagella and in particular to those aspects of their structure and function that influence or are influenced by the phenomena occurring at the ciliary or flagellar surface. Eukaryotic cilia and flagella are essentially specialized cytoplasmic domains contain• ing a highly structured incompletely bounded by a specialized domain of the plasma membrane. For that reason, they are excellent systems in which to study mem• brane-cytoskeletal interactions and indeed many of the functions of ciliary and flagellar surfaces will only be understood as deriving from a functional interaction of the mem• brane with the underlying cytoskeleton. Eukaryotic cilia and flagella also constitute ideal systems for studying a defIned plasma membrane domain because the act of deciliation or deflagellation immediately isolates a specialized domain of the plasma membrane; there is little contamination with other membrane material because these lack any internal membranes. While cilia and flagella have been recognized as motile organelles from the time of their discovery by Leeuwenhoek in approximately 1676, it is only recently that biologists have come to appreciate that cilia and flagella perform many other roles aside from the movement of cells through a liquid medium or the movement of fluid (or mucus) across a ciliated epithelium. Many of these additional functions derive from properties of ciliary

vii viii Preface and flagellar surfaces, here defined as the ciliary or flagellar membrane along with all of its associated appendages (coats, scales, mastigonemes). In fact, the first paper that dealt with the surface of eukaryotic cilia or flagella was probably that published in 1889 by Loeffler; he included a clear photograph of the mastigonemes (or Flimmergeissel) on the flagellar surface of a chrysophycean algal cell. The first person to seriously study the flagellar surface using the electron microscope was probably Manton (see Manton, 1952, for a review). One of the biggest technical advances in the study of cilia and flagella came from the ability to isolate cilia and flagella, extract them with nonionic detergents, and obtain axonemes that could be reactivated to produce patterns of bending motility strikingly similar to those seen in vivo (Gibbons, 1981). Ironically, at the same time that this opened up much detailed study of the motile functions of ciliary and flagellar axonemes, it drew attention away from the study of ciliary and flagellar surfaces. In fact, many scientists studying cilia and flagella routinely discard all of the membrane and soluble components of these organelles down the laboratory sink as so much unnecessary junk! Although the naked axoneme can exhibit motile behavior, the normal regulation of the motile activity of cilia and flagella is dependent upon the ciliary or flagellar mem• brane. An elegant combination of genetic, biochemical, and electrophysiological ap• proaches using the ciliate protozoan Paramecium has demonstrated how the behavior of the ciliary axoneme (and the entire ) is controlled by a complicated array of ion channels and pumps within the ciliary membrane (see chapter by Preston and Saimi in this volume). In fact, the general field of sensory transduction and membrane signaling is one of the more exciting ways in which ciliary and flagellar membranes are being utilized to study basic biological problems. From the avoidance response in Paramecium to the initial sexual contact in Chlamydomonas gametes to the very human appreciation of an expensive perfume, signal transduction mechanisms in cilia and flagella are at work. While the chapters in this book range widely in a phylogenetic and functional sense, there is no claim of completeness. For lack of space, a few interesting functions of ciliary and flagellar membranes have not been reviewed in this volume. For example, the ciliary membranes of olfactory receptor cells are responsible for binding the odorant molecules (Snyder et al.• 1989); this binding initiates a transmembrane signaling process involving G activation of a membrane-associated adenylate cyclase, an increase inintracili• ary cyclic nucleotide level, and a direct cyclic nucleotide gating of ciliary membrane sodium channels (Nakamura and Gold, 1987; Lancet, 1988). The adhesion of certain free• living marine invertebrates is known to occur by means of specializations of the ciliary surface (Tyler, 1973). A third topic not represented in the present volume is a considera• tion of the evolution of ciliary and flagellar surface specializations, a topic that has been studied by Bardele (1981, 1983). Despite these few omissions, the vast majority of research related to ciliary and flagellar membranes is reviewed within the present volume. My sincere thanks go to all of the chapter authors; their excellent and timely contributions have made this unique volume possible. Special thanks go to Mary Phillips Born and Susan Woolford at Plenum Publish• ing for their efforts on behalf of this volume.

Robert A. Bloodgood Charlottesville. Virginia Preface ix

References

Bardele, C. F., 1981, Functional and phylogenetic aspects of the ciliary membrane: A comparative freeze• fracture study, BioSystems 14:403-421. Bardele, C. F., 1983, Comparative freeze-fracture study of the ciliary membrane of protists and invertebrates in relation to phylogeny, J. Submicrosc. Cytol. 15:263-267. Gibbons, I. R., 1981, Cilia and flagella of eukaryotes, J. Cell Bioi. 91:107s-124s. Lancet, D., 1988, Molecular components of olfactory reception and transduction, in: Molecular Neurobiology of the Olfactory System (F. L. Margolis and T. V. Getchell, eds.), Plenum Press, New York, pp. 25-50. Loeffler, F., 1889, Eine neue Methode zum Farben der Mikroorganismen im besonderen ihre Wimperhaare und Geisseln, Zentralbl. Bakteriol. 6:209-224. Manton, I., 1952, The [me structure of plant cilia, Symp. Soc. Exp. Bioi. 6:306-319. Nakamura, T., and Gold, G. H., 1987, A cyclic-nucleotide-gated conductance in olfactory receptor cilia, Nature 325:442-444. Snyder, S. H., Sklar, P. B., Hwang, P. M., and Pevsner, J., 1989, Molecular mechanisms of olfaction, Trends Neurosci. 12:35-38. Tyler, S., 1973, An adhesive function for modified cilia in an interstitial turbellarian, Acta Zool. 54:139-151. Contents

1. Introduction to Cilia and Flagella George B. Witman 1. Introduction...... 1 2. Occurrence and Function ...... 2 3. Structure ...... 4 3.1. The Axoneme ...... 4 3.2. The Transition Zone...... 6 3.3. The and Associated Structures ...... 9 3.4. Accessory Structures of the Flagellar Shaft ...... 11 4. Production of Movement ...... 11 4.1. The Sliding Model ...... 11 4.2. Active Sliding Is Produced by Arms Acting on Adjacent Outer Doublet ...... 12 4.3. The Mechanism of Force Generation ...... 12 4.4. Internal Resistances Convert Sliding into Bending ...... 13 4.5. Coordination of Interdoublet Sliding...... 13 4.6. Central Pair Rotation ...... 15 5. Regulation of Movement ...... 16 5.1. Initiation of Motility ...... 16 5.2. Behavioral Responses ...... 17 5.3. Hyperactivation of ...... 19 6. Sensory Reception ...... 20 7. Origin ...... 22 References ...... 22

2. Linkages between Microtubules and Membranes in Cilia and Flagella William L. Dentler 1. Introduction...... 31 2. Linkage of Basal Bodies and Transition Regions to the Membrane ...... 31 2.1. Structural Studies ...... 31 2.2. Functions of the Bridges ...... 34 3. Microtubule Capping Structures Attach the Ends of Microtubules to the Membrane ...... 36 3.1. Capping Structures and Motility ...... 41 3.2. Capping Structures and Microtubule Assembly...... 43 3.3. Caps Are Bound to Assembling and Disassembling Microtubules ...... 44 3.4. Can Capping Structures Regulate Tubulin Addition to Microtubules? ... 45

xi xii Contents

3.5. Are Microtubule Caps Found in the ? ...... 46 4. Bridges Linking the Sides of Outer Doublet Microtubules to the Membrane .. 46 4.1. Bridges Linking Microtubules to Extraciliary Structures ...... 50 4.2. Sites of Attachment of the Bridges...... 51 4.3. Attachment to the Doublet Microtubules ...... 51 4.4. Sites of Membrane Attachment ...... 52 4.5. Nonciliary Microtubule-Membrane Bridges ...... 54 4.6. Functions of the Bridges ...... 54 5. Summary...... 57 References ...... 58

3. Euglena gracilis: A Model for Flagellar Surface Assembly, with Reference to Other Cells That Bear Flagellar Mastigonemes and Scales C. B. Bouck, T. K. Rosiere, and P. J. Levasseur 1. Introduction...... 65 2. The Relationship of Euglenoids to Other Organisms ...... 66 3. Flagellar Anatomy ...... 67 3.1. Mastigonemes and the Flagellar Sheath ...... 67 3.2. The Paraxial Rod ...... 71 3.3. The Flagellar Membrane ...... 72 4. Assembly of the Flagellar Surface ...... 78 4.1. Origin of Flagellar Scales, Mastigonemes, and Membranes ...... 79 4.2. Composition of Scales and Mastigonemes ...... 80 4.3. Release of Mastigonemes and Flagellar Scales at the Cell Surface ..... 81 4.4. Organizing the Flagellar Surface...... 82 5. The Control of Flagellar Surface Assembly in Euglena ...... 83 6. Summary and Prospectus...... 86 References ...... 87

4. Gliding Motility and Flagellar Glycoprotein Dynamics in Chlamydomonas Robert A. Bloodgood 1. Introduction...... 91 2. Gliding Motility ...... 93 2.1. Gliding Motility-An Overview ...... ~ ...... 93 2.2. Gliding Motility in Chlamydomonas...... 94 3. Polystyrene Microsphere Movements ...... 99 4. Flagellar Surface Motility ...... 101 5. Mating-Associated Dynamic Flagellar Surface Events ...... 101 6. Characterization of the Major Flagellar Glycoproteins in C. reinhardtii ..... 105 7. Dynamics of Flagellar Membrane Glycoproteins ...... 109 8. Flagellar Signaling in Chlamydomonas ...... 111

8.1. Signaling Related to Gametic Interactions ...... o •••••• 112 8.2. Signaling Related to Glycoprotein Redistribution in Vegetative Flagella. 113 Contents xiii

9. Use of Carbohydrate Probes in Conjunction with FACS to Isolate Mutant Cell Lines with Carbohydrate Defects ...... 115 10. Use of the L-23 Mutant Cell Line to Demonstrate That Flagellar Membrane Glycoprotein Movements Are Essential for Gliding Motility ...... 117 11. Mechanisms and Motors ...... 118 11.1. Candidates for the Motor Responsible for Flagellar Membrane Protein Redistribution and Gliding Motility ...... 118 11.2. A Proposed Mechanism for Gliding Motility ...... 120 12. Conclusions...... 122 References ...... 123

5. The Role of Flagella in the Sexual Reproduction of Chlamydomonas Gametes H. van den Ende, A. Musgrave, and F. M. Klis 1. Introduction ...... 129 2. The Agglutination Process ...... 130 3. The Agglutinins ...... 131 4. Mode of Action of the Agglutinins ...... 138 5. Longitudinal Redistribution of Agglutinins ...... 138 6. The Signaling Action of Sexual Agglutination ...... 140 7. Modulation of Sexual Agglutinability ...... 141 8. Conclusions...... 143 References ...... 143

6. The Role of Ciliary Surfaces in Mating in Paramecium Tsuyoshi Watanabe 1. Introduction...... 149 2. The Events Occurring during Conjugation in Paramecium ...... 149 3. Role of Ciliary Surfaces in the Mating Reaction ...... 150 3.1. Mating Substances in the Ciliary Membrane ...... 150 3.2. Localization of Mating-Reactive Cilia ...... 153 3.3. Attempts to Isolate Pure Mating-Reactive Cilia ...... 153 3.4. Nature of the Ciliary Interactions ...... 154 3.5. Hydrophobic Interactions between Cilia and Polystyrene Surfaces ...... 155 4. Regulation of the Expression of Mating Reactivity ...... 156 4.1. Genetic Control of the Mating Type Specificity ...... 156 4.2. Temporal Differentiation of Mating-Reactive Cilia ...... 158 5. Results of Ciliary Interactions ...... 160 5.1. Decrease in Swimming Velocity ...... 160 5.2. Early Micronuclear Migration ...... 161 5.3. Local Degeneration of Cilia and Pair Formation ...... 161 6. Biochemical and Morphological Approaches to Characterizing the Mating Substances ...... 164 7. General Discussion and Conclusions ...... 166 References ...... 167 xiv Contents

7. Calcium Ions and the Regulation of Motility in Paramecium Robin R. Preston and Yoshiro Saimi 1. Introduction...... 173 2. Voltage-Dependent Calcium Channels ...... 175 2.1. Voltage-Clamp Analysis of Calcium Currents ...... 175 2.2. Calcium Channel Mutants ...... 177 2.3. Location of Calcium Channels ...... 178 2.4. Calcium Channel Activity in Isolated Ciliary Membranes ...... 180 2.5. Curing of pawns and CNRs ...... 181 2.6. Characterization of Ciliary Membrane ...... 182 3. Enzymatic Activity Assqciated with the Ciliary Membrane ...... 184 3.1. Calmodulin and Calmodulin-Binding Proteins ...... 186 3.2. Ca-ATPases ...... 186 3.3. Adenylate Cyclase ...... 187 3.4. Guanylate Cyclase ...... 187 3.5. Protein Kinases ...... 188 4. Regulation of Cell Motility: Calcium Sensitivity ...... 189 4.1. Ciliary Responses to Hyperpolarization ...... 190 4.2. Ciliary Responses to Depolarization ...... 191 5. Perspectives...... 192 References ...... 194

8. Structure, Turnover, and Assembly of Ciliary Membranes in Tetrahymena . Norman E. Williams 1. Introduction...... 201 2. Lipid Composition ...... 201 3. Protein Components ...... 204 4. Ultrastructure ...... 205 5. Turnover ...... 208 6. Modulation ...... 209 7. Assembly ...... 211 8. Concluding Remarks ...... 213 References ...... 214

9. Ciliary Membrane Tubulin R. E. Stephens 1. Introduction ...... 217 2. Ciliary versus Flagellar Membranes ...... 217 3. Protozoan Cilia and Flagella ...... 221 4. Definitions and Origin ...... 221 5. Metabolic Relationship between Membrane and Axonemal Tubulin ...... 223 Contents xv

6. Reconstitution of Ciliary Membranes ...... 226 7. Micellarization with Detergents and Interaction with Lipids ...... 229 8. Tubulin as an Integral Membrane Structural Element ...... 231 9. Summary and Discussion ...... 233 References ...... 238

10. Lipids of Ciliary and Flagellar Membranes Edna S. Kaneshiro 1. Preparations of Cilia and Flagella and Their Membranes ...... 241 2. Lipid Composition ...... 243 2.1. Sterols...... 243 2.2. Fatty Acid Composition ...... 245 2.3. Glycerolipids ...... 247 2.4. Sphingophospholipids and Sphingophosphonolipids ...... 249 2.5. Chlorosulfolipids ...... 250 2.6. Lipoconjugates ...... 250 3. Enzymes and Lipid Metabolism ...... 251 4. Alterations in Lipid Composition ...... 253 4.1. Culture Age ...... 253 4.2. Dietary Supplementation ...... 254 4.3. Drugs and Inhibitor Compounds ...... 256 4.4. Temperature Shifts ...... 256 4.5. Mutations ...... 260 5. Conclusions...... 260 References ...... 261

11. Flagellar Surfaces of Parasitic Protozoa and Their Role in Attachment Keith Vickerman and Laurence Tetley 1. Developmental Cycles of Kinetoplastid Protozoa ...... 267 2. Flagellar Surfaces and Their Relation to Other Surface Domains ...... 271 2.1. Flagellar Structure and Function in Kinetoplastids ...... 271 2.2. Functional Aspects of the Parasite Surface: Similarity of Flagellar and Body Membranes ...... 273 2.3. Compositional Differences between Flagellar and Body Membranes .... 276 2.4. Flagellar Fractions and -Specific Antigens ...... 279 3. Flagellar Attachment to the Body or to Other Flagellates ...... 281 3.1. Attachment to the Body ...... 281 3.2. Attachment to Other Flagella ...... 283 3.3. Attachment to Cysts ...... 284 4. Flagellar Attachment to Host Surfaces ...... 284 4.1. Attachment to Chitin and Other Nonliving Surfaces ...... 284 4.2. Attachment to Living Host Cells ...... 291 xvi Contents

5. Role of Host Attachment in Parasite Development and Transmission ...... 295 5.1. Specificity of Attachment ...... 295 5.2. Parasite Reproduction and Differentiation ...... 296 6. Summary...... 297 References ...... 297

12. The Sperm Plasma Membrane: A Little More Than Mosaic, a Little Less Than Fluid Richard A. Cardullo and David E . Wolf 1. Introduction...... 305 1.1. Why Should the Mammalian Sperm Plasma Membrane Be Given Special Consideration? ...... 305 1.2. Regionalization of the Sperm Plasma Membrane ...... 306 1.3. Membrane Modification in the Absence of Macromolecular Biosynthesis ...... 307 2. Evolving a Model for Membrane Organization and Dynamics ...... 308 2.1. Lipid Diffusion in Biological Membranes ...... 308 2.2. Protein Diffusion in Biological Membranes ...... 311 3. Diffusion on Mammalian Spermatozoa ...... 312 3.1. Constraints on Sperm Geometry ...... 312 3.2. Lipid Diffusion on Mammalian Spermatozoa ...... 316 3.3. Changes in Sperm Plasma Membrane Lipid Diffusibility during , Maturation, and Capacitation ...... 318 3.4. Causes of Nondiffusing Lipid and the Question of Lipid Domains ..... 322 3.5. Protein Diffusion on Mammalian Sperm ...... 323 4. Mechanisms of Membrane Regionalization ...... 327 4.1. Regionalization by Immobilization ...... 327 4.2. Regionalization by Diffusional Barriers ...... 327 4.3. Regionalization Due to Selective Solubility ...... 329 5. How Does the Sperm Become Regionalized? ...... 329 6. Mechanisms of Redistribution ...... 329 7. Summary...... 330 References...... 331

13. Structure and Assembly of the Oviduct Ciliary Membrane Bernadette Chailley, Emmanuelle Boisvieux-Ulrich, and Daniel Sandoz 1. Introduction...... 337 2. Organization of the Ciliary Membrane ...... 338 2.1. Ultrastructural Data ...... 338 2.2. Cytochemical Data ...... 343 2.3. Ciliary Membrane-Cytoskeleton Relationships ...... 346 3. Assembly of the Ciliary Membrane ...... 347 3.1. Cytoplasmic Events ...... 347 3.2. Plasma Membrane Events ...... 349 Contents xvii

4. Deciliation...... 352 5. Functions of Ciliary Membrane in Oviduct ...... 353 References ...... ' ...... 357

14. The Surface of Mammalian Respiratory Cilia: Interactions between Cilia and Respiratory Pathogens Elaine Tuomanen 1. Introduction...... 363 2. The Structure and Cytochemistry of the Surface of Respiratory Cilia ...... 364 2.1. Structure ...... 364 2.2. Chemistry ...... 368 3. Interactions between Pathogens and Respiratory Cilia ...... 371 3.1. General Mechanisms of Adherence ...... 371 3.2. Adherence of Specific Pathogens to Respiratory Cilia ...... 372 4. Toxicity of Microbial Products for Cilia ...... 383 5. Summary: Implications of Ciliary Surface Composition to the Therapy of Infections of Cilia ...... 383 References...... 384

15. The Photoreceptor Connecting : A Model for the Transition Zone Joseph C. Besharse and Cynthia]. Horst 1. Introduction...... 389 2. Structure of Photoreceptor Cilia ...... 389 3. Microtubule-Membrane Cross-Linkers of the Connecting Cilium ...... 395 3.1. The Ciliary Surface and Its Transmembrane Assemblage ...... 395 3.2. Identification of Surface Components of the Assemblage ...... 398 3.3. The Periciliary Ridge Complex ...... 400 4. Functions of the Photoreceptor Connecting Cilium ...... 400 4.1. Delivery of Membrane Components ...... 400 4.2. The Connecting Cilium in Disk Morphogenesis ...... 404 4.3. The Connecting Cilium as a Barrier between Membrane Domains ..... 407 4.4. Delivery of Cytosolic Components to the Outer Segment ...... 409 5. Summary ...... 411 References ...... 411

Index ...... 419