No title

Current Topics in Membranes and Transport VOLUME 32

Membrane Fusion in Fertilization, Cellular Transport, and Viral Infection

Advisory Board

G . Blobel E. Carafoli 3. S. Cook D.Louvard

Cur rent Top ics in Membranes and Transport Edited by Felix Bronner D i ~ p i r r t t n ~ wot/ BioStrrrctrrrc trnd Firnc.tiori The University of Connecticut Health C r n t e r School of Dental Medicine Farrnington, Connecticut

VOLUME 32

Membrane Fusion in Fertilization, Cellular Transport, and Viral Infection

Guest Editors Nejat Duzguneg

Felix Bronner

Department qf Pharmaceutical Chemistty Cuncer Reseurch Institute and Schools of Medicine and Phnrniacy Uniuersity of Californiu San Francisco. California

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0 1 BioStrrrc.trrri, rrncl Frorc~ti~~ri The University of Connecticut Health Center School of' Dental Medicine Farrnington, Connecticirt

ACADEMIC PRESS, INC Harcourt Brace Jovanovich, Publishers

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NUMBER: 70-117091

Contents

Contributors, ix Preface, xi Peter Baker, xv Yale Membrane Transport Processes Volumes, xvii

PART I.

MEMBRANE FUSION IN FERTILIZATION AND DEVELOPMENT

Chapter 1. Sperm-Egg Fusion RYUZO YANAGIMACHI

I . Introduction. 4 11. Morphology of Sperm-Egg Fusion, 4 Ill. Specific Sites for Sperm-Egg Fusion, 13

IV. V. VI. VII.

Sperm Movement and Sperm-Egg Fusion, 18 Intermingling of Sperm and Egg Plasma Membrane, 19 Species Specificity of Sperm-Egg Fusion, 20 How Long Does the Egg Plasma Membrane Remain Capable of Fusing with Spermatozoa after the Entry of the First Spermatozoon'?, 24 VI11. Conditions Controlling Sperm-Egg Fusion. 26 IX. Effects of Miscellaneous Substances on Sperm-Egg Fusion, 31 X . Mechanism of Sperm-Egg Fusion. 34 References, 35

Chapter 2. Cortical Exocytosis in the Sea Urchin Egg ROBERT C . JACKSON AND JOSEPH H. CRABB 1. Introduction, 45 11. The Calcium Signal, 51 111. In Virro Models of Exocytosis, 56

IV. Conclusions, 73 V. Addendum. 74 References, 76 V

vi

CONTENTS

Chapter 3. Myoblast Fusion-A

Mechanistic Analysis

MICHAEL J. 0. WAKELAM 1. Introduction, 88 11. Morphological Aspects of Myoblast Fusion, 88

Ill. IV. V. VI. VII. VIII.

Kinetics of Myoblast Fusion, 92 Structure-Function Relationships in Myoblast Plasma Membranes, 94 Fusion-Induced Changes in Membrane Organization, 99 Proposed Mechanisms of Myoblast Fusion, I01 Stimulation of Myoblast Fusion, 106 Conclusions, 107 References, 107

PART 11. CELLULAR TRANSPORT-EXOCYTOSIS

AND ENDOCYTOSIS

Chapter 4. Exocytosis in Electropermeabilired Cells: Clues to Mechanism and Physiological Control PETER F. BAKER

I. Introduction, I I5 11. Morphology of Exocytosis, 117

Ill. Clues to Mechanism, 118 IV. Control of Exocytosis, 120 V. Responding to the Primary Message, 126 V1. Other Control Factors in Exocytosis, 132 References. 134

Chapter 5. Exocytosis and Membrane Recycling JACOPO MELDOLESI AND BRUNO CECCARELLI

I. Introduction, 139 11. Exocytosis, 142 111. Endocytosis, 156 1V. Conclusion, 160 References, 161

Chapter 6. Exocytosis and Endocytosis: Membrane Fusion Events Captured in Rapidly Frozen Cells DOUGLAS E. CHANDLER

I. Exocytosis, 169 11. Endocytosis, 189 111. Concluding Remarks, 194 References, 197

CONTENTS

vii

Chapter 7. Osmotic Effects In Membrane Fusion during Exocytosis KEITH W . BROCKLEHURST AND HARVEY B. POLLARD 1. Introduction. 203 11. Osmotic Properties of Isolated Secretory Granules. 204 111. Osmotic Effects in Secretion from Intact Cells. 211

I V . Osmotic Effects in Secretion from Permeabilized Chrornaffin Cells, 216 V. Conclusions, 219 References. 220

Chapter 8. Polyanionic Agents and Inhibition of PhagosomeLysosome Fusion: Paradox Lost MAYER B. GOREN I . Introduction. 228 I t . The Fluorescent Lysosomal Probes, 231 111. Nonionic Hydrocolloids in Lysosornes: Fusion Inhibitors or Gelatinous Traps?, 238 IV. Fusion Inhibition Is Incompatible with the Cells' Functional Status. 242 V. Some Residual Bodies. 246 VL. Recapitulation and Conclusions, 249 References, 250

PART 111. VIRUS-CELL FUSION Chapter 9. Fusion of Viral Envelopes with Cellular Membranes SHUN-ICHI OHNISHI I . Introduction, 257 11. Membrane Fusion Activity of Enveloped Viruses. 263 111. Mechanism of Fusion, 278

1V. Infectious Cell Entry Mechanisms. 286 References. 288

Chapter 10. Sendai Virus-Mediated Cell Fusion YOSHIO OKADA

I. 11. 111. IV.

Introduction, 297 Critical Problems in Cell-to-Cell Fusion, 298 Structure and Biological Activities of HVJ, 299 Cell-to-Cell Fusion by HVJ. 306 References, 331

viii

CONTENTS

Chapter 11. Fusion Activity of the Hemagglutinin of Influenza Virus MARY-JANE GETHING, JEAN HENNEBERRY, AND JOE SAMBROOK 1. Introduction, 337 11. Influenza Virus-Mediated Fusion: Role of the Hernagglutinin, 339

Ill. Assays for the Fusion Activity of HA, 340 IV. Expression of HA in Cultured Cells from Cloned HA cDNAs, 341 V. Genetic Approaches to Studies of HA-Mediated Membrane Fusion, 34.5 V1. Characterization of the Low pH-Induced Conformational Change in HA. 3.52 VII. Studies o n the Clcavagc Activation of HA. 3 5 3 VIII. Conclusion, 360 References. 360

Index, 365

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin

Peter F. Baker,’ MRC Secretory Mechanisms Group, Department of Physiology, King’s College London, London WC2R 2LS. England ( 1 15) Keith W. Brocklehurst, Laboratory of Cell Biology and Genetics, National Institute of Diabetes. Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 (203) Bruno Ceccarelli, Center for the Study of Peripheral Neuropathies and Neuromuscular Diseases, University of Milan, 20129 Milan, Italy (139) Douglas E. Chandler, Department of Zoology, Arizona State University, Tempe, Arizona 85287 ( 169) Joseph H. Crabb, Channing Laboratory, Harvard Medical School, Boston, Massachusetts 021 15 (45) Mary-Jane Gething, Department of Biochemistry and Howard Hughes Medical Institute, University of Texas Health Science Center, Dallas, Texas 75235 (337) Mayer B. Goren, Department of Molecular and Cellular Biology, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 (227) Jean Henneberry, Department of Biochemistry, University of Texas Health Science Center, Dallas, Texas 75235 (337) Robert C. Jackson, Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756 (45) I

Deceased. ix

X

CONTRIBUTORS

Jacopo Meldolesi, Department of Pharmacology, CNR Center of Cytopharmacology, Scientific Institute Saint Raffaele, Milan, Italy (139) Shun-ichi Ohnishi, Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606, Japan (257) Yoshio Okada, Institute for Molecular and Cellular Biology. Osaka University, Suita, Osaka 565, Japan (297) Harvey B. Pollard, Laboratory of Cell Biology and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 (203) Joe Sambrook, Department of Biochemistry, University of Texas Health Science Center, Dallas, Texas 75235 (337) Michael J. 0. Wakelam, Molecular Pharmacology Group, Department of Biochemistry, University of Glasgow, Glasgow, G12 8QQ Scotland (87)

Ryuzo Yanagimachi, Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 96822 (3)

Preface

Membrane fusion is the first step in fertilization and thus constitutes the initial event in the life of higher organisms. By mediating the cortical reaction that follows after the sperm penetrates the egg, membrane fusion prevents polyspermy and assures orderly development. It also plays a specific role in development, as when myoblasts fuse to form myotubes. As cells mature and differentiate, export of their products requires a well-defined sequence of synthesis. packaging, and routing within and, ultimately, to the outside of the cell. Membrane fusion, between organelles and between organelles and the plasma membrane, plays a prominent role in this process as well. Finally, cellular infection and disease, as brought about by lipid-enveloped viruses, involve fusion of the viral membrane with the plasma membrane or, following endocytosis in coated pits, fusion with the endosome membrane. The development in recent years of the powerful tools of cellular and molecular biology has enabled researchers to observe membrane fusion in detail and to begin studying the underlying steps of this process. Much information has accumulated on this topic, though a definitive understanding still eludes us. The search for this understanding is predicated on knowing what has been done till now, which hypotheses have proved fruitful, and which need to be discarded. The realization that there exist as yei few places where membrane fusion in biology has been described and analyzed systematically prompted us to attempt such a survey. This book is the outcome of that effort. As the title indicates, the book examines three major areas: fertilization and development, cellular transport as exemplified by endocytosis and exocytosis, and the mechanisms by which viruses penetrate cells and cause cell-cell fusion. The first chapter, by Yanagimachi, is a comprehensive survey of sperm-egg fusion in invertebrates, lower vertebrates, and mammals. It describes and analyzes sites for sperm-egg fusion, sperm movement, the intermingling of sperm and egg plasma membranes, the time the egg remains receptive to sperm fusion, and the conditions and mechanism of that event. xi

xii

PREFACE

The second chapter, by Jackson and Crabb, analyzes cortical granule exocytosis. There is now ample evidence that an increase in cytosolic free calcium precedes and is the signal for exocytosis. The calcium signal is therefore described in detail, including the regulatory role of the guanyl nucleotide binding proteins. Finally, the chapter analyzes the use of subcellular fractions of sea urchin eggs with their retained exocytotic apparatus for in uitro studies of the calcium trigger. During development mononucleate myoblasts align and fuse to form the precursors of skeletal muscle fibers. This event is dealt with in the third chapter. Wakelam recognizes that not enough is known to provide a detailed mechanistic description, but his assembly of the relevant findings should permit the reader to form a view of what approaches have or have not been useful. The second section, on the role of membrane fusion in intracellular transport and cellular export, begins with a survey of the nature of exocytosis written by the late Peter Baker. Professor Baker was among the first to have become fascinated by the role played by intracellular calcium in exocytosis and published extensively on this topic. His sudden death at a young age, shortly after he completed Chapter 4, is all the more tragic because it has deprived the scientific world of a perceptive and outstanding scholar. We therefore have asked his long-time colleague, Derek Knight, to help us commemorate him. Meldolesi and Ceccarelli, in Chapter 5, deal with the interesting problem of how membranes of intracellular vesicles that fuse with the plasma membrane are retrieved, rather than lost to the economy of the cell. They draw examples from nerve cells with which they worked extensively, but their overall survey is broad and constitutes a biologically oriented companion to the chapter by Baker. Chapter 6 by Chandler is a detailed structural analysis of the events in endocytosis and exocytosis. Chandler’s contribution is all the more unique because he has accompanied his text by revealing illustrations, some by now classics. Some years ago Pollard and colleagues, basing themselves on the ingenious chemiosmotic mechanism that Peter Mitchell had proposed for the coupling of phosphorylation to electron and hydrogen transfer, proposed an analogous mechanism to explain exocytotic membrane fusion. This hypothesis was fruitful in that it stimulated much careful, experimental work that has greatly broadened our understanding of the fusion process. Brocklehurst and Pollard, in Chapter 7, examine the evidence and conclude that the hypothesis is no longer tenable, at least in its totality. A somewhat similar situation is described in Chapter 8 by Goren. Some ten years ago Goren published findings that indicated that polyanionic

PREFACE

xiii

substances, such as dextran sulfate, when phagocytosed, inhibited the fusion of phagosomes with lysosomes. As it turned out, substances such as acridine orange that had been used as fusion markers had become immobilized, giving the appearance of no fusion, whereas the two vesicles had in fact become fused. Goren reviews the evidence in detail, and thus provides a resolution of what might otherwise have been a scientific cul-de-sac. Ohnishi in Chapter 9 introduces the third section of the book by discussing the mechanisms by which viruses bind and ultimately penetrate target cells. Even when the virus binds to its target cell membrane through interaction of the viral glycoprotein with the cell surface receptor, the two membranes are still far apart. Ohnishi describes how conformational changes in the viral glycoprotein are achieved and how these changes cause the hydrophobic segment of the glycoprotein to approach and interact with target membranes. He also describes how enveloped viruses gain entry into the cell by utilizing the machinery that cells have developed for the uptake and processing of materials useful to them. Okada, one of the first to use the Sendai virus to fuse mammalian cell membranes, reviews this field in detail in Chapter 10. His richly illustrated chapter analyzes the molecular and structural basis of this now widely utilized process. I n the final chapter, Gething, Henneberry, and Sambrook describe cell fusion mediated by influenza virus and describe genetic approaches to this process. The hemagglutinin glycoprotein is the major antigen of the influenza virus. It is the ability of these viruses to synthesize antigenically novel hemagglutinins which explains the difficulty in controlling the disease. At the same time, the advent of recombinant DNA technology, knowledge of the gene encoding the hemagglutinin, and X-ray analysis of the structure of the ectodomain of the hemagglutinin molecule have provided substantial information on its physical domains, location of antigenic and glycosylation sites, its trimeric structure, and orientation with respect to the plasma membrane. The hemagglutinin molecule, therefore, constitutes an excellent model system for the study of membrane fusion. We hope the material contained in this book will lead investigators not only to important practical solutions, such as the design of agents that will prevent entry of virions, but a step closer to understanding a fundamental biological process like fertilization. NEJATDUZGUNES FELIXB R O N N E R

PETERBAKER

Peter Baker (1939-1987)

Professor Peter Baker’s sudden death in March 1987, at the early age of 48, was a shock to those who knew him and to those in the scientific

community who knew and respected his work. Although his career was cruelly curtailed. his life was filled with challenges and was one of purpose. His achievements are now part of the foundation on which others can build. To his colleagues he was a man who succeeded in combining his exceptional scientific gifts with a practical administrative sense to create an environment in which research and achievement were initiated and nourished. To his friends in the scientific community, both at home and abroad, he was a man who carried with him a rare enthusiasm for all that he did, which he conveyed in a way that never failed to stimulate or enlighten those who came in contact with him. His resilience and strength, backed by his unwavering optimism and confidence in the unlimited scope of scientific advancements, served him in his own research and in the wider international field of life sciences. Peter Baker was born in Lincoln, England, on March 1 1 , 1939. He attended Lincoln School and from there won an Open Scholarship to Emmanuel College, Cambridge, where he read Natural Sciences. His research talents were immediately apparent and flourished in his subsequent work with Trevor Shaw and Sir Alan Hodgkin in the Department of Physiology. The most widely recognized aspect of this work was to refine the ionic theory of the nervous impulse by means of performing experiments with squid axon, the protoplasm of which had been replaced by media of accurately defined chemical composition. These studies during the 1960s led to the publication of several significant papers. While at Cambridge, he began the study of the control and maintenance of the ionic components of the intracellular environment, most notably the role of calcium. He characterized the two voltage-sensitive routes of calcium entry into the cell and then proceeded to characterize the component of calcium movement across the plasma membrane associated with the movement of sodium (sodium-calcium exchange). His involvement in xv

xvi

PETER BAKER

this area of research continued to the time of his death, when he was in the midst of organizing the first International Symposium on SodiumCalcium Exchange. His interest in exocytosis also developed at Cambridge where, with Tim Rink, he extended the link between calcium entry into cells and their secretory response. Drs. Baker and Rink provided evidence that strongly suggested that the transient nature of that response resulted from a transient calcium influx into the cell. Thus, they were able to infer that the calcium channels which gave rise to the transient calcium influx had properties similar to the ‘‘late’’ voltage-sensitive calcium channels Dr. Baker had characterized in his earlier studies with the squid axon. Peter Baker accepted the Halliburton Chair at King’s College London in 1975 and was elected a Fellow of The Royal Society a year later. At King’s College he extended his own research into the area of exocytosis, and it was there that I joined him. Together we mounted a direct attack on the problem of the mechanism of secretion by adopting and developing an electropermeabilization technique. This technique breaches the plasma membrane of cells without disturbing the structural integrity of the intracellular organelles. Thus it was possible, by diffusing solutes through these pores in the plasma membrane, to manipulate experimentally the chemical environment of the site of exocytosis. Such studies defined accurately the calcium and nucleotide requirements for secretion and provided a means of directly testing various hypotheses concerned with this mechanism. Peter Baker’s activities, however, were by no means confined to research. He exhibited equal enthusiasm in his other commitments, as a member of various committees of the Medical Research Council, Royal Society, and Agricultural and Food Research Council. He also participated in the editorial board of The Journal of Physiology. Peter Baker’s life contained many challenges. He accepted each with gladness and executed each with hope. His death is a loss, not only to me and those immediately around him, but to all those who know what it means to ‘‘live’’ science. Photo of Peter Baker, working at The Marine Biological Association of the U.K., Plymouth, was taken by T. J. Allen in January 1987.

DEREKKNIGHT Department of Physiology King’s College London

Yale Membrane Transport Processes Volumes

Joseph F. Hoffman (ed.). (1978). “Membrane Transport Processes.” Vol. I . Raven, New York. Daniel C. Tosteson, Yu. A. Ovchinnikov, and Ramon Latorre (eds.). (1978). “Membrane Transport Processes,” Vol. 2. Raven, New York. Charles F. Stevens and Richard W. Tsien (eds.). (1979). “Membrane Transport Processes,” Vol. 3 : Ion Permeation through Membrane Channels. Raven, New York. Emile L. Boulpaep (ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13 of Cirrrerzf Topics in Membranes and Trunsport (F. Bronner and A. Kleinzeller. eds.). Academic Press, New York. William H. Miller (ed.). (1981). “Molecular Mechanisms of Photoreceptor Transduction”: Volume 15 of Ciirrent Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press. New York. Clifford L. Slayman (ed.). (1982). “Electrogenic Ion Pumps”: Volume 16 of Current Topics in Membranes und Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Joseph F. Hoffman and Bliss Forbush 111 (eds.). (1983). “Structure, Mechanism, and Function of the Na/K Pump”: Volume 19 of Cirrrent Topics in Membranes and Transport (F. Bronner and A . Kleinzeller, eds.). Academic Press, New York. James B. Wade and Simon A. Lewis (eds.). (1984). “Molecular Approaches to Epithelial Transport”: Volume 20 of Current Topics in Membranes and Trurzsport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Edward A. Adelberg and Carolyn W. Slayman (eds.). (1985). “Genes and Membranes: Transport Proteins and Receptors”: Volume 23 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. xvii

xviii

YALE MEMBRANE TRANSPORT PROCESSES VOLUMES

Peter S. Aronson and Walter F. Boron (eds.). (1986). ‘“a’-H’ Exchange, Intracellular pH, and Cell Function”: Volume 26 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, Orlando. Gerhard Giebisch (ed.). ( I 987). “Potassium Transport: Physiology and Pathophysiology”: Volume 28 of Current Topics in Membranes und Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando.

Part I

Membrane Fusion in Fertilization and Develo p ment

This Page Intentionally Left Blank

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT. VOLUME 32

Chapter I Sperm-Egg Fusion RYUZO YANAGIMACHI Department of Anutotny cind Rcprodiri~tiuc~ Biolox! University of ffiinwii School o f Medic~itw Hotiolirlir , Ha Muii 96822

1.

II.

111.

IV. V. VI. VII.

VIII.

IX.

X.

introduction Morphology of Sperm-Egg Fusion A. Sea Urchins B. Some Other Marine Invertebrdte~ C. Fishes 11. Amphibians E. Birds F. Mammals Specifc Sites for Sperm-Egg Fusion A. Sperm Membrane 8 . Egg Plasma Membrane Sperm Movement and Sperm-Egg Fubion Intermingling of Sperm and Egg Plasma Membrane Species Specificity of Sperm-Egg Fusion How Long Does the Egg Plasma Membrane Remain Capable of Fusing with Spermatozoa after the Entry of the Firct Spermatozoon? Conditions Controlling Sperm-Egg Fusion A. Temperature. pH. and Ionic Composition of Medium B. Electric Potential of the Egg Plasma Membrane Effects of Miscellaneous Substances on Sperm-Egg Fu5ion A. Proteolytic Enzymes B. Antimembrane Antibodies C. Proteinase Inhibitors D. Dithiothreitol E. Erythrosine and Related Dyes F. Cytochalasin G. Other Reagents Mechanism of Sperm-Egg Fusion References

3 Copyright .I” 1988 hy Academic Press. Inc. A l l right\ 01 rrproduction in any form reserved

4

RYUZO YANAGIMACHI

I. INTRODUCTION

I would hope that readers will find sperm-egg fusion (fertilization) a fascinating example of membrane fusion. It is one of the “natural” or “spontaneous” membrane fusions that occur without the deliberate addition of exogenous fusing agents such as viruses or chemicals (Lucy, 1978; Papahadjopoulos et al., 1979; Okada, this volume, Chapter 10). Although fertilization has been studied extensively for over 100 years because of curiosity about the mechanisms that give rise to new generations (Lop0 and Vacquier, 1981), most experimental observations have been concerned with the physiological or developmental events that follow cell fusion. Few experiments have been undertaken with the specific aim to elucidate the membrane events involved in fusion per se (Papahadjopoulos et al., 1979; DuzgiineS, 1985). In this chapter, I shall discuss fertilization in a few selected classes of animals, with major emphasis on the process and mechanism of sperm-egg fusion. For comparative aspects of fertilization in a wide variety of animals, readers are referred to the books edited by Metz and Monroy (1967, 1969, 1985).

II.

MORPHOLOGY OF SPERM-EGG FUSION

A. Sea Urchins

The sea urchin egg, which is about 100 pm in diameter, is surrounded by a very thin (0.01-0.03 pm) vitelline envelope, which is further surrounded by a thick, gelatinous matrix called the jelly coat (Fig. 1A). The vitelline envelope is a glycoprotein (Glabe and Vacquier, 1977, 1978), and the jelly coat contains two major macromolecular components, a fucose sulfate-rich polymer and a sialoprotein (SeGall and Lennarz, 1979). When eggs and spermatozoa are shed into seawater, spermatozoa swarm around the jelly coat, and many of them penetrate it to reach the vitelline envelope (Fig. IB).The head of each spermatozoon binds firmly to the vitelline envelope and rotates (counterclockwise) as the tail beats vigorously (Fig. ID). Then, suddenly one of the spermatozoa (the fertilizing spermatozoon) stops its tail movement (Epel. e? al., 1977; Hinkley et al., 1986), and the vitelline envelope starts to lift up (Fig. 1E). This elevation of the vitelline envelope, due to exocytosis of cortical granules, begins around the fertilizing spermatozoon and propagates in a wavelike fashion from the point of sperm-egg fusion. During elevation of the vitelline envelope, the fertilizing spermatozoon remains almost motionless, but sporadically twitches its tail in such a way that the entire tail becomes incorporated into the perivitelline space (Fig. IF; M. Sugiyama, personal

1. SPERM-EGG FUSION

5

FIG.I . Fertilization (sperm-egg fusion) in the sea urchin. ( A ) Unfertilized egg of Tripw u s f t ~ . sg m r i / / u . surrounded by India ink to show the jelly coat around the egg. ( B ) Seven seconds after insemination. (C) Two minutes after insemination. (D-F) The fertilizing spermatozoon. shown by an arrow in E. stops its vigorous tail movement soon after fusion with the egg. ( G )The sperm head. (H-0) Successive stages of sperm-egg fusion. Abbreviations: a. acrosome; ap, acrosomal process: b. bindin: cg, cortical granules; epm, egg plasma membrane; eve. elevated vitelline envelope: hi. hyaline layer; o m . outer acrosomal membrane; pvs, perivitelline space; spm, sperm plasma membrane; ve. vitelline envelope.

6

RYUZO YANAGIMACHI

communication). The head and the entire sperm tail then gradually sink into the egg cytoplasm. The successive stages of membrane fusion between the spermatozoon and the egg are shown diagramatically in Fig. 1G through 0 (cf. Franklin, 1965; Summers and Hylander, 1974; Schatten and Mazia, 1976; Usui et al., 1980). During the passage of spermatozoa through the jelly coat (or on contact of the spermatozoa with the vitelline envelope), the acrosome opens up as a result of the fusion between the outer acrosomal membrane and the overlying plasma membrane (Fig. IH, I), while the inner acrosoma1 membrane elongates to form a process (Fig. lK, L). These changes are called the acrosome reaction (Dan, 1967). The interaction between the jelly components (or vitelline envelope components) and a sperm membrane protein (Podell and Vacquier, 1985) seems to trigger the reaction. One of the acrosomal components exposed as a result of the acrosome reaction is a protein called “bindin.” It functions in holding the acrosomal process firmly to the vitelline envelope (Fig. l K , L) (Vacquier and Moy, 1977). The mechanism by which the acrosome process penetrates through the vitelline envelope is currently unknown. It could be purely mechanical. Alternatively, it could be aided by a chymotrypsin-like enzyme released from the acrosome during the acrosome reaction (Hoshi, 1985). Membrane fusion begins between the tip of the acrosome process and the egg plasma membrane (Fig. lL, M). It should be noted that it is the acrosomal membrane, not the sperm plasma membrane, that fuses with the egg plasma membrane. Figure 2 shows sea urchin spermatozoa about to fuse (Fig. 2A) or fusing with eggs (Fig. 2B-D). B. Some Other Marine Invertebrates

Early stages of sperm-egg fusion in Hydroides hexagonus (an annelid), Saccoglossus kowalevskii (a hemicordate), Chama macerophylla (a mollusk), and Neanthesjaponica (a polychaete) are shown diagramatically in Fig. 3. Only the anterior region of spermatozoa is shown in the diagrams. The vitelline envelopes of the eggs of these species are much thicker (0.6-0.7 pm) than those of sea urchin eggs. Spermatozoa undergo the acrosome reaction on the vitelline envelope. In Hydroides, the inner acrosoma1 membrane elongates to form many acrosomal processes, each of which fuses with the egg plasma membrane. In Saccoglossus, one long process is formed, the tip of which fuses with the egg plasma membrane. The acrosomal process preexists in the spermatozoa of Chama and Neanfhes. The process, which is exposed after the acrosome reaction, fuses with one of the egg microvilli.

FIG.2 . Scanning electron micrograph5 showing successive stage5 of sperm-egg f'uaion in the w a urchin, S/ro,i~?~/oc,rn/,.c,~lrs prrrp/rr.cctrrs. (From Schatten and Mazia. 1976. with permission of Academic Press.)

8

RYUZO YANAGIMACHI

B Hemlohordate . .

.

C Mollusc

FIG.3. Sperm-egg fusion in four different marine invertebrates showing that spermegg fusion begins between the membrane covering the acrosomal process and the egg plasma membrane. Animal species and references are (A) annelid. Hvdroides lte.rugonrts (Colwin and Colwin, 1964, 1967):(B)hemichordate, Suc~coglossuskonwlruskii (Colwin and Colwin. 1964, 1967); (C) mollusk, Chuma mucerophyllu (Hylander and Summers, 1977); (D)polychaete, Neunthes juponica (Sato and Osanai. 1986). ve, Vitelline envelope.

C. Fishes

The fish egg has a thick and relatively tough vitelline envelope which is commonly called the chorion. Penetration of the spermatozoon into the egg takes place through the micropyle, a specialized narrow channel in the chorion (Fig. 4A). The relatively wide opening of the micropyle may permit the simultaneous entry or two or more spermatozoa, but its narrow bottom allows the passage of only one spermatozoon at a time.

9

1. SPERM-EGG FUSION Micropyle

FIG.4. Sperm-egg fusion in fishes. ( A ) Sagittal section of an egg before (BF) and after ( A F ) fertilization. The insert shows the relative size of the egg and spermatozoon of the herring. Clrrpeu pullasii. (B.C ) Sperm-egg fusion takes place between the plasma membrane of the sperm head and the egg plasma membrane at the bottom of the micropylar canal. Animal species and references are ( B ) carp. Cyprinrrs c,urpio (Kudo. 1980): tC) chum salmon, Oncorkynchrt.~ketu (Kobayashi and Yamamoto. 1981 1987). pvs. Perivitelline space.

Fish spermatozoa, unlike sea urchin spermatozoa, have no acrosome. In fish, it is the plasma membrane covering the sperm head that fuses with the egg plasma membrane. The manner of sperm-egg fusion in carp and salmon is shown diagramatically in Fig. 4B and C, respectively. The sturgeon is unusual among fishes in that its egg has several micropyles in the chorion (Cherr and Clark, 1982, 1985a). It is also unique in that the spermatozoon has an acrosome. The fertilizing spermatozoon appears to undergo the acrosome reaction while passing through the micropyle (Cherr and Clark, 1985b). Therefore, in the sturgeon it must be the inner acrosomal membrane covering the acrosomal process which

10

RYUZO YANAGIMACHI

fuses with the egg plasma membrane ( G . N . Cherr, personal communication). In this respect, the sturgeon is somewhat similar to the lamprey, although the egg of the latter has no micropyle (Kille, 1960). D. Amphibians

The amphibian egg has a relatively thin (3-5 pm) vitelline envelope. The envelope is further surrounded by a thick jelly coat. The probable manner of sperm-egg fusion in the bullfrog (Bufo japonica) is shown diagramatically in Fig. 5 . The acrosome opens on contact of the sperm head with the vitelline envelope (Yoshizaki and Katagiri, 1982). The acrosome is known to contain a lysin that facilitates penetration of the vitelline envelope (Iwao and Katagiri, 1982). Although no one has ever witnessed the moment of sperm-egg fusion in amphibians, it is believed to occur between the inner acrosomal membrane and the egg plasma membrane (C. Katagiri, personal communication). Sperm entry commonly occurs within a 60" circle of the animal pole (Elinson, 1975).

FIG.5. Probable manner of sperm-egg fusion in the frog, Bufo juponiru. as suggested by Dr. C. Katagiri (personal communication: Yoshizawa and Katagiri, 1982). The insert

shows the relative size of the egg and spermatozoon. cg. Cortical granules; ve, vitelline envelope.

11

1, SPERM-EGG FUSION

E. Birds

Figure 6 illustrates the manner of sperm-egg fusion in the domestic fowl (Callus gallus). The egg is surrounded by a thin (-3 p m ) vitelline envelope. The vitelline envelope is further surrounded by another coat (3-8 pm in thickness), the outer envelope. The latter is a secretory product of the oviduct. The fertilizing spermatozoon undergoes the acrosome reaction on the vitelline envelope (Okamura and Nishiyama, 1978a). The proteolytic activity of the acrosomal contents (Yanagimachi and Teichman, 1972) makes a “hole” in the envelope, through which the spermatozoon passes. Fusion begins between the inner acrosomal membrane and the egg plasma membrane (Okamura and Nishiyama, 1978b). F. Mammals

Very little is known about the process of sperm-egg fusion in the Monotremata and Marsupialia. According to Rodger and Bedford ( 1982), the opossum spermatozoon undergoes the acrosome reaction on the vitelline envelope (2-3 p m in thickness), creates a “hole” in the envelope, and then fuses with the egg plasma membrane, perhaps via the inner

I-

iooyn

.

,

.

.

.

.

.

.

. .

...

.

. ,.-

. .

FIG. 6 . Successive stages of sperm-egg fusion in the domestic fowl. G ~ U gcrll//c A (Okarnura and Nishiyarna, 1978a,b). The insert shows the relative size of the egg and spermatozoon. oe. Outer envelope: ve. vitelline envelope.

12

RYUZO YANAGIMACHI

acrosomal membrane. Eggs of eutherian mammals have a relatively thick glycoprotein envelope (usually 10-20 pm in thickness), commonly called the zona pellucida (cf. Dunbar and Wolgemuth, 1984). In most mammals, the zona is further surrounded by the cumulus oophorus at the time of fertilization. The cumulus consists of follicle cells embedded in their gelatinous (hyaluronic acid polymer) matrix. The fertilizing spermatozoon initiates the acrosome reaction (Fig. 7B) in the vicinity of, or on, the zona.

FIG.7. The acrosome reaction (A-B), zona penetration (C), and sperm-egg fusion (DGI in the cutherian mammal. The insert shows the relative size of golden hamster (Mesocric e l u ~rrurutus) and human gametes. (Dthrough G were redrawn from Bedford and Cooper, 1978, with permission of Elsevier/North Holland Biomedical Press.) Abbreviations: ac, acrosomal cap region; CG, cortical granules; eq, equatorial segment of the acrosome; iam,

inner acrosomal membrane: pvs, perivitelline space; zp, zona pellucida.

1. SPERM-EGG FUSION

13

The spermatozoon must be acrosome-reacted to pass through the zona (Fig. 7C). Sperm-egg fusion begins between the egg plasma membrane and the sperm plasma membrane over the equatorial segment of the acrosome (Fig. 7E; Bedford and Cooper, 1978; Bedford et al., 1979). The inner acrosome membrane never fuses with the egg plasma membrane. This membrane is incorporated into the egg cytoplasm in a phagocytic fashion (Fig, 7F, G) and is destined to degenerate within the cytoplasm (Yanagimachi and Noda, 1970a). Figure 8A-C shows golden hamster spermatozoa fusing with eggs. 111.

SPECIFIC SITES FOR SPERM-EGG FUSION

A. Sperm Membrane

In many animals (e.g., sea urchins, frogs, birds, and perhaps sturgeons and opossums), it is the acrosomal membrane that fuses first with the egg plasma membrane. In these animals, the sperm plasma membrane is not capable of initiating fusion with the egg plasma membrane. The fusible acrosomal membrane appears to be protected by the nonfusible plasma membrane until the spermatozoon comes close to the egg (Epel and Vacquier, 1978). In some animals, the acrosomal membrane elongates during the acrosome reaction to become the surface membrane of the acrosomal process (Figs. 1 and 3A, B). It is this surface membrane (the elongated inner acrosomal membrane) that fuses with the egg plasma membrane. It is not known whether the acrosomal membrane can undergo fusion before it elongates. The spermatozoa of some other animals have acrosomal processes even before they undergo the acrosome reaction. The inner acrosomal membrane, which is destined to fuse with the egg plasma membrane, does not elongate during the acrosome reaction (Figs. 3C, D, 5 , and 6). It is not known at present whether the membrane is capable of fusion before the acrosome reaction. Mammalian spermatozoa (with exceptions of marsupialian and perhaps monotreme spermatozoa) fuse with eggs via the head plasma membrane. The inner acrosomal membrane lacks the ability to fuse with the egg plasma membrane (for the characteristics and possible functions of the inner acrosomal membrane of mammalian spermatozoa, readers are referred to Huang and Yanagimachi, 1985). Yanagimachi and Noda (1970b), who examined fusion between zona-free hamster eggs and acrosome-reacted spermatozoa, concluded that the plasma membrane of the postacrosomal region is the first to fuse with the egg plasma membrane. However, careful examination of spermatozoa in the act of fusing with zona-intact eggs has revealed that it is the plasma membrane above the

FIG.8. Scanning electron micrographs of hamster spermatozoa fusing with eggs. [Courtesy of Dr. David M . Phillips, with permission of Plenum Press (A) and Academic Press (B, C). A is from Yanagimachi (1981); B, C are from Shalgi and Phillips (1980).]

15

1. SPERM-EGG FUSION

equatorial segment of the acrosome which fuses first with the egg plasma membrane (Bedford and Cooper, 1978: Bedford et d..1979). It is important to note that the plasma membrane above the equatorial segment can fuse with the egg plasma membrane only after the spermatozoa have undergone the acrosome reaction. The acrosome-intact spermatozoon, regardless of whether capacitated or not, cannot fuse with the egg, even when the zona pellucida is bypassed and the spermatozoon is brought directly in contact with the egg surface (Yanagimachi and Noda, 1970b). This suggests that the plasma membrane over the equatorial segment must undergo change at the same time as (or as the result of) the acrosome reaction (Fig. 9). The possibility that acrosomal material and hydrolytic enzymes released during the acrosome reaction can alter the property of the plasma membrane above the equatorial segment (hypothesis I of Fig. 9B) is unlikely, because acrosome-intact hamster spermatozoa (whether capacitated or not), when treated with crude acrosome extracts, are unable to fuse with zona-free hamster eggs (R. Yanagimachi, unpublished data). Possibly some components of the medium (e.g., monovalent and/or divalent cations) can penetrate the spermatozoon during the acrosome reaction and thereby alter the characteristics of the plasma membrane over the equatorial segment (11 of Fig. 9B). An influx of Ca’’ into acrosome-reactingheacted spermatozoa (Monroy, 1985)could be one of the factors that enables the sperm membrane to fuse with the egg plasma membrane. According to McGrath and Hillman (1980). the spermatozoa of mutant ( r L x l t L y ) mice are fully capable of undergoing the acrosome reaction and of attaching to the egg plasma membrane, yet are totally incapable of fusing with the egg plasma membrane. In this situation, either the sperm plasma membrane above the equatorial segment or the mechanism that enables this membrane to undergo fusion must be defective.

4 / *.\., I l ,. l .\

A

B

C

FIG.9. Spermatozoa before ( A ) , during (B).and after (C) the acrosome reaction. H o w does the plasma membrane over the equatorial segment become capable of fusing with the egg after the acrosome reaction‘! Hypothesis I: Some materials released from the acrosome modify the characteristics of the membrane. Hypothesis 11: Some components of the medium penetrate into the spermatozoon and modify the characteristics of the membrane directly or indirectly. ac, Acrosomal cap region; eq, equatorial segment of the acrosome.

16

RYUZO YANAGIMACHI

Freeze-fracture images of the acrosomal membrane of sea urchin spermatozoa and of the plasma membrane over the equatorial segment of guinea pig spermatozoa, before and after the acrosome reaction, are shown in Fig. 10A-D. Prior to the acrosome reaction, intramembranous particles are seen in the posterior region of the acrosomal membrane of

FIG.10. Freeze-fracture replicas of sperm membranes before and after the acrosome reaction. (A) E-face of the acrosomal membrane of an acrosome-intact sea urchin spermatozoon; note that intramembranous particles are rich in the posterior region of the acrosomal granule (ag). ( B ) The P-face of the acrosomal membrane which covers the acrosomal process (ap) has very few particles. (C)The P-face of the plasma membrane of an acrosome-intact. uncapacitated guinea pig spermatozoon has evenly distributed intramembranous particles. (D)The P-face of the membranes of an acrosome-reacted spermatozoon; note the presence of small, particle-free patches (white asterisks) in the plasma membrane of the equatorial segment. [Courtesy of Drs. Noriko Usui and lchiro Takahashi (A, B) and Fumie Suzuki (C. D).]Abbreviations: acr, plasma membrane of the acrosomal cap region: ag, acrosomal granule; ap, acrosornal process; eq, plasma membrane of the equatorial segment region: iam, inner acrosomal membrane: par, plasma membrane of the postacrosomal region.

1. SPERM-EGG FUSION

17

the sea urchin spermatozoon: the anterior region of the membrane is nearly free of the particles (Fig. IOA). The elongated acrosomal membrane covering the surface of the acrosomal process has sparsely distributed particles (Fig. IOB). According to Mann or ul. (1976), the membrane covering the tip of the acrosomal process, which is destined to fuse with the egg plasma membrane, is virtually free of the particles. In the guinea pig. particle-free patches can be seen in the plasma membrane of the equatorial region of an acrosome-reacted spermatozoon (Fig. lOD), but not in that of an acrosome-intact, uncapacitated spermatozoon (Fig. IOC). These images suggest drastic changes in the physicochemical characteristics of the membrane phospholipids (and perhaps of the cytoskeletal system under the membrane) associated with the acrosome reaction.

6 . Egg Plasma Membrane

In fish, the egg plasma membrane that normally fuses with the spermatozoon is limited to the area of the membrane beneath the micropyle (cf. Fig. 4). However, the plasma membrane in other areas of the egg seems to have the potential to fuse with spermatozoa (Fig. 1 I). In the sea urchin and mammal, the entire area of the egg plasma membrane seems to have the potential to fuse with the spermatozoon (for the mammal, see Blandau and Odor, 1952). In the sea urchin, the spermatozoon fuses with the plasma membrane of small microvilli (papillae; Fig. I). However, there is no evidence that the intermicrovillous region lacks the ability to fuse with the spermatozoon, and some investigators believe that the spermatozoon fuses with the egg plasma membrane more commonly in the intermicrovillous than in the microvillar region (N. Usui, personal communication). In mammals, it is not yet clear whether the spermatozoon fuses with the egg plasma membrane of microvilli or of the intermicrovillar region (for discussion, see Yanagimachi, 1981). The mammalian egg plasma membrane above the meiotic spindle has no microvilli. It is either "smooth," or provided with large cytoplasmic protrusions. Sperm-egg fusion does not occur o r seldom occurs in this region (Johnson el ul., 1975; Ebensperger and Barros, 1984). The reason why this region lacks the ability to fuse with spermatozoa is not clear. According to Wolf and Ziomek (1983), membrane proteins move (diffuse) more freely in this than in other regions. In the frog (Elinson, 1975) and bird (F. Okamura, personal communication), only the egg plasma membrane of the animal hemisphere (frog) o r near the animal pole (bird) appears to have the ability to fuse with the spermatozoon(oa). In the hydrozoa, the region that can undergo fusion

18

RYUZO YANAGIMACHI

seems to be limited to a very small portion of the animal pole where the second polar body was extruded (in this animal, meiosis of the egg is completed before fertilization) (Miller, 1978; Freeman and Miller, 1982). It has been known for more than 50 years that immature sea urchin eggs in the germinal vesicle stage can be penetrated by spermatozoa (Seifriz, 1927). In mammals, the plasma membrane of the immature egg at the germinal vesicle stage has the potential to fuse with spermatozoa, at least at the time when the egg is about to resume meiosis (e.g., Usui and Yanagimachi, 1976). However, it is not known when during egg growth the plasma membrane gains the ability to fuse with spermatozoa.

IV. SPERM MOVEMENT AND SPERM-EGG FUSION

Sperm-egg fusion as such does not appear to require the movement of the spermatozoon. According to Epel et al. (1977), 10 mM sodium azide completely immobilizes the spermatozoa of Lytechinus pictus (a Pacific sea urchin). Immotile spermatozoa can, nevertheless, fertilize eggs. Epel et al. (1977) did not remove the vitelline envelopes from eggs prior to insemination. Immotile spermatozoa, that were carried to the egg surface by currents, must have undergone the acrosome reaction on or near the vitelline envelope; they then must have attached to and dissolved the vitelline envelope before fusing with the egg plasma membrane. It has been shown that tailless (and therefore motionless) sea urchin spermatozoa can fertilize eggs (Vacquier, 1979a). Herring spermatozoa are almost motionless in seawater. When the spermatozoa drift to the surface of the chorion near the micropyle, they become very active and enter the micropyle one by one (Yanagimachi and Kanoh, 1953). The first spermatozoon then fuses with the egg plasma membrane. When an egg is partially denuded and then inseminated, the motionless spermatozoa can be seen to make contact with the “naked” portion of the egg (Fig. IIA) and apparently fuse with the egg membrane (see the legend of Fig. 1 I). Strong sperm movement is required for normal fertilization in mammals. Motionless or weakly motile spermatozoa are unable to pass through egg investments, particularly the zona pellucida. Once the zona is removed, however, motionless or very weakly motile spermatozoa can fuse with the eggs as long as the spermatozoa are alive and have undergone the acrosome reaction (Yanagimachi, 1981). The spermatozoa of humans with Kargagener’s syndrome present an interesting example. They are “normal,” except that they are completely motionless due to the lack of dynein arms (Afzelius et al., 1975). In a medium that allows

19

1. SPERM-EGG FUSION

A

B

C

FIG. I I . Fertilization of a partially denuded herring egg. 'l'he chorion was piirtially removed. at 4 o'clock, with the aid of a pair of iridectoniy scicsorh. By applying gentle preysure. a portion of the egg was squeeked otit of the chorion. When such ?in egg wus inseminated (A). motionless spermaloma were seen to become activated (began to move actively) near the micropyle and entered the micropyle one by one: motionless sperrniiforoa were seen attached to the naked portion of the egg ( A ) . l'he egg was activated by spermatozoa ( 8 ) and cleaved (0.The main body of the egg was fertilized monosperrnically. The protruded portion of the egg, on the other hand. wi15 fertilized polysperniically. (From Y anagimac hi. 19.57.)

capacitation and the acrosome reaction, these spermatozoa fuse with zona-free hamster eggs (Aitken et a/.. 1983). As stated already, the fertilizing spermatozoon of the sea urchin abruptly ceases its active tail movement soon after attachment to the egg surface (e.g., at 16 sec after attachment: Epel ot al., 1977). The cessation of movement is thought to occur some time after sperm-egg fusion (Epel et a / . . 1977). Hinkley el a/. (1986) have estimated that sperm immobilization occurs 4-6 sec after fusion. The fertilizing spermatozoa of mammals also cease their active tail movement almost immediately on contact with the egg plasma membrane (Yanagimachi, 1966; Sato and Blandau, 1979: Gaddum-Rosse, 1985). This can be witnessed easily by inseminating zona-free eggs in vitvo with actively motile. acrosome-reacted spermatozoa (Fig. 12). The reason for this abrupt loss of sperm tail movement is unknown, but it could be caused by an explosive release of intracellular CaITby the egg as observed by Miyazaki rt d.(1986). Although the sperm tail may display sporadic twitching or flexing for some time after the sperm head has become attached to the egg plasma membrane, active tail movement is apparently unnecessary for the migration of the sperm head (nucleus) to a position deep inside the egg.

V. INTERMINGLING OF SPERM AND EGG PLASMA MEMBRANE The sperm plasma membrane intermingles with the egg plasma membrane during fusion. This intermingling can be visualized, for example, by labeling sperm surfaces with FITC or FITC-conjugated antisperm surface

20

RYUZO YANAGIMACHI

FIG. 12. Acrosome-reacted hamster spermatozoa lose their motility soon after contact with the egg plasma membrane. An unfertilized, zona-free egg is approached by an acrosome-reacted spermatozoon (A). The spermatozoon beats its tail vigorously for a while after contact with the egg plasma membrane (B);5-15 sec after contact (C) its beat becomes By 5 min after considerably lower until it becomes motionless 15-25 sec after contact (D). insemination, the egg has many motionless spermatozoa attached to its surface (E).(From Yanagimachi, 1978a. with permission of Academic Press.)

antibodies and examining the surfaces of eggs fertilized by these spermatozoa (Gabel et al., 1979; Gundersen and Shapiro, 1984; Gundersen et al., 1986). Gaunt (1983) produced a monoclonal antibody that binds to the rat

sperm surface antigen (2D6) which is located over the entire surface of the spermatozoon (Fig. 13A, A’). The antigen to the sperm surface spreads gradually (Fig. 13B, B’) until it covers most of the surface of the fertilized egg (Fig. 13C, C’). Only traces of the antigen are detected in the two-cell embryo (Fig. 13D, D’), perhaps because the antigen has become internalized. The antigen, 2D6, is just one of the sperm surface molecules. Other molecules may also persist in the embryo for a long time. In fact, some surface proteins of sea urchin spermatozoa can be detected as late as the late gastrula stage; however, most sperm surface proteins become degraded shortly after fertilization (Gundersen and Shapiro, 1984). VI. SPECIES SPECIFICITY OF SPERM-EGG FUSION A distinct feature of fertilization is its species specificity. Generally speaking, spermatozoa of a given species cannot, or cannot readily, fertil-

FIG. 13. Fate of a rat sperm surface anigen. 2D6. incorporated into the egg plasma membrane (A-D, pha5e contrast; A'-D'. indirect immunofluorescence). ( A ) Spermatomi from the epididymal cauda. ( B ) One recently fertilized egg (center) with two unferlilired eggs. collected from immature females 14 hr after hCG injection and mating. (C) A n egg in the advanced stage of fertilization. 20 hr after hCG injection and mating. (D) A two-cell stage embryo, 40 hr after hCG injection and mating. (Courtesy of Dr. Stephen J. Gaunt. with permission of the Company of Biologists. Ltd.)

22

RYUZO YANAGIMACHI

ize the eggs of other species, although cross fertilization does occur on occasion even between gametes of distant species. An example is the cross-fertilization between sea urchin eggs and mussel or oyster spermatozoa (Longo, 1977; Osanai and Kyozuka, 1982). One of the major barriers for cross-fertilization appears to reside in the vitelline envelope. The spermatozoa of foreign (heterologous) species often fail to undergo the acrosome reaction near or on the envelope. Even if they do, they fail to bind or penetrate the envelope (Summers and Hylander, 1975). The removal of the vitelline coat is known to facilitate fertiliization (sperm-egg fusion) among different species of the sea urchin (Hultin, 1948; Jensen, 1953; Kyozuka and Osanai, 1979). The spermatozoa of most teleostean fishes do not need to undergo the acrosome reaction, as they have no acrosomes; rather their head plasma membrane is ready to fuse with the “naked” egg plasma membrane located at the bottom of the micropyle (cf. Fig. 4). Therefore, as long as the diameter of the bottom end of the micropyle is large enough to allow the passage of one spermatozoon, the chance of cross-fertilization is high. In fact, hybridization in the wild is not very rare, and cross-fertilization in the laboratory is relatively easy (Moenkhaus, 1910; Hubbs, 1955; Kobayashi, 1963; Suzuki, 1964; Nikoljuki, 1965; Ohta, 1985). In mammals, sperm penetration through the zona pellucida and spermegg fusion may occur following cross-insemination between closely related species (cf. Maddock and Dawson, 1974; Roldan e t a l . , 1985). In the case of distant species, spermatozoa usually fail to attach to (or penetrate into) the zona pellucida (Yanagimachi, 1977). Sperm-egg fusion appears to be less species specific than sperm-zona interaction. For instance, mouse spermatozoa, which seldom penetrate the zonae of rat eggs, can readily fuse with zona-free rat eggs (Hanada and Chang, 1972; Pavlok, 1979). Similarly, human spermatozoa, which cannot penetrate the zonae of golden hamster eggs, can fuse with zona-free hamster eggs (Fig. 14; Yanagimachi et al., 1976). These facts, however, should not be taken as implying that the egg plasma membrane is totally lacking in species specificity. The plasma membrane of the mouse, for example, manifests strong specificity and permits the fusion of only mouse spermatozoa (Table I). Although the hamster egg plasma membrane is unique in that it allows fusion of a variety of foreign spermatozoa (Table I), it does show some degree of specificity. For instance, when zona-free hamster eggs are inseminated in a dish containing approximately equal numbers of acrosome-reacted spermatozoa from hamster, guinea pig, and humans, the number of hamster spermatozoa that can later be found within each egg is greater than the number of guinea pig or human spermatozoa (Yanagimachi, 1981). Therefore the affinity of the hamster egg plasma membrane must be greatest

Fiti. 14. Human spermatozoa fused with tona-free hamster egg\. ( A ) Semilongitudinal section through the head of a human spermalozoon undergoing fusion with an egg. (I31 Phase-contrast micrograph of an egg fused with many spermatoroa. about I hr after inseminetion with preincubated (acrosome-reacted) spermatoroa: the egg was compreswd between a slide and coverslip before photography.

24

RYUZO YANAGIMACHI

TABLE 1 PENETRATION O F ZONA-FREE MAMMALIAN EGGS B Y SPERMATOZOA OF HOMOLOGOUS AND HETEROLOGOUS SPECIES" Zona-free eggs Spermatozoa

Golden hamster

Chinese hamster

Golden hamster Chinese hamster Mouse Deer mouse Rat Guinea pig Bat Rabbit Dog Dolphin Pig Bull Sheep Goat Horse Marmoset monkey Cynomolgus monkey Rhesus monkey Bonnet monkey Human

Yes Yesh Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yesf Yes Yes Yes Yes" Yes' Yesf Yes

Yesh Yes

-

-

No

Guinea Mouse

Rat

pig

Rabbit

YesINo Yes No YeslNo No

YesINo

Yes

No Yes Yes N0 Yes

-

-

-

No

-

Yes No Yes No

No

-

Yes

-

No

-

-

From Yanagimachi (1984). except where indicated. Roldan and Yanagimachi (unpublished data). ' Pavlok t i / . (1983); Flechon and Pavlok (1986). ci Hoffman and Curtis (19841. ' Bwatman and Bavister (1984). Warikoo ('I ctl. (1986). "

I,

for the spermatozoa of its own species. Nevertheless, it would be interesting to know what property allows hamster egg plasma membranes to fuse with spermatozoa from a wide variety of species. Clearly the structure of this membrane cannot be unique (Koehler et ul., 1985).

VII. HOW LONG DOES THE EGG PLASMA MEMBRANE REMAIN CAPABLE OF FUSING WITH SPERMATOZOA AFTER THE ENTRY OF THE FIRST SPERMATOZOON?

In some animals (e.g., elasrnobranchs, urodeles, reptiles, and birds), many spermatozoa penetrate a single egg, and their nuclei transform into

1. SPERM-EGG FUSION

25

male pronuclci. However, only one of these pronuclei unites with the female pronucleus. In these animals, the egg plasma membrane must retain the capacity to fuse with spermatozoa for an extended time period after fusion with the first spermatozoon. In the sea urchin, only one spermatozoon fuses with the egg plasma membrane. under normal conditions. All other spermatozoa are prevented from fusing with, or reaching, the egg plasma membrane. The mechanisms responsible for this block are a rapid. transient change in the membrane potential and a relatively slow, but permanent, chemical change that the vitelline envelope undergoes as a result of the action of cortical granule material (Schuel. 1984; Jaffe and Could, 1985). Both of these changes are triggered when the first spermatozoon fuses with the egg. A fertilized sea urchin egg does not allow the entry of excess spermatozoa. even if the vitelline membrane is removed and the egg is reinseminated with large numbers of spermatozoa. Does the egg plasma membrane permanently lose its ability to fuse once fertilization is complete? Sugiyama (1951) was the first to demonstrate that the egg plasma membrane remains potentially capable of fusing with spermatozoa even after the egg has reached the two-cell stage. Fertilized eggs freed from their vitelline envelopes and treated with Ca”- and Mg”-free seawater permit the entry of many spermatozoa (Sugiyama, 1951; Tyler r t al., 1956). This treatment dissolves the hyaline layer that covers the plasma membrane of the fertilized egg. Therefore, it must be the hyaline layer that renders the plasma membrane resistant to further fusion with spermatozoa. In some mammals (e.g., the rabbit), the plasma membrane of the egg becomes “refractile” to excess spermatozoa on activation by the first (fertilizing) spermatozoon (Austin and Walton, 1960; Austin, 1961). The cortical granule material that is exocytosed during activation of the egg is thought to alter the surface properties of the egg plasma membrane (Cooper and Bedford, 1971; Gordon ot d.,1975). Cortical granule material apparently does not change the properties of the zona pellucida of rabbit egg. Spermatozoa continue to penetrate the perivitelline space, often in large numbers, until a mucin coat is deposited around the zona (Austin and Walton, 1960). In some other mammals, as the golden hamster o r the mouse, the proteolytic component of the cortical granule material alters the chemical properties of zona glycoproteins in a way that prevents surplus spermatozoa from binding to, or penetrating, the zona (Gwatkin rt [ J I . , 1973; Wassarman c>t d., 1985). In the mouse, cortical granule proteinases render the egg plasma membrane incapable of fusing with excess spermatozoa (Wolf and Hamada. 1977). The plasma membrane of the hamster egg apparently remains “unaffected” by cortical granule proteinases during fertilization. It can still fuse with spermatozoa even as late as in the four-cell stage (Usui and Yanagimachi, 1976). If an

26

RYUZO YANAGIMACHI

unfertilized zona-free hamster egg is inseminated with acrosome-reacted spermatozoa, the spermatozoa can be seen t o fuse one after the other with the egg (Fig. 12). If the number of spermatozoa surrounding the egg is not limited, an excessively large number of spermatozoa will fuse with the egg. This results inevitably in cytolysis.

VIII.

CONDITIONS CONTROLLING SPERM-EGG FUSION

In experiments that aim at analyzing the mechanism of sperm-egg fusion, it is important to distinguish prefusion events from fusion itself. Consider the following experiment: Eggs with intact investments are washed and inseminated in a medium deficient in ion A and, when examined later, none is found to have been fertilized. It would be hasty to conclude that sperm-egg fusion was inhibited by the absence of ion A. The failure of the eggs to become fertilized might well have been due to the failure of spermatozoa to undergo the acrosome reaction o r to penetrate the egg investment. Sea urchin and mammalian spermatozoa, for example, will never fuse with the egg plasma membrane unless they have undergone the acrosome reaction and have penetrated the egg investments. In the case of mammals, it is advisable to free unfertilized eggs from both the cumulus oophorus and the zona pellucida before inseminating them in uitro with lively, motile spermatozoa that have undergone the acrosome reaction. Under favorable conditions, spermatozoa will fuse with eggs “instantaneously” and “synchronously.” Hinkley et a / . (1986) have reported an ingenious method to distinguish fused from unfused sea urchin spermatozoa. Sea urchin eggs are stained vitally with the DNA-specific fluorochrome Hoechst 33342 and then inseminated with unstained spermotozoa. When fusion occurs, the fluorochrome enters the spermatozoon and stains its DNA, resulting in the appearnance of a brightly fluorescent sperm head on the egg surface. The heads of unfused spermatozoa remain nonfluorescent. This simple method, which allows us to differentiate fused spermatozoa from unfused ones, without laborious electron microscopy, will be very useful in future studies of sperm-egg fusion. A. Temperature, pH, and Ionic Composition of Medium

In the golden hamster, sperm-egg fusion cannot occur at 10°C or below (Hirao and Yanagimachi, 1978a). When zona-free eggs were inseminated in uitro with acrosome-reacted spermatozoa at I0”C or below, motionless spermatozoa were seen attached to egg surfaces. There was no indication

1. SPERM-EGG FUSION

27

of sperm-egg fusion even 17-18 hr after insemination. On transfer to warm (37°C) medium, some of the spermatozoa on the egg surface became motile and sperm-egg fusion took place immediately. Interestingly, postfusion events (e.g., cortical granule exocytosis, sperm nucleus decondensation, resumption of egg meiosis, and pronuclear development) are less sensitive to low temperatures. Once sperm-egg fusion is complete, these events can proceed even at 4°C. Acidic pH of the medium reversibly inhibits sperm-egg fusion (Yanagimachi et ul., 1980). At pH 6.0, acrosome-reacted hamster spermatozoa may bind to the egg plasma membrane. but they fail to fuse with it (Fig. ISA). Fusion takes place upon transfer of eggs to more alkaline (pH 7.3) medium (Fig. ISB). Extracellular Ca2+ is essential for the fusion of mammalian (hamster, guinea pig, and human) spermatozoa with eggs (Yanagimachi, 1978b,

FIG. 15. Electron micrographs showing that acrosome-reacted hamster spermdtoioa are incapable of fusing with the plasma membrane of rona-free hamster eggs in an acid medium ( p H 6.0) ( A ) . but can readily d o so in a more alkaline medium ( p H 7 . 4 ) ( B ) . (From Yanaginiachi 6" ( I / . . 19x0. with permission of the Japanehe Society of Developmental 13iologi\t\.)

28

RYUZO YANAGIMACHI

1982). In Ca’+-deficient media, acrosome-reacted spermatozoa may attach to the egg plasma membrane, but fusion never occurs. Fusion takes place upon transfer of eggs (with attached spermatozoa) to a Ca?+-containing medium. Mg2+,Ba*+,and Sr2+can substitute for Ca?+in triggering sperm-egg fusion, although they are less effective than Ca2+(Fig. 16). In some biological systems, Mg’+ may act antagonistically to Ca?+,but in sperm-egg fusion these cations act synergistically (Yanagimachi, 1978b). Takahashi and Sugiyama (1973), Epel (1982), and Schmidt et a / . (1982) have reported that acrosome-reacted sea urchin spermatozoa can fuse with eggs in the absence of extracellular Ca2+.According to Sano and Kanatani (1980), however, the fusion cannot take place in the complete absence of Ca2+. Unlike the acrosome reaction, which requires a high concentration (5-10 mM) of extracellular Ca2+(Dan, 1954; Collins and Epel, 1977), sperm-egg fusion requires a far lower concentration of Ca’+ (Sano and Kanatani, 1980). Concentrations of Ca2+from 20 to 50 F M are enough to ensure sperm-egg fusion in 25-50% of the eggs (Fig. 17).

Cone. lmMl

FIG. 16. Relative effectiveness of various divalent cations in supporting sperm-egg fusion in the hamster. Zona-free hamster eggs were washed and inseminated with acrosomereacted spermatozoa in Ca”- and Mg’+-free media containing various concentrations of divalent cations (Ca”. Ba”, Sr2+,or Mg’+). They were examined for evidence of fertilization (sperm-egg fusion) 3-3.5 hr later. (From Yanagimachi. 1978b with permission of the Society for the Study of Reproduction.)

29

1. SPERM-EGG FUSION

In mammals, the presence of extracellular Mg?- is beneficial, but not essential, for sperm-egg fusion (Yanagimachi, 197%). In sea urchins, on the other hand, Mg?+ appears to play a significant role in sperm-egg fusion. Unless seawater contains millimolar Mg?', acrosome-reacted spermatozoa rarely fuse with eggs even in the presence of 10 mM Ca2+ (Sano ct (11.. 1980: Mohri cr ul., 1982). Extracellular K' does not appear to be essential for sperm-egg fusion in the mouse, although its presence enhances fusion (Fraser, 19x3). Guinea pig spermatozoa differ from mouse spermatozoa in that exposure to extracellular K' is essential for development of the capacity for fusion. Guinea pig spermatozoa which have been acrosome-reacted in K ' -free media are unable to fuse with the egg plasma membrane unless the insemination medium contains K' (Rogers r t (11.. 1981). Paradoxically, the presence of K' in the insemination media is not essential. Spermatozoa exposed to K ' only during (and shortly after) the acrosome reaction are capable of fusing with eggs in K+-free medium (Table 11). It thus appears that K ' triggers some important physiological change in the plasma membrane of the equatorial segment during or shortly after the acrosome reaction (Fig. 9). Once this change has taken place. extracellular K is no longer needed for sperm-egg fusion. +

loo] 80

0 0

10

20

50 Calcium

300pM

I

!

1

1

2

5

1

lOmM

COnC

Effect of extracellular Ca" concentration on fertiliration of sea urchin eggs by acrosome-reacted spermatozoa. Unfertilized egg\ of ~ ~ , / t t ; ~ , ~ , / 7pirlc r ~ ~hrrrirrrrr ~ 1 ~ / , 5~ were washed with Ca'-- and Mg?'-free seawater and inseminated with acrosome-reacted \perniatozoa in artificial seawater that cont:iined 3X.5 m M Mg?- and variou\ concentrations of CaL-, The percentage fertilization w a s determined 30-60 min after insemination by ohserving the formation of a fertilization envelope (elevated vitelline envelope) and normal cleavage. The value at 0 mM calcium represents that obtained in the Ca?--free medium conraining 1 m M EGTA. (From Sano and Kanatani. 19x0. with permission of Academic Press.) FIG. 17.

30

RYUZO YANAGIMACHI

TABLE 11

EFFECTOF T H E PRESENCE O R ABSENCE OF EXTRACELLULAR K' O N T H E FUSION OF G U I N E A PIC S P E R M A T O Z O A W I T H ZONA-FREE HAMSTEREGGS" Presence

(+)

Capacitation

+ -

-

+

or absence ( - ) of 2.7 mM K+ during Acrosome reaction

+ + +

% Eggs fused

Insemination

+ + +

-

-

-

-

with sperm (range) I00 100

I00 7 2 (44-100) 0 0

Mean number of sperm in each egg fused with sperm 9.2 6.8 6.6 1.8

" From R . Yanagimachi (unpublished data). Guinea pig yxrmatozoa were capacitated by a I-hr incubation in a Ca?+-free m T medium supplemented with 8S pgiml lysolecithin (Fleming and Yanagimachi. 1981). They were washed with albumin-saline \ohtion (0.9% NaCl with 0.1% bovine serum albumin) before transfer to the regular m T medium that contained 2 mM Ca". The majority o f spermatozoa underwent the acrosome reaction within I S min regardless o f the presence or absence o f K ' . Thirty minutes after exposure to Ca?'. the spermatozoa were washed again with the albumin-saline Solution and then mixed with zona-free hamster eggs in Ca?'-containing media with o r without K L .Sperm-egg fusion. as evidenced by the presence of swollen sperm heads or sperm pronuclei in egg cytoplasm. wa\ a\hessed I hr later.

6. Electric Potential of the Egg Plasma Membrane

Jaffe (1976) was the first to report the importance of the electric potential of the egg plasma membrane for sperm-egg fusion in the sea urchin (for reviews, see Gould-Somero and Jaffe, 1984; Jaffe and Gould, 1985; Jaffe and Cross, 1986). The resting membrane potential of the sea urchin (Strongylocentrotus) egg is about -70 mV. Within 3-30 sec after insemination, the egg membrane depolarizes to a plateau at -30 to +20 mV, and about I min later begins to repolarize (Fig. 18). This positive potential shift, triggered by the fusion of the fertilizing spermatozoon with the egg plasma membrane, prevents excess spermatozoa from fusing with the egg plasma membrane during the first minute of fertilization. Permanent mechanical barriers to excess spermatozoa are established by ( I ) transformation of the vitelline envelope to the fertilization envelope and ( 2 ) coating of the plasma membrane surface with the hyaline layer. If the membrane potential of the unfertilized egg is held negative, by applying current, during insemination, polyspermy results. On the other hand, if the potential is held positive, no fertilization takes place. Apparently, the negative resting potential of the egg plasma membrane greatly favors sperm-egg fusion. This seems to be true for other animals such as the starfish, echiuran worm (Urechis),and frog (Jaffe and Gould, 1985). The resting membrane potentials of mouse and hamster eggs are about

1. SPERM-EGG FUSION

31

FIG.IR. Fertilization potential in the w a urchin. .Yrro/i,v,vlol~ ~ n i r o r upsu r p u r o m . (From Jaffe. 1976. with Demission of McMillan Journals. Ltd.)

-40 and -30 mV. respectively. Instead of a positive shift, only either a small oscillation (mouse) or hyperpolarization (hamster) of the membrane potential has been recorded following fusion of sperm with eggs (Miyazaki and Igusa, 1985; Jaffe et d., 1983). The absence of a prominent positive shift in membrane potential in these rodent eggs is not surprising. To inhibit polyspermic fertilization these eggs rely almost exclusively on chemical modification of the zona pellucida (called the zona reaction), rather than on a change in the plasma membrane (cf. Austin and Walton, 1960: Yanagimachi, 1977). Circumstantial evidence strongly suggests that the plasma membrane of rabbit eggs becomes “refractile” to penetration by excess spermatozoa soon after fusion with the first spermatozoon. Some physical or chemical change must take place rapidly in or on the plasma membrane. In this regard. it is somewhat disappointing to find that no dramatic shift in membrane potential has been recorded following insemination of rabbit eggs in uitro (McCulloh ct i i / . , 1983). It is possible that the polyspermy block operating in the egg plasma membrane is not electrically mediated. In the rabbit, excess spermatozoa keep penetrating into the perivitelline space and continue to collide with the plasma membrane of the fertilized egg for many hours. The polyspermy block of the egg plasma membrane appears to be permanent, rather than temporary. Although changes in sialic acid and saccharide residues of the rabbit egg membrane glycocalyx have been implicated in the permanent polyspermy block (Cooper and Bedford, 1971; Gordon et d . , 1975). further studies are needed to elucidate the molecular basis of this block.

IX.

EFFECTS OF MISCELLANEOUS SUBSTANCES ON SPERM-EGG FUSION

A. Proteolytic Enzymes

The plasma membrane of the mouse egg appears to be sensitive to proteolytic enzymes. When unfertilized eggs are freed from zonae pellucidae by treatment with pronase or chymotrypsin, the resulting zona-free

32

RYUZO YANAGIMACHI

eggs are less able to undergo fusion with spermatozoa than are mechanically denuded eggs (Wolf et a / . , 1976; Quinn and Stanger, 1981; Boldt and Wolf, 1985). The egg plasma membrane of the golden hamster, on the other hand, is relatively insensitive to proteolytic enzymes. Even after a 30-min treatment of previously denuded eggs with trypsin or pronase (0.1% solution, at 25-37"C), all of the eggs remain capable of fusing with spermatozoa (Hirao and Yanagimachi, 1978b). This difference between mouse and hamster could be due to inherent differences in the sensitivity of the egg plasma membrane to the cortical granule proteinase (Yanagimachi, 1981). B. Antimembrane Antibodies

Certain antibodies raised against sperm surface components inhibit sperm-egg fusion in the hamster (Tzartos, 1979; Oikawa and Suzuki, 19791, guinea pig (Huang et ul., 1981; Yanagimachi et d., 1981; Primakoff et al., 1985), and mouse (Saling and O'Rand, 1982). Those which block fertilization by inhibiting prefusion events (e.g., sperm capacitation, acrosome reaction, and interaction with egg investments) are not considered here. According to Saling et a/.(1983, 1985), a monoclonal antibody, M29, raised against epididymal mouse spermatozoa binds specifically to the equatorial segment of the mouse acrosome (presumably with the plasma membrane over the equatorial segment). In the presence of this antibody (0.2 mg purified IgM/ml), most spermatozoa are unable to fuse with the egg plasma membrane, although they can firmly attach (bind) to it. According to Matsuda et al. (1985), three monoclonal antibodies raised against hamster eggs bing to the egg plasma membrane and impair spermegg fusion. C. Proteinase Inhibitors

In the sea urchin, the presence of proteinase inhibitors in seawater does not inhibit sperm-egg fusion. However, it renders fertilization polyspermic by disturbing cortical granule exocytosis of the egg (Schuel et al., 1976; Schuel, 1984). Sperm-egg fusion in mammals is certainly possible in the presence of trypsin inhibitors (Saling, 1981; Yanagimachi, 1981; Fraser, 1982), but some investigators have reported that proteinase inhibitors reduce the incidence of successful fusion of acrosome-reacted spermatozoa with the egg plasma membrane (Wolf, 1977; Dravland and Meizel, 1982). According to Van der Ven et al. (1985), fusion of human spermatozoa with zona-free hamster eggs is inhibited by serine proteinase inhibitors. This is probably due, at least in part, to the inhibition of the acrosome reaction by these inhibitors.

1. SPERM-EGG FUSION

33

D. Dithiothreitol

Dithiothreitol (DTT) is a disulfide-reducing and sulfhydryl-maintaining reagent. Yanagimachi o r rrl. (1983) reported that acrosome-reacted guinea pig spermatozoa are unable to bind to the egg plasma membrane in the presence of I mM DTT. Spermatozoa collide with the egg plasma membrane repeatedly. but do not bind to i t . A few spermatozoa may remain on the egg plasma membrane for some time (up to I min), but they eventually detach and swim away. Consequently, none of the eggs are fertilized as long a s DTT is present in the medium. Reduced glutathione and cysteine act similarly, but they are less effective in this respect than DTT. E. Erythrosine and Related Dyes

According to Carroll and Levitan ( 1978a,b),micromolar concentrations oferythrosine B inhibit fertilization in a wide variety of both invertebrates ( e . g . . sea urchins and annelids) and vertebrates (fishes and frogs). These authors surmise that erythrosine B and related dyes prevent sperm-egg fusion by altering the electrostatic properties of the gamete membranes. Although Carrol and Wolf (1979) reported that sperm-egg fusion in the mouse is inhibited by high concentrations of erythrosine B (0.5-1 mM), Yanagimachi (198 I ) has been unable to confirm this in the hamster-. F. Cytochalasin

According to Longo (1978), sea urchin eggs treated with cytochalasin B at concentrations of 1-10 pg/ml (2-20 p M ) are activated by spermatozoa but fail to incorporate sperm heads into the cytoplasm. It is reasonable to assume that sperm-egg fusion has taken place, but, because the cytoskeletal (actin) system has become disorganized, the fertilization cone has failed to form, so that the sperm head is not taken up (Vacquier, 197%). Mouse (Longo, 1978) and hamster eggs (Yanagimachi, 1981) treated with cytochalasin B or D remain capable of fusing with spermatozoa. Perhaps the cytoskeletal system is not directly involved in the sperm-egg fusion per se. It must be pre- or postfusion events that are sensitive to reagents like cyochalasin. G. Other Reagents Other reagents reported to inhibit sperm-egg fusion in mammals include inhibitors of glycoprotein biosynthesis, amphomycin, UDP-glucose, and 2-deoxyglucose (Ahuja, 1985), Ouabain (Talansky e l (il., 1987), lysophosphatidylserine (Fleming and Yanagimachi, 19811, and the basic

34

RYUZO YANAGIMACHI

polycationic pol yamine, compound 48/80 (Fleming and Armstrong, 1985); La3+may possibly also inhibit sperm-egg fusion (Saling, 1982). Pretreatment of egg plasma membranes with plant lectins (Yanagimachi, 1981) or inclusion of Zn2+ in the insemination media (Aonuma et al., 1981) does not disturb sperm-egg fusion. X.

MECHANISM OF SPERM-EGG FUSION

Although purified phospholipid membranes are capable of fusing with one another, fusion of biological membranes appears to be regulated closely by a variety of membrane-associated proteins (Strittmatter et al., 1985). Among these are Ca2+-binding proteins and enzymes such as ATPases, proteinases, and phospholipases. Since we do not know much about the molecular organization of gamete membranes, a molecular model of sperm-egg fusion may be premature; nevertheless, a hypothesis will at least invite debate and stimulate study. For previously published discussions and hypothesis of sperm-egg fusion, readers are referred to Vacquier (1979b), Lop0 (1983), Gould-Somero and Jaffe (1984), Monroy (1985), and particularly Jaffe and Cross (1986). When examined by the freeze-fracture technique, the area of the sperm membrane about to fuse with the egg plasma membrane is free of intramembranous protein particles or has particle-free patches (Fig. 10). This does not mean that this membrane (or particle-free region thereof) is totally lacking in proteins. Surface glycoproteins associated with the outer leaflet of the membrane lipid bilayers are presumably present in this region. The fact that acrosome-reacted (guinea pig) spermatozoa are unable to bind to the egg plasma membrane in the presence of disulfidereducing agents (Yanagimachi et a / . , 1983) would suggest that the tertiary and quaternary structures of the surface proteins of gametes are of critical importance for a close and stable contact between sperm and egg membranes. Divalent cations, particularly Ca2+,aid in establishing close apposition of two membranes by neutralizing negative surface charges of the membranes. Some membrane proteins of the spermatozoon may be inserted in the quiescent egg plasma membrane, thereby perturbing membrane lipids (Fig. 19). Such proteins could be acrosomal bindin (Glabe, 1985) and lysin (Hong and Vacquier, 1986),which have fusogenic properties. They could be enzymes, such as phospholipase which acts on membrane phospholipids to produce membrane-perturbing compounds such as lysophospholipids and fatty acids. Successful insertion of fusogenic proteins may depend on the electric potential of the egg plasma membrane. It is possible that the insertion

35

1. SPERM-EGG FUSION

I +

=f--e -

FUSION

F I G , 19. A model of the mechanism leading to the fu\ion of sperm and egg mcnibrane\. (1) Sperm and egg membranes ( S and E) carry complementary molecules Ic and c ' ) . I n addition. the sperm membrane carries fuwgenic molecule\ tf). ( 1 1 ) I f the potential of fhe egg plasma membrane i s negative and the coniplcment;iry molecule\ of \perm and egg membrane\ fit with each other. the fusogenic \perm molecule\ are in\erted into the "quic\cent" egg plasma membrane. The membrane lipid perturbance caused by the sperm molecule\ and the phase separation o f the membrane lipid\ fhrough the action o l C a 2 - leads 10the furion of the two membranes.

occurs only when the potential is negative (Jaffe and Cross, 1986). Insertion of a fusogenic protein alone may not be enough to induce membrane fusion, however. Certain membrane phospholipids may have to be latcrally separated prior to fusion. Ca?' is known to play a key role in this process (Papahadjopoulos, 1978). In some membrane systems, Mg" is totally ineffective in causing phase separation but can still induce membrane fusion or facilitate fusion induced by Ca?+ (Duzgiine$. 198s). For sperm-egg fusion. at least in mammals, Ca?' and Mg" appear to work synergistically to support the fusion. AC K N O WLEDG M E N TS The preparation o f this chapter was .;upported by National Institutes of Health Grant HD03402. I am grateful to the following persons who provided valuable information about sperm-egg fusion in various animal species: Drs. J. Michael Bedford, Gary N . Cherr. Laurinda A . Jaffe. Chiaki Katagiri. Tadayuki Ohta. Fukashi Koyanagi (Okamoto). Kiyoshi Sano. Masao Sugiyama, Robert G. Summers. Norihiko Uto, and Tadashi S. Yamamoto. I am also grateful to thme who generously supplied the original micrographs for reproduction: D r j . Stephen J. Gaunt, David M. Phillips. Gerald Schatten, Fumie Suzuki. and Noriko Usui. Finally, t h a n k are due to Dr. Ruth G. Kleinfeld. Mr. T. Timothy Smith. and Mrc. Hiroko Yanagimachi for their help i n the preparation of the manuscript.

K E F E R E N C ES Afrelius. B . A . . Eliasson. R.. Johnsen. O., and Lindholmer, C. (1975).Lack ofdynein arms in immotile human spermatozoa. J . Cdl Biol. 66, 225-232. Ahuja. K. K. (1985). Inhibitors o f glycoprotein biosynthesis block fertiliLation in the hamster. G m r r e Re.\. 11, 179-189. Ailken. K . J.. Koss. A . . and Lees. M. M . (1983).Analysis ofsperm function in Kartegener's syndrome. Fcrril. Sreril. 40, 696-698.

36

RYUZO YANAGIMACHI

Aonuma. S . , Okabe, M., Kawaguchi, M., and Kishi. Y. (1981). Zinc effects on mouse spermatozoa and in-uitro fertilization. J . Rtlprod. Fertil. 63, 463-466. Austin. C. R. (1961). ”The Mammalian Egg.” Thomas. Springfield, Illinois. Austin, C. R., and Walton, A. (1960). Fertilisation. I n “Marshall’s Physiology of Reproduction” (A. S. Parkes. ed.), Vol. I , Pt. 2, pp. 310-416. Longmans, London. Bedford, J. M., and Cooper. G . W . (1978). Membrane fusion events in the fertilization of vertebrate eggs. Cell Stir$ Reu. 5, 65-125. Bedford. J . M., Moore, H. D. M.. and Franklin. L. E. (1979). Significance of the equatorial segment of the acrosome of the spermatozoon in eutherian mammals. E.rp. Cell Rr.s. 119, 119-126. Blandau. R. J . , and Odor, L. D. (1952). Observations on sperm penetration into the ooplasma and changes in the cytoplasmic components of the fertilizing spermatozoon in rat ova. Ferfil. Stevil. 3, 13-26. Boatman, D. E.. and Bavister, B. D. (1984). Stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators. J . Reprod. Fertil. 71, 357-366. Boldt, J . . and Wolf, D. P. (1986). An improved method for isolation of fertile zona-free mouse eggs. Comere Rrs. 13, 213-222. Carrol. E. J.. and Levitan, H. (l978a). Fertilization in the sea urchin, Stlon~.vloc~r~tirrorrrs piirpurlitus, is inhibited by fluorescein dyes. Deu. Biol. 63, 432-440. Carrol, E. J . . and Levitan. H . (1978b). Fertilization is inhibited in five diverse animal phyla by erythrosin B. Deu. Biol. 64, 329-33 I . Carrol. E. J.. and Wolf, D. P. (1979). Mouse egg penetration is inhibited by erythrosin B. GutncJte Rrs. I , 293-298. Cherr. G. N . , and Clark. W. H. (1982). Fine structure of the envelope and micropyle in the eggs of the white sturgeon, Acipenser troti.stnon/unu.s Richardson. Deu. Crowrh Djffr. 24, 341-352. Cherr, G . N.. and Clark. W. H. (1985a). Gamete interaction in the white sturgeonAc,ipiwser trtin.s/nntitunu.s: A morphological and physiological review. Enuiron. Biol. Fish. 14, I I 22. Cheer. G. N., and Clark, W. H. (198%). An egg envelope component induces the acrosome reaction in sturgeon sperm. J . Exp. Z o o / . 234, 7s-85. Collins, F.. and Epel, D. (1977).The role of calcium ions in the acrosome reaction of the sea urchin sperm. Exp. Cell ROS.106, 21 1-222. Colwin, A. L., and Colwin, L. H. (1964). Role of the gamete membranes in fertilization. I n “Cellular Membranes in Development” (M. Locke, ed.), pp. 233-279. Academic Press, New York. Colwin, L. H.. and Colwin, A. L. (1967). Membrane fusion in relation to sperm-egg association. I n ”Fertilization” (C. B. Metz and A. Monroy. eds.). Vol. I . pp. 295-367. Academic Press, New York. Cooper, G. W., and Bedford. J . M. (1971). Charge density change in the vitelline surfrlce following fertilization of the rabbit egg. J . Reprod. Fertil. 25, 431-436. Dan. J. C. (1954). Studies on the acrosone. 111. Effect of calcium deficiency. B i d . Bull. 107, 335-349. Dan, J . C. (1967). Acrosome reaction and lysins. I n “Fertilization” (C. B . Metz and A. Monroy, eds.). Vol. I , pp. 237-293. Academic Press, New York. Dravland, 3. E., and Meizel, S. (1982). The effect of inhibitors of trypsin and phospholipase A? on the penetration of zona pellucida-free hamster eggs by acrosome-reacted hamster sperm. J . Androl. 3, 388-395. Dunbar, B . S . , and Wolgemuth, D. J . (1984). Structure and function of the mammalian zona pellucida, a unique extracellular matrix. In “Modern Cell Biology” (B. H. Statier, ed.), Vol. 3, pp. 77-111. Liss, New York.

1. SPERM-EGG FUSION

37

DdzguneS. N. ( 1985). Membrane fusion. .S'/rhc.c,//. Bioc,/rc3/tr.11. 195-28'3 Ehensperger. E . . and Uarros. C . (19x4). Changes at the hamster oocyte ~ r f a c efrom the germinal vesicle stage to ovulation. Gttt,ic.tc Rr\. 9, 387-397. Elinson. R. P. (1975). Site o f sperni entry and ;I cortical contraction associated with egg . 257-268. activation i n the frog Rtrt7tr pipiem\. D c v . B i ~ l 47. Epel. D. (19x2). The physiology and chemistry o f calciuni during the fertilization o f eggs. 1)i: "Calcium and Cell Function" ( W . Y. Cheung, e d . ) . pp. 355-383. Academic Press. New York. Epel. D.. and Vacquier. V . D. ( 19781. Membrane fusion event5 during invertehrate fertiliration. Cell S r t r f : R ~ J L5,J . 1-63, Epel, I)., C r o s . N . l,., and Epel. N. (19771. Flagcllar motility i s not involved in the incorporation o f t h e sperm into the egg at fertilization. D c u . Grow//iDi'pr. 119, 15-21. Flechon. J . E.. and Pavlok. A . (1986). Ultrastructural study ofthe interactions and fusion o f rani spermatoroa with zona-free hamster oocyte5. Reprod. Nitrr. Dcu. 26, 999- 1008. Fleming. A . D., and Armstrong. D. T. ( 1 9 x 5 ) . Effects o f polyaniines upon capacitation and 233, 97-100. fertilization in the guinea pig. 1.Exp. Z(JO/. Fleming. A . D.. and Yanagimachi. R. (19x1). Effects o f various lipids on the aci-osome reaction and fertiliLing capacity o f guinea pig spermatozoa. with special reference to the possible involvement o f lysophospholipids in the acrosome reaction. G ( i ) t i e / e Re\. 4, 253-273. Franklin, L. E. (1965). Morphology o f gamete membrane fusion and of sperm entry into oocytes o f the sea urchin. J . Crll Biol. 25, XI-100, Fraser. L. R. ( 1982).p-Aminobenramidine. an aci-osin inhibitor. inhibits mouse sperm penctration o f the zona pellucida but not the acrosome reaction. J . Reprod. Fcrril. 65. 185194. Fraser. L. R. ( 1983). Potassium ions modulate expression o f mouse sperm fertilizing ability. acrosome reaction and hyperactivated motility i/r uitro, J . R ~ p r ( ~Pcrril. d. 69, 539-553. Freeman, G., and Miller. R. L. (1982). Hydroroan egg can only be fertilized at the site of polar body formation. Dcu. Biol. 94, 142-152. Gabel. A,. Eddy. E. M . . and Shapiro. M. (1979). After fertilization. sperm surface components remain a s a patch in sea urchin and mouse embryos. C e l l 18, 207-215. Gaddum-Rosse. P. (19x5). Mammalian gamete interactions: What can be gained from observations on living eggs? A m . J . Anrrt. 174, 347-356. Gaunt. S. J . (1983). Spreading of a sperm surf;ice antigen within the plasma membrane of the egg after fertilization i n the rat. 1.E t t r h r y d , E.Y[J.Morpliol. 75, 259-270. Glabe. C. G. (1985). Interaction of sperm adhe\ive protein. bindin. with phospholipid v e G cles. 11. Bindin induces the fusion o f mixed phase vesicles that contain phosphatidylcholine and phosphatidylserine in u i t m . J . C d l Biol. 100, 800-806. Glabe. C. G.. and Vacquier. V. D. (1977). Isolation and characterization o f the vitelline layer o f sea urchin eggs. J . Cell Biol. 75, 410-421. Glabe. C. G.. and Vacquier. V . D. (1978). Egg surface glycoprotein receptor for sea urchin sperm bindin. Proc,. Ntrtl. Ac.trd. .S(,i. U . S . A . 5, X81-885. Cordon. M.. Fraser. L. R.. and Dandekar. P. V. (1975). The effect of ruthenium red and concanavalin A on the vitelline surface o f fertilized and unfertilized rabbit ova. Antrr. R r t . . 181, 95-1 12. Gould-Somero, M.,and Jaffe. L. A . (1984). Control o f cell fusion at fertilization by mcmbrane potential. In "Cell Fusion: Gene Transfer and Transformation" (K.F. Beers and E. G. Bassett. eds.). pp. 27-38. Raven. New York. Gundersen. G . G . , and Shapiro. B. M. (1984). Sperm surface proteins persist after fertiliiation. J . Cell Riol. 99, 1343-13.53, Gundersen, G . G.. Medili. L.. and Shapiro. B. M . (1986). Sperm surface protein5 are

38

RY UZO YANAG IMACH I

incorporated into the egg membrane and cytoplasm after fertilization. Deu. B i d . 113, 201-2 17. Gwarkin. R . B. L., Williams, D. T., Hartmann. J . F., and Kniazuk, M. (1973). The zona reaction of hamster and mouse eggs: Production in uitro by a trypsin-like protease from cortical granules. J . Reprod. Frrfil. 32, 259-265. Hanada, A, , and Chang, M. C. (1972). Penetration of zona-free eggs by spermatozoa of different species. B i d . Rrprod. 6, 300-309. Hinkley. R . E.. Wright, B. D.. and Lynn. J. W. (1986). Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342. Dru. B i d . 118, 148-154. Hirao, Y., and Yanagimachi, R . (1978a). Temperature dependence of sperm-egg fusion and post-fusion events in hamster fertilization. J . Exp. Zoo/. 205, 433-438. Hirao, Y., and Yanagimachi, R . (1978b). Effects of various enzymes on the ability of hamster egg plasma membrane to fuse with spermatozoa. Gmnere Rrs. 1, 3-12. Hoffman, M. L.. and Curtis, G . L. (1984). Prevention of monkey sperm penetration of zonafree hamster ova by sperm antibody obtained from vasectomized cynomolgus monkey. F e r / i / . Sterd. 42, 108- I 1 I . Hong, K.. and Vacquier, V . D. (1986). Fusion of liposomes induced by a cationic protein from the acrosomal granule of abalone spermatozoa. B i d m i s f r y 25, 543-549. Hoshi. M. (1985). Lysins. 111 "Biology of Fertilization" (C. B. Metz and A. Monroy, eds.), Vol. 2. pp. 431-462. Academic Press, Orlando, Florida. Huang. T . T . F., and Yanagimachi, R . (1985). Inner acrosomal membrane of mammalian spermatozoa: Its properties and possible functions in fertilization. A m . J . Ancrt. 174, 249-268. Huang, T. T. F . , Tung. K. S. K . , and Yanagimachi, R . (1981). Autoantibodies from vasectomized guinea pig inhibit fertilization in uifro. Science 213, 1267- 1269. Hubbs, C. L. (1955). Hybridization between fish species in nature. S y s f . Zoo/. 4, 1-20, Hultin. T. (1948). Species specificity in fertilization reaction. 11. Influence of certain factors on the cross-fertilization capacity of Arhuciri lixulrr. Ark. Zoo/. Scund. 40A, 1-8. Hylander, B. L . . and Summers, R . G. (1977). An ultrastructural analysis of the gametes and early fertilization in two bivalve molluscs, Chrrrncr mrrc~erop/iy//uand S p i s r h S(J/idksirncr with special reference to gamete binding. Cell T i s s ~ eR P S .182, 469-489. Iwao. Y., and Katagiri, C. (1982). Properties of the vitelline coat lysin from toad sperm. J . Exp. Zoo/. 219, 87-95. Jaffe. L. A. (1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature (London) 261, 68-71. Jaffe, L. A., and Cross, N . L. (1986). Electrical regulation of sperm-egg fusion. Annrr. R c u . Physiol. 48, 191-200. Jaffe, L. A., and Gould. M. (1985). Polysperniy-preventing mechanisms. In "Biology of Fertilization" (C. B. Metz and A. Monroy, eds.). Vol. 3, pp. 223-250. Academic Press, Orlando, Florida. Jaffe, L. A. , Sharp, A. P., and Wolf, D. P. (1983). Absence of an electrical polyspermy block in the mouse. Deu. B i d . 96, 317-323. Jensen. A. B. (1953). The effect of trypsin on the cross-fertilizability of sea urchin eggs. Exp. Cell Res. 5 , 325-328. Johnson, M. H . , Eager, D., Uggleton-Harris, A., and Grave, H. M . (1975). Mosaicism in organization of concanavalin A receptors on surface membranes of mouse eggs. Nrrtrtre (London) 257, 321-322. Kille, R . A. (1960). Fertilization of the lamprey egg. Exp. C r / /Res. 20, 12-27. Kobayashi, H. (1963). Some cytological observations on fertilization in the Ioach (female)funa (male) cross. J p n . J . Genet. 38, 113-122. Kobayashi, W., and Yamamoto, T. S . (1981). Fine structure of the micropylar apparatus of

1. SPERM-EGG FUSION

39

the chum salmon egg, with ii discussion of the mechanism for blocking polyspermy. J , E x p . Zoo/. 217, 265-275. Kobaya5hi. W . . and Yamamoto. T. S. (1987). I*ight and electron microscopic observations of sperm-entry in the chum salmon egg. J . E.vp. Zoo/. 243, 31 1-322. Koehler, J.. Clark. J . M., and Smith, D. (1985). Freeze-fracture observations on mamnialian oocytes. A m . J . Anu!. 174, 317-329. Kudo, S. ( 19x0). Sperm penetration and the formation of a fertilimtion cone in the common carp egg. D P U .Gron.th DiffCJr. 22, 403-414. Kyoruka, K., and Osanai. K. (1979). Heterospermic fertilization of sea urchin eggs pretreated with pancreatin. B i t / / . Mar. Biol. S t n . AS L J ~ ?J M Tohoku .S/I~ Univ. 16, 179- 188. Longo, F. J . ( 1977). An ultrastructural study of cross-fertilization (Arbuciu female x M.~ti/trs male). J . Cell Eiol. 73, 14-26. Longo, F. J. ( 1978). Effects of cytochalasin B on sperm-egg interactions. Deu. Biol. 67, 249-265. Lopo, A. C. (1983). Sperm-egg interactions in invertebrates. I n “Mechanism and Control of Animal Fertilization” (J. F. Hartmann. ed.). pp. 269-324. Academic Press. New York. Lopo, A. C.. and Vacquier, V. D. (1981).Gamete interaction in the sea urchin. I n “Fertilization and Embryonic Development in Virro” ( L . Mastroianni and J . D. Biggers. edh.), pp. 199-232. Plenum. New York. Lucy. J . A. (1978). Mechanisms of chemically induced cell fusion. Cell Surf: R P U .5, 268304. Maddock, M. B . . and Dawson. W . D. (1974). Artificial insemination of deermice with sperm from other rodent species. J . Emhryol. Exp. Morphol. 31, 621-634. McCulloh. D. H.. Rexroad. C. E.. and Levitan. A. (1983). Insemination of rabbit egg in associated with slow depolarization and repetitive diphasic membrane potentials. Dcu. Bin/. 95, 372-377. McGath, J . . and Hillman, N. (1980). Sterility in mutant ( i l “ / i l ‘ )male mice. 111. In uiiro fertilization. J . Emhnol. Exp. Morphol. 59, 49-SX. Mann. S . . Schatten. G . , Steinhardt. R . , and Friend, D.S. (1976). Sea-urchin sperm: oocyte interaction. J. Cell Biol. 70, I10a (Abstr. No. 330). Matsuda. T . , Komori, K . . Funauchi. H.. Shigeta. M.. Koyama. K.. and Isojima. S. (1985). Blocking effect of monoclonal antibodies to oocyte (zona pellucida and plasma membrane) on in uitro fertilization in hamster. Ac.fc4 Ohsre!. Gyrrwol. Jpn. 37, 2615 (Abstr. No. 398). Metz, C. B.. and Monroy. A , , eds. (1967). “Fertilization.” Vol. I. Academic Press, New York. Metz, C . B., and Monroy. A , . eds. (1969). “Fertilization.” Vol. 2. Academic Press. New York. Metz, C. B., and Monroy, A., eds. (1985). ”Biology of Fertilization,” Vols. 1-3. Academic Press, Orlando, Florida. Miller, R. L. (1978). Site-specific sperm agglutination and time related release of a hpermchenio-attractant by the egg of the Leptomedusan, 0rihopy.ri.s (.u/icu/u!u.J . E.rp. Z o o / . 205, 385-392. Miyazaki. S.. and Igusa. Y. (1981). Fertilization potential in golden hamster eggs consists of recurring hyperpolarization. Nurrtrc (London) 290, 702-704. Miyazaki. S . , Hashirnoto. N., Yoshimoto. Y.. Kishimoto, T., Igusa, Y., and Hiramoto. Y. ( 1986). Temporal and spatial dynamics of the periodic increase in intracellular free calcium at fertilization of golden hamster egg. Deu. B i d . 118, 259-267. Moenkhaus, W. J . (1910). Cross-fertilization among fishes. Proc. Indiun Al’Nd.Sc,i. 353-393. Mohri. H., Usui. N . , and Sano, K. (1982). Magnesium ions in fertilization of sea urchins. Cell Difier. 11, 259-260.

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Sano. K.. and Kanatani. H. (1980). External calcium ions are requisite for fertilization o f w a urchin eggs by spermatoroa with reacted acrowmes. Drv. Uiol. 78, 242-246. Sano. K., Usui. N.. Ueki. K.. Mohri. T.. and Mohri. H. (1980). Magnesium ion-requiring step in fertilization of sea urchins. DcrI. G r o i i ~ t l iDiffcr. 22, 531-541. Sato. K.. and Blandau. R. J . (1979). Time and process of sperm penetration into cumulusfree mouse eggs fertilized in uitro. G e i m ~ t cRas. 2, 295-304. Sato. M . . and Osanai, K. (1986). Morphological identification of sperm receptor\ above egg microvilli in the polychaete. N W I I / / W ,Sj q ~ o t i i w Doti. . Biol. 113, 263-270. Schatten. G . . and Mazia. D. (1976). The penetration of the spermatozoon through the wii urchin egg surface at fertilimtion. Exp. Cell Rc~s.98, 325-337. Schmidt, T., Patten, C.. and Epel. D. (1982). Is there a role for the Ca'* inHux during fertilization of the sea urchin egg'? Dcu. Riol. 90. 284-290. Schuel, H. (1984).The prevention of polyspermic fertilization in sea urchins. B i d . Bull. 167, 27 1-309. Schuel, H., Longo. F. J . , Wilson. W. L... and Troll. W. (1976). Polyspermic fertilziation of \ea urchin eggs treated with protease inhibitors: Localization of sperm receptor \ires at the egg surface. Deu. Biol. 49, 178-184. SeGall, G . K . . and Lennarz. W. L. (1979). Chemical characterization of the component of thejelly coat from sea urchin eggs responsible for induction of the acrosome reaction. f)<Ju. B i ~ l71, . 33-48. Seifriz. W. ( 1927). Protoplasmic papillae of Ec~/ii~iurrr~./iniit.s oocytes. Pro/oplrr.snicr 1. 1-4. Shalgi. R . , and Phillips, D. (1980). Mechanic5 of sperm entry in cycling hamsters. J . U//ru,stritct. Res. 71, 154-161. Strittmatter. W. J.. Couch. C. B . , and Mundy. 0 . I . (1985). Role of proteins in the fusion of biological membranes. Mev?hr. Flitid. Biol. 4. 259-291. Sugiyama. M . (1951). Re-fertilization of the fertilized eggs of the sea urchin. R i d . Bttll. 101, 335-344. Summers, R. G.. and Hylander. B . L. (1974). A n ultrastructural analysis of early fertiliution in the sand dollar, Echincrrrrc./iniu.s ptrrniti. Cell Tis.\ur Rc,.s. 150, 343-368. Summers. R . G., and Hylander. B. L. (1975). Species-specificity of acrosome reaction and primary gamete binding in echinoids. Exp. C P / /R P S .96, 63-68. Suzuki, R. (1964). Hybridization experiments in cyprinid fishes. VI1. Reciprocal crosses between Psrudogohio esocinrts and B i i i , ~ i ( :i r z r r ~ ~ (Jip. n . J . f c , / i t / i y o / .12, 18-22. Takahashi, Y . M., and Sugiyama. M. (1973). Relation between the acrosome reaction and fertilization in the sea urchin. 1. Fertilization in Ca-free seawater with egg-water-treated spermatozoa. Dru. Gron~rhD(fjrC.r.15, 261 -267. Talansky, B. T . , Berg, P. E., and Gordon, J . E. (1987). Ion pump ATPase inhibitors block the fertilization of zona-free mouse oocytes by acrosome-reacted spermatozoa. J . Reprod. Frrtil. 19, 447-455. Tyler, A , , Monroy. A.. and Metz. C. B. (1956). Fertilization of fertilized sea urchin egg. Biol. B i t / / . 110, 184-195. Tzartoz, S. J . (1979). Inhibition of in uitro fertilization of intact and denuded hamster eggs by univalent anti-sperm antibodies. J . Rrproil. Fertil. 55, 447-455. Usui. N . . and Yanagirnachi, R. (1976). Behavior of hamster sperm nuclei incorporated into eggs at various stages of maturation, fertilization and early development. J . L///rci.s~rircr. Rr.s. 57, 276-288. Usui, N..Sano, K., and Mohri, H. (1980). The surface events at fertilization of the sea urchin egg. I . Events on the surface of the vitelline coat. Dru. Grow//i DIffrr. 22, 461473. Vacquier, V. D. (1979a). The fertilizing capacity of sea urchin sperm rapidly decreases after induction of the acrosome reaction. Deu. Growt/i Differ. 21, 61-69.

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proteinase in mammalian and avian spermatoroa by ;I silver proteinate method. Biol. R l > [ l i l J d . 6 , 87-97. Yanagimachi. R.. Yanagimachi. H.. and Rogers. B . J . (1976). The use of rona-free animal o v a as a test system for the assessment of the fertiliLing ctipacity of human spermalo/.oil. Biol. Rcprod. 15, 47 1-476. Yanagimachi. R.. Miyashiro, L. H . . and Yanagimachi. H . (1980). Reversible inhibition of ~ i h 22, 281-288. sperm-egg fusion in the hamster by low pH. I1r.u. G i o ~ ~Dlffir. Yanagimachi. R.. Okada. A , . and 'rung. K. S . K. (1981). Sperm autoantigens and fertilization. 11. Effects of anti-guinea pig autoantibodie\ o n sperm-ovum interaction>. Biol. ~ r p ~24, d 5. I 2-5 I 8 . Yanagimachi, R..Huang. T. 'I. F..Fleming. A . D.. Kosower. N . S . , and Nicolson. 6. 12, (1983). Dithiothreitol, a disulfide-reducing agent. inhibits capacitation. iicrosome reaction and interaction with eggs by guinea pig spermatozoa. GcitniJreRrs. 7, 145-154. Yoshizaki. N . . and Katagiri. C. (1982). Acrosonie reaction in sperm of the toad BI&J h j i ) ,jrr/xJnic~cc.(;trtiirri~Rc>.s.6, 343-352.

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CURRENT TOPICS I N M E M B R A N E S A N D TRANSPORT. VOL.CMC.

i?

Chapter 2 Cortical Exocytosis in the Sea Urchin Egg ROBERT C . JACKSON A N D JOSEPH H . CRABB' Deprirttnttit OJ' Bioclietwistry Dcirttnorrth Mcdicwl School Hutiourr, Nenj Hlitnpshirc, 037.56

I. Introduction A. Exocytosis in Perspective

B. Cortical Exocytosis in the Sea Urchin Egg The Calcium Signal A . Phosphoinositide Metabolism and Signal Transduction B . The Role of G Proteins 111. I,r Vitro Models of Exocytosis A . Permeabilized Cell Sy\tems B . Cell-Free Systems C. Egg Cortex as an ;,I uiiro Model of Ca!--Triggered Exocytosis D. Hypothetical M ~ d e l s 1V. Conclusions V . Addendum A. Sea Urchin Egg B. Other Cell Types References 11.

I.

INTRODUCTION2

The initiation of new life at fertilization is an event of profound significance. for both the individual and the species. For the individual, fertilization marks a beginning; for the species, it represents continuance: for the I Present address: Channing Laboratory, Harvard Medical School. Boston. Mahsachusetts 011 15. Abbreviations: CL, cortical lawn: CSC. cell surface complex: CV. cortical vesicle: DAG, diacylglycerol; FE. fertilization envelope: IP,, inositol 1,4.5-1risphosphate: NEM. N ethylmaleimide: PI, phosphatidylinositol; PIP. phosphatidylinositol 4-phosphate: PIP?. phosphatidylinositol 4.5-bisphosphate; PM. plasma membrane.

45

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ROBERT C. JACKSON AND JOSEPH H. CRABB

cell biologist, it comprises a unique and often advantageous system for analyzing basic cellular phenomena. Exocytosis, the phenomenon in which secretory proteins and stored effector molecules are delivered to the cell surface and beyond, is a process that can be conveniently studied during fertilization, particularly in organisms (e.g., the sea urchin) that produce an abundance of gametes. In this chapter we focus on the process of cortical exocytosis in the sea urchin egg. Two areas will be examined in detail: (1) the mechanism of generation of the intracellular calcium signal and (2) the use of egg cortex as an in uitro model for investigation of the molecular mechanism of the terminal stages of exocytosis. We have not attempted to present an exhaustive review of the literature on cortical exocytosis, rather we have tried to emphasize results obtained within the last 5 to 7 years in relation to what is known from other exocytotic systems. Other recent reviews have dealt with the biology of fertilization in the sea urchin (Guidice, 1986; Shapiro et al., 1981), and with the structure of cortical secretory vesicles and their role in formation of the fertilization envelope (Epel, 1978, 1982; Epel and Vacquier, 1978; Kay and Shapiro, 1985; Schuel, 1985). A. Exocytosis in Perspective

Exocytosis is, of course, the final step in the secretory pathway; however, it is also representative of the more global phenomenon of vesicular transport, in which molecules entering and exiting the cell, as well as those destined for particular organelles, are transported within membrane-bound vesicles. This process of vesicular transport insures that these molecular “passengers” do not admix with cytoplasmic “residents,” and thus allows remarkably efficient segregation and sorting of incoming (endocytic) and outgoing (exocytic) traffic. Vesicular transport occurs in three stages: budding of a vesicle from the compartment of origin, translocation of that vesicle through the cytoplasm, and fusion of the vesicle with the destination compartment. 1. CONSTITUTIVE VERSUS REGULATEDSECRETION

During exocytosis, the compartment of destination is the extracellular space. Virtually all eukaryotic cells use this mechanism to externalize newly synthesized secretory and plasma membrane (PM) proteins (Kelly, 1985; Tartakoff and Vassalli, 1977, 1978). Secretion from cells on a bulkflow basis, as the proteins are synthesized, is termed constitutive secretion, while exocytosis in direct response to a stimulus at the cell surface is

47

2. CORTICAL EXOCYTOSIS

called regulated secretion. The regulated form of secretion seems to have evolved for the sole purpose of rapidly externalizing large quantities of physiologically important mediators such as hormones. enzymes, neurotransmitters, and immunological effectors in response to specific stimuli. It is marked by the accumulation and storage of secretory vesicles within the cytoplasm. During storage, the secretory vesicles develop a characteristic dense core of secretory product, concentrated some 10- to 200fold over that of constitutive secretory vesicles (Bendayan et d,, 1980; Salpeter and Farquar, 1981). 2. STIM U L US-SECRETION COUPLING The role of Ca2+in regulated secretion was first recognized in the early 1960s by Douglas, who coined the term stimulus-secretion coupling to denote its similarity to the Ca?+-dependentprocess of excitation-contraction coupling (Douglas and Rubin, 1961, 1963). Douglas' description of stimulus-secretion coupling, in conjunction with ultrastructural studies of Palade and co-workers (1975) established the general scheme for regulated secretion shown in Fig. I : Upon stimulation, the cytoplasmic concentration of free calcium ion ([Ca?+],) transiently rises, from resting levels of approximately lo-' M to several micromolar (Rubin, 1982). At about the same time, the secretory vesicles are translocated from the cytoplasm to the plasma membrane and become closely apposed (or at-

@ FIG. I . Regulated secretion. ( I ) Stiumulation or activation through receptor-ligand interaction. (2) Rise in intracellular [Ca?+].(3) Vesicle translocation. (4) Vesicle attachment (close apposition). ( 5 ) Membrane fusion. (6) Discharge and dispersal of vesicle contents.

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ROBERT C. JACKSON AND JOSEPH H. CRABB

tached) to it. Next, the bilayers of the vesicle and the plasma membrane fuse, establishing continuity between the two membranes and joining the intravesicular and extracellular compartments. This allows the vesicle contents to be discharged and dispersed, while maintaining the membrane topology and the all-important barrier between the cytoplasm and the extracellular milieu. While it is apparent that this process occurs via an interdependent progression, there are basically three discrete stages which can be recognized. These are signal generation, vesicle translocation, and fusion; corresponding to the initial, intermediate, and final stages. It has proved difficult to assign particular biochemical requirements to discrete steps in this pathway, since the stages cannot be independently manipulated in whole cell studies. This is where an in vitro approach can be most useful. In uitro reconstitution of the individual steps along this pathway should permit unambiguous assessment of the requirements and properties of each step. B. Cortical Exocytosis in the Sea Urchin Egg

For discussion purposes, fertilization in the sea urchin can be thought of as a three-step process. The first step consists of sperm activation and gamete binding. Gamete fusion comprises the second step. The third step involves activation of the metabolism of the dormant egg cell. The cortical reaction of the egg occurs immediately after gamete fusion. It is one of two exocytotic events that occur during the fertilization sequence; the other, the acrosome reaction of the sperm, occurs during sperm activation. The unique morphology of the sea urchin egg is the feature that makes it such a useful system for studying the terminal events in exocytosis. The secretory vesicles of most cells must move from the cytoplasm to the plasma membrane before they can fuse and release their contents into the extracellular medium. In the sea urchin egg, as in the egg cells of most other species (Anderson, 1974; Gulyas, 1980), specialized secretory vesicles called cortical vesicles (CVs) become firmly attached to the PM during oogenesis (Anderson, 1968; Detering et al., 1977; Longo, 1981; Vacquier, 1975). This specialization of the egg has two practical consequences: it simplified exocytosis by effectively eliminating the translocation and attachment steps (steps 3 and 4 in Fig. l ) , and it permits the isolation of egg cortex fractions in which the CVs remain bound to the PM and retain their exocytotic capacity. The morphology of the cortical region of a sea urchin egg is shown in Fig. 2. Cortical vesicles in this particular species (Strongylocentrotus prrrpuratus) are approximately 1 p m in diameter and consist of a mem-

FIG.2. Electron micrograph of the cortical region of an unfertilized sea urchin egg (S. p / o p r m u s ) . Cortical vesicles lie in a monolayerjust beneath the plasma membrane and have a characteristic spiral lamellar core. The vitelline layer (arrow)is the thin. fuzzy structure coating the extracellular surface of the plasma membrane. Bar. 0.5 ~ I I I (Reproduced . from Chandler and Heuser, 1979. with permission. )

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ROBERT C.JACKSON AND JOSEPH H. CRABB

brane-encapsulated spiral lamellar core of condensed proteins and mucopolysaccharides. The entire cytoplasmic surface of the PM is covered with a densely packed array of CVs. This can best be appreciated in light micrographs of fragments of egg cortex (see Fig. 3B in Section lll,C,l). It has been estimated that a single egg cell contains approximately 15,00018,000 CVs and that fusion of the CVs with the PM during cortical exocytosis more than doubles the surface area of the egg (Schroeder, 1979; Vacquier, 1975; Zimmerberg and Whitaker, 1985). The egg PM is comprised of a typical phospholipid bilayer, but it is covered with the vitelline layer, an extracellular glycocalyx, which contains the sperm receptors of the egg (Shapiro et al., 1981). Filamentous structures are abundant on the cytoplasmic surface of the egg cortex, but the specific nature of the linkage between the CVs and the PM is not known (see Section III,C,6). When an egg is penetrated by a sperm at fertilization, the CVs undergo a synchronous Ca*+-regulated exocytotic reaction and release their contents into the perivitelline space (i.e., the narrow channel between the plasma membrane and the vitelline layer). The primary function of the cortical reaction is to produce the fertilization envelope (FE), a tough, extracellular investment that establishes a permanent block to polyspermy and shields the developing embryo from environmental insult. The FE is formed by the combination of CV content proteins with the vitelline layer (Kay and Shapiro, 1985). Cortical vesicles contain a variety of constituents, most of which are thought to participate in FE formation. A serine protease(s) contained within the CV may be responsible for destroying sperm receptors and detaching the vitelline layer from the PM (Alliegro and Schuel, 1985; Carroll and Epel, 1975; Fodor et ul., 1975; Sawada et al., 1984). Proteoliaisin, a recently purified CV protein, is thought to form a major structural component of the FE (Weidman et al., 1984). Ovoperoxidase, a major CV enzyme, cross-links FE proteins via their tyrosine side chains and thereby hardens the so called “soft” FE that is initially formed (Deits et al., 1984; Foerder and Shapiro, 1977; Hall, 1978). Hydration of CV mucopolysaccharides within the perivitelline space is thought to elevate the detached FE from the egg surface (Schuel, 1978; Schuel et al., 1974). Hyalin, a high molecular weight CV glycoprotein, comprises the major constituent of the hyaline layer which forms in the space between the egg and its FE. The hyaline layer is important in blastomer adhesion in the early embryo (Hylander and Summers, 1982; Kane, 1970; McClay and Fink, 1982; Stephens and Kane, 1970). P-Glucanase, a CV enzyme capable of hydrolyzing terminal p-1,3-linked glucosides, has been purified, but

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its function remains unknown (Epel et al., 1969; Muchmore er ul., 1969; Talbot and Vacquier, 1982). II. THE CALCIUM SIGNAL There is ample evidence that exocytosis is the direct result of an increase in the cytoplasmic concentration of free Ca2+to the micromolar level. Exocytosis can be initiated by treating intact cells with Ca?? ionophores (Chambers er d., 1974; Forman et ul., 1973; Rasmussen and Goodman, 1977; Steinhardt and Epel. 1974), by the direct intracellular microinjection of CaZ+buffers (Gilkey. 1983; Hamaguchi and Hiramoto, 1981; Hollinger and Schuetz, 1976; Hollinger et a l . , 1979; Kanno et ul.. 1973; Miledi, 1973), or by bathing permeabilized cells in a Ca?+-containing medium (Baker and Knight, 1981; Dunn and Holz, 1983; Knight and Baker, 1982; Knight and Scrutton, 1980; Wilson and Kirshner, 1983). Exocytosis has been shown to be preceded by increases in [Caz+],,and inhibition of the [Caz+],transient by microinjection of EGTA has been shown to block exocytosis in medaka and sea urchin eggs (Gilkey. 1983; Zucker and Steinhardt, 1978). The “wave” of elevated [Ca2+],that accompanies exocytosis was directly detected for the first time by microinjection of the Car+-sensitive photoprotein aequorin into egg cells. In the egg of the medaka fish, the wave of increased [CaZ+], was shown to begin at the point of sperm entry and proceed across the egg within about 2 min (Gilkey et al., 1978). The maximal [Ca”], reached was estimated to be approximately 30 p M . In similar studies with the sea urchin egg. a peak [CaZ+J, of approximately 5 p M was reached within I min after fertilization (Steinhardt el a / . , 1977). In a more recent study, Busa and Nuccitelli (1985) used microelectrode methodology and found that the subcortical [Ca?’], in Xenopus eggs rose from resting levels of 0.40 pM to a peak concentration of 1.2 5 0. I5 p M within 2 min, and returned to prefertilization values within 12 min. In recent years, the development of Ca’+-sensitive fluorescent dyes (quin2 and fura2) that can be loaded into cells as acetoxymethyl esters, and trapped in the cytoplasm by the action of cellular esterases, has revolutionized the measurement of intracellular Car+in smaller cells that cannot be easily microinjected (Grynkiewicz et a l . , 1985; Tsien, 1980, 1983; Tsien et u l . , 1984).This technology has stimulated the accumulation of a wealth of data demonstrating that intracellular Ca?+transients, with peak [Car+],in the micromolar range, are associated with regulated secretion in a wide variety of cells, including platelets, neutrophils, adrenal

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ROBERT C. JACKSON AND JOSEPH H. CRABB

chromaffin cells, GH3 and primary pituitary cells, PC12 cells, mast cells, and pancreatic cells (Tsien et al., 1984; White et u l . , 1984). The use of quin2 and fura2 in invertebrate eggs has been limited because the relatively low temperatures needed to maintain healthy eggs apparently interferes with the efficient loading of these cells with the “membrane permeant” acetoxymethyl derivatives (Tsien, 1983). Thus, it has been necessary to microinject quin2 and fura2 into eggs. Utilizing this technique Poenie et al. (1985) were able to trace [Caz+Iifluctuations in Lytechinus pictus eggs. They found that the Caz+transient at fertilization peaked at 1.95 -+ 0.16 puM, a value consistent with the earlier aequorin studies of Steinhardt et al. (1977) and the recent microelectrode results of Busa and Nuccitelli ( 1985). A. Phosphoinositide Metabolism and Signal Transduction

An explosion of information has occurred in the last 5 years on the role of a minor class of plasma membrane phospholipid, the phosphoinositides (PIS) [including phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol4,S-bisphosphate(PIPI)],in conversion of receptor-mediated signals into intracellular messengers. The PI cycle has been exhaustively reviewed in recent literature (Berridge, 1985; Berridge and Irvine, 1984; Nishizuka, 1984a), and interested readers are directed to these sources. While the details of the PI cycle are beyond the scope of this chapter, it is worth noting that, in brief, PIP and PIP, derive from PI via sequential phosphorylations by specific kinases (Nishizuka, 1984a).Their hydrolysis by receptor-linked activation of PI-specific phospholipase C (also called polyphosphoinositide phosphodiesterase) initiates a metabolic cycle, the products of which serve as intracellular messengers before they are reutilized to replenish PI in the plasma membrane. PIP2,which represents only a few percent of the total PIS in the plasma membrane, is probably the most important of the PIS with regard to intracellular signaling. I . PI-DERIVED SECOND MESSENGERS Receptor-mediated hydrolysis of PIPz by PI-specific phospholipase C generates two bona fide second messengers, inositol 1,4,5-trisphosphate (IPJ and diacylglycerol (DAG). Nishizuka and co-workers (1984b) established that DAG was the endogenous analog of the tumor-promoting phorbol esters and acted intracellularly by activating a Ca2+and phospholipidsensitive protein kinase (protein kinase C). Recent results suggest that

2. CORTICAL EXOCYTOSIS

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protein kinase C may act synergistically with Ca2+to maximize the exocytotic response (Baker, 1984). In fact, studies combining the use of quin2 and phorbol esters revealed that in certain cells, particularly platelets and neutrophils, treatment with phorbol esters can obviate the requirement for a rise in [Ca2+Jito elicit exocytosis (Baker, 1984; Di Virgilio et al., 1984; Kaibuchi et a / . , 1983; Rink rt a / . , 1983). This indicates that, while elevated [Ca2+]iseems to be sufficient to elicit exocytosis in most cells, it may not be necessary in all cases. Berridge, working on the soluble constituents of PIPz breakdown, first showed that IP3was generated with sufficient rapidity to precede the Ca2+ transient (Berridge. 1983). Next, it was demonstrated that IP3was capable of causing the release of Cali from intracellular stores in permeabilized pancreatic acinar cells and from rat insulinoma microsomes (Prentki r t a l . , 1984; Streb et d., 1983). These results laid the groundwork for subsequent elucidation of the pivotal roles played by PI-derived second messe ngers . The involvement of PIP2 turnover in the generation of the Ca” transient during fertilization was confirmed in a series of reports on activation of sea urchin and Xerioprrs eggs. Turner et a / . (1984) obtained early hints in this regard by demonstrating that the amount of PIP and PIP2increased within IS sec after fertilization, preceding the cortical exocytotic wave. Kame1 et cil. (1985) confirmed Turner’s results, and showed in addition that the amount of PI in an egg declines by about SO95 within 30 sec of fertilization, then rapidly returns to its original value by 2-5 min postinsemination. Concomitant with this decrease in PI. there is a 5-fold increase in [!H]IP3 production (in cells prelabeled with [3H]inositol)during the first 10 min postinsemination. Both sets of results are consistent with the interpretation that PIS are becoming ”primed” for use in egg activation. Whitaker and Irvine (1984) established that IP3 acts as a second messenger in egg activation, by demonstrating that microinjected 1P3 was capable of initiating a cortical exocytotic wave identical to that seen during fertilization. Moreover, they observed that micromolar Ca2+induced PIP, hydrolysis in egg cortex preparations. Clapper and Lee (1985) and Oberdorf et a / . (1986) have since shown that 1P3 is able to stimulate the release of stored Ca2+in egg homogenates and cortex preparations, respectively. Together, these results suggest that the exocytotic wave following sperm binding is the result of an autocatalytic event beginning with receptor-mediated generation of IPj, followed by local Ca” release, further IP3 generation. etc., leading to propagation of the signals around the circumference of the egg.

ROBERT C. JACKSON AND JOSEPH H. CRABB

54

Utilizing double-barreled electrode technology, Busa et al. (1985) were able to directly demonstrate increased [Ca2+]ifollowing IP3 microinjection into Xenopus eggs. Examination of the spatial IP, requirements within the egg indicated that IP3 was most effective when injected into the cortex as opposed to deep within the cytoplasm (Busa et al., 1985). This latter result is consistent with the observation that Xenopus eggs have an extensive cortical reticulum that is thought to contain the Ca2+stores used in cortical exocytosis (Charbonneau and Grey, 1984). Sardet (1984) has observed a similar, though less extensive cortical reticulum in S . purpuratus egg cortex preparations. 6. Role of

G Proteins

While the evidence implicating the PI cycle in intracellular signaling was growing to encompass an increasing variety of cells, independent lines of research were being conducted to determine the link between cell surface receptors and activation of phospholipase C. These studies recognized similarities between PI-mediated signaling and the adenylate cyclaselcyclic AMP system which involves the action of guanyl nucleotidebinding regulatory proteins (G proteins) (Berridge, 1985; Gilman, 1984). In the adenylate cyclase system, there are two G proteins, G, and Gi, which operate through receptors that cause the stimulation and inhibition of adenylate cyclase activity, respectively (Gilman, 1984). These G proteins share many biochemical features. They both consist of three subunits, a,p, and y , with molecular weights of 39,000-45,000, 35,000, and 10,000, respectively. The a subunit is responsible for interaction with adenylate cyclase and contains a high affinity binding site for the guanine nucleotides GTP and GDP as well as a latent GTPase activity. The a subunits also contain sites for NAD-dependent ADP ribosylation by either cholera toxin or islet-activating protein (IAP, a Bordetefla pertussis toxin). The p subunit serves as a regulatory subunit, maintaining the (Y subunit in the resting, or deactivated, state, while the y subunit is uncharacterized at present. The generally accepted chain of events in the cyclase system is as follows. The G proteins contain a tightly bound GDP in the resting state. On receptor stimulation, the a subunit catalyzes the exchange of GTP from the cytosol for bound GDP, and concomitantly releases the p subunit. This a subunit, in the activated conformation, dissociates from the receptor and can then interact with adenylate cyclase and modulate its activity. The latent GTPase activity of a results in hydrolysis of the bound GTP, allowing the p subunit to reassociate, and returning the system to the ground state. Nonhydrolyzable analog of GTP (such as GTPyS,

2. CORTICAL EXOCYTOSIS

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GppNp. or GppCp) are capable of causing the persistent activation of the G proteins, since the GTPase activity is blocked. Cholera toxin-mediated ADP ribosylation blocks the GTPase activity of G, and results in its persistent activation. IAP-mediated ADP ribosylation of Gi blocks GTP/GDP exchange, thereby blocking activation of Gi and consequently preventing its inhibition of adenylate cyclase (Berridge, 1985). Efforts over the past 2 years have established a role for a G protein in the transduction of Ca?+ signals in a variety of mammalian cell types (reviewed by Gomperts et ml., 1986; Joseph. 1985). The specifics of these studies are beyond the scope of this chapter, but, in brief, the following has been demonstrated: ( I ) GTP analogs (particularly GTPyS) can induce secretion and potentiate exocytosis at low [Ca2‘Ii (Gomperts, 1983; Haslam and Davidson, 1984). (2) Application of GTPyS to plasma membrane preparations has been shown to elicit PIP2.turnover (Cockcroft and Gomperts, 1985; Wallace and Fain, 1985). (3) IAP treatment of intact mast cells resulted in ADP ribosylation of a 41-kDa protein, and blocked agonist-mediated release of histamine, but had no effect on release by A23187 (Nakamura and Ui, 1985). These studies provide strong support for the involvement of a G protein in the P1-linked signal transduction mechanism, but they fall short of demonstrating whether the G protein is authentic Gi or a G protein unique to PI turnover. Is a G protein also involved in the generation of Ca?.’ signals in the sea urchin egg? Turner et uf. (1986) investigated this question and determined that a G protein is indeed involved, at a step prior to IP, production. Using microinjection techniques, they confirmed Whitaker and Irvine’s (1984) result that injection of 1P3resulted in cortical exocytosis (measured as elevation of fertilization envelopes). In addition they demonstrated that injection of EGTA buffers to maintain [Caz+Iiat lo-’ M prevented exocytosis in response to subsequent IP3 injection. This result established that IP3acts at a step prior to generation of the Ca?’ signal. Injection of 28 p M GTPyS also elicited exocytosis at a step prior to Ca?’, generation, since preinjection of EGTA (as above) blocked this response. GDPpS, a competitive inhibitor of GTP exchange in the G proteins, inhibited cortical exocytosis in response to fertilization but not in response to IP3 microinjection. Thus the G protein-dependent step must precede both the 1P3 and Ca2+signals. These results are consistent with the interpretation that a G protein links sperm receptor occupation to activation of phospholipase C and Ca’+ mobilization. In a broader context, they suggest that G proteins may be an integral part of the regulated secretory pathway in invertebrates as well as in vertebrates.

ROBERT C. JACKSON AND JOSEPH H. CRABB

56

111.

In Vitro MODELS OF EXOCYTOSIS

As stated previously, molecular information on the terminal (membrane fusion) stage of exocytosis, is “embarrassingly” sparse (Tartakoff, 1985). The reason is obvious: Membrane fusion is the least accessible step in the exocytotic pathway. It occurs within the cell and is dependent on all previous steps in the process. The difficulty of isolating the fusion stage from the translocation/attachment step is particularly troublesome; for although the cell’s interior can be accessed through microinjection or permeabilization, the fusion stage, in most secretory cells, is absolutely dependent on the translocation/ attachment step. Thus, for example, it is impossible to discern whether the energy requirements observed i n most permeabilized systems (Knight and Baker, 1982) are at the level of translocation/attachment, fusion, or both. A faithful in virro model of exocytosis should surmount this difficulty and yield information on the molecules involved in fusion. To be valid, such an experimental model must meet the morphological and biochemical criteria established for in uiuo exocytosis. So little is known about terminal exocytosis, however, that there are only two well-defined criteria that must be strictly met in order to satisfy the known in viuo requirements. These are ( I ) a [Ca”] threshold in the physiological range, and (2) membrane fusion-mediated vectorial transport of the vesicle contents into a compartment corresponding to the extracellular space. This latter characteristic must be rigorously demonstrated, since release of the vesicle contents can occur merely by vesicle lysis in the absence of any membrane fusion. A. Permeabilized Cell Systems

Permeabilized cell systems represent an intermediate between in uiuo studies and cell-free systems. Since the morphological features of a permeabilized cell remain essentially intact, release of vesicle contents has, in most cases, been assumed to occur by exocytosis without rigorous proof of membrane fusion. However, these important assumptions have been validated by the careful studies of Knight and Baker (1982) on adrenal chromaffin cells that had been permeabilized by the technique of high voltage electric discharge (permeabilized cell systems are reviewed by Baker in Chapter 4, this volume). A particularly important use of the permeabilized cell systems has been

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the demonstration of the free calcium concentration required for secretion. Half-maximal exocytosis was found to occur at lCa-”li of I p M in the chromaffin cell (Baker and Knight, 1978). Remarkably similar thresholds have been found in other cell types using this technique (Baker, 1984). This technology has also revealed important requirements regarding metabolic energy and protein involvement in the process. Specifically, Baker’s group has found an absolute requirement for metabolic energy, in the form of Mg-ATP. Omission of Mg-ATP resulted in complete inhibition of exocytosis, even at [Ca2+Iiapproaching 100 p M (Knight and Baker. 1982). Restoration of exocytosis in response to physiologic [Ca2*Ii was achieved by addition of 5 mM Mg-ATP. Other nucleotides could not support the reaction. That the ATP might be needed to maintain an intragranular membrane potential or pH gradient was ruled out, because exocytosis proceeded reasonably well in the presence of trimethyltin, FCCP. and NH,CI, agents that block the granular ATPase and collapse the membrane potential, or pH gradient, respectively. I n a survey of a variety of chemical compounds, Knight and Baker (1982) discovered that N-ethylmaleimide (NEM. a thiol-modifying reagent) at 1OV‘ M abolished exocytosis in response to 10 p M Ca?’. This result provided strong evidence in favor of the involvement of protein(s) in the response to calcium. Moreover, they (and many others) observed inhibition by trifluoperazine. a drug known to interfere with the actions of calmodulin. However, this class of drug inhibits a variety of cellular processes, and is a potent membrane perturbant, so cautious interpretation of this type of data is in order (Mori rt d., 1980; Shier, 1977; Takai rt d., 1981). A variety of other techniques have been developed to transiently or permanently permeabilize cells. These include detergent methods (digitonin or saponin) (Dunn and Holz, 1983: Wilson and Kirshner, 1983).ATP4permeabilization (exploited by Gomperts, 1983. in the GTP studies of mast cell secretion), Sendai virus (at nonfusogenic concentrations) (Barrowman er d.,1986). patch-clamp-type techniques (Lindau and Fernandez. 1986), and staphylococcal a toxin (Ahnert-Hilger rt u1.. 1985). These techniques have their own particular sets of advantages and disadvantages and are reviewed elsewhere (Gomperts and Fernandez. 1985). In short, when these techniques were applied to regulated secretory cells, the requirements observed by Baker’s group (with few exceptions) have been reproduced. The limitations of this general approach are, as discussed previously, that the translocation/attachment step and the fusion step cannot be distinguished.

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ROBERT C. JACKSON AND JOSEPH H. CRABB

B. Cell-Free Systems

Calcium ion has long been known to fuse liposomes with each other, and with planar phospholipid bilayers (Papahadjopoulos, 1978). These can be considered the simplest in uitro models of membrane fusion, and are oft cited as evidence that exocytosis need not involve protein(s). Unfortunately, the [Ca*‘] requirements for these events are unphysiologically high (typically in the millimolar range), and even the best-tailored systems cannot achieve fusion below 100 pM [Ca2+](Wilschut and Hoekstra, 1984; Diizgiineg 1985). Nevertheless, these studies illustrate an important role that calcium ion may play in exocytosis, i.e., removal of the hydration barrier between phospholipid head groups, thereby promoting close apposition, a prerequisite for fusion (LeNeveu et al., 1976; Parsegian, 1977; Portis et al., 1979; Diizgiineg et ul., 1981; Rand, 1981). There are three or four varieties of in uitro models for exocytosis that utilize cellular fractions: vesicle-vesicle systems (Creutz et al., 1978; Ekerdt et al., 1981), vesicle-plasma membrane systems (Davis and Lazarus, 1979; Konings and DePotter, 1982; Lelkes et al., 1980), vesicleliposome systems (Bental r t ul., 1984), and vesicle lysis, which some consider as a model (Hoffman et al., 1976; Pazoles and Pollard, 1978). The phenomena studied in these systems include Ca?+-inducedvesiclevesicle aggregation (Creutz et a[., 1378), vesicle-vesicle fusion detected by electron microscopy (Ekerdt et al., 1981), vesicle content release on incubation with plasma membranes in the absence (Lelkes et al., 1980) or presence (Davis and Lazarus, 1979; Konings and DePotter, 1982) of Ca*+, and vesicle lysis induced by ATP and anions (Hoffman et al., 1976; Pazoles and Pollard, 1978). The results obtained with these in uitro systems have been generally disappointing. The principal difficulties associated with these systems include (1) inadequate demonstration of membrane fusion-mediated vectorial discharge, (2) Ca2+requirements which are nonexistent of or questionable relevance to the in uiuo situation, and (3) lack of an in uiuo correlate in which to test information derived from the particular cell-free system. The disappointment of investigators with attempts to reconstitute exocytosis in uifro has been succinctly summarized by Burgoyne (1984): “Until an in uitro system is developed which can readily be shown to undergo a Ca2+-dependent release by a demonstrably exocytotic mechanism, the significance of the existing in uitro systems to the in uiuo situation must remain in doubt.”

59

2. CORTICAL EXOCYTOSlS

C. Egg Cortex as an in Vitro Model of Ca2+-TriggeredExocytosis

The principal difficulties associated with the permeabilized cell systems and the aforementioned in v i m models have been largely resolved by the finding that, in sea urchin eggs, subcellular fractions can be obtained which retain their exocytotic apparatus and maintain their capacity to undergo an exocytosis-like reaction in response to applied micromolar Ca? i-

1. EGGCORTEXPREPARATIONS

There are two types of cortex preparation. Cortical lawn (CL) preparations, developed by Vacquier (197.0, consist of fragments of egg cortex 1i.e.. CVs, plasma membrane (PM),and vitelline layer]. A CL is obtained by directing a jet of Ca!+-free buffer across the surface of a polylysinecoated microscope slide that contains a monolayer of adherent egg cells. This procedure shears the eggs and produces a “lawn” of cortical frdgments uniformly oriented with their cytoplasmic face accessible to the medium (Fig. 3). The other preparation is known as the cell surface complex (CSC). This preparation, developed by Detering et ul. (19771, consists of a suspension of purified egg cortex. CSC is prepared by homogenization of a suspension of dejellied eggs, followed by several rounds of differential centrifugation to remove cytoplasmic constituents (Fig. 4).

2 . MORPHOLOGY OF CORTICAL

VESICLE

DISCHARGE

The CL and the CSC preparations are responsive to Ca”. Half-maxima1 release of CV contents, in both preparations, occurs at approximately 3 p M Ca!’ (Moy et a l . , 1983; Whitaker and Baker, 1983) and is accompanied by dramatic morphological changes. The change most readily observed in the light microscope is disappearance of the CVs (compare B and C in Figs. 3 and 4). A fertilization envelope-like structure is concomitantly formed in the CSC preparation. Electron microscopy showed that the morphology of this structure varies to some extent with the buffer composition, but in a buffer closely resembling seawater it is nearly identical to the fertilization envelope of inseminated eggs (Schon and Decker, 1981).

Direct electron microscopic evidence for Ca?+-mediated fusion between CVs and the plasma membrane has been obtained by several investigators. Whitaker and Baker (1983) examined transversely sectioned

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ROBERT C. JACKSON AND JOSEPH H. CRABB

A

dejellied eggs

I

cortical lawn

(CL1

Fic. 3. Cortical lawn preparation. (A) Procedure for preparing CL. (B) Phase-contrast micrograph of a single cortical fragment in a cortical lawn preparation. (C) Same fragment as in B, after treatment with a buffer containing 12 p M free Ca?'. Bar, 10 prn.

preparations of Echinus esculentus CL that had been bathed in Ca*+containing buffers. They observed that discharge results in the release of CV contents on the extracytoplasmic face of the PM, and the formation of a perivitelline-like space that is both topologically and morphologically equivalent to the perivitelline space of the fertilized egg. Utilizing rotary shadowed replicas of Ca*+-stimulatedS. purpurutus CL, Chandler (1984) and Zimmerberg et al. (1985) observed the formation of structures (membranous domes) indicative of the incorporation of CV membrane into underlying plasma membrane. Both sets of images suggest that CV discharge in CL preparations is the in uirro equivalent of exocytosis. The

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cel I surface complex

(CSC) FIG.4. Cell surface complex preparation. ( A ) Procedure for preparing CSC. ( B ) Phasecontrast micrograph of a single cortical fragment in a csc preparation. (c)A different cortical fragment from the same CSC preparation. after treatment with a buffer containing 12 p M free Ca”. Bar. 10 pm.

thin-sectioned samples emphasize the vectorial nature of exocytosis while the rotary shadowed samples emphasize membrane fusion. Two factors complicate electron microscopic observation of CV discharge in these preparations. First, the high density of CVs on the sutface of the CL results in a massive reaction that often obscures the behavior of individual vesicles. Second, because an individual CV must be favorably

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ROBERT C. JACKSON AND JOSEPH H. CRABB

oriented in time and space in order to visualize fusion, the number of CVs that can be clearly shown to fuse with the PM is a small fraction of the total, Consequently, it is not possible to ascertain, from electron microscopic data alone, whether exocytosis (i.e., CV-PM fusion) is the mechanism by which the bulk of the CVs discharge their contents. That the images observed in the electron microscope are indeed representative of the mechanism by which the bulk of the CVs release their contents has been strengthened by evidence obtained with an immunofluorescence-based vectorial transport assay (Crabb and Jackson, 1985). Analysis of “sparse” CLs (i.e., CLs from which most CVs have been removed by shear, so that discrete CVs can be observed) showed that CV contents are vectorially discharged into a membrane-bounded compartment (beneath the plasma membrane) where they were incapable of combining with antihyalin antibodies (Fig. 5A). Detergent disruption of this plasma membrane-bounded compartment permitted the CV contents to combine with the antibodies (Fig. 5B). The fact that essentially all the CVs in the sparse CL preparations released their contents by the same mechanism ( i s . , CV-PM fusion and vectorial discharge) suggests that the isolated instances of vectorial discharge observed by electron microscopy are representative of the mechanism by which the bulk of the CVs react. Based on these results it seems probable that CV discharge in the CL and CSC preparations is the in uitro equivalent of exocytosis; thus we have chosen to use these terms interchangeably in the remainder of this chapter. However, to prove the equivalence of CV discharge and exocytosis it will ultimately be necessary to identify molecules responsible for the in uitro reaction and to determine whether the same molecules are also essential for exocytosis in uiuo. 3. ASSAYSFOR CORTICAL VESICLEDISCHARGE

The identification of essential molecules will require reliable, quantitative assays for in uitro exocytosis in the CSC and CL system. Several are available. Two groups independently established turbidimetric assays for exocytosis (Haggerty and Jackson, 1983; Sasaki and Epel, 1983). Both variations are based on the observation that CV discharge results in a sharp decrease in the turbidity of a CSC suspension. Since the magnitude of the decrease in turbidity has been shown to be directly proportional to the extent of CV exocytosis, the turbidity assay can be used quantitatively (Haggerty and Jackson, 1983). The turbidity assay is both quick and easy to perform; however, it is compatible only with the CSC preparation, and care must be exercised in its use since conditions that result in aggre-

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FIG. 5 . Cortical vesicle contents are vectorially transported across the plasma membrane. Sparse CLs (i.e., CLs from which most CVs had been removed) were treated with a buffer containing 44 @free Ca?' and then fixed with 1% glutaraldehyde. Lawns with intact membranes (A) and lawns whose membranes had been disrupted with 0 5 % ( v h ) Triton X100 after fixation (B)were processed for irnniunofluorescence with a CSC-absorbed polyclonal antihyalin antiserum, at a dilution of 1 : 20. The paired micrographs in A and B are phase-contrast (left) and immunofluorescent (right) images of the same field. Bar. 10 pm. (Reproduced from Crabb and Jackson, 1985.)

gation or fragmentation of CSC (e.g., vigorous shaking) interfere with the assay. CV exocytosis can also be followed by the release of CV enzymes. Two such assays have been developed. In one assay, the amount of released ovoperoxidase (and hence exocytosis) is quantitated by radioiodination of endogenous substrates (Haggerty and Jackson, 1983; Jackson e t al., 1985). In the other, the amount of released p-1,3-glucanaseis quantitated

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by hydrolysis of an exogenous substrate (Moy et ul., 1983). Although the P-glucanase assay was devised for use with CLs, and the ovoperoxidase assay for use with CSC, both assays could, in principle, be used with either preparation. The enzyme release assays are more tedious than the turbidimetric assay, and substances that inhibit ovoperoxidase or P-glucanase will, of course, interfere. A microscopic assay, devised by Zimmerberg ef d.(1985), can be used to quantitate the extent of CV exocytosis in CLs. The assay is based on the observation that the individual CVs in a CL preparation are essentially the only structures that scatter light when viewed by dark-field microscopy. Fusion of CVs with the plasma membrane reduces the number of scattering centers and decreases the amount of scattered light. The magnitude of the CaZt-triggered decrease in light scattering has been shown to be directly porportional to the extent of CV exocytosis (Crabb and Jackson, 1986). This microscopic assay has two disadvantages: it requires specialized equipment, and it can be very tedious because only one sample can be processed at a time. On the other hand, this is the only available assay that allows simultaneous visualization and quantitation of the sample. 4. INHIBITORS Attempts to unravel the molecular mechanism of exocytosis in the CL and CSC preparations have not yet produced substantial results. A variety of inhibitors have been identified, but the process of elucidating their molecular targets has just begun. u. Thiol Reagents.

NEM and other thiol-modifying reagents have been shown to block exocytosis in the CL and CSC preparations (Haggerty and Jackson, 1983). NEM also blocked the cortical reaction in intact eggs, whereas 5,5’-dithiobis(2-nitrobenziocacid) (DTNB), a membrane-impermeant thiol reagent, did not (K. K. Ward and R . C. Jackson, unpublished). These results demonstrate that cortical exocytosis can be inhibited by modification of a sulfhydryl(s), accessible at the cytoplasmic surface of the CSC. They suggest that the protein containing this sulfhydry1 group(s) may play a role in exocytosis. The mode of inactivation with NEM is interesting. Under mild conditions, NEM inhibits cortical exocytosis by increasing the threshold Ca2+ concentration required for exocytosis, without affecting the magnitude of the response at higher Ca2t concentrations. When the NEM modification conditions are more severe, exocytosis is blocked even at high Ca2+concentrations (Jackson et ul., 1985). This mode of inhibition suggests that

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the sulthydryl group(s) responsible for inactivation may be involved in the mechanism that imparts sensitivity to micromolar Ca” .

6 . Protecrses. Like NEM, trypsin and pronase also inhibit exocytosis by progressively increasing the threshold Ca?‘ concentration required to initiate exocytosis (Jackson ef 01.. 1985). Mild tryptic digestions have little effect on both exocytosis and CSC morphology (i.e., CVs are not released from the CSC), whereas prolonged digestions, of the sort required to totally inactivate the CSC, result in the appearance of individual CVs (Jackson et d.,1985). It remains to be determined whether these observations are merely coincidental, or whether the inhibition of exocytosis is the direct result of CV detachment. While investigating the possibility that proteolytic inactivation could be due to digestion of an essential sulfhydryl-containing protein, it was discovered that NEM-treated CSC could be reactivated by mild tryptic digestion (Jackson et d., 1985). Although a straightforward molecular interpretation of this phenomenon is currently not possible. it may prove useful in identifying the NEM target protein. c . Polycutions. Polycations (e.g., polylysine) have recently been shown to inhibit exocytosis in the CL preparation (Crabb and Jackson, 1986). Since polycations inhibit a wide range of cellular processes, this observation does not help elucidate the molecular mechanism of exocytosis. However, it may explain the finding that “aged” CLs (which are prepared on polylysine-coated surfaces) lose their sensitivity to microcmolar Ca” (Moy et al., 1983). Regarding the use of CL preparations. it is important to note that “aging” requires a period of 15-20 min before a noticeable increase in the Ca?’ threshold can be detected (Crabb and Jackson, 1986). Thus, observations made soon after CL preparation should not be affected by the “aging” phenomenon. d . Phenofhiuzines. Several investigators have demonstrated that the phenothiazine drug. trifluoperazine (TFP, a calmodulin antagonist), inhibits exocytosis in the CL and CSC preparations (Baker and Whitaker, 1979: Haggerty and Jackson, 1983; Moy ef ( I / , , 1983). However, as noted above, TFP inhibition data must be interpreted with caution since phenothiazine drugs are potent membrane perturbants (Naccache rf d., 1980; Seeman, 1972) and have been shown to inhibit enzymes that are not calmodulin regulated (Chau and Tai. 1982; Mori ef NI., 1980; Shier, 1977; Takai et a / . , 1981). Additional evidence in support of the calmodulin hypothesis was provided by the report (Steinhard and Alderton, 1982) that exocytosis in CL

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C.JACKSON AND JOSEPH H. CRABB

preparations can be inhibited by incubation with high concentrations of an affinity-purified anticalmodulin antibody. However, since this work was performed before it was realized that cortical exocytosis is inhibited by the polylysine used in CL preparation (Crabb and Jackson, 1986), the potential for artifact must be reconsidered. We have determined that exocytosis in the CSC preparation is not inhibited by melittin or R 24571 (Crabb and Jackson unpublished), two of the most potent calmodulin antagonists currently available (SellingerBarnette and Weis, 1984; Van Belle, 1981). We have also been unsuccessful in our attempts to inhibit exocytosis in CSC with several samples of anticalmodulin antisera (Stacy and Jackson, unpublished). In addition, Gilkey (1983) has reported that microinjected anticalmodulin IgG failed to inhibit exocytosis in medaka eggs. e. KCI Extraction. Sasaki (1984) has reported that a KCI-extractable proteinaceous factor is required for exocytosis at micromolar Ca2+ in Hemicentrotus pulcherrimus (but not S . purpuratus) CSC. The molecular weight of the crude factor (approximately 100,000, by gel filtration chromatography) suggests that it is not calmodulin. 5. SUBFRACTIONATION OF T H E EGGCORTEX

A second approach to understanding the mechanism of CV discharge entails subfractionating the cortex into its component parts (i.e., plasma membrane and CVs), analyzing each, and reassembling them into active cortex. a . Purified Cortical Vesicles. Several CV purification procedures have been published. Schuel et al. (1972) used zonal centrifugation to prepare CVs from egg homogenates. The purified CV fraction obtained by this method was enriched in /3-1,3-glucanase activity, and it has been used in determining the chemical and enzymatic composition of CV contents (reviewed by Schuel, 1978, 1985). Recent CV purification procedures have utilized egg cortex as starting material. Kopf et al. (1982) prepared CVs from S.purpuratus eggs. Their method involves mechanically dislodging CVs from large cortical lawns (prepared in petri dishes) by repeatedly squirting a jet of a high ionic strength, dissociative buffer across the surface of the lawn. Dislodged CVs are collected by centrifugation. Decker and Kinsey (1983) prepared CVs from Lytechinus variegatus eggs. Their procedure is based on dislodgement of CVs from CSC by gentle homogenization in a low ionic strength, isotonic buffer. CVs are separated from other components of the

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homogenate by a two-step procedurc involving ultracentrifugation on a sucrose step graident. I n the procedure reported by Crabb and Jackson (1985). CVs are dislodged from S . pirrpirrutrrs CSC by gentle homogenization in an isotonic buffer with a pH of 9.1, and purified by low speed centrifugation. Marker enzyme analysis indicated that all three procedures yielded CV fractions that were enriched in CV proteoesterase activity and had substantially reduced levels of a plasma membrane marker (Na‘ ,K+-ATPase). Decker and Kinsey (1983) have analyzed the protein and phospholipid Compositions of their CV preparation. CV proteins were found to be rich in high molecular weight glycoproteins. CV lipids were enriched in arachidonic acid and contained 2.3 times as much cholesterol as egg plasma membrane. h. Piirijied P/usmu Membrane. Crabb and Jackson ( 1985)and Kinsey et ul. (1980) have devised plasma membrane purification procedures. The procedure of Crabb and Jackson (1985) is a microprocedure, designed specifically for use in reconstitution experiments (see below). CVs are mechanically disloged from CL preparations with a jet of isotonic buffer. The resulting PM “lawns” are made up of CV-free fragments of PM attached via their vitelline layer to a polylysine-coated slide or coverslip. The procedure of Kinsey et al. (1980) produces a suspension of CV-free plasma membrane sheets. The PM fraction was substantially enriched in Na ,K+-ATPaseand contained reduced levels of CV proteoesterase. The egg PM has an unusually high content of phosphatidylinositol (25%. on a molar basis) and, like the CV membrane, is enriched in arachidonic acid (Decker and Kinsey, 1983). +

6. RECONSTITUTIONOF ACTIVECORTEX PLASMAMEMBRANE

FROM PURIFIED

CVS A N D

Although the CSC and CL preparations are exocytotically competent, their usefulness in investigations of the molecular basis of exocytosis is limited because the CVs and the PM cannot be independently manipulated. Recently, Crabb and Jackson ( 1985) have reported that exocytotically active egg cortex can be reconstituted from purified CVs and egg PM. Reconstituted cortex was prepared by recombining purified CVs with a P M lawn to form a reconstituted lawn preparation (Fig. 6). CV discharge in reconstituted lawn preparations required a 3- to 4-fold higher concentration of Ca’+ than control cortical lawns. Purified CVs that were not attached to a PM lawn were shown to remain intact, even in buffers containing high concentrations of Ca?’ ( I .O mM).

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ROBERT C. JACKSON AND JOSEPH H. CRABB

FIG.6. Reconstituted cortical lawns and their response to 44 pM free Ca?'. A PM lawn was prepared by dislodging CVs from a CL with a jet of isotonic buffer. A representative field, containing a single PM fragment, was selected and photographed (A). Subsequent steps were performed with the sample in place on the microscope stage so that the same field could be followed throughout the experiment. (B) Reconstituted lawn formed when a suspension of purified CVs was applied to the PM lawn, allowed to bind for 10 rnin, and thoroughly washed with an isotonic buffer. (C, D) Same reconstituted lawn 5 and 60 sec, respectively, after addition of the Ca?'-containing buffer. Bar, I 0 pm. (Reproduced from Crabb and Jackson, 1985.)

Using the immunofluorescence-based vectorial transport assay described above (Section III,C,2), it was shown that CV discharge from a reconstituted lawn resulted in the vectorial transport of hyalin from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane (Fig. 7). These results constitute strong evidence in support of the contention that Ca2+-stimulateddischarge of CV contents in a reconstituted lawn is the in v i m equivalent of exocytosis. Nevertheless, it will be necessary to reconfirm vectorial transport with another technique (ens.,electron microscopy). It will also be necessary to determine whether a CV can fuse

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Fic,. 7 . Cortical vesicle contents are vectorially transported across the plasma membrane in reconstituted lawns. Reconstituted lawns were prepared. reacted with it buffer containing 44 wh4 free Ca?*. and tixed with 1% glutaraldehyde. Antihyalin immunofluorescence was performed with CSC-absorbed antihyalin antiserum at a diluation of I : 40. ( A ) Paired phasr-contrast and immunotluorescent images of a reconstituted cortical fragment with intact membranes. (B)Paired phase-contrast and immunofluorescent images of a reconstituted cortical fragment with membranes disrupted by Triton X-100.Bar. 10 pn. (Reproduced from Crabb and Jackson, 198s.)

with any membrane to which it binds, or whether there is specificity in the reassociation and/or Ca”-dependent fusion stages. As a first step in this direction, a method of quantitating CV binding has been devised which was used to show that S. pi4rpirrrrtirs CVs do not reassociate with PM lawns prepared from human red blood cells (R.C. Jackson and P. Modern, unpublished).

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7. THECORTICAL VESICLE-PLASMA MEMBRANE JUNCT~ON

Elucidation of the structures responsible for binding CVs to the PM should shed light on the function of junctional proteins in exocytosis, and may help unravel the roles of ATP and Ca2+in exocytosis. The possibility that microfilaments or other cytoskeletal structures anchor CVs to the PM is attractive. Actin and other cytoskeletal proteins have been localized to the egg cortex (Bryan, 1982; Burgess and Schoeder, 1977; Otto et al., 1980; Whitaker and Baker, 1983), and filamentous structures approximately 6 nm in diameter have been observed in association with the CV and PM surfaces in both intact eggs and isolated cortex (Chandler, 1984). The similarity of the extraction conditions that have proved effective in releasing CVs from isolated CSC [i.e., 0.6 A4 KI (Vacquier, 1976); elevated pH (Crabb and Jackson, 1985; Hylander and Summers, 1981); low ionic strength sucrose buffers (Decker and Kinsey, 1983)l and the conditions used to disrupt microfilaments (Carraway et al., 1982; Pollard, 1982; Pollard et al., 1974) is also suggestive. On the other hand, it has been reported that cytochalasin B does not disrupt the filamentous connections between CVs and PM (Chandler, 1984), and attempts to inhibit cortical exocytosis in isolated CLs with microfilament and microtubule toxins have been unsuccessful (Whitaker and Baker, 1983). Clearly, it is too early to draw any conclusions regarding the nature or identity of the structures that comprise the CV-PM junction

D. Hypothetical Models

Several hypothetical models for the terminal events in exocytosis have been propsed, but none has gained universal acceptance. Four of the more prominent hypotheses and their relationship to cortical exocy,tosis are considered below.

I . OSMOTIC FORCES The osmotic hypothesis is based on the observation that exocytosis is generally accompanied by a concomitant swelling of the secretory vesicle (reviewed by Finkelstein et al., 1986). The hypothesis suggests that osmotic swelling of the secretory vesicle provides the driving force for vesicle-plasma membrane fusion. Using mammalian cells, Pollard and co-workers have amassed a substantial amount of circumstantial evidence in favor of this hypothesis

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(Pollard of a/.. 1979; see also Brocklehurst and Pollard, Chapter 7, this volume). Cohen et t i / . (1982) and Zimmerberg Pf a / . (1980) have presented evidence supporting a role for osmotic forces in the fusion of liposomes with planar phospholipid bilayers. Using sea urchin eggs, Zimmerberg and Whitaker (1985) have shown that CV discharge both in intact eggs and in CL preparations is inhibited by hyperosmotic buffers. Membrane capacitance experiments with intact eggs indicated that increased osmotic strength had inhibited membrane fusion and not just dispersal of CV contents. Since CLs prepared in hyperosmotic, Ca”-containing buffer underwent CV discharge when returned to an isosmolar buffer, (even in the absence of Ca”), it was suggested that Ca?+may produce an irreversible change that primes CVs for exocytosis. Support for this proposal derives from the observation that the CVs in a CL prepared in hyperosmotic, Ca”-containing buffer had a mean vesicular diameter slightly larger than &‘+-free controls (Zirnmerberg and Whitaker, 1985). On the other hand, Holz and Senter ( 1986) have reported that digitonin-permeabilized chrornaffin cells incubated in hyperosmotic Ca?’ -containing buffer do not undergo exocytosis when returned to an isosmotic &?+-free buffer. Furthermore, depsite considerable effort, the search for a mechanism that might be responsible for unleashing intravesicular osmotic forces prior to exocytosis has not been fruitful. In fact, most mechanisms have been eliminated. Thus, in chromaffin cells it has been shown that ( I ) the absence of anions and monovalent cations does not inhibit exocytosis in intact or permeabilized cells, ( 2 ) reagents that collapse the H’ electrochemical gradient across the chromaffin granule membrane are not inhibitory, and (3) hyposrnotic buffers do not induce or enhance exocytosis from intact or permeabilized cells (see Baker and Knight, 1984, and Holz, 1986, for recent critiques of the osmotic hypothesis). In brief, while the osrnotoic hypothesis is attractive, it remains difficult to determine whether hydration and swelling of secretory vesicles occurs before or after membrane fusion. 2 . METABOLIC ENERGY The observation that metabolic poisons inhibit exocytosis in a variety of secretory cells (Becker and Henson, 1973; Feinstein and Schramm, 1970; Jamieson and Palade, 1971; Peterson, 1974) has long suggested that exocytosis is an energy-requiring process. This hypothesis is supported by the fact that most (Bader et a / . , 1986; Barrowman r f u / . , 1986; Dunn and Holz, 1983; Ronning and Martin, 1986; Wilson and Kirshner, 1983), but not all (Ahnert-Hilger et al., 1985; Ruggiero rt al., 1985; Smolen r f u / . ,

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1986), permeabilized mammalian cell systems require ATP for exocytosis. Exocytosis in the sea urchin egg has also been reported to be inhibited by metabolic poisons (Baker and Whitaker, 1978; Okazaki, 1956); however, unlike most mammalian cells, permeabilized eggs, prepared either from poisoned or control eggs, did not require ATP for exocytosis (Baker and Whitaker, 1978). Likewise ATP is not required for CV discharge in the CL preparation (Baker and Whitaker, 1978). ATP has been reported to slow “aging” and maintain the micromolar Ca2+ sensitivity of CL preparations (Moy er ul., 1983), but as discussed above the “aging” phenomenon is probably caused by the polycations used in preparing CLs and, therefore, may not be physiologically relevant. Inconsistencies among the reported ATP requirements of the various permeabilized mammalian cell types, as well as the “aging” phenomenon, prevent a straightforward assessment of these results. However, there appear to be two paradoxes: (1) Permeabilized mammalian cells seem to require ATP for exocytosis, but permeabilized eggs do not. (2) Permeabilized eggs as well as the CL and CSC fractions do not require metabolic energy for exocytosis, yet intact eggs are inhibited by metabolic poisons. The first of these paradoxes may be the result of the unique morphology of the egg. The vesicle translocation and attachment steps of exocytosis (Fig. 1, steps 3 and 4) are not required for cortical exocytosis in the egg; in mammalian cells they are. If these steps are responsible for the ATP requirement of mammalian cells the paradox can be dispelled. The second paradox could be explained if metabolic poisoning of the egg produced an inhibitory substance that was lost on permeabilization or subfractionation of the egg. Clearly, the role of metabolic energy in exocytosis is an area that warrants further study.

3. THEVIRALMODEL The instance of membrane fusion that is best understood at the molecular level is the fusion of an enveloped virus with its host cell. In the acidic environment within a host cell endosome (pH 5.0-5.5), one of the surface proteins of enveloped viruses undergoes a pH-induced conformational change that exposes a hydrophobic domain, resulting in fusion of the viral and host cell membranes (Skehel et al., 1982; White er al., 1983; see also Chapters 9-1 1, this volume). If exocytotic fusion is comparable to viral fusion, a Ca2+-inducedconformational change in a secretory vesicle or plasma membrane protein might be responsible for membrane fusion. This is an attractive model, but it is also one that is difficult to investigate

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experimentally. Thus, there is currently no direct evidence either for or against it. 4. PHOSPHOLIPASES The phospholipase hypothesis suggests that fusogenic molecules generated by Ca”-stimulated phospholipase hydrolysis of membrane phospholipids are required for fusion of secretory vesicles with the plasma membrane. Both phospholipase A? and phospholipase C have properties that are compatible with a role in exocytosis. Phospholipase A: requires Ca?+ for activity (Van Den Bosch, 1980) and produces known fusogens: lysophospholipids (Lucy, 1978) and free fatty acids (Creutz, 1981). Phospholipase A? activity has been detected in egg homogenates, and it has been reported that quinacrine, a phospholipase inhibitor, blocks the cortical reaction in intact eggs (Ferguson and Shen, 1984). As described above, phospholipase C is intimately involved in the production of the Ca2+ signal that is responsible for exocytosis. DAC, a product of phospholipase C hydrolysis of PIP?, is known to satisfy one requirement for membrane fusion, i.e.. it distorts the molecular packing of phospholipid bilayers (Das and Rand, 1984). Another way that phospholipase C activity may promote exocytosis is through reduction of the membrane surface charge. Hydrolytic release of multiply charged inositol phosphate groups from the membrane surface should reduce hydration of the polar phospholipid head groups and thereby decrease the energy barrier to close apposition of lipid bilayers (LeNeveu ef NI., 1976; Parsegian, 1977; Rand, 1981). Whitaker and Aitchison (1985) have shown that &!+-activated CV discharge in CL preparations is accompanied by PI turnover. Both PI turnover and CV discharge had comparable Ca?’ requirements, and neomycin (a polycationic inhibitor of polyphosphoinositide hydrolysis) was shown to inhibit exocytosis in the CL preparation. Additional experiments will be required to determine whether neomycin inhibition results from decreased PI turnover or from other actions of the drug. Nevertheless, the possibility that DAG is involved in exocytosis is attractive: It is known to destabilize membranes and is generated in a timely manner at the appropriate cellular location by a Ca”-stimulated enzyme. IV.

CONCLUSIONS

The fundamental mechanism of Ca?’ signal generation in the sea urchin egg and in mammalian cells appears to be virtually identical: Receptor

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ROBERT C. JACKSON AND JOSEPH H. CRABB

occupation results in the sequential activation of a G protein and P1specific phospholipase C. IP3 produced by phospholipase C-catalyzed hydrolysis of PIP2acts to release Ca2+from internal stores. The details of the various steps in this sequence (i.e., the linkage between sperm receptor occupation and activation of G protein, the mechanism by which activated G protein stimulates phospholipase C, and the details of Ca2+ gating by IP3) need to be resolved. It is hoped that similarities between signal transduction in the 1P3 and CAMPsystems will continue to facilitate these studies. Studies of the molecular mechanisms of the terminal steps in exocytosis are in their infancy. Several models have been proposed, but the fundamental mechanism of membrane fusion remains unknown. The egg cortex constitutes a particularly advantageous model system for studying exocytotic membrane fusion. The available data strongly suggest that CV discharge in the CSC and CL preparations is the in vitro equivalent of exocytosis in the egg: however, ultimate validation of this in vitro approach will come only through the identification of proteins that are required for exocytosis both in vitro and in vivo. V.

ADDENDUM

Since completing work on this review a number of pertinent articles have been published or have come to our attention. A. Sea Urchin Egg

Turner et al. (1987) have shown that microinjection of cholera toxin into unfertilized sea urchin eggs stimulates cortical exocytosis, via a CAMP-independent mechanism. Cholera toxin treatment of egg CSC resulted in ADP ribosylation of a 47-kD polypeptide; whereas pertussis toxin catalyzed the ADP-ribosylation of a 40-kD polypeptide. These results suggest that the signal transduction mechanism for exocytosis in the egg relies on a G protein that has a different toxin sensitivity than observed in most mammalian secretory cells. Oinuma et al. (1986) have partially purified the pertussis toxin substrate protein from sea urchin eggs, and shown that its 37-kD beta subunit binds antibodies raised to the betdgamma subunits of rat brain G-proteins. A pertussis toxin substrate protein has also been identified in sea urchin sperm (Kopf et al., 1986). Trimmer and Vacquier (1986) have published a review on the activation

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of sea urchin gametes that includes sections on exocytosis in both the sperm (acrosome reaction) and egg (cortical reaction). B. Other Cell Types

Based on results obtained with neutrophils (Barrowman e f al., 1986), Gomperts (1986) has proposed that a GTP-binding protein may participate in the terminal stages of exocytosis (i.e.?after the increase in cytoplasmic concentration of Ca?'). Vallar e f ul. (1987) have obtained similar results with an insulin-secreting cell line. Knight et al. (1985) have shown that adrenal chromaffin cells that have been treated for several days with botulinum toxin will not secrete in response to various secretagogues, or when permeabilized and challenged with Ca2+buffers. The long incubation time required for inactivation was apparaently due to the slow rate of entry of the toxin into the cells, since Penner et al. (1986)were able to obtain inactivation within 1 hr by directly microinjecting botulinum or tetanus toxins into cells. The mechanism of action of botulinum and tetanus toxins is not known; however, related bacterial toxins inactivate cellular processes through ADP ribosylation of GTP-binding proteins. Ohashi and Narumiya ( 1987) have observed that treatment of a crude membrane faction from bovine adrenal gland results in ADP-ribosylation of a 21-kD polypeptide. The relationship of this polypeptide to GTP-binding proteins and to exocytosis remains to be determined. Perrin et al. (1987) demonstrated that the exocytotic release of catecholamine from digitonin-permeabilized adrenal chromaffin cells can be partially inhibited by preincubation with anti-fodrin antibodies. Their results suggest that rearrangement of the cytoskeletal network of the cell may be required for efficient exocytosis. With regard to the various models for exocytosis, two groups (Zimmerberg et al., 1987; Breckenridge and Almers, 1987) have presented cogent data in opposition to the osmotic hypothesis. By simultaneously monitoring capacitance and observing the morphology of mast cells from the beige mouse, it was demonstrated that the capacitance increases (i.e., membrane fusion) precede swelling of secretory vesicles. Plattner and colleagues (Vilmart-Seuen e f al., 1986) have divised an in uitro system for investigating the exocytotic release of trichocysts from Parumecirrm. Paramecium is similar to the sea urchin egg in that secretory vesicles are firmly attached to the plasma membrane; hense, Parumecium cortex, like sea urchin egg cortex, is exocytotically active. Evidence from both in uiuo and in uitro experiments (Momayezi e f a l . , 1987)

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suggest that the exocytotic release of trichocysts may involve a protein phosphatase, perhaps calcineurin. ACKNOWLEDGMENTS We would like to thank Paul Modern and Mary Harrington for helping to prepare the manuscript. The authors' research was supported by Grant GM 26763 from the National Institutes of Health. REFERENCES Ahnert-Hilger, G., Bhakdi. S . . and Gratzl, M . (1985). Minimal requirements for exocytosis. J . B i d . Chern. 260, 12730-12734. Alliegro, M . C.. and Schuel. H. (1985). Characterization of a soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs. Biochemistry 24, 3926-393 I . Anderson, E. ( 1968). Oocyte differentiation in the sea urchin, Arhacitr punctitlotu. with particular reference to the origin of cortical granules and their participation in the cortical reaction. J . Cell Biol. 37, 514-539. Anderson, E. (1974). Comparative aspects of the ultrastructure of the female gamete. fnr. Rev. C y t d . Sitppl. 4, 1-70. Bader, M.-F., Thierse. D.. Aunis. D., Ahnert-Hilger, G., and GrdtZl. M. (1986). Characterization of hormone and protein release from ru-toxin-permeabilized chromaffin cells in primary culture. J . Biol. Chetn. 261, 5777-5783. Baker. P. F. (1984). Multiple controls for secretion'? Nuture (London) 310, 629-630. Baker, P. F.. and Knight, D. E. (1978). Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Nutitre (London) 276, 620-622. Baker. P. F.. and Knight, D. E. (1981). Calcium control of exocytosis and endocytosis in bovine adrenal medullary cells. Philos. Truns. R . Soc. London Ser. B . 296, 83-103. Baker, P. F . . and Knight, D. E. (1984). Chemiosmotic hypothesis of exocytosis: A critique. Biosci. Rep. 4, 285-298. Baker, P. F., and Whitaker. M. J . (1978). Influence of ATP and calcium on the cortical reaction in sea urchin eggs. Nutiire (London) 276, 513-Sl5. Baker. P. F., and Whitaker. M. J. (1979). Trifluoperazine inhibits exocytosis in sea urchin eggs. J . Physiol. (London)298, 55P. Barrowman, M. M.. Cockcroft. S . , and Gomperts, B. D. (1986). Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils. Nuture (London) 319, 504-507. Becker, E. L . , and Henson. P. M. (1973). I n uirro studies of immunologically induced secretion of mediators from cells and related phenomena. Adu. fmmrrnol. 17, 93-193. Bendayan, M., Roth, J . , Perrelet. A,, and Orci, L. (1980). Quantitative immunocytochemical localization of pancreatic secretory proteins in subcellular compartments of the rat acinar cell. J . Histochrm. Cytochem. 28, 149- 160. Bental, M., Lelkes, P. 1.. Schloma. J., Hoekstra, D.. and Wilschut. J. (1984). Ca'+-lndependent, protein-mediated fusion of chromaffin granule ghosts with liposomes. Biochim. Biophys. act^ 774, 296-300. Berridge, M. J. (1983). Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyze polyphosphoinositides instead of phosphatidylinositol. Biochem. J . 212, 849-858.

Berridge, M. J . (1985). The molecular basis of communication within the cell. Sei. A m . 253, 142- 152. Berridge, M. J . , and Irvine, R. F. (1984). Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nuture (London) 312, 315-321,

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stimulating role for GTP, has been reached by Barrowman Pt ul. (1986) from their studies of histamine secretion in permeabilized mast cells. The nature of the GTPyS binding site is unknown; but it is an attractive possibility that G proteins may be involved directly in exocytosis. Speculating a little, it is possible that when protein kinase C becomes associated with the plasma membrane, it also becomes subject to modulation by inhibitory (or stimulatory) G proteins, and, if this were true, it would not be a very large step to hypothesize that G proteins might mediate transmem-

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brane control of protein kinase C and exocytosis. A direct effect of this kind might, for instance, underlie a number of presynaptic control mechanisms. One specific case where this might apply is the inhibition of insulin release from pancreatic /3 cells following sympathetic nerve stimulation. This effect persists following application of noradrenaline to permeabilized /3 cells, and a direct effect on exocytosis looks a real possibility (Jones ef ul., 1986). Inhibition by botulinum toxin is particularly exciting as it persists for many days and may lead ultimately to isolation of one of the components of the secretory machinery. So far, the mechanism by which botulinum toxin acts has resisted exposure, but many related toxins act via ADP ribosylation of different GTP-binding proteins. A fascinating feature of botulinum action is that, after exposure to the toxin, adrenal medullary cells accumulate catecholamines and behave in all respects as if they have “forgotten” how to secrete despite exposure to the appropriate messages. Endogenous agents having actions similar to botulinum toxin could play a key role in the long-term alteration of synaptic efficacy, opening up the possibility that direct effects on the machinery of exocytosis may contribute to the long-term regulation of synaptic function. REFERENCES Bainton, D. F., and Fdrquhar, M. G . (1966). Origin of granules in polymorphonuclear leukocytes. Two types derived from opposite faces of the Golgi complex in developing granulocytes. J . Cell B i d . 28, 277-301. Baker, P. F. (1972). Transport and metabolism of calcium ions in nerve. Prog. Biophys. M o l . Biol. 24, 177-223. Baker, P. F. (1974). Excitation-secretion coupling. Recenr Adu. Physiol. 9, 51-86. Baker, P. F. (1986). Protein kinase C and exocytosis. Prog. Zoo/. 33, 265-274. Baker, P. F. and Knight, D. E. (1978). Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Nature (London)276, 620-622. Baker, P. F. and Knight, D. E. (1981). Calcium control of exocytosis and endocytosis in bovine adrenal medullary cells. Philos. Trans. R . Soc. London Ser. B 296, 83-103. Baker, P. F. and Knight, D. E. (1984). Chemiosmotic hypotheses ofexocytosis: A critique. Biosci. R e p . 4, 285-298. Baker, P. F. and Whitaker, M. J . (1978). Influence of ATP and calcium in the cortical reaction in sea urchin eggs. Nature (London) 276, 513-515. Baker, P. F., and Whitaker, M. J. (1980). Trifluoperazine inhibits exocytosis in sea urchin eggs. J . Physiol.(London) 298, 5SP. Baker, P. F. Knight, D. E., and Whitaker, M. J. (1980). The relation between ionized calcium and cortical granule exocytosis in eggs of the sea urchin Echinus e.sculentus. Proc. R . Soc. London Ser. B 207, 149-161. Baker, P. F., Knight, D. E., and Umbach, J . A. (1985). Calcium clamp of the intracellular environment. Cell Calcium 6 , 5-14. Barrowman, M. M., Cockcroft, S . , and Gornperts, B. D. (1986). Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils. Nature (London) 319, 506-507.

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Bartfai. T. (1985). Presynaptic aspects of the co-existence of classical neurotransmitters and peptides. Trends Pharmacol. Sci. 6, 33 1-334. Berridge, M. J. (1984). Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J . 220, 345-360. Berridge, M. J . , and Irvine, R. F. (1984). Inositol trisphosphate: A novel second messenger in cellular signal transduction. Nuticrc4London) 312, 315-321. Brooks. J . C.. and Treml. S. (1983). Catecholamine secretion by chemically-skinned cultured chromaffin cells. J. Neurochem. 40,468-473. Ceccarelli, B., and Hurlbut, W. P. (1980). Vesicle hypothesis of the release of quanta of acetylcholine. Physiol. Rev. 80, 396-441. Chandler, D. E. and Heuser, J . E. (1980).Arrest of membrane fusion events in mast cells by quick freezing. J. Cell Biol. 86, 666-674. Cockcroft, S. and Gomperts, B. D. (1980). The ATP- receptor of rat mast cells. Biochem. J . 188, 789-798. Cole, K. S . (1935). Electric impedance of Hipponoe eggs. J . Gen. Physiol. 18, 877-887. Creutz, C. E., Pazoles, C. J., and Pollard. M. B . (1978). Identification and purification of an adrenal medullary protein (synexin) that causes calcium-dependent aggregation of isolated chromaffin granules. J. B i d . Chem. 253, 2858-2866. Creutz. C. E . , Dowling, L. G., Sands J . J . . Vilan-Palasi, C.. Whipple, J . H.. and Zaks. W. J . (1983). Characterization of trans-lindins: Soluble proteins that bind to the chromaffin granule membrane in the presence of Ca”. J . B i d . Chem. 258, 14664414674, Di Virgilio, F., Lew, D. P.. and Pozzan. T. (1984). Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cylosolic Ca” levels. Nature (London) 310, 691-693. Dolly. J . 0.. Black, J.. Williams, R. S. and Melling, J . (1984). Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization. Narure (London) 307,457-460. Douglas, W. W. (1968). Stimulus-secretion coupling: The concept and clues from chromaffin and other cells. Br. J . Pharmcicol. 34, 451-474. Dreyer, F., Peper. K . , Akert, K., Sandri. C.. and Moor. H. (1973). Ultrastructure of the active zone in the frog neuromuscular junction. Bruin Res. 62, 373-381. Dunn. L. A., and Holz, R. W. (1983). Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells. J. B i d . Chem. 258, 4989-4993. Edwards, C., Dolezal. V., Tucek, S., Zemkova. H.. and Vyskocil, F. (1985). Is an acetylcholine transport system responsible for non-quanta1 release of acetylcholine at the mouse myoneural junction’?Pruc. N u t . A u l d . Sci. U . S . A . 82, 3514-3519. Fernandez. J. M., Neher. E. and Gomperts, B. D. (1984).Capacitance measurements reveal stepwise fusion events in degranulating mast cells. Natirre (London) 312, 453-455. Gillespie, J . 1. (1979). The effect of repetitive stimulation on the passive electrical properties of the presynaptic terminal of the squid giant synapse. Proc. R . SOC. London. S e r . B 206, 293-306. Gomperts. B. D., and Fernandez. J . M. (19x5). Techniques for membrane permeabilization. Trends Biochem. Sci. 10, 414-417. Gomperts. D. D.. Baldwin. J . M., Micklem. K . J. (1983). Rat mast cells permeabilized with Sendai virus secrete histamine in response to Ca?* buffered in the micromolar range. Biochem. J . 210, 737-745. Haslam, R. J., and Davidson. M. M. L. (1984). Receptor induced diacylglycerol formation in permeabilised platelet; possible role for a GTP binding protein. J. Receptor Res. 4,605629. Hess, P., Fox, A. P., Lansman, J. B., Nilius. B., Nowycky, M. C., and Tsien, R . W. (1986).

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Calcium channel types in cardiac, neuronal and smooth muscle-derived cells: Differences in gating, permeation and pharmacology. I n "Ion Channels in Neural Membranes," pp. 227-252. Liss, New York. Heuser, J. E., Reese, T. S . , Dennis, M. J., Jan, Y.. Jan, L.. and Evans. L. (1979). Synaptic vesicle exocytosis captured by quick-freezing and correlated with quanta1 transmitter release. J . Cell B i d . 81, 27.5-300. Holmsen, H . , Dangelmaier. C. A,. and Holmsen, H.-K. (1981). Thrombin-induced platelet responses differ in requirement for receptor occupancy. Evidence for tight couplings of occupancy and compartmentalized phosphatidic acid formation. J . Eiol. Cheni. 256, 9393-9396. Holz, R. W. (1986). The role of osmotic forces in exocytosis from adrenal chromaffin cells. Annu. Rev. Physiol. 48, 175-189. Jones. P. M., Stutchfield. J . , and Howell, S . L. (1985). Effects ofCa?' and a phorbol ester on insulin secretion from islets of Langerhans permeabilized by high-voltage discharge. FEBS L e f f .191, 102-106. Jones. P. M., Fyles, J . M.. and Howell, S . L. (1986). Regulation of insulin secretion by CAMP in rat islets of Langerhans permeabilized by high voltage discharge. FEBS Lett. 205, 205-209. Judah, J. D., and Quinn. P. S . (1978). Calcium ion-dependent vesicle fusion in the conversion of proalbumin to albumin. Nuturc (London)271, 384-385. Katz, B. (1966). "Nerve. Muscle and Synapse." McGraw-Hill. New York. Katz, B., and Miledi, R. (1977). Transmitter leakage from motor nerve endings. Pror. R . Soc. London. Ser. B 196, 59-72. Kelly, R. B. (1985). Pathways of protein secretion in eukaryotes. Science 230, 25-31. Kerr, L. M., and Yoshikama, D. (1984). A venom peptide with novel presynaptic blocking action. Nurrrre (London) 308, 282-284. Knight, D. E., and Baker, P. F. (1982). Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields. J . M e m h r . Eiol. 68, 107-140. Knight, D. E., and Baker, P. F. (1983). The phorbol ester TPA increases the affinity of exocytosis for calcium in "leaky" adrenal medullary cells. FEES Lett. 160, 98-100. Knight, D. E.. and Baker, P. F. (1985). The chromaffin granule proton pump and calciumdependent exocytosis in bovine adrenal medullary cells. J . Membr. Biol. 83, 147-156. Knight. D. E., and Kesteven. N. T. (1983). Evoked transient intracellular free Ca?' changes and secretion in isolated bovine adrenal medullary cells. Proc. R . Soc. London, Ser, E 218, 177-199. Knight, D. E.. and Koh. E. (1984). Ca?' and cyclic nucleotide-dependence of amylase release from isolated rat pancreatic acinal cells rendered permeable by intense electric fields. Cell Calcium 5, 401-418. Knight, D. E., and Scrutton, M. C. (1980). Direct evidence for a role for Ca2* in amine storage granule secretion by human platelets. Throtnh. Res. 20, 437-446. Knight, D. E.. and Scrutton, M. C. (1984a). The relationship between intracellular second messengers and platelet secretion. Eiochem. Sot,. Trcins. 12, 969-972. Knight, D. E.. and Scrutton, M. C. (1984b). Cyclic nucleotides control a system which regulates Ca?+-sensitivityof platelet secretion. Nrttrtre (London) 309, 66-68. Knight. D. E., Niggli, V . , and Scrutton. M. C. (1984). Thrombin and activators of protein kinase C modulate secretory responses of permeabilized human platelets induced by CaZT.Eur. J . Biorhem. 143, 437-446. Knight, D. E . , Tonge, D. A., and Baker, P. F. (1985). Inhibition of exocytosis in bovine adrenal medullary cells by botulinum toxin type D. Nutitre (London) 317, 719-721. Knopf, J . L., Lee, M.-H., Sultzman, L. H.. Kriz, R. W., Loomis, C. R., Hewick, R. M.,

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and Bell. K. M. (1986). Cloning and expression of multiple protein kinase C cL>NAs. C’o// 46. 491-502. Kraft. A . S.. and Anderson. W. U . (1983). Phorbol e\ters increase the amount of C:i>*. phospholipid-dependent protein kinase associated with pla\ma membrane. Ntrrrrw (LOt7dOtJ)301,

621-623.

Lingg. C.. Fischer-Colbrie. K.,Schmidt. W. and Winkler. H . (19x3). Expo5ure o f an antigen of chromaftin granules in cell surface during eaocytosis. Ntrrrrre (Lotrdori)301, 610-61 I . Llinas, R . , Steinberg. 1. Z., and Walton. K . (19x1). Pre\ynaptic calcium currents i n squid giant synapse. Biop/r,v.\. J . 33, 289-32 I . Llinas. K.. McGuineah. T. L. M.. Leonard. c‘. S..Sugimori. M . . and Greengard, P. ( 19x5). lntraterminal injection o f synapsin I or calciumic;rlmodulin-dependent protein kinase I I alters neurotransmitter release at the squid giant synapse. Proc.. N o r / . A ( rrd. .Sr.i. U. S . A .82. 3035-3039. Lucy. 1 . A . . and Ahkong. 0 . F. ( 19x6). An osmotic model tor the fusion of hiological membranes. FEBS L r / r . 199, 1 - 1 I . Lundberg. J . M . . and Hokfelr. T. ( 1983). (‘0-existence of peptide\ and classical neurotransi. .6, Y ~375-333. mitter\. 7wrrd.c N c u ~ ( J Meldolesi. J.. Scheer. H.. Madeddu. L . . and Wank. E. (1986). On the mechanism of action of cu-latrotoxin. the presynaptic stimulator)) toxin o f the black widow spider venom. Trt,trtl.r P/rtrr.trlrrc~o/.sc.i. 7, 151-155. Navone. F.. Greengard. P . . and l>e Caniillo. 1’. (19x4). Synap\in I in nerve terminals: Selective aswciation with \mall synaptic vesicles. .Sr.ietii.c, 226, 1209- 12 I I. Neher. E. and Almers. W . (1986). Fast calcium transients in rlit peritoneal mast cells are not sufficien~t o trigger- e x o c y t o k . E M B O J . 5, 5 1-53, Neher. E. and Marty, A . (1982). Discrete changes o f c e l l membrane capacitance ohserved under conditions of enhanced secretion in bovine adrenal medullary cells. Proc . Ntrrl. A> SITS, pyridoxal phosphate >> probenecid with isethionate being inactive, whereas the corresponding series for inhibition of chromaffin granule lysis was SITS > probenecid > suramin > pyridoxal phosphate >> isethionate. Conceivably, the differences in activity of the drugs in the two systems relate to anion specificity. 5-Hydroxytryptamine release from platelets was suppressed by increasing extracellular osmotic strength, and the relationship between suppression and osmotic strength was quantitatively similar to that observed for the inhibition of chromaffin granule lysis. FCCP also inhibited platelet secretion. Pollard et al. (1977) therefore concluded that platelets and chromaffin granules were similar in terms of the osmotic basis of the release reaction, but differed with respect to the anions involved. The chemiosmotic hypothesis of secretion was also investigated in dissociated parathyroid cells (Brown et al., 1978) from which parathyroid hormone (PTH) secretion could be elicited by exposure to a low extracelMar Ca2+concentration (Brown et al., 1976). Both SITS and probenecid were found to inhibit PTH secretion almost completely, whereas replacement of NaCl by either sucrose or sodium isethionate caused a 70% inhibition of secretion. The inhibition of secretion by SITS and probene-

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cid was competitive with respect to CI-, with each drug having a K , between 400 and 600 pM. Increasing the osmotic strength of the medium completely inhibited secretion. Various cation replacements for Na' had no effect on PTH release; FCCP blocked secretion. The observation that anion transport blockers inhibited PTH secretion almost totally, whereas omision of C1- or its replacement with isethionate only inhibited release by 70%. suggested the possibility that another anion beside CI- might also play a role. Lowering the medium pH inhibited PTH secretion, and probenecid was found to be a competitive inhibitor of release with respect to O H - . Thus, OH- might be another permeant anion in this system. Lysosomal enzyme release from human neutrophils stimulated with immune complexes was inhibited by SITS and 4,4'-diisothiocyano-2,2'stilbene disulfonic acid (DIDS), as was A23 187-induced release (Korchak et ul., 1980). Neither the nature of the permeant anion(s) nor the role of anion influx in degranulation was identified, however. Influx of CI-, OH-, or PO:- did not seem to be important. Permeant anions supported antigen-induced histamine release from human basophils in the following order: acetate > Br-, I- > CI- (Hook and Siraganian, 1981). lsethionate and SO:- did not support histamine release. SITS and probenecid did not inhibit IgE-mediated histamine release from these cells, and isosmotic solutions of several sugars were capable of supporting antigen-or antiIgE-induced histamine release in the presence of Ca2+.Increasing the osmolarity of the medium by adding more NaCI/KCI actually enhanced antigen- or anti-IgE-induced histamine release. These results do not support the hypothesis that exocytosis from basophils depends on anions or results from osmotic lysis. Finally, predictions based on the chemiosmotic hypothesis have been applied to the process of insulin secretion from pancreatic islet cells. Insulin release induced by glucose or a-ketoisocaproate from isolated rat pancreatic islets was inhibited when extracellular CI- was replaced with isethionate or SO:-; when the extracellular osmotic strength was raised by the addition of sucrose; and when the islets were exposed to probenecid or DIDS (Orci and Malaisse, 1980; Pace and Smith, 1983; Somers ef a / . , 1980). The inhibition of glucose-induced insulin release by C1- substitution was associated with a small decrease in glucose oxidation but no significant change in glucose-stimulated net 45Ca2tuptake by the islets (Somers e f a / . , 1980). In the isolated perfused rat pancreas the isethionate- or sucrose-induced inhibition of glucose-stimulated insulin release was shown to be a rapid and rapidly reversible phenomenon (Somers P t a / . , 1980). However, the substitution of C1- by isethionate in this system inhibited the second phase of the glucose-induced secretory response more than the first phase; it also failed to inhibit the insulin response to gliclazide. Similarly, in perfused rat islets, CI- substitution by isethionate

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did not inhibit the first phase of glucose-induced insulin secretion nor did it inhibit tolbutamide-stimulated secretion (Tamagawa and Henquin, 1983). Thus, although the chemiosmotic hypothesis for exocytosis may apply to the process of insulin secretion, further evidence is required to substantiate this mechanism. IV. OSMOTIC EFFECTS IN SECRETION FROM PERMEABILIZED CHROMAFFIN CELLS

In an effort to gain direct access to the secretory machinery of the cell and thus bypass the permeability barrier imposed by the plasma membrane, chromaffin cells have been permeabilized by subjecting them to high-voltage discharge. Catecholamine release from such cells can be induced by low Ca2+concentrations (in the low micromolar range) and requires ATP (Baker and Knight, 1978, 1980, 1981; Knight and Baker, 1982). The standard medium used to study catecholamine release from these cells contains glutamate as the principal anion. When glutamate was replaced with CI-, Ca2+-dependentrelease was inhibited. The effectiveness of different anions at inhibiting Ca2+-dependentrelease was in the order SCN- > Br- > CI- > acetate > glutamate, a sequence that follows the lyotropic series. Inhibitory anions may therefore bind to and disrupt some part of the release machinery which may regulate exocytosis in uiuo. Neither SITS nor DIDS (at concentrations of 0. I mM) had an effect on Caz+-evokedcatecholamine release. Glutamate (in the form of sodium glutamate) supported catecholamine release from intact chromaffin cells approximately 75% as well as CI- in the form of NaCl; glutamate also supported ATP-dependent chromaffin granule lysis, but significantly less well than C1- (Pollard ef al., 1984). Ca2+-activationof release from permeabilized cells was essentially normal in buffered isotonic sucrose. When the osmotic pressure of the medium was increased by raising the sucrose concentration, Ca2+-dependentrelease was inhibited. This observation may reflect the involvement of an osmoticially active step in the secretory process. The internal pH of chromaffin granules in electrically permeabilized cells was estimated to be 5.8, based on [*4C]methylamineuptake by the cells (Knight and Baker, 1985). Methylamine accumulation was greatly reduced in the presence of NH: or monensin (a protodcation exchanger), but was unaffected by the proton pump blocker trimethyltin or FCCP. SI4CN- accumulation was also used as a measurement of the granule membrane potential. The mean membrane potential was reduced in the absence of exogenous ATP and in the presence of trimethyltin or FCCP.

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Neither the intragranular pH nor the granule membrane potential was affected by Ca?' over the range of free Ca2+concentrations that activate secretion, except when large amounts of catecholamine were released. In that case both the methylamine and SCN- spaces were reduced. This is consistent with accumulation of these compounds in secretory granules and their release on exocytosis. Exposure of leaky cells to NH: concentrations that alkalinize the granule core failed to block Ca2t-dependent release, although there was a small reduction in this release at NHd concentrations that greatly reduced the pH gradient between the vesicle core and the cytosol. Both Ca2+-dependent release and S14CN- accumulation were dependent on ATP, but the latter was more sensitive than the former. There were also differences in the nucleotide specificity of these two processes, CaZ'-dependent release being very specific for ATP, and SI4CN- accumulation being almost equally activated by ATP, GTP, UTP, and ITP. Ca2+Dependent release still occurred when the granule proton pump and SCN- accumulation were inhibited by trimethyltin, although high trimethyltin concentrations did cause a small reduction in Ca?'-dependent release. Ca?+-Dependent release also persisted at FCCP concentrations that collapsed the granule membrane potential; however, very high FCCP concentrations caused a modest inhibition of Ca2+-dependent release. There were no significant alterations in the Ca2+activation curve of catecholamine release under conditions where either the vesicle pH gradient or the membrane potential was collapsed. Ca?+-dependentrelease also took place when both the granule pH gradient and membrane potential were collapsed with NHd and trimethyltin or FCCP, respectively. Therefore, Ca?+-dependent release from high-voltage-permeabilized cells appears to be largely unaffected by the pH gradient across the secretory vesicle or its membrane potential. However, as the authors point out. this conclusion is heavily dependent on the interpretation of SI4CN- and [14C]methylaminespaces in terms of the membrane potential and internal pH of the secretory vesicles. Although contributions to these spaces from other compartments such as lysosomes cannot be ruled out, it is likely that chromaffin granules make a significant contribution to these spaces. As Cat+-dependent release persists with both methylamine and SCNspaces close to the 3H20 space, it would seem that the pH gradient and membrane potential of the chromaffin granule are not necessary for this process. However, the reduction in Ca?+-dependentrelease seen at high concentrations of agents used to collapse the pH gradient and membrane potential suggests that these two processes may play a small role in exocytosis. It must be remembered, however, that in permeabilized cells neither a membrane potential nor ion gradients exist across the plasma

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membrane. Therefore, the release event in these cells may not be representative of what occurs in intact cells. The possibility that an “osmotic event” plays a role in the exocytotic process has also been investigated with chromaffin cells that were permeabilized with the detergent digitonin. As is true of electrically permeabilized cells, digitonin-permeabilized cells require Ca2+and ATP for catecholamine secretion (Dunn and Holz, 1983; Wilson and Kirshner, 1983). Glutamate and acetate were most effective in supporting Ca2+dependent secretion in these cells, with Br- and CI- less so (Wilson and Kirshner, 1983). Sucrose and glycine support Ca2+-evokedcatecholamine release at about 80% of that seen with sodium acetate (Wilson and Kirshner, 1983); potassium isethionate also supports release (Holz and Senter, 1985). Evidence from studies of [3H]noradrenaline uptake into digitonin-permeabilized cells suggests that an ATP-induced proton electrochemical gradient exists across the membrane of chromaffin granules within these cells (Holz and Senter, 1985). The observation that digitonin-treated cells, unlike intact cells, undergo little or no cell shrinkage in solutions made hyperosmotic with potassium glutamate or various carbohydrates indicates that molecules as large as tetrasaccharides have free access to the cell interior. Also, experiments in which the cells are placed in hyposmotic solutions have shown that chromaffin granules inside digitonin-permeabilized cells have osmotic stability similar to that of intact cells (Holz and Senter, 1985). The inhibition by hyperosmotic solutions of catecholamine secretion from digitonin-permeabilized chromaffin cells has been investigated by Holz and Senter (1986). These cells were much more resistant to increases in medium osmolality due to sucrose or other carbohydrates than were intact chromaffin cells. On the other hand, the permeabilized cells proved to be more sensitive to hyperosmotic solutions of potassium glutamate and sodium isethionate than to hyperosmotic carbohydrate solutions. Increases in salt concentrations inhibited secretion even if osmolality was kept constant with sucrose. The inhibition of secretion from intact cells by hyperosmotic solutions may therefore be mediated by increased intracellular ionic concentrations resulting from cell shrinkage. The fact that the sensitivity of electrically permeabilized chromaffin cells to hyperosmotic solutions seems to fall between that of intact and digitoninpermeabilized cells may be attributed to the differences in permeability characteristics, the digitonin procedure apparently allowing a more rapid equilibration of sucrose across the plasma membrane. Thus, a component of the hyperosmotic inhibition of Ca2+-dependentsecretion from electrically permeabilized chromaffin cells could be due to cell shrinkage and increased intracellular ionic strength. However, this interpretation as-

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surnes that chromaffin granules in digitonin-perrneabilized cells are irnperrneable to the osmoticants used and that significant granule shrinkage can occur under hyperosmotic conditions. While these findings do not rule out the possibility that an osmotically regulated process is involved in exocytosis, they place real constraints on such a process. Again, questions can be raised regarding the validity of using.permeabilized cells as a model system. In the course of membrane fusion during exocytosis in intact cells, ion gradients separate the granule interior from the extracellular medium at the site of fusion, and other ion gradients separate the granule interior from the cytosol. In permabilized cells, on the other hand, these two sets of ion gradients are essentially the same.

V.

CONCLUSIONS

The chemiosmotic hypothesis of secretion was formulated following the discovery of the chemiosmotic properties of isolated chromaffin granules. However, subsequent results obtained with intact and permeabilized cells revealed large differences between the predicted and observed secretory properties of these cells. An interesting finding to emerge from these studies was that isolated chromaffin granules display properties that differ from those exhibited by granules present in intact or perrneabilized cells. Perhaps granules undergo a “preparation catastrophe” during their isolation which causes them to express characteristics normally revealed only under stress. Studies are needed to elucidate the reasons why isolated granules show properties different from granules in their natural environment. Although the chemiosmotic hypothesis as originally proposed is no longer valid, several alternative hypotheses have been advanced according to which the osmotic swelling of secretory vesicles constitutes the force that drives the fission step in exocytosis. One such hypothesis, proposed by Geisow and Burgoyne (1982), is based on the observation that the exposure of isolated chrornaffin granules to the cation ionophore monensin in Na+- or K+-containing isotonic media resulted in granule lysis. Lysis occurred equally well in media containing the K’ or Na+ salt of the irnpermeant anion isethionate or in media containing the salt of the permeant anion C1-. Lysis was prevented by hyperosmotic media or by lowering the medium pH to 5.5. Since monensin also causes intracellular lysis of chrornaffin granules, the authors proposed that in the cell monensin probably exchanges cytosolic K+ ions for intragranular protons. The efflux of protons from the granule may cause the dissociation of intragran-

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ular impermeant weak acid species; the resulting K+ salt formation would lead to entry of H 2 0 into the granule. Grinstein et al. (1982) have proposed a model of secretory granule swelling during exocytosis in which the countertransport of H+/alkali metal cation across the granule membrane is mediated by an exchanger. More recently, Zimmerberg and Whitaker (1985) presented data that are consistent with the notion that a secretory granule must swell in order to fuse with the plasma membrane during the exocytosis of cortical granules in eggs of the sea urchin, Lytechinus pictus (see Jackson and Crabb, Chapter 2, this volume). Their experiments showed that granules shrink on exposure to the high osmolality stachyose medium and appear to swell slightly when exposed to Ca2+.Thus, it was proposed that under normal conditions Ca2+causes granule swelling which leads to fusion, and, in the presence of solutions of high osmolality, the Ca*+-induced swelling is not sufficient to result in fusion. The effects of Ca2+,however, are thought to be irreversible such that when the granule is returned to a solution of normal osmolality swelling continues and fusion occurs. The mechanism by which Ca2+promotes granule swelling is not known. Evidence that such a mechanism does not appear to be involved in exocytosis from the chromaffin cell, however, has been presented by Holz and Senter (1986). Using digitonin-permeabilized chromaffin cells, it was shown that pretreatment of cells (in which secretion was inhibited) with Ca2+in a solution made hyperosmotic with stachyose did not result in enhanced catecholamine secretion when cells were returned to normal osmolality. Finally, a model for exocytosis based on the opening of Ca2+-activated K+ channels in secretory vesicle membranes has been proposed (Stanley and Ehrenstein, 1985). The opening of these channels coupled to anion transport across the vesicle membrane could result in the influx of K+ and anions and subsequent osmotic lysis of the vesicles. At the moment, however, there is little evidence to support such a model. Thus, the idea that the fission step of exocytosis follows osmotic swelling of the secretory granule still appears to be accepted by many. However, the mechanism by which osmotic swelling is achieved, if indeed it occurs, remains to be elucidated. REFERENCES Abrahamsson, H . , and Gylfe, E. (1980). Demonstration of a proton gradient across the insulin granule membrane. Acra Physiol. Scand. 109, 113-114. Apps, D., and Schatz, G. (1979). An adenosine triphosphatase isolated from chromaffingranule membranes is closely similar to Ft-adenosine triphosphatase of mitochondria. Eur. J . Biochem. 100,411-419.

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Apps. D. K.. Pryde. J . G . . and Phillips, J . H. (1980). Both the transmemhrane pH gradient and the membrane potential are important in the accumulation of amines by resealed chromaffin-granule “ghosts.” FEES Lett. 111, 396-390. Arvan. P., Rudnick, G . , and Castle. J . D. (1984). Osmotic properties and internal pH of isolated rat parotid secretory granules. J . B i d . C‘hPm. 259, 13.567- 13572. Baker. P. F . , and Knight. D. E. (1978). Calcium-dependent exocytosis in bovine adrenal medullary cells with leaky plasma membranes. Nature. ( h n d o w ) 276, 620-622. Baker. P. F.. and Knight, D. E. (1980). Gaining access to the site of exocytosis in bovine adrenal medullary cells. J . Plrysiol. (Prrris) 76, 497-504. Baker, P. F.. and Knight, D. E. 11981). Calcium control ofexocytosis and endocytosis in bovine adrenal medullary cells. Philo.\. Trun.~.R . SOC. London. S e r . B 296, 83-103. Bashford, C. L.. Casey. R. P., Radda. G. K . , and Ritchie. G . A. (1976). Energy-coupling in adrenal chromaffin granules. Nf,ro.osc.ic.,rc,c,I, 399-412. Brown. E. M.. Hurwitz, S., and Aurhach, G. D. (1976). Preparation of viable isolated bovine parathyroid cells. E n d ~ c r i n ~ l ~99, ~ g1582y 1588. Brown. E. M.. Pazoles. C. J . , Creutz. C. E.. Aurbach. G . D.. and Pollard. H. B. (1978). Role of anions in parathyroid hormone release from dispersed bovine parathyroid cells. PI.OC.Nut/. Acrid. Sci. U . S . A . 75, 876-880. Cabantchik, Z. I., Knauf. P. A,, and Rothstein. A. (1978).The anion transport system of the red blood cell. The role of membrane protein evaluted by the use of “probes.” Biochim. Biophys. Actrr 515, 289-302. Carty, S. E . . Johnson, R. G . , and Scarpa. A. (1981). Serotonin transport in isolated platelet granules. Coupling to the electrochemical proton gradient. J . Biol. Chetn. 256, 11244I 12.50. Carty. S. E., Johnson. R. G . , and Scarpa, A. (1982). Electrochemical proton gradient in dense granules from anterior pituitary. J . Biol. Cltrm. 257, 7269-7273. Casey, R. P., Njus. D., Radda, G. K.. and Sehr. P. A. (1976). Adenosine triphosphateevoked catecholamine release in chromaffin granules. Osmotic lysis as a consequence of proton translocation. Biochrm. J . 158, 583-588. Casey, R. P., Njus, D.. Radda, G . K., and Sehr, P. A . (1977). Active proton uptake by chromaffin granules: Observation by amine distribution and phosphorus-3 I nuclear magnetic resonance techniques. Biocliemisrry 16, 972-977. Cidon. S., and Nelson, N. (1983). A novel ATPase in the chromaffin granule membrane, J . B i d . Chem. 258, 2892-2898. Cidon. S.. Ben-David. H., and Nelson, N . (1983). ATP-driven proton flux across membranes of secretory organelles. J. Biol. Clietn. 258, 11684-1 1688. Cohen, F. S., Zimmerberg, J.. and Finkelstein. A. (1980). Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. 11. Incorporation of a vesicle membrane marker into the planar membrane. J . Grn. Physiol. 75, 251-270. Cohen, F. S., Akabas, M. H., and Finkelstein. A. (1982). Osmotic swelling of phospholipid vesicles causes them to fuse with a planar phospholipid bilayer membrane. Science 217, 458-460, Creutz, C. E., and Pollard, H . B. (1980). A biophysical model of the chromaffin granule. Accurate description of the kinetics of ATP and CI dependent granule lysis. B i c p h y ~ . J . 31, 25s-270. Dean, G. E.. Fishkes, H., Nelson, P. J., and Rudnick, G . (1984).The hydrogen ion-pumping adenosine triphosphatase of platelet dense granule membrane. Differences from FIFO. 259, 9569-9574. and phosphoenzyme-type ATPases. J . B i ~ l Chem. Dolais-Kitabgi, J . , and Perlman, R. L. (1975).The stimulation of catecholamine release from chromaffin granules by valinomycin. Mol. Pharmnc,ol. 11, 745-750.

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Dunn, L. A., and Holz, R. W. (1983). Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells. J . Biol. Chem. 258,4989-4993. DiizgiineS. N. (1985). Membrane fusion. Subcell. Biochern. 11, 195-286. Ekholm. R., Ericson, L. E., and Lundquist, 1. (1971). Monoamines in the pancreatic islets of the mouse. Subcellular localization of 5-hydroxytryptamine by electron microscopic autoradiography. Diubetologiu 7 , 339-348. Geisow, M. J., and Burgoyne, R. D. (1982). Effect of monensin on chromaffin cells and the mechanism of organelle swelling. Cell Biol. I n r . Rep. 6, 933-939. Grinstein, S.. Meulen, J. V., and Furuya, W. (1982). Possible role of H+-alkali cation countertransport in secretory granule swelling during exocytosis. FEBS L e f t . 148, 1-4. Hampton, R. Y., and Holz, R. W. (1983). Effects of changes in osmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis. J . Cell B i d . 96, 1082-1088. Hellman, B., Lernmark, A., Sehlin, J . , and Taljedal, I.-B. (1972). Transport and storage of 5-hydroxytryptamine in pancreatic p-cells. Biochem. Pharmucol. 21, 695-706. Hoffman, P. G., Zinder, 0..Bonner, W. M., and Pollard, H. B. (1976). Role of ATP and fl,y-iminoadenosine triphosphate in the stimulation of epinephrine and protein release from isolated adrenal secretory vesciles. Arch. Biochem. Eiophys. 176, 375-388. Holz, R. W. (1978). Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential. Proc. Nurl. Acad. Sci. U.S.A. 75, 5190-5194. Holz, R. W. (1979). Measurement of membrane potential of chromaffin granules by the accumulation of triphenylmethylphosphonium cation. J. Biol. Chem. 254, 6703-6709. Holz, R. W., and Senter, R. A. (1985). Plasma membrane and chromaffin granule characteristics in digitonin-treated chromaffin cells. J . Neurochem. 45, 1548-1557. Holz, R. W., and Senter, R. A. (1986). Effects of osmolality and ionic strength on secretion from adrenal chromaffin cells permeabilized with digitonin. J . Neurochem. 46, 18351842. Holz, R. W., Senter, R. A., and Sharp, R. R. (1983). Evidence that the H+ electrochemical gradient across membranes of chromaffin granules is not involved in exocytosis. J . Biol. Chem. 258, 7506-7513. Hook, W. A . , and Siraganian, R. P. (1981). Influence of anions, cations and osmolarity on IgE-mediated histamine release from human basophils. Immunology 43, 723-73 1. Hutton, J. C. (1982). The internal pH and membrane potential of the insulin-secretory granule. Biochem. J . 204, 171-178. Hutton, J. C., and Peshavaria, M. (1982). Proton-translocating Mg2+-dependent ATPase activity in insulin-secretory granules. Biochem. J . 204, 161-170. Izumi, F., Kashimoto, T.. Miyashita, T., Wada, A., and Oka, M. (1977). Involvement of membrane associated protein in ADP-induced lysis of chromaffin granules. FEBS Letr. 78, 177-180. Johnson, R. G . , and Scarpa, A. (1976a). Internal pH of isolated chromaffin vesicles. J . Biol. Chem. 251, 2189-2191. Johnson, R. G., and Scarpa, A. (1976b). Ion permeability of isolated chromaffin granules. J. Cen. Physiol. 68, 601-631. Johnson, R. G., and Scarpa, A. (1979). Protonmotive force and catecholamine transport in isolated chromaffin granules. J . Biol. Chem. 254, 3750-3760. Johnson, R. G., Scarpa, A,, and Salganicoff, L. (1978). The internal pH of isolated serotonin containing granules of pig platelets. J . Eiol. Chem. 253, 7061-7068. Johnson, R. G., Pfister, D., Carty, S. E.,and Scarpa, A. (1979). Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients. J. Biol. Chem. 254, 10963-10972. Johnson, R. G., Carty, S. E., and Scarpa, A. (1981). Proton: substrate stoichiometries

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during active transport of biogenic amines in chromitffin ghosts. J . Biol. Chem. 256, 5773-5780. Knight, D. E., and Baker, P. F. (1982). Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields. J . Mm7hr. &id. 68, 107-140. Knight, D. E., and Baker, P. F. (1985). The chromaffin granule proton pump and calciumdependent exocytosis in bovine adrenal medullary cells. J . Mrmhr. B i d . 83, 147-156. Knoth, J . . Handloser. K.. and Njus, D. (1980). Electrogenic epinephrine transport in chromaffin granule ghosts. Bioc,hemis/ry 19, 2938-2942. Korchak. H. M.. Eisenstat. B. A., Hoffstein. S . T.. Dunham. P. B., and Weissmann, G . ( 1980). Anion channel blockers inhibit lysosomal enzyme secretion from human neutrophils without affecting generation of superoxide anion. Proc.. Nut/. A c a d . Sci. U . S . A . ?7, 2721-2725. Lishajko, F. (1969). Influence of chloride ions and ATP-Mg?’ on the release of catecholamines from isolated adrenal medullary granules. A c f a Phvsiol. Scand. 75, 255-256. Lorenson, M. Y., Lee, Y.-C., and Jacobs. L. S. (1981). Identification and characterization of an anion-sensitive Mg”-ATPase in pituitary secretory granule membranes. J . B i d . Ckem. 256, 12802-12810. Mitchell, P. (1961). Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nrrturt (London) 191, 144-148. Njus. D., Sehr, P. A., Radda, G . K., Ritchie, G . A., and Seeley. P. J . (1978). Phosphorus-31 nuclear magnetic resonance studies of active proton translocation in chromaffin granules. Biochemistry 17, 4337-4343. Ogawa, M . . and Inouye. A. (1979). Responses of the transmembrane potential coupled to the ATP-evoked catecholamine release in isolated chromaffin granules. Jpn. J . Phy.\iol. 29, 309-325. Oka, M.. Ohuchi, T., Yoshida, H . , and Imaizumi, R . (1965). Effect of adenosine triphosphate and magnesium on the release of catecholamines from adrenal medullary granules. Biochim. Biophvs. Acfu 97, 170-171. Orci. L., and Malaisse. W. (1980). Single and chain release of insulin secretory granules is related to anionic transport at exocytotic sites. Dicrheres 29, 943-944. Pace, C. S . . and Smith, J . S . (1983). The role of chemiosmotic lysis in the exocytotic releare of insulin. Endocrinology 113, 964-969. PaLoles, C. J . (1982). Anion and proton transport in chromaffin granules. Fed. Proc., Fed. Atn. Soc. Exp. Biol. 41, 2769-2774. Pazoles, C. J . , and Pollard, H. 8 . (1978). Evidence for stimulation of anion transport in ATP-evoked transmitter release from isolated secretory vesicles. J . Eiol. C h e m . 253, 3962-3969. Pazoles. C. J.. Creutz. C. E., Ramu. A.. and Pollard. H. B. (1980). Permeant anion activation of Mg-ATPase activity in chromaffin granules. Evidence for direct coupling of proton and anion transport. J . Bio(. C h e m . 255, 7863-7869. Percy, J. M., Pryde, J . G., and Apps. D. K. (1985). Isolation of ATPase I, the proton pump of chromaffin-granule membranes. BiochcJm.J . 231, 557-564. Phillips. J . H. (1977). Passive ion permeability of the chromaffin-granule membrane. B ~ o chem. J . 168,289-297. Phillips, J . H., and Allison, Y. P. (1978). Proton translocation by the bovine chromaffingranule membrane. Biochem. J . 170, 661-672. Poisner, A. M., and Trifaro, J. M. (1967). The role of ATP and ATPase in the release of catecholamines from the adrenal medulla. I . ATP-evoked release of catecholamines. ATP, and protein from isolated chromaffin granules. Mot. Phrumacol. 3, 561-571. Pollard, H. B., Zinder. O . , and Hoffman, P. G. (1976a). Occurrence and properties of

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chromaffin granules. I n "Biological Handbook, I: Cell Biology" (P. L. Altman and D. D. Katz, eds.). pp. 358-362. Fed. Am. SOC.Exp. Biol., Bethesda, Maryland. Pollard. H.B., Zinder, O., Hoffman, P. G., and Nikodijevik, 0. (1976b). Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogs, and external pH. J. Eiol. Chem. 251, 4544-4550. Pollard, H. B., Tack-Goldman, K.. Pazoles, C. J., Creutz, C. E., and Shulman, N. R. (1977). Evidence for control of serotonin secretion from human platelets by hydroxyl ion transport and osmotic lysis. Proc. Nut/. Acud. Sci. U.S.A. 74, 5295-5299. Pollard, H . B . , Pazoles, C. J . , Creutz, C. E., and Zinder, 0. (1979a). The chromaffin granule and possible mechanisms of exocytosis. I n t . Rev. Cytcd. 58, 159-197. Pollard. H . B., Shindo. H., Creutz, C. E., Pazoles, C. J.. and Cohen, J. S. (1979b). Internal pH and state of ATP in adrenergic chromaffin granules determined by "P nuclear magnetic resonance spectroscopy. J. B i d . Chem. 254, 1170-1 177. Pollard. H . B., Pazoles, C. J . , Creutz, C. E., Scott, J. H.. Zinder, O., and Hotchkiss, A. (1984). An osmotic mechanism for exocytosis from dissociated chromaffin cells. J. B i d . Ch6T.m. 259, 1114-1121. Pollard. H. B., Ornberg, R., Levine, M.. Kelner, K.. Morita, K., Levine, R., Forsberg, E., Brocklehurst, K. W., Duong, L., Lelkes, P. I.. Heldman, E., and Youdim, M. (1985). Hormone secretion by exocytosis with emphasis on information from the chromaffin cell system. Vitum. Horm. 42, 109-196. Russell, J. T. (1984). ApH, H' diffusion potentials, and Mg2*-ATPase in neurosecretory vesicles isolated from bovine neurohypophyses. J . B i d . Chem, 259, 9496-9507. Russell, J . T., and Holz, R. W. (1981). Measurement of ApH and membrane potential in isolated neurosecretory vesicles from bovine neurohypophyses. J . B i d . Chem. 256, 5950-5953. Saermark, T.. Andersen. N . M., Atke, A., Jones, P. M., and Vilhardt, H. (1986). Processing and secretion in the neurohypophysis. Stability of isolated secretory vesicles and role of internal pH. Biochem. J . 236, 77-84. Salama, G.. Johnson, R. G., and Scarpa, A. (1980). Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules. J . Gen. Physiol. 75, 109-140. Scherman. D., Nordmann, J., and Henry, J.-P. (1982). Existence of an adenosine 5'-triphosphate dependent proton translocase in bovine neurosecretory granule membrane. Eiochemistry 21, 687-694. Schuldiner, S., Fishkes, H., and Kanner, B. I. (1978). Role of a transmembrane pH gradient in epinephrine transport by chromaffin granule membrane vesicles. Proc. Nurl. Acad. Sci. U.S.A. 75, 3713-3716. Somers, G., Sener, A., Devis, G., and Malaisse, W. J . (1980). The stimulus-secretion coupling of glucose-induced insulin release. XLV. The anionic-osmotic hypothesis for exocytosis. Pfluegers Arch. 388, 249-253. Stanley, E. F., and Ehrenstein, G. (1985). A model for exocytosis based on the opening of calcium-activated potassium channels in vesicles. Life Sci. 37, 1985-1995. Tamagawa, T., and Henquin, J.-C. (1983). Chloride modulation of insulin release, nhRb+ efflux, and 4sCa2+fluxes in rat islets stimulated by various secretagogues. Diabetes 32, 4 16-423. Taugner, G. (1972). The effects of univalent anions on catecholamine fluxes and adenosine triphosphatase activity in storage vesicles from the adrenal medulla. Biochem. J. 130, 969-973. Toll, L., and Howard, B. D. (1978). Role of MgZ+-ATPaseand a pH gradient in the storage of catecholamines in synaptic vesicles. Biochemistry 17, 25 17-2523.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 32

Chapter 8 Polyanionic Agents and Inhibition of Phagosome-Lysosome Fusion: Paradox Lost M A YER B . GOREN' Department o j Molecular and Celliilar Biology Notional Jewish Center for Immunology cind Reqiratory Medicine Denvm, Colorudo 80206 and Department of Microbiology und 1rnmiinolog.v University of Colorado Health Sciences Center Denver. Colorado 80220 I.

11.

111.

1V.

V.

VI.

Introduction A . Historical Background B. Why Question the Polyanionics Hypothesis? C. Procrustes and an Alternative Interpretation The Fluorescent Lysosomal Probes A. Concerning Acridine Orange B. The Sulfonated Fluors: Mobile but Not Fickle Nonionic Hydrocolloids in Lysosomes: Fusion Inhibitors or Gelatinous Traps? A . Developing the Gelatinous Trap Model B. Differential Delivery of Lysosomal Constituents C. Differential and Sequential Transfer of Two Fluorescent Lysosornal Markers Fusion Inhibition Is Incompatible with the Cells' Functional Status A. Indigestion? Not at All B. Microbicidal Activities Some Residual Bodies A. Concerning Mechanisms: In Search of a Phenomenon'? B. Some Vesicles Are More Equal than Others Recapitulation and Conclusions References

'

Visiting Scientist, Department of Biophysics, The Weizmann Institute of Science, Rehovot, Israel.

227 Copyright 8 IYRB hy Academic P r e w Inc. All nghts of reproduction i n any form r e w v e d .

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When two texts contradict each other, the meaning run be determined only when u third texi is found which harmonizes them. Rabbi Ishmael, Introduction to Sifru

This anecdotal and editorial account is concerned with a hypothesis advanced by myself and colleagues from Mill Hill, London, about the antagonistic effects of endocytosed polyanionic substances on phagosome-lysosome fusion in cultured macrophages (Goren et al., 1976). It is a hypothesis that has been embraced by others and extensively studied. Still, I am now convinced that it is invalid. This chapter is an analysis of a host of studies-principally those of others-which, in our interpretation, support this conviction. The bulk of experimental evidence from our own studies is detailed in recent reports (Goren et al., 1987a,b). Because of its special relevance to the sulfatides of Mycobacterium tuberculosis, the “polyanionics hypothesis” is one to which I had an understandably strong commitment (Goren, 1977; Goren and Brennan, 1979). However, when I was unable to confirm the results of the earlier collaborative studies, the complication led us to recognize certain important artifacts of methodology. Based on these findings, I expressed my early doubts and apprehensions about polyanionic agents and fusion inhibition at the Third Leiden Conference on Mononuclear Phagocytes (Goren et al., 1980), but I believe the weight of unquestionably impressive contradictory opinions. also presented at the conference, was persuasive (Hart and Young, 1980; Kielian and Cohn, 1980a). Indeed, ultimately we were able to bring our results into agreement with those of others. But our more recent findings are for us convincing that the “polyanionics hypothesis” is flawed and invalid. For a participant in the creation and development of an attractive and seemingly fruitful concept, there is little satisfaction to be derived from its dismantling. That is a “pleasure”-a Schadenfreude-usually reserved for others! For me the dismantling is doubly disappointing, for it potentially denies the fusion-inhibiting activity of the sulfatides (“sulfolipids”) of Mycobacterium tuberculosis (see Goren and Brennan, 1979) and revokes their role as “virulence agents,” enjoyed for a brief period when they seemed to be implicated in protecting the intracellular pathogen from assault by the macrophage lysosomal hydrolases (Armstrong and Hart, 1971). For the present, the agent, if any, that mediates this behavior (see below) remains unknown. 1.

INTRODUCTION

A. Historical Background Certain viable intracellular pathogens, acting from within phagosomes, can prevent the fusion of the entrapping phagosorne with lysosomes. The

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phenomenon has been viewed as a potentially powerful mechanism in pathogenesis (Armstrong and Hart, I97 I ; Davis-Scibienski and Beaman, 1980: Friis, 1972; Horwitz, 1983; Jones and Hirsch, 1972; Wyrick and Brownridge, 1978; also see reviews by Densen and Mandell, 1980; Edelson, 1983; Goren, 1977; Goren c f al., 1980; Horwitz, 1982). Interest in identifying specific substances that might be implicated in antagonizing fusion led to the recognition of several structurally defined lysosomotropic (de Duve et a / . , 1974) polyanionic compounds that appeared to impose such a block to phagosome-lysosome (P-L)’ fusion from the lysosomal side. The original studies, exciting at the time, showed that when an active polyanionic such as suramin (Hart and Young, 1975),mycobacterial sulfolipids, or dextran sulfate (Goren r t a / . , 1976) was incorporated into macrophage secondary lysosomes, together with specific markers that also accumulate in these organelles (fluorescent acridine orange or electron-opaque colloidal ferritin), transfer of the lysosomal labels to newly formed phagosomes was largely prevented as compared with their prompt and abundant delivery to phagosomes in control cells. Viable Baker’s yeast, Saccharornyces crreuisiae, was a phagocytosis target of choice. Absence of the markers in the phagosomes after yeast uptake was logically viewed as evidence of P-L fusion inhibition. The resulting “polyanionics hypothesis” (Goren et d . , 1976; Goren, 1977) that linked polyanionic structure with an ability to induce the fusion dysfunction was subsequently “confirmed” by other investigators using similar techniques. And it was strengthened and broadened further in perhaps a score of publications that followed (Draper et a/., 1979; Geisow et d . , 1980, 1981; Hart, 1982; Hart and Young, 1978, 1979, 1980; Hart e l a / . , 1983; Kielian and Cohn, 1980a,b, 1982; Kielian ct a / . , 1982). But our own studies confronted us with a disturbing paradox. We consistently confirmed polyanion-induced fusion inhibition as judged from the behavior of the lysosomal acridine orange (AO) marker, whose transfer to yeast-containing phagosomes was almost abolished. But this was consistently denied by electron microscopic (EM) evidence: transfer of the particulate colloidal marker (Thorotrast) was izot inhibited-a finding also reported by Pesanti (1978), who used ferritin. The dilemma led us to recognize and describe serious artifactual limitations of the A 0 methodology stemming principally from the dye’s weakly basic properties (see below). We therefore largely abandoned A 0 as a lysosomal marker and concentrated on EM methodology and on fluorescent labeling with highly

’

Abbreviations: P-L phagosome-lysosome: AO, acridine orange: LR. lissamine rhodamine: S R , sulforhodamine; EM, electron microscopy: DS, dextran sulfate; FD, fluoresceinated dextran; SL, mycobacterial sulfolipids; FITC, fluorescein isothiocyanate: LY, lucifer yellow; TCA, trichloroacetic acid; KLH, keyhole limpet hemocyanin; HRP, horseradish peroxidase.

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ionized “nonpermeant” markers such as lissamine rhodamine, lucifer yellow, and sulforhodamine (see below) (Goren, 1983; Goren et al., 1984a). Our recent studies relieved, but still did not explain, the earlier dilemma of the contradictory A 0 versus EM results (Goren et a[., 1987a), but they allowed us to reestablish that polyanionic substances indeed can prevent transfer of both A 0 and the electron-opaque Thorotrast to phagosomes. In the main, we accepted this behavior as confirmation of the original hypothesis that we had long sought, and it brought our results into agreement with those of others. Nevertheless, 1 was not able to relieve a persisting suspicion that polyanion-induced fusion inhibition may be more artifactual than real. 8. Why Question the Polyanionics Hypothesis?

Is not the concordant evidence from both vital fluorescence acridine orange studies and from electron microscopy with markers such as ferritin or Thorotrast adequate to affirm that polyanions in fact antagonize P-L fusion? Certainly the apparent inhibition documented by both methods seems undeniable. We suggest, however, that most of the data heretofore reported for “pol yanion” cells as obtained with acridine orange may legitimately be questioned (see Section III,A) The fickle behavior of this mobile fluor has too often been ignored in a surrender to the dye’s seductive aesthetic allure. On the other hand, it seems reasonable that the transfer of Thorotrast from secondary lysosomes to phagosomes, whether in control or polyanion-containing cells, is probably a valid indication that the labeled lysosomes have participated in some kind of fusion process to effect delivery. But absence of the particulate marker from phagosomes is not unequivocal evidence that lysosomal fusion has been abolished. It implicates only a failure of delivery of a specific electron-opaque marker from lysosomes to phagosomes and provides no evidence about fusion behavior (a membrane phenomenon) or about the transfer, or not, of other lysosomal contents. Our conviction that the “polyanionics hypothesis” is invalid rests principally on the argument that no important function of macrophages seems to be disturbed by the supposed fusion inhibition, surely a strange finding if the activity of such a prominent cytoplasmic compartment is abolished in cells in which it must find almost constant use. This then is the “text” that contradicts the text of “no marker delivery, therefore no fusion.” C. Procrustes and an Alternative Interpretation

Studies in macrophages of phenomena that would be expected to be notably affected by an inhibition of phagolysosome formation have con-

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tinued to uncover puzzling features-ever more difficult to reconcile with a profound lysosomal dysfunction. In my opinion, this has required invoking increasingly tenuous and convoluted interpretations in order that a growing number of disparate and paradoxical experimental findings can all be accommodated into a polyanionic “Procrustean bed,” another recurrent theme. Our analysis suggests that a conjunction of methodologic and physicochemical pitfalls transformed the “bed” into an “iron maiden” in which all of us who have utilized the established techniques for studying the phenomena were embraced. Almost all of the accumulated evidence may be accounted for by the alternative interpretation that we propose, namely, that fusion is not inhibited, in contradiction of conclusions deduced solely from the retention of specific markers in lysosomes. We suggest instead that the polyanionic agents accumulate in lysosomes as gelatinous, sluggishly moving hydrosols that p h y s i c d v entrap electron-opaque markers and ionicul/y trap the usual alternative marker, the weakly basic acridine orange, in a matrix that may not transfer to phagosomes for many hours. But there are good reasons to believe, as described in succeeding sections, that more mobile lysosomal contents are still delivered to the fusion “partners,” after which the partly depleted lysosome probably pinches off and reseals itself. Evidence supporting this interpretation is presented later. In Section I1 (admittedly pedantic), the behavior of various fluorescent lysosomal labels will be examined in order to simplify analysis of phenomena that are observed by these techniques. Taken together with our model of lysosomal polyanionic “gels” that trap both EM markers and A 0 but do not inhibit P-L fusion, a plausible interpretation of earlier unexplainable A 0 data is then developed. The behavior of nonionic hydrocolloids as “fusion inhibitors” is then discussed, followed by sections that review and buttress arguments for rejecting the “polyanionics hypothesis.” The fertility of the alternative model is illustrated by the valid predictions and elementary interpretations that it allows.

II. THE FLUORESCENT LYSOSOMAL PROBES

A. Concerning Acridine Orange

An elementary analysis of the properties of acridine orange (Fig. I ) and its behavior as a lysosomal marker will provide an understanding of why it may so rarely be used with confidence. Its movement can be especially misleading in the presence of polyanionic agents. For additional, more detailed analyses, see de Duve et a / . , (1974), Allison and Young (1969), and Goren et al. (1984a).

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232

FIG. I .

Structure of acridine orange, a weakly basic permeant lysosomotropic dye.

1. PERMEATION OF MEMBRANES

Acridine orange is a weak base that in its uncharged free base form is sufficiently lipid soluble to traverse biological membranes fairly freely. As depicted in Fig. 2 (after de Duve et al., 1974), in lysosomes, of considerably lower pH than the cytoplasm, the base becomes protonated and trapped within these organelles because the membrane is a more formidable barrier to passage of the charged salt form: R R

I I

R-N:+HA

K

I I

L R - N + : H

A

R

Free base form Protonated salt form (traverses membrane freely) (traverses membrane poorly)

Incubation of normal macrophages with a weak base of appropriate pK ( A 0 in this instance) results in concentrating the dye within lysosomes (pH -4.5) up to several hundred-fold over its concentration in the cytoplasmic or extracellular space (pH -7.2). This concentration process is promoted by proton pumping at the lysosomal membrane, (de Duve et al., 1974; Ohkuma and Poole, 1978). The process is not instantaneous; achieving the final concentration within lysosomes may require several hours (de Duve et al., 1974). When a macrophage monolayer is exposed to 5 pg AO/ml in Hanks buffer for about 15 min, the lysosomes are usually adequately labeled to provide good transfer of fluor to a phagocytosed target such as a yeast cell. B Y ANIONIC SUBSTANCES 2. COMPLEXING

Acridine orange, a basic dye, complexes with and is bound by acidic substances: the mycobacterial sulfolipids (SL), cation-exchange resins (Lerman, 1961), microorganisms with acidic surfaces (i.e., tubercle bacilli), dead microorganisms (Strugger, 1947; Freidlin et d., 1977; Pantazis and Kniker, 1979; Goren et al., 1984a),and DNA. Accumulation of A 0 in macrophage lysosomes is powerfully reinforced when these contain pol yanionic substances such as suramin, SL, dextran sulfate, or polyglu-

8. POLYANIONIC AGENTS AND PHAGOSOME-LYSOSOME FUSION

pH4.5

233

2

W

FIG.2. Uptake of acridine orange by a macrophage. Protonation of the free base inside lysosomes traps the dye and concentrates it in these organelles. (Afterde Duve rr ul.. 1974.)

tamic acid. From our observations, a semiquantitative order of affinity of these substances or domains for A 0 is as follows: Dowex-50 or equivalent Polyanions within lysosomes Sulfolipids of M. tuberculosis Viable or killed tubercle bacilli Normal lysosomes; macrophage nuclei Killed and digesting microorganisms (yeasts, Escherichia coli) Viable microorganisms (yeasts) Accordingly, in a closed system containing elements as enumerated above, an excess of acridine orange will provide dye to all of the components. But if A 0 is available in only limited amounts, only the elements of highest affinity for the dye may be colored.

3 . L’ACRIDINA k MOBILE Because of the equilibrium between protonated and free base forms of AO, mobile free base is always present even if only in minute amounts and is free to traverse membranes. Therefore, in a closed system, A 0 from any compartment can distribute to any other compartment where it is more tightly bound. When a macrophage monolayer labeled with A 0 is exposed in medium to sufficient Dowex-SO (Na+.form), essentially all of the dye is transferred to the extracellular cation-exchange resin (Fig. 3) Goren, 1983; Goren r t a / . , 1980, 1984a). A second monolayer with lysosomes containing sulfoli-

MAYER 8.GOREN

234

HA=(

Dowex 50

\

FIG.3. Redistribution of acridine orange by membrane permeation from lysosomes to an external trapping agent, Dowex-SO.

pids or dextran sulfate may be substituted for the Dowex-50 beads. From the above affinity scale, one would infer that, if there is insufficient A 0 to saturate all domains, dye present in a compartment or organelle of lower affinity can redistribute to a domain of higher affinity, for example-and notably-to lysosomes sequestering polyanions (even in neighboring macrophages), if they are not saturated with the dye. 4. ARTIFACTSAND

A PARADOX

In employing acridine orange as the lysosomal label for assessing P-L fusion, most investigators expose the cells to about 5 pg AO/ml for about I5 rnin (Hart and Young, 1975; Kielian and Cohn, 1980a,b, 1982; Goren et al., 1984a,b). As noted before, this seems adequate for appropriate labeling and for subsequent delivery of the dye to (yeast) phagosomes (Fig. 4a). Penetration of the phagosome by A 0 to give a uniform bright green, yellow, or orange color is good evidence that the yeast or other target has been either severely injured or killed and is undergoing digestion (reviewed by Goren ef d . , 1984a). The macrophage nuclei are also bright green with acquired dye. Curiously, this is not a trivial observation. We have noted and described before (Goren et al., 1980, 1984a) that when “polyanion” macrophages sequestering, e.g., sufficient rnycobacterial sulfatides, dextran sulfate, or polyglutamic acid are similarly exposed FIG. 4. (a) Normal control macrophage prelabeled by exposure to 5 pg AO/ml and allowed to phagocytose viable yeasts. The yeast phagosomes (Y)show a variety of colors. Uniform yellow to orange colorations indicate the yeasts to be at least severely injured or dead, and undergoing digestion. The macrophage nuclei (N)are green with acquired dye, a typical fusion pattern, Bar, 10 pm. (b) Macrophages with mycobacterial sulfatide sequestered in lysosomes after exposure to 5 pg AO/ml for 15 min. The dye is entirely trapped in lysosomes (L) where the complexing capacity for A 0 has not been reached. The nuclei are devoid of dye. Bar, 5 Fm.

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for 10-15 min to 5 Fg AO/ml, the lysosomes become brilliant. But in contrast with the aspects of Fig. 4a, the nuclei are essentially devoid of dye (Fig. 4b). When such cells ingest yeasts, the phagosomes remain dark against a background of brilliantly fluorescent lysosomes. The nuclei still remain dark (Fig. Sa). Kielian and Cohn (1980a) reported the same behavior (see also Fig. 2b of Kielian ef u l . , 1982). This is a typical “nonfusion” pattern, for absence of dye from the phagosomes has been taken as definitive evidence that fusion has been suppressed. The apparent fusion inhibition is also supported by EM evidence: invariably, transfer of the Thorotrast lysosomal marker to the phagosomes is also highly suppressed. Surely this concordance would appear to be unequivocal evidence for fusion inhibition. Instead, we suggest on the basis of the alternative “gelatinous trap” interpretation that the “unequivocal evidence” is only a compounding of two artifacts: trapping of the markers and a deficiency of acridine orange.

5. PARADOX RESOLVED? We earlier described, but were not able to explain, how labeling of “polyanion” cells with IS-30 pg AO/ml (instead of 5 pg) for the usual IS min, followed by yeast phagocytosis, changes the “nonfusion” pattern to one of abundant fusion (Goren ef al., 1980, 1984a). Figure Sb shows such a polyanion monolayer (that was also prelabeled with Thorotrast) after a IS-min exposure to 25 p g AO/ml followed by yeast phagocytosis. The macrophage nuclei are green. The phagosomes are now aglow with dye. The yeasts are very likely dead and being digested, undeniable evidence of P-L fusion and delivery of both lysosomal A 0 and lysosomal hydrolases. But, paradoxically, the secondary lysosomes are evidently still unchanged-engorged and also brilliant with dye; this is ordinarily taken as evidence that fusion is inhibited (Goren rt al., 1980. 1984a). And this interpretation is still supported by EM evidence: the exposure to the higher concentration of acridine orange did not result in increased release FIG.5 . (a) Macrophage as in Fig. 4b 2 hr after ingestion of viable yeasts. Phagosomes

(Y)remain dark against a background of engorged brilliant lysosornes. The nucleus (N)is also dark because the A 0 exposure was inadequate to saturate the ”polyanion” lysosornes. Bar. 10 p n . (b) “Polyanion” macrophages labeled by exposure to 25 pg AOlml for 15 min. Viable yeasts were fed after the cells were washed and chased. Reversal of the inhibition of dye transfer seen in Fig. 5a is apparent. All of the phagosomes are brilliant with dye; the nuclei are green. But the polyanion lysosornes still retain the engorged status, with dye complexed by gelatinous polyanions. Thorotrast in these cells was not transferred to the phagosomes. Bar, 10 pm.

236

MAYER 6.GOREN

of thoria marker to the phagosomes (M. B. Goren and A. E. Vatter, unpublished). This situation can be resolved merely by invoking a “Procrustean bed” interpretation, namely, that the undeniable intrusion of lysosomal contents into the yeast phagosomes stems solely from the activities of primary lysosomes, for the secondary lysosomes are still clearly intact based on both EM and visual evidence. We suggest that contributions from primary lysosomes would indeed be expected, and will be discussed later. However, the “gelatinous trap” model taken together with the very high affinity of the polyanions for A 0 allows for a more convincing explanation of this curious behavior. Although the lysosomes of the “polyanion” cells exposed to 5 pg AO/ ml (Figs. 4b and 5a) have acquired abundant dye, the nuclei remain unilluminated. Since the polyanion lysosomes represent the domain of greatest affinity for A 0 in this system, it is evident then that the lysosomal complexing capacity has not been satisfied during the exposure to the usual 5 pg AO/ml. The appearance suggests that the gelatinous polyanion traps not only the Thorotrast but evidently all of the dye present in the cells. None is free to equilibrate with nuclei; none is free to be delivered to phagosomes. However, the capacity of the polyanion for weak bases is not infinite. It is evidently saturated by exposure to the higher concentration of A 0 (or might be by longer exposure to lower concentrations), and the evidence for this is found in the nuclei (Fig. 5b): they too have now acquired the fluor. Free dye is also accumulated by the lysosomes, probably by the usual passive diffusion and protonation, aided by proton pumping. It is largely this increment of free AO, then, that is delivered to phagosomes. The gelatinous polyanion with its complexed dye and entrapped thoria remains in the secondary lysosomes. And so the illusion of inhibited fusion of secondary lysosomes as judged by both EM and fluorescence microscopy is sustained-by a dual deception. B. The Sulfonated Fluors: Mobile but Not Fickle

From considering the sulfonated heterocyclic structures shown in Fig. 6, i.e., highly ionized, polar fluors of relatively low molecular weight, it may be inferred that lissamine rhodamine B (LR) (Goren, 1983; Goren et a!., 1984a), lucifer yellow (LY), and sulforhodamine (SR) should behave as impermeant fluorescent labels for macrophage secondary lysosomes (see also Goren et al., 1987a). Indeed, we found them to be taken up adequately from the culture medium by pinocytosis during prolonged exposure (usually more than 24 hr). Thus, the mechanism of uptake is

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so, H FIG. 6. Structure of the sulfonated heterocyclic fluors: ( a ) lissarnine rhodarnine. (b) lucifer yellow. and ( c ) sulforhodamine. These are strongly ionized in solution, mobile, irnperrneant, and accumulate in lysosomes. They are not cornplexed by polyanionic substances.

clearly different from the rapid passive diffusion, and trapping by protonation, that governs labeling by acridine orange. After uptake, all of the sulfonated probes showed a quite similar punctate-to-vacuolar appearance characteristic of secondary lysosomes, and these exhibited dramatic saltatory motions when viewed by means of time-lapse video recordings. Wang and Goren (unpublished) found no evidence in such time-lapse recordings that the saltatory motions were inhibited by either sequestered dextran sulfate or by 10 mM NH4CI in the medium. (Compare Hart er ul., 1983). The lysosomal localization of these fluors has since been well established for LY (Miller et al., 1983; Swanson et a / . , 1985) and for LR and SR (Wang and Goren, 1987).

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MAYER B. GOREN

Following phagocytosis of heat-killed yeasts, all of these dyes are delivered with about the same facility to the phagosomes and there penetrate and color the targets. Since we established that the behavior of viable and of heat-killed yeasts is identical in polyanion-containing macrophages (Goren rt al., 1984b, 1987a), the killed yeasts are legitimate targets for PL fusion studies using these highly ionic probes as lysosomal labels. When viable yeasts are used as targets, killing and especially digesrion evidently do not proceed rapidly enough for significant penetration of the targets by these fluors in less than about 6 hr (Goren et al., 1984a). They are mobile but, in our experience, not so freely so as acridine orange. Neither are they fickle: we found no evidence of any redistribution behavior such as we earlier documented for AO. This is in accord with their structures: because they are ionized salts of strong (sulfonic) acids, they are not complexed by lysosomally sequestered polyanionic substances or by macrophage nuclei, so there is no “demand” for these fluors in these domains. Neither is it plausible that they can be delivered from lysosomes to phagosomes (or elsewhere) by a facile traversal of biological membranes. As described before, various intracellular organelles can be essentially depleted of the permeant A 0 by, e.g., extracellular Dowex-SO. We found, however, that SR, as an example, although strongly adsorbed from solutions by anion-exchange resins, is not subject to such “robbing” from lysosomes by either strong acid or strongly basic ion-exchange resins. It is therefore impermeant. Hence polar fluors are not complexed and trapped by either polyanionic (DS, polyglutamic acid) or by nonionic hydrocolloids (Goren, 1983; Goren et al., 1987a). Although the monochromatic fusion figures that the sulfonated fluors exhibit are not so psychedelically exhilarating as those obtained with AO, the polar probes are judged to be much more reliable and trustworthy in their behavior. Use of FITC-labeled heat-killed yeasts as targets (Goren, 1983) enhances the aesthetic qualities and provides a palette of fusion figures (with SR lysosomes) from pure green (unfused) to orange-red that unequivocally demonstrates that P-L fusion is quantitatively the broad spectrum that would be expected for a biological system-not all black or white, but rather including many shades of gray. 111.

NONlONlC HYDROCOLLOIDS IN LYSOSOMES: FUSION INHIBITORS OR GELATINOUS TRAPS?

A fortuitous conjunction of circumstances led to an explosive multiplication in the number of documented polymeric materials that antagonize

8. POLYANIONIC AGENTS AND PHAGOSOME-LYSOSOME FUSION

239

transfer of lysosomal EM markers to phagosomes and so would be judged to inhibit P-L fusion. We asked (probably naively) if the quadrivalent positive thorium oxide marker might become cross-linked with adsorbed polyanionics into a hydrocolloidal gelatinous matrix that was effectively “immobilized” within lysosomes even if they were not inhibited in fusion with phagosomes. For us, the answer to this question provided the elusive harmonizing “third text” to which Rabbi Ishmael referred. A. Developing the Gelatinous Trap Model

The resulting search, detailed in recent publications (Goren rt d., 1987a,b), led to the surprising finding that a variety of nonionic waterdispersible polymers behaved remarkably like dextran sulfate (DS)in macrophage lysosomes. Exposure of cells to much higher concentrations was required, for unlike DS the neutral polymers are not endocytosed by an accelerated uptake that can concentrate the polyanions in lysosomes to surprisingly high levels (Cohn and Parks, 1967; Kielian et al.. 19821, and it is likely that the polyanions within lysosomes are much more viscous than the counterpart nonionic polymers (see below). The effects of the nonionics on secondary lysosomes were much the same as induced by DSmorphologically, and in interfering with transfer of EM marker to phagosomes (Goren ~t a l . , 1987a). However, in direct contradiction of the fusion inhibition implicit in the EM results,.pirorescent Iysosomul mcirkcrs used for simultaneous labeling were readily delivered to the phagosomes. not only the sulfonated fluors of the preceding section, but, of course, acridine orange as well. None of these are complexed by the nonionic polymers. A “Procrustean bed” analysis nevertheless would argue that the neutral nonionic polymers cilso must inhibit P-L fusion. and would dismiss the fluorescent marker transfer as being due to membrane permeation. It is of interest that Kielian et a / . (1982) and Kielian and Cohn (1980b), from comparing neutral dextran with the polyanionic dextran sulfate. concluded that cells exposed to 30 mg dextran/ml (instead of 10-20 pg DS/rnl) were nor inhibited in P-L fusion. But, as judged from their description, the behavior was evidently assessed only by fluorescence microscopy with A 0 and not by E M , and, as we noted, A 0 would not be trapped by dextran. Clearly the “Procrustean” interpretation embracing all of the neutral polymers as inhibitors would require a considerable dismantling of various hypotheses (described later) offered to account for the fusion-inhibiting behavior of the polyunions. We suggest instead that all of the polymers described, whether neutral, nonionic, or polyanionic, form “hydrosols” in water (Weiser, 1939)and in sufficiently high concen-

240

MAYER B. GOREN

trations will acquire a gelatinous or jellified structure that moves only sluggishly within (or out of) lysosomes. Indeed, fluorescence polarization measurements with fluoresceinated dextran (molecular weight 40,000; FD 40) indicate that the polymer behaves in lysosomes as if dissolved in 90% glycerol. Thus, its “motion in the normal intralysosomal environment was relatively restricted by comparism with free solution” (Geisow et al., I981). We would predict that lysosomal fluorescinated chlorite-oxidized amylose (“COAM”) (Geisow et ul., 1980) would exhibit an even more aberrant fluorescence polarization than FD 40 because of its much higher molecular weight and anionic (carboxylic) qualities. It has been described as a potent inhibitor of P-L fusion. B. Differential Delivery of Lysosomal Constituents We further deduce that the hydrosols are viscous and trap the colloidal EM markers physically, and we have presented EM evidence that supports this conviction (Goren et ul., 1987a). The neutral hydrosols cannot ionically trap any of the fluorescent mobile markers, which are therefore delivered to phagosomes with relative facility. This behavior is thus viewed as a diffPrentia/ delivery of lysosomal constituents. Polyanions, on the other hand, should trap only acridine orange ionically but should not trap the sulfonated probes. In accord with this expectation, when “dextran sulfate cells” were labeled with both Thorotrast and any one of the ionic sulfonated fluors, the dyes were transferred to phagosomes, whereas Thorotrast was not. This is still another example of differential delivery-and we cannot account for this by other than a fusion process. “Inhibition” of movement of EM markers must be understood not as an absolute abolition of marker transfer, but rather as a shift toward much lower amounts of label found in phagosomes as compared with those seen in control macrophages. The difference has been assessed by stereologic analysis (Kielian and Cohn, 1980a,b; Kielian et a/., 1982) or by visual “scoring” (scale 0-4) of the amount of marker delivered (Goren et ui., 1980, 1984b, 1987a). We have suggested, however, that because of the swelling of hydrocolloid lysosomes and distortions in distribution of the EM markers even these methods of comparing marker delivery in control and in experimental cells, although “sensitive” (Kielian et ul., 1982) may not be entirely valid (Goren et al., 1987a). As noted before, the ionic fluors are not as freely mobile as AO. Their transfer in the presence of DS requires about 3-4 hr for abundant delivery as compared with 1-2 hr in the presence of the neutral hydrocolloids. This probably reflects the consequences of a “cross-linking” of DS by bivalent cations and even, to an extent, by multiple cationic sites on proteins-to

8. POLYANIONIC AGENTS AND PHAGOSOME-LYSOSOME FUSION

24 1

increase the effective molecular weight of polymer and to promote its gelatinous qualities. “DS” cells that allow considerable transfer of sulforhodamine during 3-4 hr may still trap Thorotrast marker even after 24 hr (Goren et a/., 1987a). C. Differential and Sequential Transfer of Two Fluorescent Lysosomal Markers The differential transfer of the impermeant sulfonated fluors from lysosomes in which Thorotrast is presumed to be “immobilized” in a gelatinous nonionic hydrosol logically leads to predictions about derivative systems that allow for testing of the “gelatinous trap” model. If dextran is the polymer in the “jelly,” and is also rendered fluorescent, as by fluoresceination, then the green gel should be visihly immobilized in the lysosoma1 compartment. And it would be anticipated that if lysosomes are labeled with both the red mobile fluor and the green dextran then the red fluor of low molecular weight should transfer preferentially to phagosomes. We found the behavior to be essentially as predicted when cells were simultaneously labeled with sulforhodamine and fluoresceinated dextran (FD), molecular weight 40,000. The SR was delivered almost to the point of disappearing from the lysosomal domain during about 3 hr, to leave only the green FD behind. But afterward, this hydrophilic polymer of modest molecular weight, not surprisingly, then became transferred slowly to phagosomes over a period that lasted about 24 hr, when, for the most part, it was found in the same phagosomes to which SR had been delivered earlier. We have interpreted this behavior as supporting a piecemeal or “nibbling” process to describe the interactions of phagosomes with these particular kinds of lysosomes, i.e., containing components of grossly different mobilities (Wang and Goren, 1987). This is an important result: the FD 40 behavior is the same if it is used alone to label the macrophages. It also trapped Thorotrast, but only transiently (M. B. Goren, unpublished), probably because of insufficiently high molecular weight. Accordingly, if the functional status of such cells is judged only from the delivery of the FD 40 to phagosomes during a 4-5 hr period, then the cells would be considered “inhibited,” when in fact they are not! (see Goren et ul., 1984a, for an example). Based on these studies we anticipate that FD of considerably higher molecular weight or perhaps even DS conjugated with an appropriate fluor would remain immobilized in lysosomes for much longer periods, while mobile markers of low molecular weight are transferred differentially. Fluoresceinated “COAM,” an oxidized starch described as a remarkably potent “inhibitor of fusion” by Geisow et al. (1980), might

242

behave in degraded. molecular prolonged

MAYER B. GOREN

such a fashion for a prolonged period if it is not lysosomally It is a true polyanionic substance and of considerably higher weight than FD 40. Both characteristics should contribute to lysosomal immobilization.

IV. FUSION INHIBITION IS INCOMPATIBLE WITH THE CELLS’ FUNCTlONA L STATUS

As noted earlier, it is strange that no important function of macrophages seems to be disturbed by the supposed fusion inhibition, e.g., phagocytosis, microbicidal activity, but especially paradoxical is the welldocumented unaltered intracellular digestion. A. Indigestion? Not at All!

Data from our own fairly extensive quantitative studies, published in part (Goren, 1983; Goren et al., 1985), as well as more limited data from others indicate that the course of intracellular digestion is unaffected in “polyanion” macrophages. Hart and Young (1980) found digestion of S. cereuisiue “to proceed” in such cells and reconciled fusion inhibition with this behavior by invoking the participation of primary lysosomes. Indeed, they stated that lysosomal acid phosphatase was delivered “in only slightly reduced amounts into yeast-containing phagosomes” (in polyanion-inhibited cells). Kielian et al. (1982) reported that the enzymatic half-life of endocytosed horseradish peroxidase (HRP) was the same in control and in dextran sulfate (DS)-inhibited cells. Choosing among several interpretations, these investigators postulated that the normal digestion might be attributed to “a population of primary lysosomes and/or secondary lysosomes that have not yet taken up very much DS and that are able to fuse and degrade pinocytosed HRP.” In examining EM profiles of Thorotrast-labeled “polyanion” cells or cells in which the marker was trapped by a nonionic polymer, we have regularly recognized yeasts digesting in phagosomes in which no trace of marker was discernible (see Fig. 7). And we earlier described the entirely unaltered digestion of various radiolabeled soluble and particulate substances by “polyanion” cells. The surprising results were also interpreted as possibly being due to the activities of primary lysosomes or to incompletely inhibited secondary lysosomes (Goren, 1983). As stated earlier, participation of primary lysosomes would be expected: by definition they could not have accumulated any polyanionic substances. More recently we compared the digestion of yet another target--”SI-labeled heat-killed

FIG.7. Macrophage labeled with a colloidal gold preparation stabilized with gelatin and given heat-killed yeast cells to ingest. The gelatin hydrosol traps the gold and prevents transfer of the label to phagosomes. Nevertheless, the yeasts show signs of extensive digestion. Bar. 0.5 Fm.(Electron micrograph courtesy of Dr. N . Mor.)

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MAYER B. GOREN

yeasts-in control cells and in monolayers exposed to more than adequate dextran sulfate to inhibit Thorotrast transfer to yeast phagosomes. The release of trichloroacetic acid (TCA)-soluble radiolabel from these cells over 10 hr is depicted in Fig. 8 (Goren et al., 1985, 1987b). The conclusions are the same as reached earlier: digestion is essentially unaffected by the polyanionics residing in secondary lysosomes. To account for these kinds of data we suggested that perhaps secondary lysosomes of resident macrophages may be less functional than would have been thought (Goren et al., 1985). However, we abandoned this interpretation for it seemed weak and unconvincing (Goren et al., 1987b). 6. Microbicidal Activities

At one time the presumed fusion inhibition seemed to be a potential tool for dissecting the role of P-L fusion in the microbicidal activities of

\,. \*-

Residual IZsl

r

0

*

10

-

Normal Cells 0---0 Dextran Sulphate Cells &A

1

5

I 10

How$ FIG.8. Digestion of '2sI-labeled heat-killed yeasts by control macrophages compared with cells that have accumulated dextran sulfate in more than sufficient quantities to inhibit transfer of Thorotrast marker to the yeast-containing phagosomes. Monolayers in quadruplicate were pulsed with 1 x 106 opsonized radioiodinated yeasts for 45 min then washed free of uningested yeasts, and the cells were incubated in culture medium during the succeeding 10 hr. At the indicated intervals, the supernatants were collected, and their total radioactivity and TCA-soluble activity were assessed. The rate of release of soluble label during 10 hr was substantially identical for the two kinds of cells (data courtesy of Dr. W. J. Bruyninckx). (Reproduced from Goren et al., 1987b with permission of Alan R. Liss, Inc.)

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mononuclear phagocytes. And yet no convincing evidence has emerged that polyanion-induced P-L fusion “dysfunction” in macrophages antagonizes fungicidal or bactericidal behavior for S. cweuisiue, Li.stc.riu monocytogenes, Staphylococcus uiireiis, or Proteirs miruhilis, or affected even related activities, e.g. phagocytosis (Pesanti, 1978; Hart and Young, 1980; Geisow et ul., 1981; Kielian et al., 1982; Warr and Jakab, 1983). Our studies also showed that microbicidal activity for E . c d i or for Streprococciis fuecalis was the same in control and in “dextran sulfate” macrophages (Goren et al., 1985). The ambitious early study by Pesanti was stimulated by an extremely important goal: “If phagolysosome formation could be blocked, then the role of lysosomes in macrophage antimicrobial activity could be more accurately assessed.” Pesanti found that suramin pretreatment of the cells “inhibited transfer of acridine orange to phagosomes in macrophages that had interiorized either Snc.charornyces or L . tnonoc~ytogetie.s” (virulent or avirulent for mice). “However, electron micrographs of ferritin-loaded macrophages indicated that . . . there was fusion of lysosomes with phagosomes following ingestion of living Listeriu of either strain. All interiorized organisms in both control and suramin-treated macrophages werc contained within vacuoles in which ferritin was present . . . . ,. Pesanti simply accepted the EM evidence with L . monocytogc’nrs as definitive-for uninhibited fusion. However, Pesanti’s results can be explained away so as not to violate the “polyanionics hypothesis.” To account for the unrestricted fusion of phagosomes containing L. monocytogenes (in the presence of lysosomal suramin), Hart and Young (1980; see also Draper et a / ., 1979) invoked an overwhelming “fusion-promoting power of the intracellular targets” which surpassed the fusion-inhibiting power of the suramin; they dismissed the disparate A 0 data as being “due to optical limitations of the system.” Some data from Alexander (1981) suggest that “inhibition” of P-L fusion by poly-D-glutamic acid promoted the survival of Leishmania mexicuna mexicanu amastigotes and promastigotes in infected macrophages, and that chloroquine caused a marked reduction in parasite growth. It has been claimed that this and other lysosomotropic weak bases accelerate the P-L fusion process and also relieve the fusion block imposed by polyanions (Hart and Young, 1978, 1979, 1980). Although the evidence from Alexander is impressive, only a circular argument can link the effects with fusion “inhibition” on one hand and “fusion promotion” on the other. If there are components in macrophage lysosomes or in nonlysosomal granules that are cidal or inhibitory for Leishmuniu, might they not be inactivated or antagonized by polyanions? There is abundant evidence that the latter can inhibit a variety of enzymes (for review see Ginsburg and Sela, 1976). Considering the obverse side of

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MAYER B. GOREN

the coin, the lysosomotropic chloroquine will surely reach phagosomal targets either by permeation or via P-L fusion. But the rise in pH that this base elicits dramatically antagonizes the activity of the lysosomal acid hydrolases; this is reflected in an equally dramatic fall in digestive processing (Seglen el d., 1979; Ziegler and Unanue, 1982). Is it not then more likely that chloroquine by itself is the specific agent that damages intraphagosomal Leishmania rather than that the damage is due to a facilitated but probably ineffectual P-L fusion? The same analysis can be applied to the intracellular behavior of virulent M . tuberculosis. The normal nonfusion pattern for this pathogen is described as being reversed by “coating” the target with either normal or immune rabbit serum before phagocytosis. But this had no effect on the pathogen’s intracellular survival (Armstrong and Hart, 1975). The nonfusion pattern may also be reversed with chloroquine, but now with a concomitant intracellular killing or stasis of the pathogen (Hart and Young, 1980). If relief of the fusion block by (digestible) serum coating-which cannot have long lasting effects in protecting tubercle against any hydrolytic onslaught-fails to alter the intraphagosomal fate of the bacilli, how can a putative relief of the block by chloroquine, which simultaneously deranges lysosomal hydrolase activity, serve to kill or suppress the bacteria? Again, it seems probable that the behavior is due to a direct action of the amine on the pathogen. The facility of some weak bases to promote P-L fusion or to relieve a polyanionic “block” is not unequivocal. Kielian et a / . (1982) found that chloroquine “failed to modify inhibited P-L fusion.” Our (limited) tests with chloroquine on polyanion cells labeled with acridine orange gave similar negative results (Goren, 1983). Still, it seems plausible that chloroquine could cooperate with A 0 to saturate the complexing capacity of lysosomal polyanions for weak bases. An increment of complexed A 0 might then be released and transferred to phagosomes. Hence the chloroquine would substitute for the additional A 0 of Fig. 5b.

V.

SOME RESIDUAL BODIES

The resolution of the acridine orange paradoxes, the correct anticipation concerning the differential deliveries of any of the mobile fluorescent probes in the presence of nonionic hydrosols and of delivery of the sulfonated probes from dextran sulfate lysosomes, and the valid prediction of the behavior of colocalized low molecular weight red sulforhodamine and polymeric yellow-green fluorescein-dextran all seem to presage a utility of the “gelatinous trap” model to provide simple answers to some

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complex questions. There are. of course, exceptions-“residual bodies”-that have so far defied dissolution. The suggested antagonism between polyanions and the weak bases (above) is one example. Another might be the classification of microorganisms as “fusers” or “nonfusers” on the basis of their appearance within polyanion cells labeled for electron microscopy. But the assignment to the “fuser” category in effect endows the microorganism with an ability to influence from within the phagosome the response to the lysosoma1 polyanionic “inhibitors” (see Hart and Young, 1980; Draper et al., 1979). It is a facility that defies prediction and which can be assigned only after the fact. If EM marker delivery is recognized with a given target in cells that are “inhibited” when tested with viable yeasts, then the target in question becomes a “fuser”: otherwise it is a “nonfuser.” Current EM studies by N . Mor and M. Goren (in preparation) suggest that these “fuser”-“nonfuser” assignments are probably not warranted. However, the phenomenon alluded to earlier, of fusion inhibition from the phagosome by a limited selection of viable intracellular pathogens in normul macrophages. seems established, although the mechanisms are uncertain.

A. Concerning Mechanisms: In Search of a Phenomenon?

1 . LYSOSOMAL pH; MEMBRANE FLUIDITY The search for a convincing mechanistic interpretation to account for the “fusion-inhibiting” qualities of the polyanions has not yielded any profound insights. It has been neither a road to Damascus nor an Archimedes submerged. In a study by Kielian and Cohn (19821, “fusion inhibition” by dextran sulfate could not be correlated with a slight lowering of intralysosomal pH (from about 4.76 to about 4.36). and “increasing the lysosomal pH with . . . chloroquine failed to modify the inhibition of fusion.” But Geisow et al. (1981) found the intralysosomal pH of cells powerfully “inhibited’ with the polyanionic “COAM” to be increased to about 5.0. It is, therefore, unlikely that P-L fusin could be inhibited by an increased lysosomal pH on one hand and also by a decreased pH on the other. Fluorescence polarization studies with isolated DS-containing lysosomes labeled withp-parinaric acid, a membrane probe, led to the inference that direct interaction of lysosomal membranes with polyanions decreases the fluidity of the membranes and thereby antagonizes fusion (Kielian and Cohn, 1982). The postulated direct interactions are, of course, ionic. Accordingly, since the nonionic hydrocolloids mimic the behavior of DS in inhibition of Thorotrast transfer, then the modest influence of DS on membrane fluidity would appear to be of marginal significance.

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2. CALCIUM ION The inhibition of P-L fusion by polyanions is curiously insensitive to manipulations of the internal Ca2+concentration. It is well known that this ion has important roles in many membrane fusion processes (for review see Papahadjopoulos, 1978). Therefore, an appealing mechanistic interpretation for the presumed inhibition would take into account the capacity that the polyanionics surely have for complexing calcium. Moderate affinity of “COAM” for divalent cations has been described. Therefore, a relief of the block might be expected if a putative Ca2+deficiency were alleviated. However, according to Geisow et ul. (1980), although levels of both Ca2+and Mg2+in macrophages were artificially controlled with the ionophore A23187, neither high nor low concentrations of the cations appeared to affect P-L fusion in “COAM” cells that were fusion inhibited. T h i s j n d i n g should not be surprising. If, as we suggest, P-L fusion in these cells is actually not inhibited, but only marker is not transferred to phagosomes, then changes in intracellular Ca2+concentration would have no effect on the appearance of the yeasts. Marker would not be delivered in any event. In the absence of convincing alternatives, most investigators have concluded that the basis for “polyanion-induced fusion dysfunction” probably lies in membrane perturbations caused by interaction between the polyanion and multiple cationic sites in membrane proteins. Even this interpretation seems to be denied by the evidence. It is relevant that either sequestration of calcium or membrane perturbations might be fulfilled by particles of cation-exchange resins, which are, of course, particulate insoluble polyanions. It seemed to us, therefore, that these should be able to impose the fusion block from within the phagosomal confines. Our early studies in acridine orange-labeled cells with strong acid ionexchange particles served only to reveal important pitfalls of the A 0 technique (Goren et al., 1980b, 1984a). In recent EM studies we found that neither carboxylated nor sulfonated microscopic cation-exchange particles prevent P-L fusion: phagocytosis of these substances elicits a prompt and abundant fusion response (Goren et a l . , 1987a). B. Some Vesicles Are More Equal than Others

If polyanions indeed block fusion of biological membranes, then it is paradoxical that they accumulate in macrophage secondary lysosomes. Should not the polyanions inhibit fusion of the pinosome that carries it into the cell? Perhaps the pinosomal concentration is insufficient for this.

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But how can the delivery then be maintained in the face of a growing concentration of polyanions in the lysosomes that ultimately renders the latter “incapable” of fusing with phagosomes? Lysosomes heavily laden with dextran sulfate still undergo fusion with pinosomes that carry either horseradish peroxidase (HRP) or Thorotrast markers into the cells (Kielian et al., 1982). C. L. Swendsen and M. B. Goren (unpublished data) also have documented the labeling of “polyanion” cells with Thorotrast and have interpreted the results to mean that pinosomes are truly free to fuse with ‘‘inhibited” lysosomes. Accordingly, at the time we abandoned soluble materials (iodinated KLH) in favor of particulate targets (KLH-anti-KLH; radiolabeled yeasts, zymosan, etc.) as substrates for intracellular digestion studies in “polyanion” cells (Goren, 1983; Goren et al., 1985, 1987a).How than can we account for what is surely an awkward dilemma if the lysosomes are presumed to be unable to fuse? We now believe that no unusual properties of the pinosomal membrane need be invoked (see Kielian ef al., 1982)to relieve this paradox. Accepting unhindered fusion of polyanion lysosomes with both types of endocytic vesicles, we suggest that only “one-way” traffic of marker is allowed: HRP and Thorotrast are surely free to move from ordinary pinosomes into pol yanion-containing lysosomes following fusion, but lysosomal Thorotrast, trapped in a matrix of gelatinous dextran sulfate (polyanion), is not free to move out. VI.

RECAPITULATION AND CONCLUSIONS

In summary, no important macrophage function has been discerned that is antagonized by lysosomal accumulation of polyanions: phagocytosis, intracellular digestion, microbicidal activity. The apparent block to fusion is indifferent to Ca2+concentrations and cannot be imposed from the phagosomal compartment by particles of cation-exchange resins. If the “phenomenon” depends on perturbations in vesicle membranes, then pinosomal membranes would appear to be embarrassing exceptions to the inhibition; instead they are very likely behaving like phagosomal membranes, both allow fusion with “polyanion” lysosomes-but with oneway blocks to traffic. Neutral hydrocolloids mimic the behavior of polyanions with Thorotrast markers, but still allow delivery of permeant or nonpermeant mobile lysosomal fluors to phagosomes. We have documented similar behavior in “dextran sulfate” cells doubly labeled with mobile ionized fluors and Thorotrast; that is, the fluors are transferred to phagosomes, but the Thorotrast, and presumably the polyanion, are not. In addition, we have recognized a differential and sequential delivery that

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distinguishes between mobile impermeant fluors and fluoresceinated dextran of even moderate molecular weight. Finally, the gelatinous trap model allows understanding of the behavior of acridine orange in the presence of polyanions and why it can be manipulated. These findings justify the conclusion that polyanionic agents do not inhibit phagosomelysosome fusion. ACKNOWLEDGMENTS Original studies from my laboratory described here were supported by Grant A1 17509 from the National Institutes of Health and by Grant A1 08401 from the U.S.-Japan Cooperative Medical Science Program administered by the National Institute of Allergy and Infectious Diseases. I thank Nadia destackelburg for illustrations and Shirley Downs and Marjorie McCormick for careful preparation of the manuscript. M.B.G. is the Margaret Regan Investigator in Chemical Pathology, National Jewish Center for Immunology and Respiratory Medicine. REFERENCES Alexander, J. (1981). Leishmania mexicana: Inhibition and stimulation of phagosome-lysosome fusion in infected macrophages. Exp. Parasirol. 52, 261-270. Allison, A. C., and Young, M. R. (1969). Vital staining and fluorescence microscopy of lysosomes. In “Lysosomes in Biology and Pathology” (J. T. Dingle and H. B. Fell, eds.), Vol. 2, pp. 600-628. Elsevier, Amsterdam. Armstrong, J . A., and Hart, P. D. (1971). Response of cultured macrophages to Mycobacterium tuberculosis. with observations on fusion of lysosomes with phagosomes. J . Exp. Med. 134, 713-740. Armstrong, J. A., and Hart, P. D. (1975). Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual non-fusion pattern and observations on bacterial survival. J . Exp. Med. 142, 1-16. Cohn, Z. A., and Parks, E. (1967). The regulation of pinocytosis in mouse macrophages. 11. Factors inducing vesicle formation. J . Exp. Med. 125, 213-232. Davis-Scibienski, C., and Beaman, B. L. (1980). Interaction of Nocardia asteroides with rabbit alveolar macrophages: Association of virulence, viability, ultrastructural damage, and phagosome-lysosome fusion. Infect, Immun. 28, 610-619. de Duve, C., de Barsy, T., Poole, B., Trouet, A,, Tulkens. P., and Vanhoof. F. (1974). Lysosomotropic agents. Biochem. Pharmacol. 23, 2495-253 I . Densen, P., and Mandell, G. L. (1980). Phagocyte strategy vs. microbial tactics. Rev. Infect. Dis. 2, 817-838. Draper, P., Hart, P. D., and Young, M. R. (1979). Effects of anionic inhibitors of phagosome-lysosome fusion in cultured macrophages when the ingested organism is Mycobacterium lepraemurium. Infect. Immun. 24, 558-561. Edelson, P. J . (1982). lntracellular parasites and phagocytic cells: Cell biology and pathophysiology. Rev. Infect. Dis. 4, 124-135. Freidlin, I. S ., Khavkin, T. N., Artemenko, N. K., and Sakharova, I. Ya. (1977). Vital fluorescence microscopy of lysosomes in cultured mouse peritoneal macrophages during their interactions with microorganisms and active substances. 11. Interactions of macrophages with a non-pathogenic strain of Escherichia coli. Acta Microbiol. Acad. Sci. Hung. 24, 293-302.

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25 1

Friis, R. R. (1972). Interaction of L cells and Chlarnydiu p.yittu(.i-Entry of the parasite and host responses to its development. J . BLic,tcriol. 110, 706-721. Geisow. M. J.. Beaven. G . H., Hart, P. D., and Young. M. R . (1980). Site of action of a polyanion inhibitor of phagosome-lysosome fusion i n cultured macrophages. E x p . Cell Res. 126, 159-165. Geisow, M. J . , Hart, P. D., and Young, M. R. (1981). Temporal changes of lysosome and phagosome pH during phagolysosome formation i n macrophage$: Studies by Huore+ cence spectroscopy. J . Cell Biol. 89, 645-652. Ginsburg. I., and Sela. M. (1976). The role of leukocytes and their hydrolases in the persistence, degradation, and transport of bacterial constituents in tissues: Relation to chronic inflammatory processes in staphylococcal. streptococcal, and mycobacterial infections and in chronic periodontal disease. Crit. Rev. Mic,robiol. 4. 249-332. Goren. M. B. ( 1977). Phagocyte lysosomes: Interaction with infectious agents, phagosomes. and experimental perturbations in function. Annu. Reu. Micmhiol. 31, 507-533. Goren, M. B. (1983). Some paradoxes of macrophage functions. I n “Host Defenses to Intracellular Pathogens” (T. K. Eisenstein. P. Actor. and H . Friedman. eds.), pp. 3150. Plenum. New York. Goren, M. B., and Brennan, P. J. (1979). Mycobacterial lipids: Chemistry and biologic activities. In “Tuberculosis” (G. P. Youmans, ed.), pp. 64-193. Saunders, Philadelphia. Goren, M. B . . Hart, P. D.. Young, M. R.. and Armstrong. J . A . (1976). Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides of Myc~ohoc~teriurn tuhrrculosis. Proc. N a t l . Acud. Sci. U . S . A . 73, 2510-2514. Goren, M. B., Swendsen, C. L., and Henson, J. (1980). Factors modifying the fusion ot phagosomes and lysosomes: Art, fact, and artefact. I n “Mononuclear Phagocytes: Functional Aspects, Part 11” (R. van Furth. ed.). pp. 999-1038. Martinus Nijhoff. The Hague. Goren, M. B.. Swendsen. C. L., Fiscus. J.. and Miranti. C. (1984a). Fluorescent markers for studying phagosome-lysosome fusion. J . Lcrrkocvrc, Biol. 36, 273-292. Goren. M. B.. Vatter. A. E., and Fiscus, J . (1984b). Mycobacterial sulfolipids and polyanionics as inhibitors of phagosome-lysosome fusion: Resolution of an enigma. I n “Proceedings, Joint Tuberculosis and Leprosy Symposium,” pp. 33-42. US-Japan Cooperative Medical Science Program. Tokyo. Goren, M . B., Bruyninckx. W . J . , Leung. K.-P., Swendsen, L. S.. Heifets, L., and Fiscus. J . ( 1985). Functionality of secondary lysosomes in murine resident peritoneal macrophages. I n “Proceedings, Joint Leprosy and Tuberculosis Symposium.” pp. 9-30. U.S.-Japan Cooperative Medical Science Program. Bethesda. Maryland. Goren, M . B . , Vatter, A . E., and Fiscus, J . (1987a). Polyanionic agents as inhibitors of phagosome-lysosome fusion in cultured macrophages: Evolution of an alternative interpretation. J . Leukocyte Biol. 41, I 1 1-121. Goren. M . B.. Vatter, A. E., and Fiscus. J . (1987b). Polyanionic agents do not inhibit phagosome-lysosome fusion in cultured macrophages. J . Leukocyte B i d . 41, 122- 129. Hart, P. D. (1982). Lysosome fusion responses of macrophages to infection: Behavior and significance. In “Phagocytosis-Past and Future” (M. L. Karnovsky and L. Bolis, eds.), pp. 437-447. Academic Press. New York. Hart, P. D., and Young, M. R . (1975). Interference with normal phagosome-lysosome fusion in macrophages using ingested yeast cells and suramin. Nature (London) 256, 47-49. Hart, P. D., and Young, M. R. (1978). Manipulations of the phagosome-lysosome fusion response in cultured macrophages. Enhancement of fusion by chloroquine and other amines. Exp. Cell Res. 114, 486-490.

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Hart, P. D., and Young, M. R. (1979). The effect of inhibitors and enhancers of phagosomelysosome fusion in cultured macrophages on the phagosome membranes of ingested yeasts. Exp. Cell. R e $ . 118, 365-375. Hart, P. D., and Young, M. R. (1980). Manipulation of phagosome-lysosome fusion in cultured macrophages: Potentialities and limitations. In “Mononuclear Phagocytes: Functional Aspects. Part 11’’ (R. van Furth, ed.), pp. 1039-1055. Martinus Nijhoff, The Hague. Hart, P. D., Young, M. R., Jordan, M. M., Perkins, W. J., and Geisow, M. J . (1983). Chemical inhibitors of phagosome-lysosome fusion in cultured macrophages also inhibit saltatory lysosomal movements. A combined microscopic and computer study. J . Exp. M e d . 158, 477-492. Horwitz, M. A. (1982). Phagocytosis of microorganisms. Reu. Infect. Dis. 4, 104-123. Horwitz, M. A. (1983). The legionnaires’ disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J . Exp. Med. 158,2108-2126. Jones, T. C., and Hirsch, J . G. (1972). The interaction between Toxoplasmn gondii and mammalian cells. 11. The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J . Exp. M e d . 136, 1173-1 194. Kielian, M. C., and Cohn, Z. A. (1980a). Phagosome-lysosome fusion. Characterization of intracellular membrane fusion in mouse macrophages. J . Cell Biol. 85, 754-765. Kielian, M. C., and Cohn, Z. A. (1980b). Determinants of phagosome-lysosome fusion in mouse macrophages. In “Mononuclear Phagocytes: Functional Aspects. Part 11” (R. van Furth, ed.), pp. 1077-1095. Martinus Nijhoff, The Hague. Kielian. M. C., and Cohn, Z. A. (1981). Phorbol myristate acetate stimulates phagosomelysosome fusion in mouse macrophages. J . Exp. Med. 154, 101-1 1 I . Kielian, M. C., and Cohn, Z. A. (1982). Intralysosomal accumulation of polyanions. 11. Polyanion internalization and its influence on lysosomal pH and membrane fluidity. J . Cell Biol. 93, 875-882. Kielian, M. C., Steinman, R. M., and Cohn, Z. A. (1982). Intralysosomal accumulation of polyanions. 1. Fusion of pinocytic and phagocytic vacuoles with secondary lysosomes. J . Cell Biol. 93, 866-874. Lerman, L. S. (1961). Structural considerations in the interaction of DNA and acridines. J . Mol. Biol. 3, 18-30. Miller, D. K., Griffiths, E., Lenard, J., and Firestone, R. A. (1983). Cell killing by lysosomotropic detergents. J . Cell Biol. 97, 1841-1851. Ohkuma, S., and Poole, B. (1978). Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Narl. A c a d . Sci. U . S . A . 15, 3327-3331. Pantazis, C. G., and Kniker, W. T. (1979). Assessment of blood leukocyte microbial killing by using a new fluorochrome microassay. J . Reticuloendothel. SOC.26, 155-170. Papahadjopoulos, D. (1978). Membrane fusion. Calcium-induced phase changes and fusion in natural and model membranes. Cell Surf. R e v . 5, 766-790. Pesanti, E. L. (1978). Suramin effects on macrophage phagolysosome formation and antimicrobial activity. Infecf Immun. 20, 503-51 1. Seglen, P. E., Grinde, B., and Solheim, A. E. (1979). Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur. J . Biochem. 95, 215-225. Strugger, V. S. (1947). Die Vitalfluorochromierung des Protoplasmas. Naturwissenschaften 34, 267-273. Swanson, J. A,, Yirinec, B . D., and Silverstein, S. C. (1985). Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J . Cell Biol. 100, 851-859.

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Wang, Y.-l., and Goren, M . B. (1987). Differential and sequential delivery of lysosomal fluorescent probes into phagosomes in mouse peritoneal macrophages. J . Cell Biol. 104, 1749-1754. Warr. G. A . , and Jakab, G. J . (1983). Lung macrophage defense responses during suramininduced lysosomal dysfunction. E x p . M o l . Parhol. 38, 193-207. Weiser. H. B. (1939). “Colloid Chemistry.” Wiley. New York. Wyrick. P. B., and Brownridge, E. A. (1978). Growth of Chltrmydiu p . s i r / ~ c in i macrophages. Infecr. I m m u n . 19, 10.54-1060. Ziegler. K.. and Unanue, E. R. (1982). Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells. Pvac. Narl. Acud. Sci. U . S . A .79, 17.5-178.

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Part Ill

Virus-Cell Fusion

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 32

Chapter 9

Fusion of Viral Envelopes with Cellular Membranes SH UN-ICHI OHNISHI Depctrtment of Biophysics Fucirlty of Science Kyoto University Sak-vo-kid. Kyoto 606, Jnpcin

Introduction Membrane Fusion Activity of Enveloped Viruses A . HVJ or Sendai Virus B . Influenza Virus C. Semiliki Forest Virus D. Vesicular Stomatitis Virus 111. Mechanism of Fusion A . Binding and Close Apposition B. Interaction of the Hydrophobic Segment with the Target Cell Membrane Lipid Bilayer LV. lnfectious Cell Entry Mechanisms References I.

11.

1.

INTRODUCTION

Many families of viruses have an envelope wrapping their genome, RNA o r DNA. The envelope is a lipid bilayer membrane with the virusspecific glycoproteins spanning it. The bilayer originates from the host cell membrane and has a lipid composition and transbilayer distribution quite similar to the host's. The viral glycoproteins have the functions of binding to the target cell surface and fusion with the cell membranes (see Table I). The two functions are carried by a single glycoprotein in influenza virus (HA), VSV (G), and SFV (E).' In HVJ, the functions are 257 Copyright 0 19811 by Academic Pre\\. Inc All rights of reproduction in any form reserved.

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SHUN-ICHI OHNlSHl

TABLE I VIRALENVELOPE GLYCOPROTEINS

Virus Hemagglutinating virus of Japan (HVJ) Influenza virus Semliki Forest virus (SFV) Vesicular stomatitis virus (VSV) Mouse mammary tumor virus (MMTV) La Crosse virus Mouse hepatitis virus

Molecular weight (K) Glycoprotein

HN

61

Function

Refs.u

Binding and neuraminidase

I

F2 + FI 52, I I HA1 + HA2 44, 30 48-63 NA E3, E2, 1I, 52, El 51 G 61

Fusion Binding and fusion Neuraminidase Binding and fusion

2, 3 4, 5

Binding and fusion

8

gp52 + gP36 G I , G2 E2 + 90A + 90B

52, 36

Fusion

9

120, 34 120 90, 90

Binding and fusion (?) (GI)

6 7

10

II Binding and fusion (90A)

Key to references: (I)Kohama er 01. (1978). (2) Homma and Ohuchi (1973). (3) Scheid and Choppin (1974). (4) Lazarowitz and Choppin (1975). ( 5 ) Klenk el ul. (1975). (6) Bucher and Pdlese (1975). (7) Garoff el u!. (1980). (8) Rose and Gallione (1981). (9) Redmond and Dickson (1983). (10) GonzalezScarano (1985). (11) Sturman ef ul. (1985).

carried by separate glycoproteins, HN for binding and F for fusion. When viruses encounter target cells, they first bind to the cell surface through interaction of the viral glycoprotein with receptors (Fig. la). Sialoglycoproteins andlor sialoglycolipids are known to be the receptors for H N in HVJ and HA in influenza virus. Fusion of the virus envelope with target cell membranes is an essential initial step in infection since the virus can transfer its genome into the cytoplasm by this event (Fig. lc). Envelope fusion is induced by the action of the virus glycoprotein on target membranes. Some fusion proteins are initially produced in precursor forms and then cleaved posttranslationally by proteolytic enzymes. F in HVJ and HA in influenza virus are such examples. They are produced as precursor forms FO and HA0 and then cleaved into F2 and F1 (Homma and Ohuchi, 1973; Scheid and Choppin, 1974) and HA1 and HA2 (Klenk et al., 1975; LazaroI Abbreviations: HVJ, hemagglutinating virus of Japan, a synonym of Sendai virus; SFV, Semliki Forest virus; VSV, vesicular stomatitis virus; MMTV, mouse mammary tumor virus; HA, hemagglutinin; BHA, HA released from influenza virus by bromelain treatment; IMPS, intramembrane particles; PC, phosphatidylcholine; PE, phosphatidylethanolarnine; PS, phosphatidylserine.

9. FUSION OF VIRAL ENVELOPES WITH CELLULAR MEMBRANES

259

"4 ff

t

FIG. I . (a) Binding of an enveloped virus to a target cell membrane through interaction of the viral glycoproteins with receptors. (b) Close apposition of the two membranes and disturbance in the target cell membrane lipid bilayer by the hydrophobic segment of the virus fusion glycoprotein. ( c ) Fusion of the two membranes. resulting in release of the viral nucleocapsid into the target cell cytoplasm. The nucleocapsid is a complex of virus genome. RNA or DNA, with proteins. Matrix proteins underneath the envelope probably bind to both the membrane and nucleocapsid.

witz and Choppin, 19751, linked by disulfide bonds between the subunits, respectively. The precursors are inactive, but the cleaved forms are active in both fusion and infectivity, strongly suggesting a causal relationship between them. Other examples are gPr73 in MMTV and E2 in mouse hepatitis virus which are cleaved into gpS2 and gp36 (Redmond and Dickson, 1983) and 90A and 90B (Sturman et al., 1983, respectively, to become active. E in S F V is cleaved into E3, E2, and El (Garoff et d.,1980). On the other hand, G in VSV is not subject to cleavage. The amino-terminal segment of HA2, F1, and gp36 consists of 20 or more hydrophobic amino acid residues (Table 11). That of HA2 also contains two or three acidic residues. These segments are produced on posttranslational proteolytic activation. They are assumed to be responsible for fusion activity because of the hydrophobicity and also because of the conservation of sequence among various strains of HVJ and influenza virus (Gething el a!., 1978). However, the sequence homology is not always observed; for example, influenza C virus and pneumovirus have sequences different from other viruses except for the hydrophobicity. E l in SFV and G in VSV do not contain such amino-terminal hydrophobic segments but have internal hydrophobic stretches. Residues 80-100 in El and residues 100-131 in G, which have sequence homology among the strains, may be such stretches though not strongly hydrophobic (Table 11). They also contain a few acidic residues within the sequence. In the fusion reaction, the two membranes should come close together,

TABLE I1 HYDROPHOBIC SEGMENTSOF VIRALFUSIONGLYCOPROI-EINS"

fu

8

Pararny xoviridae Paramyxovirus F1 HVJ' sv5=

NDV' Pneurnovirus FI RS3 Ort hornyxoviridae Influenza virus HA2 A/PR/8/34 (H I )4 NJapan1305157 (HW A/Aichi/2/68 (H3F NFPV/Rostock/34 (H7)' BIL.ee/W Influenza C virus C/CaI/l 89

FFGAVIGTIALGVATSAQITAGIALAEARFAGV VIGLAALGVATAAQVTFIGAIIGGVALGVATAAQITFLGFLLGVGSAIASGVAVSK-

GLFGAIAGFIEGGWTGMIDGWYGYHGLFGAIAGFIEGGWQGMVDGWYGYH-

GLFGAIAGFIENGWEGMIDGWYGFRGLFGAIAGFIENG WEGLVDGWYGFRGFFGAIAGFLEGGWEGMIAGWHGTY-

IFGIDDLIIGLLFVAIVETGIGGYLLGSR-

Togaviridae Alphavirus El SFV (79-1 10)"' Sindbis (79-1 Rhabdoviridae Vesiculovirus G VSV Ind (100-132)" VSV NJ (100-132)'3 VSV Ind ( 1 74-200)12 VSV NJ ( 174-200)'2 Retroviridae Type B oncovirus MMTV gp3614 Type C oncovirus MoMLV plSE' Akv p15Ei6 F-MUIVp15E'ATLV p20E18

-KVYTUVYPFMWGGAYCFCDSENTQLSEAYVDR-KVFGGVY PFMWGGAQCFCDSENSQMSEAYVEL-

-KQGTWLNPG FPPQSCGYATVTDAEAVIVQVTPH-

-KDGVSFNPGFPPQSCGYGTVTDAEAHIVTVTPH-KGLCDSNLISMDITFFSEDGELSSLGK-ESVCSQLFTLVGGIFFSDSEEITSMGL-

FVAAIl LGlSALl AIITSFAVATTALVK-

EPVSLTLALLLGGLTMGGIAAGIGTGTTALMATQQFQQLQAA VQDDLREPVSLTLALLLGGLTMGGlAAGVGTGTTALVATQQFQQLQ.4AMHDL)LKEPVSLTLALLLGGLTMGG1AAGVGTGTTALVATQQFQ~LHAAVQL)DLKAVPVAV WLVSALAMGAGVAGGITGSMSLASGK-

" Key 10 superscript reference numbers: I I ) Blumberg e f ctl. (19851: Hsu and Choppin (1984). ( 2 ) Richardson

r r / . (IYXO). ( 3 1Collins (1984).(4) Winter rf ul. (1981). ( 5 ) Gething " f ul. (1980). (6) Verhoeyen e r d . (1980). (71 Porter C I d . (197Y). ( X I Kr! stal c*/ d.(1982). (9) Nakada ef ul. (1984). (10) Garoff el 01. (1980). ( 1 1 ) Rice and Strauss (19x1). (12) Rose and Gallione 11981). (13) Gallione and Rose (1983). (14) Redmond and Dickson (1983). ( 1 0 Shinnick ef crl. 11981). (16) Lenz ef ol. 119821. (17) Koch c r ol. (19831. (181Seki c d . (1983). (21

ef ul.

(21

262

SHUN-ICHI OHNlSHl

and the putative fusogenic segment should be able to interact with the target membrane, inducing some disturbance eventually leading to fusion (Fig. Ib). Generally, proteins may have similar hydrophobic segments in them. Even acqueous enzymes have such segments: two in porcine trypsin and one in hepatic alcohol dehydrogenase (Asano and Asano, 1984). Virus fusion proteins can also have more than one hydrophobic segment. For example, residues 175-199 in G is another hydrophobic sequence (Table 11). These hydrophobic segments can be fusogenic only when they approach and interact with target membranes. Envelope fusion was first shown to occur for HVJ by electron microscopy (Howe and Morgan, 1969). Such fusion was not clearly demonstrated for other viruses, however, and, instead, uptake of viruses into intracellular coated vesicles and smooth vesicles was observed. This raised a question of the mechanism of virus genome transfer into the target cell cytoplasm for these viruses. Ten years later, the induction of envelope fusion activity in mildly acidic media was discovered for SFV (VMnanen and Kaariainen, 1979, 1980), influenza virus (Maeda and Ohnishi, 1980; Huang ef al., 1981; Lennard and Miller, 19811, and later VSV (White et al., 1981; Mifune et al., 1982; Matlin et al., 1982). The pH dependence of fusion was markedly different; while HVJ can fuse at neutral as well as acidic pH values, SFV, influenza virus, and VSV can fuse only at acidic pH (Fig. 2). A new virus entry mechanism was proposed for these viruses on the basis of these findings, SFV by Helenius et ul. (1980), influenza virus by Maeda and Ohnishi (1980), Matlin et al. (19811, and Yoshimura et al. (1982), and VSV by Matlin et al. (1982). After uptake into intracellular vesicles, these viruses fuse with vesicle membranes when the intravesicular lumen becomes acidic and release their genome into the cytosol. At first, lysosomes were proposed as the acidic compartment, since they are well known to have a pH of 4.8 (Ohkuma and Poole, 1978). After the proposal, however, a rapid acidification of prelysosomal endocytic vesicles (endosomes) was discovered in 1982 by Tycko and Maxfield (1982) and van Renswoude et al. (1982). Genome transfer by fusion with endosomes was shown shortly afterward by Marsh et al. (1983b) for SFV and Yoshimura and Ohnishi (1984) for influenza virus. In this chapter, I first review some characteristic features of membrane fusion activity for each virus and then discuss the mechanisms of membrane fusion, especially low pH-induced membrane fusion. I concentrate on the interaction of the hydrophobic segment with the target cell membrane lipid bilayer and suggest the entrance of the segment into the lipid bilayer hydrophobic core as a key step in fusion. For the hydrophobic segments containing a few acidic residues, I emphasize protonation of those residues as a requirement for the entrance. Finally, I briefly review

9. FUSION OF VIRAL ENVELOPES WITH CELLULAR MEMBRANES

~ H V J

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263

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o

b IFV

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PH Fic. 2. pH dependence of membrane fusion activity of enveloped viruses: envelope fusion (13). hemolysis ( 0 )and . cell fusion (A). Curves were derived from the following data: inlluenra virus envelope fusion and cell f u \ i o n with MDCK cells IYoshimur'u c'f ( I / . . 19x2): SFV envelope fusion with BHK-21 cells (White c! o / , , 1980) and fusion with BHK-21 cell\ (White c ~ f [ I / . . 19x1): VSV envelope fu5ion and hernolysis with lrypsinized erythrocytes (Yamada and Ohnishi. 1986) and fusion with MDCK cells (White et ( I / . , 1981). (Modified from Ohnishi and Yoshimura. 19x4.)

the entry pathway of virus into cells leading to infection. Several review articles have been published on virus membrane fusion activity (White r t a / . ,1983; Ohnishi and Yoshimura, 1984; Asano and Asano, 1984; Ohnishi, 1985a) and virus entry mechanisms (Helenius t r f i l . , 1980: Dimmock, 1982; Marsh, 1984). II. MEMBRANE FUSION ACTIVITY OF ENVELOPED VIRUSES

A. HVJ or Sendai Virus

I . ENVELOPE FUSION Fusion of HVJ with erythrocyte membranes was first observed by electron microscopy as a dispersal of viral antigen into the cell membrane (Howe and Morgan, 1969). Virus-induced hemolysis has been used as a

264

SHUN-CHI OHNlSHl

rapid, sensitive, and convenient assay for envelope fusion. However, this assay is indirect since it measures a result of envelope fusion. A different approach based on intermixing of viral lipids with target cell membrane lipids was developed (Maeda et al., 1975; Ohnishi, 1985a). Virus is incubated with spin-labeled phospholipids to incorporate them into its envelope. The spin-labeled virus is then incubated with target cell membranes. The ESR peak height increases on fusion due to dilution of spin-labeled phospholipids with cell membrane lipids. Assays of fusion using fluorescent probes based on the same principle were also developed (Wyke at al., 1980; Struck et al., 1981). A recent example is to use octadecyl rhodamine B chloride loaded into virus envelopes and to measure the relief of fluorescence quenching on fusion (Hoekstra at al., 1984). These spectroscopic methods provide rapid, continuous, and quantitative information on fusion. A drawback is the difficulty in discriminating fusion from exchange of lipids between two membranes without fusion, if it occurred. Envelope fusion is a rapid reaction reaching a saturation level within 12 min at 37°C. It does not require Ca2+or Mg2+.It has a characteristic temperature dependence; practically no fusion below 20°C and progressively faster fusion at higher temperatures. The temperature dependence was correlated with the onset of the segmental and rotational motion of the viral glycoproteins in the envelope as measured by the decay of transient dichroism of eosine triplet probes (Lee et al., 1983). A discontinuity in the fluidity of target erythrocyte membranes was also observed near 20°C using spin-labeled PC (Tanaka and Ohnishi, 1974). The fusion reaction follows first-order kinetics. The rate constant of fusion with erythrocyte membranes was obtained as 0.84 min-' at 37°C by the spin-label assay (Kuroda et al., 1985). Kinetic analysis suggested that not all virus particles bound on cells fuse at this rate constant but only about 1-2 particles per cell do. Kinetic analysis of the fluorescence dequenching data based on the mass action law gave 2.4-4.2 min-' for the rate constant (Nir et al., 1986b), which is around 3-5 times greater than that obtained by the spin-label method. The pH dependence of fusion is rather broad in the range from pH 8 to 5 (Fig. 2a). The fluorescence assay also showed a broad pH dependence, although the rate constant as well as the efficiency of fusion are considerably low on either sides of neutral pH (Hoekstra et al., 1985). HVJ can fuse with liposomes containing as well as not containing receptors (glycophorin or gangliosides). Fusion with liposomes containing anionic phospholipids is more efficient. The effect of cholesterol on fusion is controversial. While Haywood and Boyer (1984) observed no effect of cholesterol, Hsu et al. (1983) showed a requirement in fusion and Kundrot

9. FUSION OF VIRAL ENVELOPES WITH CELLULAR MEMBRANES

265

et a / . (1983) demonstrated a requirement in virus-induced lysis of lipo-

somes containing glycophorin. The role of cholesterol in viral fusion activity has been reviewed recently (DuzgiineS, 1988). Liposomes reconstituted with the viral glycoproteins HN and F have hemolytic and fusogenic activities (Hosaka and Shimizu, 1972; Volsky and Loyter, 1978a). Ozawa and Asano (1981) showed that cholesterol was required for the functional reconstitution.

2. HEMOLYSIS HVJ causes hemolysis. The virus appears to have an inherent defect in the envelope membrane which creates pores in the cell membrane after fusion. Accelerated water inflow causes cell swelling and lysis after a certain threshold. Pore formation is observed as the permeability to low molecular weight (. Prioc.. N t d . A c d . Sci. U.S.A. 79, 4800-4808. Kundrot, C. E.. Spangler. E. A.. Kendall. D. A . , MacDonald, R. C . . and MacDonald, R. I. ( 1983). Sendai virus mediated-lysis of liposomes requires cholesterol. Proc. Nu//. Actid. Sc?. U . S . A . 80, 1608-1612. Kuroda, K . , Kawasaki, K., and Ohnishi. S. (19XS). Kinetic analysis of fusion of hemaggluti-

292

SHUN-ICHI OHNlSHl

nating virus of Japan with erythrocyte membrane using spin-labeled phosphatidylcholine. Biochernisrry 24, 4624-4629. Lazarowitz, S., and Choppin, P. W. (1975). Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virolog? 68, 440-454.

Lee, P. M.. Cherry, R. J., and Blchi. T. (1983). Correlation of rotational mobility and flexibility of Sendai virus spike glycoproteins with fusion activity. Virology 128, 65-76. Lenard, J., and Miller, D. K. (1981). pH-dependent hemolysis by influenza. Semliki Forest virus, and Sendai virus. Virology 110, 479-482. Lenz. J., Crowther, R.,Straceki, A., and Haseltine, W. (1982). Nucleotide sequence of the AKV enu gene. J . Virol. 42, 519-529. Madshus, 1. H., Olsnes, S., and Sandvig, K. (1984). Mechanism of entry into the cytosol of poliovirus Type 1: Requirement of low pH. J . Cell Bid. 98, 1194-1200. Maeda, T.. and Ohnishi, S. (1980). Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes. FEES Lett. 122, 283-287. Maeda, T.. Asano, A.. Ohki, K., Okada, Y . , and Ohnishi, S. (1975). A spin-label study on fusion of red blood cells induced by hemagglutinating virus of Japan. Biochemistry 14, 3736-3741.

Maeda, Y., Kim. J., Koseki, I., Mekada, E., Shiokawa, Y.. and Okada, Y. ( 1977). Modification of cell membrane with viral envelopes during fusion of cells with HVJ (Sendai virus). Ill. Effects of mono- and disaccharides on cell fusion and membrane movement of fused cells. Exp. Cell Res. 108, 95-106. Maeda, T.. Kawasaki, K., and Ohnishi. S. (1981). Interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at pH 5.2. Proc. Natl. Acud. Sci. U.S.A. 78, 4133-4137. Marsh, M. (1984). The entry of enveloped viruses into cells by endocytosis. Biochem. J. 218, 1-10.

Marsh, M., Bolzau, E., White, J., and Helenius, A. (1983a). Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells. J. Cell B i d . 96, 45.5461.

Marsh, M.. Bolzau, E., and Helenius, A. (1983b). Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell 32, 93 1-940. Matlin, K. S . , Reggio, H., Helenius, A., and Simons, K. (1981). Infectious entry pathway of influenza virus in a canine kidney cell line. J. Cell &a/. 91, 601-613. Matlin, K. S . . Reggio, H.. Helenius, A.. and Simons, K. (1982). Pathway of vesicular stomatitis virus entry leading to infection. J . M o l . &I/. 156, 609-63 I . Metsikko, K., van Meer. G., and Simons, K. (1986). Reconstitution of the fusogenic activity of vesicular stomatitis virus. EMBO J. 5, 3429-3435. Mifune. K., Ohuchi, M.. and Mannen, K.(1982). Hemolysis and cell fusion by rhabdovirus. FEBS L e t t . 137, 293-297. Murata, M., Nagayama, K., & Ohnishi. S. (1987a). Membrane fusion activity of succinylated melittin is triggered by protonation of its carboxyl groups. Biochemistry 26, 40564062.

Murata, M., Sugahara, Y.,Takahashi, S., & Ohnishi, S . (1987b). pH-Dependent membrane fusion activity of a synthetic twenty amino acid peptide with the same sequence as that of the hydrophobic segment in influenza virus hemagglutinin. J . Biochem. 102, 957962.

Nakada, S., Creager, R. S., Krystal, M., Aaronson, R. P.. and Palese, P. (1984). Influenza C virus hemagglutinin: Comparison with influenza A and B virus hemagglutinins. J . Virol. 50, 118-124. Nir. S., Stegmann, T.. and Wilschut, J. (1986a). Fusion of influenza virus with cardiolipin

9. FUSION OF VIRAL ENVELOPES WITH CELLULAR MEMBRANES

293

liposomes at low pH: Mass action analysi\ of kinetics and extent. Biochcmistr? 25, 257266.

Nir. S.. Klappe. K..and Hoekstra. [I. (1986b). Kinetics and extent offusion between Sendai virus and erythrocyte ghosts: Application of ti mas\ action kinetic model. Bioclwrnisrry 25, ? I 5 5 - 2 l 6 l . Ohki. S. (IY87). Surface tension. hydration energy and membrane fusion. In “Molecular Mechanisms of Membrane Fusion” (S. Ohki. D. Doyle, T. Flanagan. S. W. Hui. and E. Mayhaw. eds.), in press. Plenum. New York. Ohkuma. S.. and Poole. B. (1978). Fluorescence probe measurement of the intl-alysosomal pH in living cells and the perturbaof pH by various agents. Proc. Null. Acrrd. Sci. U . S . A . 75, 3317-3331. Ohnishi. S . (l985a). Membrane fwion: Mechanism and role in cellular response to enveloped virus. In “Biomolecules: Electronic Aspects” tC. Nagata. M. Hatano, J. Ttinaka. and H. Suruki. eds.). pp. 227-252. Japan Scientific Societies Press. Tokyo; Elsevier. Amsterdam. Ohnishi, S . ( I985b). Interaction of hemagglutinin with target membranes. V a c c i n e 3, 104. Ohnishi. S.. and Yoshimura. A. (1984). Infectious cell entry mechanism of enveloped v i ruses. Virus (Jpn.) 34, 11-24. Ohuchi. M., Ohuchi. R.. and Mifune, K . (1982). Demonstration of hemolytic and fusion activities of influenza C virus. J . Virid. 42, 1076-1079. Okada, Y. ( 1958). The fusion of Ehrlich’s ascites tumor cells caused by HVJ virus in u i ~ r o . ~ i / i J ~. 1, , ~103-110. Okada, Y . . Koseki, I . . Kim. J., Maeda, Y., Hashimoto. T.. Matsui. Y . , and Kanno. Y. (1975). Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus) I . Interaction between cell membranes and virus in the early stage. k i p . C P / /R C S .93, 368-378. Ozawa, M., and Asano. A. ( 198 I).The preparation of cell fusion-inducing proteoliposomes from purified glycoproteins of HVJ (Sendai virus) and chemically defined lipids. J . B i d . C’licrn. 256, 5954-5956. Parsegian. A. ( 1969). Energy of an ion cros\ing B low dielectric membrane: Solutions to four relevant electrostatic problems. Narrrre ~Lonrlr~n) 221, 844-846. Pahternak. C. A. ( 1984). How viruses damage cells: Alterations in plasma membrane function. J . Biosci. 6, 569-583, Porter. A. G . . Barber. C.. Carey, N. H . . Hallewell. K. A., Threlfall. G . . and Emtage, J. S. ( 1979). Complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned DNA. Ncrlure (Lonclon)282, 471 -477. Kedmond. S. M. S., and Dickson, C. (1983). Sequence and expression of the mouse mammary tumor v i r w enu gene. E M B O J . 2, 125-131. Kedmond, S., Peters, G., and Dickson, C. (1984). Mouse mammary tumor virus can mediate cell fusion at reduced pH. Virology 133, 393-402. Rice. C. M.. and Strauss. J. H. (1981). Nucleotide sequence of the 26s mRNA of Sindbis virus and deduced sequence of the encoded virus structural proteins. Proc. Nirtl. Acod. 3’c.i. U.S.A. 78, 2062-2066. Richardson, C. D.,and Choppin, P. W.(1983). Oligopeptides that specifically inhibit membrane fusion by paramyxoviruses: Studies on the site of action. Virology 131, 518-532. Richardson. C. D., Scheid. A,, and Choppin. P. W. (1980). Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F, or H A 2viral polypeptides. Virology 105, 205-222. Kose. J . K., and Gallione. C. J. (1981). Nucleotide sequences of the mRNA’s encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions. J . Virol. 39, 519-529.

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Rott. R., Orlich, M.. Klenk, H.-D., Wang, M. L., Skehel, J. J., and Wiley. D. (1984). Studies on the adaptation of influenza viruses to MDCK cells. EMEO J. 3, 3329-3332. Ruigrok, R. W. H.,Wrigley, N. G . , Calder, L. J . , Cusack. S . , Wharton, S . A., Brown, E. B.. and Skehel, J. J. (1986). Electron microscopy of low pH structure of influenza virus huemagglutinin. EMBO J . 5, 41-49. Sandvig, K.. and Olsnes, S . (1980). Diphtheria toxin entry into cells is facilitated by low pH. J . Cell Bird. 87, 828-832. Sato, S. B.. Kawasaki, K.. and Ohnishi, S . (1983). Hemolytic activity of influenza virus hemagglutinin glycoproteins activated in mildly acidic environments. Proc. Null. Arud. Sci. U . S . A . 80, 31534157. Scheid. A., and Choppin, P. W. (1974). Identification of biological activities of paramyxovirus glycoproteins. Activation of cell Fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus. Virufogy57,475-490. Schlegel, R., and Wade, M. (1984). A synthetic peptide corresponding to the NH2 terminus of vesicular stomatitis virus glycoprotein is a pH-dependent hemolysin. J. B i d . Chem. 2S9,469 1-4694.

Schlegel, R..and Wade. M. (1985). Biologically active peptides of the vesicular stomatitis virus glycoprotein. J . Virol. 53, 319-323. Schlegel. R.. Tralka, T. S., Willingham, M . C., and Pastan, I. (1983). Inhibition of VSV binding and infectivity by phosphatidylserine: Is phosphatidylserine a VSV binding site? Cell 32, 639-647. Schloemer, R. H., and Wagner, R. R. (1975). Cellular adsorption function of the SkdlOglyCOprotein of vesicular stomatitis virus and its neuraminic acid. J . Virol. 15, 882-893. Schmidt, M. F. G. (1983). Fatty acid binding: A new kind of posttranslational modification of membrane protein. C w r . Top. Microhio/. fmmunol. 102, 101-129. Schmidt, M. F. C . , and Lambrecht, B. (1985). On the structure of the acyl linkage and the function of fatty acyl chains in the influenza virus haemagglutinin and the glycoproteins of Semliki Forest virus. J . Cen. Virrd. 66, 2635-2647. Seki, M., Hattori. S . , Hirayama, Y.,and Yoshida, M. (1983). Human adult T-cell leukemia virus: Complete mucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc. Natl. Acud. Sci. U.S.A. 80, 3618-3622. Sekiguchi, K.,and Asano, A. (1978). Participation of spectrin in Sendai virus-induced fusion of human erythrocyte ghosts. Pror. Null. Aciid. Sri. U.S.A. 75, 1740-1744. Shibata, M., Maeno, K., Tsurumi, T.. Aoki, H.,Nishiyama, Y., Ito, Y.,Isomura. S.. and Suzuki, S. (1982). Role of viral glycoproteins in hemolysis by influenza B viruses. J. Gen. Virol. 59, 183-186. Shimizu, Y. K., Shimizu, K., lshida, N., and Homma. M. (1976). On the study of Sendai virus hemolysis 11. Morphological study of envelope fusion and hemolysis. Virology 71, 48-60.

Shinnick, T. M., Lerner, R. A., and Sutcliffe, J. G. (1981). Nucleotide sequence of Moloney murine leukemia virus. Nufure ( h n d O n ) 293, 543-548. Skehel, J. J.. Bayley, P. M.,Brown, E. B.,Martin. S. R., Waterfield, M. D., White, J. M.. Wilson, 1. A,. and Wiley, D. C. (1982). Changes in the conformation of influenza virus haernagglutinin at the pH optimum of virus-mediated membrane fusion. Proc. Nod. A c u ~ Sci. . U.S.A. 79, 968-972. Stegmann, T.,Hoekstra, D., Scherphof. G., and Wilschut. J. (1985). Kinetics of pH-depen, dent fusion between influenza virus and liposomes. Biochemistry 79,968-972. Stegmann, T.. Hoekstra. D.,Scherphof, G.,and Wilschut, J. (1986). Fusion activity of influenza virus. A comparison between biological and artificial target membrane vesicles. J. B i d . Chem. 261, 10966-10969.

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Struck, I). K., Hoekstra. 0.. and Papano. P. E. (1981). Use of resonance energy tr;insfer t o monitor membrane fusion. Bioc./ierni.trr.y 20, 4093-4099. Sturman, L. S.. Kicard, C . S..and Holmeh. K. V . (1985). Proteolytic cleavage of the E? glycoprotein of murine Coronavirus: Activation of cell-fusing activity of virions by trypsin and separation o f two different YOK cleavage fragments. J . V i r ( ~ /56, . 904911. 'l'anaka. K.. and Ohnishi, S. ( 1974). Heterogeneity in the tiuidity of intact erythrocyte membrane and i t s homogenization upon hemolysih. Bioclrirt?. B i c ~ p hA~ i.m 426, 218-

231. Tsci.ji. A , . and Ohnishi. S . (1986). ReWiction o f t h e liitcriil motion o f hand 3 in the erythrocyte membrane by the cytoskcletiil network: Dependence on speclrin association state. BicJf'/iCr!li.Y/r\ 25, 6 133-6 139, Tycko, B., and Maxfield. F. R. ( 19x2). Rapid iiciditication of endocytic vesicles containing a~-inacroglobulin.Ci4/ 28, 6 4 - 6 5 I . Viiinanen, P.. and Kiiirilinen. L. ( 1979). Haemolysis hy two iilphaviruses: Semliki Forest and Sindhis virus. J . Giw. Viral. 43, 593-601. Viiiiniinrn. P.. and Kiiiiriiiinen. L . i 19x0). Fuhion hind haeniolysih of erythrocytes caused by three togaviruses: Semliki Forest Sindbih, ;ind rubella. J . Gou. V i r o / . 46, 467-475. van Kenswoude. J . . Bridges. K . R..Harford. J . B . . and Klausner. K. I). (19x2). Receptoi-mediated endocytosis o f transferrin and the uptake of Fe in KS62 cell\: Identification of a nonlysosomal acidic compartment. Pro(.. Nor/. Actrd. S c i . U.S.A. 79. 6186-6190. Verhoeyen, M.,Fang. K..Min Jou. W., Devih, K., Huylehroeck. D.. Saman. E.. and Fiers, W. ( 1980). Antigenic drift between the heemagglutinin of the Hong Kong influenza striiins AiAichii2i68 and AiVictoriai3i75, Nirrrrrc. ( L o t i d o n ) 286, 771-776. Vtrlsky. D. J.. and Loyler, A . i 197Ra). An cflicient method for r e a w m h l y o f fusogenic Sendni virus envelopes after solubili/ation of intact virions with Triton X- 100. FERS LtJII.92, 190-194. Volsky. I).J . . and Loyter, A. (1978h). Role ofC;i'* in virus-induced membrane fu\ion. Ca" accumulation and ultrastructural changes induced by Sendai viru\ in chicken erythrocytes. J . c 1 4 / B i o / . 78, 465-47'). von Heijne. G . (1981). On the hydrophobic nature of signal sequence. Ero.. 1.Bioc./it,/n. 116, 419-422. Wharton, S. A., Skehel. J. J . . and Wiley. 1). C. ( IYX6). Studies ofinlluenza haemagglutininmediated membrane fusion. V ~ ~ ( J / O149, R X 27-35. White. J . . and Helenius. A. (1980). pH-dependent fusion between the Semliki Forest virus membrane and liposomes. /'roc. N d . A m / . .Y(.i. U.S.A. 77, 3273-3277. White, J . . Kartenbeck. J . . and Heleniw. A. (19x0). Fusion olSemliki Forest virus with the plasmit membrane can be induced by low pH. 1.Coil E i ~ l 87, . 264-272. White. J., Matlin, K.. and Heleniu5. A . (1981). Cell fuhion by Semliki Forest. influenzit kind vesicular stomatitis viruh. J . Cr//Biol. 89, 674-679. White, J.. Kartenbeck. J . , and Heleniub, A. (1982). Membrane fusion activity of influenza virus. EMBO J . I , 217-222. White, J . , Kielian. M.,and Helenius, A. (1983). Membrane fusion protein5 of enveloped animal viruses. 0. Rev. Bioplrv.\. 16, 151-195. Wilson, I . A , . Skehel. J . J . , and Wiley. I). C. (19x1). Structure o f the haemagglutinin memhrane glycoprotein of influenza virus at 3 A resolution. Nurrtrr~( f m i c l m i )2H9, 366373. Winter. G . , Fields, S.. and Brownlee, G . G . (1981). Nucleotide sequence o f the haemagglutinin gene of a human influenza virus H I subtype. Ntrrrrrr (fAJlidOt/)292, 72-75. Wyke, A . M.. Impraim. C. C.. Knutton. S..iind Pasternak, C. A. (1980). Components

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involved in virally mediated membrane fusion and permeability changes. B i o c h m . J. 190, 625-638. Yamada. S.. and Ohnishi. S . (1986). Vesicular stomatitis virus binds and fuses with phospholipid domain in target cell membranes. BiocRemistrV 25, 3703-3708. Yamamoto, K., Suzuki, K., and Shimizu, B. (1981). Hemolytic activity of the envelope glycoproteins of Western equine encephalitis virus in reconstitution experiments. Virology 109,452-454. Yasuda, Y., Hosaka, Y., Fukami, Y., and Fukai, K. (1981). Immunoelectron microscopic study on interactions of noninfectious Sendai virus and murine cells. J . Virol. 39, 273281. Yoshimurd, A . ( 1985). Adenovirus-induced leakage of co-endocytosed macromolecules into the cytosol. Cell Strcrc/. Fun a

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large numbers of individual clones by radioimmune assay (Sambrook et d.,1985). This was feasible for cell lines such as C127 and NlH-3T3 that were efficiently transfected by the BPV-HA vector but did not prove successful with other cell types. However, use of a fluorescence-activated cell sorter to identify cells that bind FITC-conjugated anti-HA antibodies (or hemagglutinate FITC-labeled red blood cells) has facilitated the rapid

344

MARY-JANE GETHING ET AL.

selection of rare HA-expressing cells and has also allowed us to clone out those cells that display the greatest amounts of HA at the cell surface. The Japan or Aichi (X-31) HA genes have now been introduced into cell lines derived from a number of different species including mouse (C127, NIH-3T3, and MME cells), dog (MDCK cells), pig (PK cells), and hamster (CHO cells). The level of expression of HA varies between cell types: up to 10’ molecules of HA per cell can be obtained in C127, NIH-3T3, or CHO cell lines, while somewhat lower levels (104-106 molecules per cell) are obtained in the other, more differentiated cell types. Obviously, the development of an increasing variety of HA-transformed cell lines of different types and species coupled with the ability to deliver efficiently into the cytoplasm probes such as antibodies or oligonucleotides provides great opportunities for studies of intracellular events and architecture in living cells. Figures 2 and 3 characterize the synthesis, cellular location, and functional activities of the wild-type Japan HA protein expressed in a continuous line of NIH-3T3 cells transformed with the BPV-HA vector, pBVIMTHA (Sambrook et al., 1985). Comparison of HAS produced from SV40-HA and BPV-HA vectors in CV-1 cells and NIH-3T3 cells, respectively (Fig. 21, indicates that the nonglycosylated forms of HA synthesized in each cell type in the presence of the drug tunicamycin are identical in size, as are the core-glycosylated forms of the proteins synthesized during a 15-min pulse. However, variation between these cell types in the trimming and modification of the oligosaccharide side chains results in differences in the terminal glycosylation patterns of the HA molecules, and thus in differences in their mobilities on SDS-polyacrylamide gels. Figure 2 also illustrates the quantitative cleavage by exogenous trypsin of terminally glycosylated precursor HA0 to HA1 and HA2 subunits. Figure 3 indicates the cell surface location of the HA synthesized in NIH-3T3 cells and demonstrates that the protein is active in hemagglutination, cellcell fusion, and red blood cell-mediated delivery assays. The availability of cell lines that express HA at different levels has allowed us to estimate the level of expression required on a per cell basis for cell-cell fusion to occur. Table I compares, for six different transformed NIH-3T3 cell lines, the number of HA molecules per cell and the cell-cell fusion and hemagglutination activities. The results indicate that a minimum expression level of approximately 3 x 106molecules per cell is necessary for the manifestation of fusion activity. A similar result was obtained when red blood cell-mediated delivery experiments were performed (results not shown).

11. HEMAGGLUTININ OF INFLUENZA VIRUS

345

FIG.3. Analysis of HA expressed from BPV-HA recombinant vectors. A continuous NIH-3T3 cell line transformed with the pBVI-MTHA vector (Sambrook ef n l . , 1985) was characterized using the following assays: immunofluorescence on nonpermeabilized cells (A), hemagglutination (B), cell-cell fusion (C), and erythrocyte-cell fusion measured by delivery of HRP and staining with diaminobenzidine (D). The details of the experimental protocols have been described previously (Gething and Sambrook, 1981. 1982: Sambrook et al., 1985; Doxsey et al., 1985).

V.

GENETIC APPROACHES TO STUDIES OF HA-MEDIATED MEMBRANE FUSION

Two genetic approaches have been employed to analyze the mechanism of the fusion reaction mediated by HA. The first involves studies of variant influenza viruses that induce fusion with raised pH thresholds. Sequence analyses of HAs from the variant viruses have identified altered

346

MARY-JANE GETHING ET AL.

TABLE 1 CORRELATION OF THE LEVELOF EXPRESSION OF HA IN MURINE BVi-MTHA CELLLINESWITH THE CAPACITY TO UNDERGO LOW PH-MEDIATED CELL-CELLFUSION Cell line

TRI-I TRI-4 TR 1-5 NTRI-4 NTRI-5 NTRI-I I

HA rnolecules/cell" 6 I 1 3 I 8

x x x x x

lo0 I06 106

I@ 106

x 106

Fusion activityh

Hemagglutination'

+++

++t ++t

+ + +++ -

+++

+++ +++ +++ +++

" Cell extacts were prepared from a known number of cells and assayed by solid-phase radioimmunoassay (Cething and Sambrook. 1981), Ir Fusion activity was measured by polykaryon formation following treatment of cell monolayers with trypsin and neurdminidase (Sambrook P I a / . . 1985) and repeated ( 2 x ) brief exposure to buffer at pH 5.0 (PBS containing 10 mM HEPES). Symbols: -, no polykaryons observed; +. small numbers of polykaryons containing 4-5 nuclei: + + +. massive formation of polykaryons containing large numbers of nuclei (see. for example. Fig. 3 0 . Hemagglutination assays were performed after treatment of cell monolayers with trypsin and neuraminidase (Sambrook el d.,1985) using a IW solution of guinea pig erythrocytes. ++ +. Erythrocytes bound to SO-IOO%, of cells in the monolayer (see, for example. Fig. 3 B ) . 1

amino acids that play a role in the pH dependence of fusion. The second approach ulitizes oligonucleotide-directed, site-specific mutagenesis of a cloned HA gene to alter the nucleotide sequence encoding selected amino acids in the HA molecule. Expression of the mutant genes in simian cells has confirmed the central role of the fusion peptide and provided insights into the mechanism of the fusion reaction. A. Studies on Variant Influenza Vlruses That Induce Fusion at Elevated pH

To gain insight into the molecular mechanism underlying the pH dependence of HA-mediated fusion, influenza virus variants have been isolated that induce fusion at threshold pH values higher than those of their parent viruses. Rott et al. (1984) showed that variants of the X-31 strain, selected for their ability to undergo activation cleavage and grow in MDCK cells, also displayed an elevated threshold pH for fusion that was approximately 0.7 pH units higher than the wild type. Variants of the X-31 and Weybridge virus strains have been selected for by growth in the presence of amantadine, a compound that raises endosomal pH (Daniels et af., 1985). Variant viruses were obtained that mediated fusion at pH values 0.1-0.7

1 1 . HEMAGGLUTININ OF INFLUENZA VIRUS

347

unit higher than the parental strain. Finally, we have isolated and analyzed a naturally occurring variant ofthe X-31 strain whose pH threshold for fusion was elevated by 0.2-0.3 units (Doms P t al., 1985). Sequence analysis of the HAS from these variant viruses has identified individual amino acids that appear to play a role in the low pHmediated conformational change in the molecule. Some of these residues are located along the interface between the subunits of the trimer. while others stabilize the unexposed location of the fusion peptide at the amino terminus of HA2 (see Fig. 4A). It has been proposed that these amino acids participate in interactions that stabilize the structure of the wild-type molecule at neutral pH (Daniels et d., 1983. Substitution of these residues would lower the energy barrier necessary for the low pH-induced conformational transition to the fusion-active state. All the results are consistent with the widely held theory that dissociation of the HA trimer at low pH is a necessary and early step in the fusion mechanism. 6. Site-Directed Mutagenesis of the Fusion Peptide of HA

Although the majority of the amino acids that were altered in the fusion variants were located along the trimer interface (Fig. 4A), three altered residues were located within the hydrophobic fusion peptide at the amino terminus of the HA2 subunit. The fact that these three residues have undergone conservative substitutions probably reflects the fact that mutant viruses unable to mediate the fusion reaction at any pH could not enter and infect cells and therefore could not be propagated. To obtain mutant HAS that might be inactive or disabled for fusion and to probe the consequences of altering the length and hydrophobicity of the fusion peptide. we have used oligonucleotide-directed mutagenesis of the Japan HA cDNA to introduce single base changes into the sequence that encodes this peptide (Gething ef ul., 1986a). Three mutants were constructed that introduce single, nonconservative amino acid changes in the fusion peptide (Fig. 4B). When the mutants were assayed for fusion activities and for the low pHmediated conformational change and acquisition of lipid binding capacity (Table 11), three fusion phenotypes were observed: 1 . Substitution of glutamic acid for the glycine residue at the amino terminus of HA2 abolished all fusion activity although the mutant HA could still undergo a conformational change (at lower pH than the wild type) that resulted in protease sensitivity and lipid binding capability. Study of this mutant has provided the first indication that the conformational change can be temporally separated from lipid binding, and also

FIG.4. Schematic diagrams of the ectodomain of the HA monomer showing the location of amino acids that have been altered in the fusion variants (A) or the site-directed mutants (B). The schematic diagram of the three-dimensional structure of the HA monomer is taken from Wilson et a / . (1981). The data summarized in A are drawn from Rott er al. (1984),

348

- GLYl

-

GLY4

GLU

-

GLU

1- GLY

Daniels et a / . (1989, and Doms et u / . (1986), while that in B is from Gething ct u / . (1986a). In A the residues marked with an asterisk are from the HA1 subunit; all other residues are from the HA2 subunit.

349

350

MARY-JANE GETHING ET AL.

TABLE I1 FUSION PHENOTYPES OF T H E WILD-TYPE A N D MUTANT HA

SUMMARY OF THE ~~

~

~

Threshold pH Efficiency at pH 5 of erythrocyteof cellcell fusion cell fusion (%)

Threshold pH of erythrocytecell fusion

WT

PROTEINS"

~

Efficiency at pH 5 of cell-cell fusion (%)

pH at which 50% of BHA is converted to protease sensitivity and lipid binding

5.3

5.3

90

90

5.3

MI

-

-

M4 MI1

5.7 5.3

5.6 4.6

0 50

0 50

5.0 5.5

90

0

5.3

~~

~

This table is adapted from and summarizes results described in Cething

PI

ul. (1986a).

allows distinction of the stages of lipid binding and bilayer fusion, indicating that HA does more than simply bring the two membranes close together. 2. Substitution of glutamic acid for the glycine residue at position 4 in HA2, which decreased the length of the apolar stretch to 6 amino acids, raised the threshold pH both for the conformational change and for fusion and also reduced the efficiency of fusion. It appears that the mutation has destabilized the neutral conformation of the HA trimer in a similar fashion to the amino acid alterations identified in the variant viruses described above. The results suggest that the amino acid at position 4 in HA2 may play two roles: a structural role in maintaining the fusion peptide in its neutral conformation and another role in the stage of bilayer destabilization. 3 . Extension of the hydrophobic stretch by replacement of the glutamic acid at position 11 with glycine yielded a mutant protein that underwent the conformational change and induced fusion of erythrocytes with cells with the same efficiency and pH profile as the wild-type protein. However, the ability of this mutant to induce polykaryon formation was greatly impaired. This phenotype provides the evidence for a distinction between cell-cell fusion and erythrocyte-cell fusion. The mutant HA is competent to mediate fusion of the cell and erythrocyte membranes over the small area necessary for injection of HRP into the cytoplasm, but, except at very low pH, the mutant HA is unable to induce bilayer destabilization over areas sufficient to cause polykaryon formation. Analysis of these mutants has allowed us to delineate several stages of the mechanism of HA-mediated membrane fusion which were not separated in previous studies. These include (1) the low pH-induced conformational change in HA that exposes the fusion peptide; (2) the interaction

11. HEMAGGLUTININ OF INFLUENZA VIRUS

351

of the fusion peptide and/or other regions of the HA molecule with the target lipid bilayer; ( 3 ) destabilization of the lipid bilayer and membrane coalescence over small areas; and (4) membrane coalescence and separation over large areas, resulting in polykaryon formation. The amino terminus of the HA2 subunit is the most highly conserved region of HA (Gething P I a / . . 1980; Kawaoka rt a / . , 19841, indicating that this region of the molecule must be of great importance for the structure and function of HA and the life cycle of the virus. Nevertheless, although the HAS of field strains never show amino acid changes in the fusion peptide, it is possible, either by selection of variant viruses by growth in amantadine or by in vitro mutagenesis, to introduce alterations into this peptide that do not inhibit the assembly, intracellular transport, or fusion activity of this protein. Presumably variant viruses carrying these altered HA molecules would be at a disadvantage in a competition with field strains, either because occurrence of the conformational change or the fusion reaction at a higher pH is not desirable or because the mutant HA proteins are more unstable or thermolabile. Whatever their defects would be in the real world, HA mutants generated in uitro provide the opportunity to study the role of this hydrophobic peptide in the fusion reaction. C. Possible Involvement of Other Regions of HA in the Process of Fusion

Although these studies have confirmed a function for the amino terminus of HA2 in the fusion mechanism, they have not ruled out a role for other regions of the molecule in the interaction with the target lipid bilayer. Very promising candidates for other structures that may be involved in fusion are the two amphipathic helices of the stalk domain, which are made up of sequences from the HA2 subunit (Wilson el ul., 1981). In the neutral trimer, hydrophobic amino acids which lie along one face of the top half of the long helix form extensive contacts between the individual subunits. Following the low pH-induced conformational change when the subunits ofthe trimer separate, the helices will no longer interact with each other and may well be available to interact instead with the lipid bilayer. The short helix is also amphipathic. Physiologically important interactions of amphipathic surfaces (Q helices or p sheets) with membranes have been described (Kaiser and Kezdy, 1984) with the amphilicity of the structure being more conserved than specific amino acid sequence. Design and analysis of mutations in this region of HA will be quite complex, since it is clear that the long helix in HA2 is intimately involved in the folding and final structure of the trimeric molecule (Wilson et ul.,

352

MARY-JANE GETHING ET AL.

1981). It will be necessary to design mutations that do not affect the folding and transport of the protein but that might interfere with the interaction with the target bilayer following the low pH-induced conformational change. Candidates for oligonucleotide-directed mutagenesis would include residues with aliphatic or aromatic side chains on the hydrophobic face of the helix, but which do not appear to be so intimately involved in the interactions that connect the subunits in the trimer. Conservative substitutions would test the role of individual amino acids in the fusion mechanism, while nonconservative substitutions would test the importance of amphilicity rather than specific sequence. Alteration of the pH threshold for fusion would indicate that the substituted amino acid plays a structural role in the conformational change, while alteration of the efficiency of fusion would suggest that the amphipathic helices might indeed interact with the target lipid bilayer during the fusion reaction. VI.

CHARACTERIZATION OF THE LOW-pH INDUCED CONFORMATIONAL CHANGE IN HA

A current view of the low pH-induced conformational change in HA is that protonation of one or more amino acid side chains in the subunit interface causes the dissociation of the HA trimer. Because the quaternary interactions between the three globular domains are few in number in the neutral conformation of the trimer (Wilson et al., 1981), dissociation of the subunits may simply occur by separation of the globular domains at the top of the molecule. This would then be followed by breaking of the interactions between the amphipathic helices in the stalk which leads to exposure of the previously buried fusion peptide. Recent experiments, however, have suggested that a more complicated series of conformational changes may occur. White and Wilson (1987) have used antipeptide antibodies, directed against individual sequences located near the fusion peptide and along the subunit interface in both the globular and stalk domains, to probe the temporal sequence of separate conformational alterations that take place following treatment at low pH. Surprisingly, their results indicated that the conformational change begins with exposure of a loop (HA1 residues 14-52) which in the neutral structure is embedded in the trimer interface, followed by exposure of the fusion peptide and then the carboxy terminus of HAl. The separation of the subunits, and in particular of the globular domains, appears to be a relatively late event in the process. Thus, although these results must be interpreted with the caveat that the experiments were carried out using HA solubilized in

11. HEMAGGLUTININ OF INFLUENZA VIRUS

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detergent, it appears that conformational changes around the stem of the molecule may precede dissociation of the subunits. Neither scenario is contradicted by the data from the fusion variant studies which identified amino acids important in stabilizing the neutral positions of both the HA1 carboxy terminus and the fusion peptide, as well as the intersubunit connections, but could not define any order of events in the conformational change. Whichever model is the more correct, we still need to understand better the events that lead to disruption and reestablishment of the lipid bilayer. It is to be hoped that elucidation of the role of individual amino acids of HA in the fusion reaction might lead to a physiochemical model for the rearrangements that occur within the fusing membranes.

VII. STUDIES ON THE CLEAVAGE ACTIVATION OF HA

As described above, the fusion activity of HA and thus the infectivity of influenza virus requires that the HA0 precursor be processed by a posttranslational proteolytic cleavage to an active form of the molecule. The HAS from human type A influenza viruses contain a single arginine residue at the processing site, and activation involves cleavage on the carboxyl side of this amino acid by a trypsinlike protease, followed by remove1 of the arginine by a carboxypeptidase activity (reviewed by Rott and Klenk, 1986). Cellular proteases that carry out these cleavages are expressed only in primary epithelial cells, a situation which normally restricts viral replication to the respiratory epithelium during influenza virus infections of humans. Virulent avian influenza viruses encode HAS that have several arginine and lysine residues at the processing site, so that the HA is cleaved in most cell types (Rott and Klenk, 1986), leading to systemic infections. The pathogenicity of these viruses tends to correlate with the numbers of basic amino acids at the cleavage site of HA, although other structural features of HA such as the positioning of carbohydrate side chains can modulate the virulence of the infection (Kawaoka et al.. 1984). In addition, pathogenicity has proven to be a multigenic trait (Schulman, 1983). In cells in culture, HAS derived from human viruses are not cleaved so that it is the precursor HA molecule that is displayed at the cell surface; treatment with exogenously added trypsin is required to activate the protein for fusion. HAS derived from pathogenic avian viruses are activated by cellular proteases in cultured cells; it is as yet uncertain whether cleavage takes place before or after arrival of the protein at the cell surface.

354

MARY-JANE GETHING ET AL.

To study the structural requirements for activation cleavage of HA we have used oligonucleotide-directedmutagenesis to alter the amino acid sequence at the processing site of X-31 HA. A single nucleotide change, AGA- ACA, wasintroduced togenerate the cleavage-minus mutant, C-T, in which the arginine residue at the cleavage site is substituted by a threonine residue. Loop-in mutagenesis of 3 or 15 nucleotides was performed to generate the other two mutants. The first, C+RR, contains an additional arginine residue at the cleavage site. The second, C+RRKKR, contains two extra arginines and two extra lysine residues at the cleavage site (reproducing the cleavage site in the HA from the A/Turkey/Ontario/ 66 strain of influenza virus). Figure 5 shows the results of DNA sequence analysis to confirm the nucleotide sequences encoding the region around the cleavage sites in the wild-type and mutant HA cDNAs. The nucleic acid and corresponding amino acid sequences of the relevant segment of the wild-type and mutant proteins are shown in Fig. 5B.Following confirmation of the desired mutant sequences, the mutated X-31 cDNAs were used to replace the corresponding wild-type fragments in the SVEXHA recombinant viral genome (Doyle ef d.,1986).The recombinant genomes were transfected into CV-I cells, and high titer virus stocks were developed (Doyle et al., 1985) and used to infect fresh monolayers of CV-I cells for analysis of the biosynthesis, intracellular transport, and functional activities of the wild-type and mutant HA proteins. An analysis of the transport and proteolytic cleavage of these proteins is shown in Fig. 6. Fifteen minutes after synthesis, the wild-type HA is present predominately as the core-glycosylated HA0 species which appears as a sharper, faster migrating band. Following a 2-hr chase period, most of the protein has been converted to the slightly slower migrating, terminally glycosylated form of the precursor and has been transported to the cell surface where it is available for cleavage by exogenously added trypsin. The C-T protein is also quantitatively converted to the terminally glycosylated form during the 2-hr chase period. However, although this protein can be demonstrated to be at the cell surface by immunofluorescence and hemagglutination assays (results not shown), the mutant protein cannot be cleaved by exogenously added trypsin. The C+RR mutant is indistinguishable from the wild-type protein in this experiment, demonstrating that the presence of an additional arginine residue at the cleavage site does not result in endogenous cleavage of HA in CV-I cells. Finally, the C+RRKKR mutant is cleaved into HA1 and HA2 subunits in the absence of any exogenous protease. The addition of trypsin to the medium above the cells does not result in any further cleavage of the remaining (core-glycosylated) precursor protein. This result demonstrates that the presence of multiple arginine and lysine residues (five total) at the

11. HEMAGGLUTININ OF INFLUENZA VIRUS

355

cleavage site facilitates the cleavage of the HA precursor by a protease endogenous to CV-I cells. The data shown in Fig. 6 do not provide evidence that this mutant protein reaches the cell surface; however, immunofluorescence and erythrocyte binding experiments reveal the presence of this protein at the plasma membrane. Each mutant protein was then tested for a requirement for in uirro proteolytic activation for mediation of low pH-induced cell-cell fusion. Figure 7 shows that (as previously reported; Gething c't a / . , 1986a) cells expressing the wild-type HA protein can be fused by treatment at low pH only after the HA0 precursor at the cell surface has been treated with exogenous trypsin, thereby cleaving the molecule into disulfide-bonded HA1 and HA2 subunits. However, the C T mutant cannot mediate low pH-induced fusion even after treatment with trypsin, presumably because alteration of the cleavage site prevents cleavage activation of the molecule. The phenotype of the C'RR mutant is identical to that of the wildtype protein, requiring pretreatment with trypsin to activate the molecule for fusion. The C+RRKKR mutant is active in low pH-mediated cell fusion without prior treatment with exogenous trypsin because the HA molecules at the cell surface have already been activated for fusion via cleavage by an endogenous protease. In summary, a mutant in which the single arginine residue at the cleavage site was altered to threonine was not cleaved under any circumstances, even after treatment with exogenous protease. This cleavageminus mutant was transported normally to the cell surface and displayed hemagglutinating activity although it could not mediate low pH-induced fusion. The second mutant contained two arginine residues at the processing site; this protein was indistinguishable from the wild-type HA in its transport and biological activities and was cleaved only after treatment with external trypsin. The final mutant reproduced the processing site of the HA from a pathogenic avian virus, i.e., . . . Arg.Arg.Lys.Lys.Arg. . . . This mutant HA was cleaved by a cellular protease in CV- I cells and mediated low pH-induced fusion without prior treatment with trypsin. Further experiments are planned to probe the precise structural requirements for cleavage activation. In addition to testing whether endogenous processing occurs when the site contains three or four basic amino acids, it will be of interest to probe any differences betwen arginine and lysine as protease substrates and to study the role of the conserved nonpolar residues that both precede and follow the processing site. The processing site mutants that are already available will be useful for a number of purposes. First, the cleavage-minus mutant will provide an opportunity to produce precursor HA0 for crystallographic studies. A significant, although possibly localized, conformational change must take

WT

C'T

WT

C+RR

C+RRKKR

FIG. 5. Nucleic acid and corresponding amino acid sequences around the cleavage activation sites of wild-type and mutant HA molecules. The Clal-BarnHI restriction fragment that encompasses the entire coding sequence of X-31 HA was cloned into an M13 mp18 vector. Single-stranded phage DNA was used as the template for oligonucleotide-directed mutagenesis to generate mutants that encode altered sequences at the activation cleavage site of HA. The autoradiographs (A) show the results of DNA sequence analysis to confirm the nucleotide sequences encoding the region around the cleavage sites in the wild-type and mutant HA cDNAs. The arrowheads and dashed lines show the positions and extent of nucleotide changes or insertions. The amino acids corresponding to each codon are shown in single letter code: R, Arg; K, Lys. (B) The nucleic acid and corresponding amino acid sequences of the relevant segment of the wild-type and mutant proteins. The vertical arrows show potential cleavage sites.

357

11. HEMAGGLUTININ OF INFLUENZA VIRUS

B

1HA2

XHA-WT

..Asn.Val.Pro.Glu.Lys.Gln.Thr Arg.Gly.Leu.Phe.Gly. AAT GTA CCA GAG AAA CAA ACT AGA GGC CTA TTC GGC

XHA-C-T

..Asn.Val.Pro.Glu.Lys.Gln.Thr

XHA-C'RR

,

XHA-C'RRKKR

. . A m .Val .Pro.Glu.Lys.Gln.Thr.Arg.Arg. 1 1Lys. Lys.Arg.Gly.Leu.Phe.Gly.. I IHA2

ihr.Gly.Leu.Phe.Gly. ACA

1

.Asn.Val .Pro.Glu. Lys.Gln.Thr.Arg.Arg. /HA* .Gly.Leu.Phe.Gly.. AGG AGA

I

AGG AGA AAG AAG AGA

FIG.5 .

(Con~in~ed)

place on cleavage activation since the carboxy terminus of the HA1 chain and the amino terminus of the HA2 chain, which are separated by only one amino acid in the precursor polypeptide, end up separated by a gap corresponding to approximately 10 residues in the cleaved molecule (Wilson PI a/.. 1981). The only satisfactory way to analyze this conformational change is by comparison of the known three-dimensional structure of the cleaved molecule with that of the precursor form. Uncleaved HA produced in tissue culture systems has not been a suitable source of precursor because it undergoes activation cleavage during harvesting from the cell surface with bromelain. Furthermore, the anchor-minus precursor HA that is secreted in large amounts from CV-1 cells that have been infected with the SVEHA20-A- recombinant virus (Gething and Sambrook, 1982) also is not useful for structural studies. Although the anchor-minus HA is initially assembled into trimeric structures, the oligomers are not stable and fall apart into monomers over time. By contrast, the solublized trimers of wild-type HA (BHA) that can be released from the virion or cell surface with bromelain (Brand and Skehel, 1972) are very stable. Preliminary studies indicate that soluble BHAO trimers can be prepared from the cleavage-minus variant of X-3 1 HA. A second application involves the C+RRKKR mutant which undergoes endogenous activation cleavage in cultured cells. We have constructed a composite mutant X-31 HA cDNA that encodes a protein containing both the cleavage site insertion and the substitution of asparagine for aspartic acid at residue 132 (Dams et al., 1985), which results in a higher pH threshold for fusion activity. When this cDNA was expressed in CV-I

358

MARY-JANE GETHING ET AL.

FIG.6. Analysis of the transport and proteolytic cleavage of wild-type and mutant HA proteins. CV-1 cells infected for 36 hr with SV40-HA viruses encoding the wild-type or mutant HA proteins were labeled for I5 min with [Y5]methionine. Cell extracts were prepared immediately (A) or following further incubation for 2 hr in medium containing an excess of nonradioactive methionine (B,C). In C trypsin at a concentration of 10 j&ml was included in the medium above the cells for the final I5 min of the 2-hr period. Proteins were precipitated from the cell extracts with anti-HA serum, separated by SDS-PAGE, and autoradiographed. The positions of the precursor H A 0 molecules and of the HA1 and HA2 cleavage products are shown to the right.

cells from an SV40-HA vector, the expected phenotype was obtained of fusion activity at pH 6.0 in the absence of prior treatment with trypsin. The double-mutant cDNA has also been inserted into a BPV-based vector, and continous cell lines (NIH-3T3and CHO) that express the variant HA are being selected by fluorescence-activated cell sorting. Because these cell lines will undergo cell-cell or erythrocyte-cell fusion under very mild and simple conditions (only moderately low pH and no protease treatment), they should provide excellent experimental systems for studies of fusion or as universal recipients for any materials of biological interest (antibodies, enzymes, oligonucleotides, etc.) that can be loaded into erythrocyte ghosts (Doxsey et al., 1985).

FIG.7. Low-pH-induced cell-cell fusion of CV-I cells expressing wild-type or mutant HA proteins: requirement for proteolytic cleavage activation. CV-I cells were infected with SV40-HA vectors containing the wild-type or mutant HA genes. Fifty hours after infection. the medium was aspirated from the monolayers which were then washed 3 times with medium lacking serum. Following incubation at 37°C for IS min with medium alone (top row) or with medium containing trypsin (10 &ml) (bottom row). the cells were briefly treated with buffer at pH 5.3 and then incubated further in medium containing serum for 8 hr to permit visualization of the formation of polykaryons with clustered nuclei. The cell monolayers were then fixed with formaldehyde, stained with Giemsa. and photographed.

360

MARY-JANE GETHING ET AL.

VIII.

CONCLUSION

The studies described in this chapter illustrate how the use of recombinant DNA techniques can advance our knowledge of the mechanism of membrane fusion. Cloning of cDNA copies of genes encoding wild-type and variant fusogenic proteins provides information on their primary amino acid sequences. Site-directed mutagenesis of the cloned genes can be used to probe the importance of chosen domains of a protein in the fusion mechanism. Expression of wild-type and mutant genes in mammalian cells using various types of eukaryotic vectors facilitates the analysis of their fusion phenotypes and in addition provides cell lines that can be used for cell fusion experiments or for delivery of macromolecules into the cell cytoplasm. Future experiments that combine the power of recombinant DNA techniques with the elegance of the model system provided by influenza hemagglutinin should reveal further details of the molecular mechanism of the fusion reaction induced by this particular membrane protein. The availability of cloned copies of a number of other viral fusion proteins (for references, see Gething et al., 1986a) will now permit similar analyses in other systems. Such studies should reveal any common or unique features of the mechanisms by which these different fusogenic proteins mediate membrane fusion. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health to M.J.G. and J.S. REFERENCES Bachi, T.,Gerhard, W., and Yewdell, J. W. (1985). Monoclonal antibodies detect different forms of influenza virus during viral penetration and biosynthesis. J. Virol. 55,307-313. Braciale, T. J., Lukacher, A. E., Morrison, L., Braciale, V. J., Smith, G., Moss, B., Gething, M. J., and Sambrook, J. (1986). Influenza viral antigen recognition by Class I and Class 11 MHC restricted cytolytic T lymphocytes. In "Options for the Control of Influenza Virus" (P. A. Kendal and P. A. Patriarca, eds.), pp. 407-421. Liss, New York. Brand, C. M., and Skehel, J. 3. (1972). Crystalline antigen from the influenza virus envelope. Nature (London) New Biol. 238, 145-147. Copeland, C., Doms, R. W., Bolzau. E. M., Webster, R. G., and Helenius, A, (1986). Assembly of influenza hemagglutinin trimers and its role in intracellular transport. J. Cell Biol. 103, 1179-1 191. Daniels, R. S., Downie, J. C., Hay, A. J., Knossow, M., Skehel, J. I., Wang, M. L., and Wiiey. D. C. (1985). Fusion mutants of the influenza virus hemagglutinin glycoprotein. Cell 40,431-439. Doms, R. W., Helenius, A., and White, J. M. (1985). Membrane fusion activity of the

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influenza virus hemagglutinin. The low pH-induced conformational change. J . Biol. Chem. 260, 2913-298 I . Doms. R. W., Gething, M.-J.. Henneberry, J . H., White, J . . and Helenius, A. (1986). A variant influenza hemagglutin that induces fusion at elevated pH. J. Virol. 57,603-613. Doyle, C.. Roth. M. G., Sambrook, J . , and Gething, M-J. (1985). Mutations in the cytoplasmic domain of the influenza virus hemagglutin affect different stages of intracellular transport. J . Cell B i d . 100, 704-714. Doyle, C., Sambrook, J . , and Gething, M.-J. (1986). Progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin. J. Cell B i d . 103, 11931204. Doxsey. S. J., Sambrook, J . . Helenius, A.. and White. J. M. (1985). An efficient method for introducing macromolecules into living cells. J. Cell Biol. 101, 19-27. Drzenick, R., Seto, J. T., and Rott. R. (1966). Characterization of neuraminidases from myxoviruses. Biwhim. Biiplrys. Acru 128, 547-558. Garten, W., Bosch, F.-X., Linder, D., Rott, R., and Klenk. H.-D. (1981). Proteolytic activation of the influenza virus hemagglutinin: The structure of the cleavage site and the enzymes involved in cleavage. Virok>gy115, 361-374. Gething. M . 4 . . and Sambrook, J . (19811. Cell-surface expression of influenza haemag glutinin from a cloned DNA copy of the RNA gene. Nuture (London)293, 620-625. Gething. M.-J.. and Sambrook, J. (1982). Construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein. Nuture (London)300,598603. Gething. M.-J.. and Sambrook, J. (1983). Expression of proteins on the cell surface using mammalian vectors. In “Genetics of Influenza Viruses” (P. Palese and D. W. Kingsbury, eds.), pp. 169-191. Springer-Verlag. Berlin and New York. White, J. M., and Waterfield, M. D. (1978). Purification of the fusion protein Gething, M.-J.. of Sendai virus: Analysis of the amino terminal sequence generated during precursor activation. Proc. Natl. Acad. Sci. U.S.A. 75, 2137-2140. Gething, M.-J., Bye, J., Skehel, J. J., and Waterfield. M. D. (1980). Cloning and DNA sequence of double stranded copies of haemagglutinin genes from H2 and H3 strains elucidates antigenic shift and drift in human influenza virus. Nuture (London)287,301306. Gething, M.-J., Doms, R. W., York, D.. and White, J . (1986a). Studies ofthe mechanism of membrane fusion: Site-specific mutagenesis of the hemagglutinin of influenza virus. J . Cell Biol. 102, 11-23. Gething, M.-J., McCammon, K., and Sambrook. J . (1986b). Expression of wild-type and mutant forms of influenza hemagglutinin: The role of folding in intracellular transport. Cell 46, 939-950. Gething. M. J . . Doms, R. W.. White, J. M., and Helenius, A. (1986~).Studies on the mechanism of membrane fusion. I n ”Protein Engineering: Applications in Science, Medicine and Engineering” (M. Inouye and R. Sarma, eds.). pp. 193-215. Academic Press, New York. Hirst. G. K. (1941). The agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus. Science 94, 22-23. Huang, R. T., Rott, R., and Klenk, H.-D. (1981). Influenza viruses cause hemolysis and fusion of cells. Virology 110, 243-247. Kaiser, E. T., and Kezdy, F. J . (1984). Amphiphilic secondary structure: Design of peptide hormones. Science 223, 249-255. Kawaoka. Y., Naeve, C. W., and Webster, R. G . (1984). Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? Virology 139, 303-316.

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Index

A

4-Acetamido-4’-isothiocyanostilbene 2.2’-disulfonic acid inhibition of anion transport in erythrocyte membranes, 208 chromaffin granule ATPase, 209 5-hydroxytryptamine secretion. 2 14 parathyroid hormone secretion, 2 14-2 IS Acetate. inhibition of Ca2+-dependent catecholamine release in permeabilized chromaffin cells, 2 16-2 18 Acetylcholine release from nerve terminals, 143 at neuromuscular junction. 116-1 17. 145 N-Acetylglucosamine, effect on myoblast fusion. 95 Acidic residues. protonation in hydrophobic segment, 281 Acridine orange, 229 complexing by anionic substances, 2 32 -2 33 deficiency of, 235 marker trapping, 235 mobility of forms, 233-234 permeation of membranes, 232 suramin pretreatment effect, 245 transfer to phagosomes. 229, 245 uptake of sulfonated fluors and. 237 Acrosomal membranes intramembranous particles. 16- 17 sperm-egg fusion site, 13 Acrosome reaction freeze-fracture images of acrosomal membrane, 16

membrane phospholipid changes and, 17 sea urchins, 6 sturgeon spermatozoa and, 9-10 Acrylamide, fluorescence quenching, 272 Adenosine triphosphate analogs, chromaffin granule lysis and. 206 and Ca2+,requirements in chromaffin cells and platelets. 149 lysis induced in isolated chromafin granules, 208 removal, secretory system sensitivity, 126 Adenovirus, cell entry route. 288 Adenylate cyclase system, G proteins, 54-55 Adrenal chromaffin cells, botulinum toxin effect, 75 Adrenal medullary cells, permeabilized, exocytosis inhibition, 132-134 Aequorin Ca” role in stimulus-secretion coupling and, 120 microinjection, in sea urchin eggs. 51 Aldehyde fixation blebs, 175 exocytosis and, 170 vesicular structures, 175 Alphavirus E l glycoproteins, hydrophobic segments, 260 Amiloride, protein kinase C inhibition, I30

Amino acid residues, hydrophobic. HA2, FI. and gp36 glycoproteins, 259 Aminopeptidase M,effect on FI amino terminus, 267 Amino terminals, HA2, FI. and gp36 glycoproteins. 259

365

366 Ammonia chromaffin granule lysis mediated by, 208 effect on methylamine accumulation in permeabilized chromaffin cells, 216-2 I7 veratridine-induced catecholamine secretion in chromaffin cells, 216-219 Amphibians eggs, vitelline envelope, 10 sperm-egg fusion morphology, 10 Amphipathic helix, in hemagglutinin, 351-352 Amphomycin, effect on sperm-egg fusion, 33 Anion permeability, pH in isolated chromaffin cells and, 205 Anion permeability series, 207, 212 Anion transport blockers anion transport mechanisms in chromaffin granules and, 208 inhibition of 5-hydroxytryptamine secretion, 214 Antibodies anticalmodulin cortical exocytosis inhibition, 66 exocytosis and, 152 antihemagglutinin, 342-344 antimembrane, sperm-egg fusion and, 32 delivery into cytoplasm, 344 Antigenic properties, influenza virus, 337-338 Antigenic structure, hemagglutinin, 338 Artifacts with fluorescent lysosomal probes, 234-235 in rapid freezing, 173-175 ATP, see Adenosine triphosphate ATPase chromaffin granules, potentiation and inhibition, 209 clathrin-dependent, clathrin coat removal and, 194 F , - , mitochondrial, in chromaffin membrane preparations, 204-205 proton-translocating, see Proton-translocating ATPase

INDEX

ATPase I, chromaffin granule membranes, 204-205 ATPase 11, chromaffin granule membranes, 204-205 AtT20 cells, a-latrotoxin effect. 155 Azurophilic granules, polymorphonuclear leukocytes, 142-143

B Band 3 proteins, mobilization by influenza virus, 275 Basophils, human, antigen-induced histamine release, permeant anion role, 215 BAY-K 8644, calcium agonist, 123 BHA, see Bromelain hemagglutinin BHK-21 cells, Semliki Forest virus envelope fusion, 263 Bilayers, membrane, role in membrane fusion, 101-102 Bindin acrosomal process and, 6 in sperm-egg fusion, 34 Birds, sperm-egg fusion morphology, I I Blebs in glutaraldehyde-fixed cells, formation, 181-182 production by aldehyde fixation, 175 Botulinum toxin effect on adrenal chromaffin cells, 75 capacitance, 125 exocytosis inhibition, 124-125, 132, 134, 153-1 54 type D, effect on exocytosis, 125 Bovine papilloma virus vectors, 341-345 Bromelain hemagglutinin (BHA) circular dichroism spectrum, 274 three-dimensional structure, 272 trypsin action on, 274 Bromide anion Class 1, 208 inhibition of Ca*+-dependent catecholamine release in permeabilized chromaffin cells, 216-218 permeability, 207 use of blocker-sensitive anion transport site, 209

367

INDEX Er& juponica. sperm-egg fusion, 10 BV I-MTHA cell lines. hemagglutinin expression and low pHmediated cell-cell fusion, correlation. 346 C

C127 cells bovine papilloma virus-transformed, hemagglutinin expression, 342 X-3 I gene introduction. hemagglutinin expression, 344 Calcium ion agonists and antagonists, 123 and ATP. requirements in chromaffin cells and platelets, 149 channels agonist-gated, 123 in nervous tissue. 123 voltage-sensitive, 123 clamping. 150 in cortical exocytosis (sea urchin), 51-52 G proteins and, 54-55 phosphoinositide cycle, 52-54 phospholipase C, phosphatidylinositol-specific, 52. 74 signal transduction. 52-54 effect on hemagglutinating virus of Japan-mediated cell fusion, 310-31 I elevation. transiency of release responses induced by. 149 endocytosis regulation and, 159-160 exocytosis control and, 120-121 extracellular, in sperm-egg fusion, 27-28 fusogenic process and, 126 granule swelling and, 220 influx in spermatozoa. I5 inhibition of phagosome-lysosome fusion by polyanionics and, 247-248 pore formation in virus-induced hemolysis, 265 myoblast fusion dependence on, 89, 91-93, 99-101, 105 permeability in secretory cells, 120-121 phase separation of phospholipids and, 35 as primary messenger in exocytosis, 122- 123

protein kinase C activators and, I50 in regulated exocytosis. 147-151 in regulated secretion, 47-48 release from endoplasmic reticulum, 124 requirement for protein kinase C. 129- I30 requirement in secretory systems, 122 stimulation of proteolysis, membrane fusion and, 103-104 Calcium ionophore A23187 lysosomal enzyme release induced by, inhibition by 4-acetamido4’-isothiocyanostilbene 2.2‘disulfonic acid, 215 stimulation of 5-hydroxytryptamine secretion, inhibition by anion transport blockers, 2 14 Carboxyl groups, protonation, 282-284 Catecholamine secretion (chromaftin cells) digitonin-permeabilized.2 18-219 osmotic effects, 216-219 permeabilized cells. 216-219 proton gradient and, 213 Cation-exchange resins acridine orange complexing, 232-233 phagosome-lysosome fusion and. 248 Cations, impermeability of chromaffin granules, 206 Cell-free secretory systems, 149 Cell lines. myogenic. 89 Cell lysis. hemagglutinating virus of Japan-mediated in Erlich ascites tumor cells. 309-3 1 1 Cell specificity. of myoblast fusion. 89-91 Cell surface complex preparations, 59 Chumu mucerphyllu, sperm-egg fusion morphology, 6-8 Chemically fixed cells, exocytosis. 170175 Chemiosmotic hypothesis, 203-204 alternative hypotheses. 219-220 Chloride anion Class 1. 208 CI-.ATP-induced lysis of isolated chromaffin granules, 207-208 inhibition of Ca”-dependent catecholamine release in permeabilized chromdffin cells, 2 16-21 8 permeability, 207

INDEX

use of blocker-sensitive anion transport site, 209 Chloroquine, phagosome-lysosome fusion and, 245-246 CHO cells, 31 gene introduction, hemagglutinin expression, 344 Cholera toxin, cortical exocytosis stimulation, 74 Cholesterol effect on fusion of hemagglutinating virus of Japan, 264-265 membrane distribution during exocytosis, 185 in target membranes, binding of FI amino-terminal segment, 267 Chromaffin cells digitonin-permeabilized, catecholamine secretion, 218-2 19 exocytotic fusion-fission, GTP-binding protein role, 152 permeabilized membrane recycling rate and Ca2+, 160 osmotic effects on secretion, 216-219 rapidly frozen, exocytosis, 177-179 regulated exocytosis, ATP and Ca2' requirements, I49 secretion, osmotic effects, 21 1-214 catecholamine secretion, 213-214 osmotic strength of media effects, 212 permeant anion effects, 212 probenecid effects. 212 proton electrochemical gradient and, 213-214 pyridoxal phosphate effects, 212 p-trifluoromethoxyphenylh ydrazone effects, 2 12-2 I4 Chromaffin granules ghosts, 204-205, 210-21 I ionic permeabilities, 206-209 isolated electrochemical proton gradient, 205 lysis, anion groups and, 208 pH. intragranular. 205 proton-translocating ATPase, 204-205 valinomycin,K+-induced lysis, 208 osmotic fragility in vitro, 213 osmotic lysis, 206-209 Chromobindin 9, membrane fusion-fission in exocytosis and, 152

Chymotrypsin, effect on sperm-egg fusion, 3 1-32 Circular dichroism bromelain hemagglutinin (BHA) spectrum, 274 measurement of influenza virus hemagglutinin, 279 Clathrin basket, 191. 194 curvature, enzymatic activities and, 194 Cleavage, proteolytic, hemagglutinin precursor, 339, 358 Clostridium toxins, exocytosis inhibition, 153-155

Clustering, intramembrane particle arrays, 323-32.5 "C-NMR, protonation of carboxyl groups, 282-284 Coated vesicles in endocytosis, 157, 159 virus uptake, 262, 286 Complementary DNA, encoding influenza virus hernagglutinin, 341-345 Concanavalin A, effect on myoblast fusion, 94-95 Conformational change, hemagglutinins, 352-353 Constitutive secretion, in exocytosis. 46-47 Cortical exocytosis (sea urchin egg), 48-51 calcium signal, 51-55 cell surface complex preparations, 59 cortical lawn preparations, 59 cortical vesicle discharge, 59-62 cortical vesicle-plasma membrane junction, 70 G proteins and, 54-55 inhibitors, 64-66 metabolic energy and, 71-72 osmotic forces and, 70-71 phosphoinositide cycle, 52-54 phospholipases and, 73 vectorial nature of, 61 viral model, 72-73 Cortical granules, proteolytic component, 25 Cortical lawn preparations, 59 Cortical vesicles (sea urchin egg), 48-51 discharge assays, 62-64 morphology, 59-62

369

INDEX

junction with plasma membrane, 70 purification. 66-67 purified, reconstitution of active cortex from. 67-69 Cross fertilization species specificity of sperm-egg fusion. 20-24

in various species. 24 vitelline envelope and, 22 CV-I cells anchor-minus precursor hemagglutinin, 357

hemagglutinins produced from SV40-HA and BPV-HA vectors in,

macrophage lysosome\ and, 232-233

hehavior of nonionic water-dispersihle polymers and. 239-240 cells labeled with Thorotrast and sulfonated fluors. dye transfer to phagosomes. 240 fusion inhibition. lysosomal pH and. 247 phagosome-lysosome fusion and. 229 Diac ylgl ycerol analogs. effect on exocytosis. 128 in cortical exocytosis, 73 myoblast fusion and. I02 as primary messenger in exocytosis.

344

122-123

low pH-induced fusion. 3S9 mutant hemagglutinin protein activity. 354

Cyclic AMP effect on exocytosis in parotid cells. I50 exocytosis regulation in parotid acinar cells, 153 hemagglutinating virus of Japanmediated fusion and, 317 levels after myoblast fusion, 106 in salivary and pancreatic exocrine secretion, 120 Cytochalasin B . inhibition of hemagglutinating virus of Japan-mediated fusion, 320-323 Cytochalasin D inhibition of Ehrlich ascites tumor cell fusion, 331 hemagglutinating virus of Japan-mediated fusion. 320-323 Cytochalasins effect on sperm-egg fusion, 33 inhibition of hemagglutinating virus of Japan-induced cell fusion, 266 Cytoplasmic organelles, alteration, hemagglutinating virus of Japan-mediated fusion and, 317-320

D 2-Deoxyglucose. effect on sperm-egg fusion, 33 Dextrans, exocytosis inhibition. 186 Dextran sulfate acridine orange accumulation in

signal transduction and, 52-54 Diaphragms, freeze-fracture imaging. 17 I Dicyclohex ylcarhodiimide effect on secretion from intact chromaffin cells, 213 reactive peptide in ATPase I. 205 Differential delivery, lysosomal constituents. 240-241 Digestion, intracellular. polyanionics and. 242-244

Digitonin-permeahilized chromaffin cells, catecholamine secretion, 218-219 Dihydropyridines, calcium antagonists. 123 Diphtheria toxin cell entry route, 288 fusion. hemagglutinating virus of Japan-mediated, 303-305 Disaccharides, inhibition of hemagglutinating virus of Japan-induced cell fusion. 266 S.S’-Dithiohis(2-nitrohenzoicacid]. inhibition of cortical exocytosis. 64 Dithiothreitol. effect on sperm-egg fusion, 33

Dowex-SO. affinity for acridine orange, 233 Dynein arms, sperm motility and, 18

E Ectodomain fragment. hemagglutinin, 340 Efficiency of fusion, influenza virus mutants, 350 Egg cortex (sea urchin) cortical vesicle purification, 66-67

370 plasma membrane purification, 67 reconstitution, 67-69 Egg plasma membrane electric potential, 30-3 1 intermingling with sperm plasma membrane, 19-20 membrane potential. 25 sea urchin, 48 junction with cortical vesicles, 70 purification, 67 purified, reconstitution of active cortex from, 67-69 sperm-egg fusion sites, 17-18 E glycoproteins, Semliki Forest virus, 276 EGTA, microinjection, in sea urchin eggs, 51

Ehrlich ascites tumor cells hemagglutinating virus of Japanmediated fusion cell lysis and, 309-3 I I close attachment of cell membranes, 325-329 clustering of intramembrane particle arrays, 323-325 concentration of virus required for. 308-309 cyclic AMP levels and, 317 cytoplasmic organelles, alteration, 3 17-320 effect of cations, 310-31 1 freeze-fracture imaging, 326-327 incubation at 37°C. 314-317 inhibition by cytochalasins B and D, 33 I , 320-323 membrane potential and, 315 model, 329-331 perturbation of cell membrane structure, 323-325 pH range required for, 308-309 viral envelope fusion and cell fusion. 320-323 virus-cell interaction at low temperature, 312-314 Elaidic acid, effect on myoblast fusion, 98 Electronectin, synthesis before myoblast fusion, 95-% Electron microscopy Ca2'-mediated fusion, 59-62 exocytosis monitoring by, 117-1 18 exocytosis rates and, 140

INDEX

illusion of inhibited fusion of secondary lysosomes, 236 markers, hydrosol trapping of, 240 transfer of acridine orange and Thorotrast to phagosomes, 229-230 viral envelope fusion, 262 Electropermeabilizdtion,122 Endocytic vesicles, prelysosomal, rapid acidification, 287 Endocytosis, 189- 194 clathrin basket, 191, 194 exocytosis and, 196 mechanisms, fast and slow, 158-159 membrane sorting, 156-159 regulation, 159-160 rotary-shadow method, 189 synaptic vesicle depletion, 160 triggered, 156- 160 Endoplasmic reticulum CaZ+release, 124 hemagglutinin localization, 341 Endosomes, Semliki Forest virus and influenza virus genome transfer, 262 Eosine. transient dichroism, 264 Epinephrine secretion chromaffin cells, 212 isolated chromaffin granules, 208 Erythrocyte membranes anion transport, inhibition, 208 band 3 proteins, mobilization by influenza virus, 275 fusion, viral envelope fusion and. 331 hemagglutinating virus of Japan fusion. 263-265 influenza virus effects, 274-275 influenza virus fusion, 269, 339 trypsinized, vesicular stomatitis virus envelope fusion and hemolysis, 263 Erythrosine B, effect on sperm-egg fusion, 33 Escherichiu coli

affinity for acridine orange, 233 microbicidal activity, phagosomelysosome fusion and, 244-246 N-Ethylmaleimide, inhibition of cortical exocytosis, 64 Exocrine gland cells, membrane recycling in, 159 Exocytosis biochemical study, limitations, 140

371

INDEX

CaZ+permeability in secretory cells. 120-12 I in chemically fixed cells, 170-17.5 constitutive secretion, 46-47, 142 continuous, I16 cortical. see Cortical exocytosis electron microscopy, 117-1 18 electrophysiological approach, 140-141 endocytosis and, 196 guanosine 5'4riphosphate binding-protein role in fusion-fission. I52 inhibition by botulinum toxin. 132, 134 Closrridium toxins, 153-155 dextrans. 186 GTPyS. 132 hyperosmotic forces, 186 inhibition in permeable adrenal medullary cells. 132-134 in v i m models, 56-73 Caz'-triggered, egg cortex and, 59-70 cell-free systems, 58 permeabilized cell systems, 56-57 membrane capacitance and, 117-1 18 membrane fusion-fission in, 151-1.53 Mg-ATP role. 126-129 permeabilized-cell studies, 121- 123 primary message generation. 123-124 protein kinase C and, 129-130 in rapidly frozen cells, 175-189 rates, activation of protein kinases and Ca", 152 regulated ATP and CaZ+requirements, 149 release, 142-143 second messenger control, 147-15 I secretion, 46-47 stimulation by a-latrotoxin, 155-156 stimulus-secretion coupling. 47-48 toxins and, 124-125 triggered, 116, 118, 120-121 types, 128-129 viral fusion model, 1 18- I20 Exo-endocytotic coupling, 141-142

F FI-ATPase, mitochondrial, in chromaffin membrane preparations, 204-205

Fatty acids, see ulso specific w i d effect on myoblast fusion. 98-99 in sperm-egg fusion, 34 cis-unsaturated. requirement for vesicular stomatitis virus. 277 Ferririn, 229 Fertilization envelope, sea urchin egg, SO Fibroblasts, in myoblast cultures. 88-89 Field strains, influenza virus, 351 Fishes, sperm-egg fusion morphology, 8-10 Fission. in exocytotic process, 206. 220 Fluoresceinated dextran, and mobile impermeant Huors, differential and sequential delivery, 241-242 Fluorescein isothiocyanate. in pH measurements, 287 Fluorescence, relief of quenching, 264 Fluorescence microscopy, illusion of inhibited fusion of secondary lysosomes, 236 Fluoride anion Class 111, 208 permeability, 207 Fluors, sulfonated, see Sulfonated Huors Free energy of transfer, virus hydrophobic segment peptide from aqueous to lipid bilayer phases, 279-280 Freeze-fracture electron microscopy myoblast fusion, 101 particle-free patches in sperm membrane, 34 particle-free regions in myoblast membrane at fusion sites, 99 Freeze-fracture imaging cell membranes induced by hemagglutinating virus of Japan, 327-328 diaphragms, 171 intramembrane particle arrays. 171-173 sperm membrane before and after acrosome reaction, 16 Freeze-thawing, hemagglutinating virus of Japan, 265 Fura2 Ca" measurement with. 149 microinjection, in sea urchin eggs, 5152 Fusers, microorganisms as, 246-247 Fusion-fission. membrane, in exocytosis, IS 1-153

372

INDEX

G Gullus gullus, sperm-egg fusion, 1 I Gangliosides, toxin binding, 154 Gap junctions, myoblast fusion and, 100 Gelatinous structure gelatinous trap model. 239-240 residual bodies, 246-249 testing, 241-242 in lysosomes, 239 @-I ,3-Glucanase, cortical vesicle exocytosis assay, 63-64 in sea urchin cortical vesicles, 50 Glutamate, effect on Ca”-dependent catecholamine release in permeabilized chromaffin cells, 2 16-218 Glutaraldehyde-fixed cells, bleb formation, 181 -182 Glycerol, dehydration, induction of intramembrane particle arrays, 173-175 GI ycoproteins myoblast fusion and. 94-96 viral envelope, see speciJic envelope glvcoprotein

Golgi regions, hemagglutinin localization, 34 I Granule membranes cholesterol distribution during exocytosis, 185 intermixing with plasma membrane constituents during fusion. 183 mobility before and during fusion, 183-185 GTP-binding protein, role in exocytosis, 75 Guanosine 5‘-diphosphate, role in exocytosis. 54-55 Guanosine 5’-triphosphate analogs exocytosis inhibition, 132 role in exocytosis, 54-55 GTPyS, 124 phospholipase C stimulation, 124 role in exocytosis, 54-55, 123

H Hemagglutinating virus of Japan (HVJ) Ca2+ inhibition of pore formation, 265

cholesterol effect on fusion, 264-265 clustering of intramembrane particle arrays, 266 effect on target membranes, 266 envelope structure, 301-303 FI glycoprotein amino-terminal hydrophobic segment, 267 amino-terminal segment, 259 binding to cholesterol in target membrane, 267 free energies for transfer, 280 molecular weight and function, 258 posttranslational cleavage, 301 precursor forms, 258 F2 glycoprotein molecular weight and function, 258 posttranslational cleavage, 301 precursor forms, 258 freeze-thawing, 265 fusion of Ehrlich ascites tumor cells, 306-308 cell lysis and, 309-3 I 1 close attachment of cell membranes, 325-329 clustering of intramembrane particle arrays, 323-325 concentration required for, 308-309 cyclic AMP levels, 317 cytoplasmic organelles, alteration, 3 17-320 effect of cations, 310-311 freeze-fracture imaging, 326-327 incubation at 37”C, 314-317 inhibition, 320-323 model, 329-33 I membrane potential and, 315 perturbation of cell membrane structure, 323-325 pH range required for, 308-309 viral envelope and cell fusion separation, 320-323 virus-cell interaction at low temperatures, 3 12-3 14 fusion cell, 266, 300-301 envelope, 263-265 with erythrocyte membranes, 263-265 factors required for, 303-306 lipid intermixing, 264

INDEX

with liposomes. 264-265 rate constant. 264 harvests, early and late, 265 hemolysis, 265 hemolytic activity. 300-301 HN glycoprotein, molecular weight and function. 258 infectivity, 300-301 intramembrane particle arrays, 265-266 nucleocapsid strands. 265 proteins, 299-300 sequence homology, 259 sonication, 265 structure. 299-300 Hemagglutinin amphipathic helix role in fusion. 351-352 anchor-minus precursor secreted from CV-I cells, 357 antigenic structure, 338 assays for fusion activity, 340-341 circular dichroism measurements. 279 cleavage activation, 353-359 cloned genes, site-specific mutagenesis. 346 conformational change, low pH-induced. 352-353 ectodomain fragment, 340 expression in cells from cloned hemagglutinin cDNAs, 341-345 fusion peptide, site-directed mutagenesis. 347-351 HA I . 258, 339-340 interaction with liposomes. 271-272 molecular weight and function. 258 HA2. 258, 339-340 amino-terminal segment, 259 hydrophobic segment, 272 secondary structure formation and. 284 free energies for transfer. 280 hydrophobic segments. 260 interaction with liposomes. 271-272 molecular weight and function, 258 primary sequence, as a factor in interaction with lipid bilayers, 284 interaction with liposomes, 271-272 low pH-induced conformational change, 272-274 mutants C'RR, 354

373 C'RRKKR, 354-355, 357-358 C T, 354-355 precursor forms, 258 posttranslational cleavage, 339, 358 reconstituted vesicles, 272 rosettes. 270 synthetic peptide from. 271 synthesized from SV40-HA and BPV-HA vectors, comparison, 343 three-dimensional structure. 338 transport, 358 vectors based on bovine papilloma virus, 34 1-345 Hemolysis, hemagglutinating virus of Japan-induced. 265, 300-301 Histamine release, from human basophils, permeant anion role, 215 Hoechst 33342, sea urchin egg staining, 26 Horseradish peroxidase, endocytosed, in dextran sulfate-inhibited cells, 242 Host-controlled modification, hemagglutinating virus of Japan, 300-301 HVJ virus, see Hemagglutinating virus of Japan Hyaline layer barriers to excess spermatozoa and. 30 resistance to fusion with spermatozoa and, 25 in sea urchin cortical vesicles, 50 Hydration repulsion, myoblast fusion and, 102-103 Hvdroides he.rugonits, sperm-egg fusion morphology, 6-8 Hydrophobicity, HA2, FI. and gp36 amino-terminal segments, 259 Hydrophobic segment entrance into lipid bilayer, 279-280 lipid bilayer as target, 279 protonation of acidic residues, 281-282 viral fusion glycoproteins, 260-261 Hydrophobic stretches, internal. Semliki Forest virus El and vesicular stomatitis virus G glycoprotein, 259 Hydrosols accumulation of polyanionic substances in lysosomes and, 23 I formation in lysosomes. 239-240 movement in lysosomes, 239-240

INDEX

structure in lysosomes, 239-240 trapping of colloidal electron microscopic markers, 240 5-Hydroxytryptamine, transport in porcine anterior pituitary, 210-21 1 Hyperosmotic buffers, inhibition of cortical vesicle discharge, 7 I Hyperosmotic forces, exocytosis inhibition, 186 I

Immune response, cellular, to influenza virus, 338 Immunocytochemistry, secretogranin 11 identification, 143 lmmunofluorescence indirect, hemagglutinin localization, 341 surface changes during myoblast fusion, 96-97 Infectivity, hemagglutinating virus of Japan, 300-301 Influenza C virus glycoproteins, hydrophobic segments, 260 sequence homology, 259 Influenza virus antigenic properties, 337-338 binding to liposomes lacking receptors, 279 brornelain hemagglutinin (BHA) three-dimensional structure, 272 trypsin action on, 274 cellular immune response to, 338 in coated pits and coated vesicles, 268 conformational change in viral glycoprotein, 278-279 effect on erythrocyte membranes, 274-275 endocytosis in MDCK cells, 268 in endosomes, 268 entry mechanism, 262 envelope fusion in acidic media, 262 and cell fusion with MDCK cells, 263 fusion with cultured cells, 339 erythrocytes, 269, 339 liposomes, 272, 339 genome transfer by fusion with endosomes, 262

interaction with erythrocytes and MDCK cells, 268 mutants pH modification of fusion activity, 285 threshold pH and efficiency of fusion, 350 NA glycoprotein, molecular weight and function, 258 pH dependence of viral fusion, 270 variants inducing fusion at elevated pH, 346-347 X-31 strain, 269 Inositol phospholipids, breakdown, myoblast fusion and, 102, 104 Inositol 1,4,5-triphosphate, signal transduction and, 52-54 Inositol trisphosphate, Ca2' release from endoplasmic reticulum and, 124 Insulinoma cells, exocytotic fusion-fission, GTP-binding protein role, 152 Insulin secretion, pencreatic islets, chemiosmotic hypothesis-based predictions, 215-2 16 Intermediates, in virus membrane fusion, 285 lntramembrane particle arrays, 171-173 acrosomal membranes, 16-17 clustering, in hemagglutinating virus of Japan-mediated fusion, 266, 323-325 formation, 182 glycerol dehydration and, 173-175 hemagglutinating virus of Japanmediated fusion, 265 influenza virus-induced, in erythrocyte membranes , 274-275 Iodide anion Class I, 208 use of blocker-sensitive anion transport site, 209 Ionic permeability, isolated chromaffin granules, 206-209 Isethionate, Class I1 anion, 208

K Kargagener's syndrome, spermatozoa-in, 18

L La Crosse virus cell entry route, 288

INDEX

GI glycoprotein, molecular weight and function. 258 G2 glycoprotein, molecular weight and function, 258 low pH-induced fusion activity, 278 pH dependence of viral fusion, 270 Lanthanum ion, effect on sperm-egg fusion, 33 a-Latrotoxin Ca'+-free effect, 156 exocytosis stimulation, 155-156 Leishmania mexicana. survival, phagosome-lysosome fusion effect. 245-246 Limulus amebocytes exocytosis, 177 plasma and granule membrane boundary during exocytosis, 183-185 Linoleic acids. effect on myoblast fusion, 98. 106 Lipid bilayers entrance of hydrophobic segment into, 279-284 interaction with HA2 primary sequence, 284 mobility, 180- 182 target for hydrophobic segment, 279 Lipidic particles, point defects and, 179-180 Lipids binding capacity of hemagglutinin mutants, 347-3.50 exchange, virus-cell fusion and, 264 intermixing, in virus-cell fusion, 264 role in myoblast fusion, 97-99, 101 targets for vesicular stomatitis virus, 277 Lipid-soluble ions, chromaffin granule pH and. 204-205 Liposomes ganglioside-containing, as hemagglutinin receptor, 271 hemagglutinating virus of Japan fusion, 264-265 influenza virus binding, 279 influenza virus fusion, 272, 339 interaction with influenza virus hemagglutinin, 271-272 vesicular stomatitis virus binding and fusion, 277 Lissamine rhodamine, label for secondary lysosomes, 236-237

375 Lucifer yellow, label for secondary lysosomes, 236-237 Lysin, in sperm-egg fusion, 34 Lysis CI ,ATP-induced in isolated chromaffin granules, 207-208 osmotic, chrornaffin granules. 206-209 Lysophosphatidylserine. effect on sperm-egg fusion, 33 Lysophospholipids. in sperm-egg fusion, 34 Lysosomal probes, fluorescent, set> speriJjc probe

Lysosomes constituents. differential delivery. 240-24 1 differential and sequential delivery of mobile impermeant fluors and tluoresceinated dextran, 241-242 free dye accumulation, 236 gelatinous structure in. 239 hydrosol formation, structure, and movement, 239-240 in macrophage antimicrobial activity. 245-246 nonionic hydrocolloids in, 238-242 pH. fusion-inhibiting qualities of polyanionics and, 247 primary accumulation of polyanionic substances and. 242 contents, intrusion into yeast phagosomes, 236 fusion inhibition and digestion of S. cereuisiue. 242 in yeast phagosornes, 236 secondary, 229 accumulation of polyanionics, 248 digestion, polyanionic substances in, 244 effects on nonionic water-dispersible polymers, 239 impermeant fluorescent labels. 236-238 intactness after phagosome-lysosome fusion, 236 Thorotrast transfer from, 230 in viral infection, 288 Lysosomotropic reagent, effect on viral replication, 287

376

INDEX

M Macrophages, nuclei, affinity for acridine orange, 233 Magnesium ion extracellular, in sperm-egg fusion, 29 phase separation of phospholipids and, 35 Mammals, sperm-egg fusion morphology, 11-13 Marsupialia, sperm-egg fusion, 11-13 Mast cells exocytosis, 122 intermixing of granule and plasma membrane constituents during fusion, 183 membrane recycling i n , 159 rapidly frozen, exocytosis. 175-177 small vesicles fused with granule membranes, 183 MDCK cells influenza virus envelope fusion and cell fusion, 263 growth in, 346-347 interaction, 268 X-3 I gene introduction, hemagglutinin expression, 344 Melittin, succinylated, pH-dependent fusion activity, 282-284 Membrane capacitance, 149 botulinum toxin effects, 125 electrophysiological approach, 141 endocytosis and, 158-159 exocytosis monitoring by, 117-1 18, 122 tetanus toxin effects, 125 Membrane fluidity inhibition of phagosome-lysosome fusion by polyanionics and, 247 membrane fusion and, 100-101 Membrane fusion in chemically fixed cells, 170-175 initiation at point defects, 179-180 microdomains and, 17 1- I73 Membrane perturbations, polyanioninduced fusion dysfunction and, 248 Membrane potential egg plasma membrane, 25 hemagglutinating virus of Japanmediated, 3 I5

Membrane retrieval, see Exo-endocytotic coupling Membranes pentalaminar and trilaminar structures, 170- 175 permeation by acridine orange, 232 Metabolic energy hypothesis, cortical exocytosis and, 71-72 Metal ions, role in myoblast fusion, 92-93 Metalloendoproteases, inhibition of myoblast fusion, 104 Me thy lamine accumulation in permeabilized chromaffin cells, 216-218 effect on veratridine-induced catecholamine secretion in chromaffin cells, 216-219 Mg-ATP, role in exocytosis, 126-129 Microbicidal activity, role of phagosome-lysosome fusion, 244-246 Microdomains, membrane fusion and, 171 - 173 Microscopic assay, for cortical vesicle exocytosis, 64 MME cells, X-31 gene introduction, hemagglutinin expression, 344 MMTV virus. see Mouse mammary tumor virus Mobility acridine orange, 233-234 sulfonated fluors, 238 Monensin chromaffin cell exposure in Na+- or K+-containing media, granule lysis. 2 19-220 effect on methylamine accumulation in permeabilized chromaffin cells, 216-2 I7 Monoclonal antibodies, surface changes during myoblast fusion, 96 Monosaccharides, inhibition of hemagglutinating virus of Japan-induced cell fusion, 266 Monotremata, sperm-egg fusion. 11-13 Motility, spermatozoa, sperm-egg fusion and, 18-19 Mouse hepatitis virus 90A glycoprotein, molecular weight and function, 258 E2 glycoprotein, molecular weight and function, 258

377

INDEX Mouse mammary tumor virus cell entry uncoating route, 288 gp36 glycoprotein amino-terminal segment. 259 conformational change, 28 1 free energies for transfer. 280 molecular weight and function. 258 gp52 glycoprotein, molecular weight and function. 258 low pH-induced fusion activity, 278 pH dependence of viral fusion, 270 Mucopolysaccharides, in sea urchin cortical vesicles, 50-51 Muscarinic receptors, activation. Ca?' and, 150 Mutagenesis site-directed, of hemagglutinin fusion peptide, 347-35 1 site-specific, of cloned hemagglutinin genes, 346 Mycobucr~~riitrn ruhercitkosis. sulfatides 228 Myconectin, synthesis before myoblast fusion. 95-96 Myoball cell cultures, 89 Myoblast fusion aggregation and adhesion stages, 92 Ca2+dependence, 89. 91-93 cell specificity, 89-91 lineage and, 89-91 lipids and, 97-99 mechanisms bilayer structure changes, 101-102 Ca'+-stimulated proteolysis, 103I04 I 2-diacylglycerol effects, 102-103 hydration repulsion and, 102- 103 protein phosphorylation role, 104 membrane changes, 105 membrane fluidity and, 100-101 precursor cell heterogeneity, 89-91 primary cultures. 88-89 CaZ+dependence, 89 cell lines, 89 fibroblast reduction, 88 myoball cultures. 89 receptor-stimulated, 106 recognition stage. 91-92 role of proteins, 94-96 stimulation, 106-017 temperature effects, I00 I

time dependence. 94 ultrastructural changes, 99-100 Myoblast plasma membranes antibody studies, 98-97 glycoproteins. 94-95 lipids, 97-99 protein phosphorylation and, 96 Myristic acid, effect on myoblast fusion, 98

N Neiinrhes j u p o n i w . sperm-egg fusion morphology. 6-8 Neuromuscular junction exocytosis acetylcholine release, 116-1 17, 145 Ca2' role, 147 effect of protein kinase C activators. I50 electrophysiological approach, 141 inhibition by botulinum toxin. 154 a-latrotoxin effect, 155-156 endocytosis Ca2+ and, I60 models, 157 Neurosecretory vesicles, intragranular pH, 210 Neurotransmitters acetylcholine release, 143 conventional. I16 peptide. 116. 143 Neutrophils, lysosomal enzyme release. inhibition by 4-acetamido-4'isothiocyanostilbene 2.2'-disulfonic acid, 215 Nigericin chromaffin granule lysis mediated by. 208 effect on veratridine-induced catecholamine secretion in chromaffin cells. 216-219 NlH-3T3 cells bovine papilloma virus-transformed, hemagglutinin expression, 342 cell surface location of hemagglutinin synthesized in, 344 hemagglutinins produced from SV40-HA and BPV-HA vectors in, 344 X-3 I gene introduction, hemagglutinin expression, 344

378

INDEX

Nuclear magnetic resonance spectroscopy, I3C-, protonation of carboxyl groups, 282-284 Nucleocapsid strands, hemagglutinating virus of Japan, 265 0

Octadecyl rhodamine B chloride, 264 Oleic acid, effect on myoblast fusion, 98 Oligonucleotides, delivery into cytoplasm, 344 Omega toxin, effect on exocytosis, 124 Osmotic effects in secretion intact cells chromaffin cells, 21 1-214 neutrophils, human, 2 I5 pancreatic islets, 215-216 parathyroid cells, dissociated, 214-215 platelets, human, 214 permeabilized chromaffin cells, 216-2 19 Osmotic forces cortical exocytosis and, 70-71, 75 role in fusion process, 185-186 Osmotic properties isolated chromaffin granules electrochemical proton gradient, 205 ionic permeabilities, 206-209 osmotic lysis, 206-209 proton-translocating ATPase, 204-205 Ouabain, effect on sperm-egg fusion, 33 Ovoperoxidase assay for cortical vesicle exocytosis, 63 in sea urchin cortical vesicles. 50

P Pancreatic acinar cells, exocytosis, 150 Pancreatic islets insulin secretion, chemiosmotic hypothesis-based predictions, 21 5-216 insulin secretory granules, intragranular pH, 209-210 P arumecium, trichocyst discharge, 186- 187 Paramyxovirus, fusion with cell surface plasma membrane, 286 Paramyxovirus FI, glycoproteins, hydrophobic segments, 260

Parathyroid cells, parathyroid hormone secretion, inhibition by 4-acetamido-4’-isothiocyanostilbene 2,2’-disulfonic acid and probenecid, 214-2 I5 Parotid cells acinar cells, regulation by CAMP, 147, 153 CAMPeffect on exocytosis, 150 Parotid gland, intragranular pH, 210 Particle-free patches in acrosome-reacted spermatozoa, 17 in myoblast membranes at fusion sites, 99 in sperm membrane, 34 Patch clamp technique, 149 Pathogenicity, influenza virus, 353 pBVI-MTHA vector, 344 PC12 cells ATP requirement, 150 a-latrotoxin effect, 155 regulated exocytosis requirements, 150 Pentalaminar structure, of membranes, 170- 175 Permeability, ionic, isolated chromaffin granules, 206-209 Permeabilized cells detergent, 122 electropermeabilization, 122 techniques in secretion studies, 149 Pertussis toxin, substrate protein in sea urchin eggs, 74 PH acidic, protonation at, 281 conformational change in hemagglutinin, 272-274 effects on sperm-egg fusion, 27 elevated, influenza virus variant fusion induction at, 346-347 endosomal, lysosomotropic reagents and, 287-288 extracelluar medium, virus replication and, 287 fluorescein isothiocyanate in measurement of, 287 fusion activity dependent on HA2 N-terminal peptide, 282 HA2 peptide, 282 influenza virus, 340

INDEX succinylated melittin, 282-284 viral envelope, 262 virus membrane, 285-286 gradient. catecholamine release in permeabilized chromaffin cells and. 2 17-218 hemolytic activity of Semliki Forest virus and, 275-276 vesicular stomatitis virus, 276-277 intragranular insulin secretory granules, pancreatic islets, 209-210 isolated chromaffin cells. ADP effects. 206 isolated chromaffin granules, 205 neurosecretory vesicles, 210 parotid gland, 210 porcine anterior pituitary granules, 2 10 low conformational change induced in hemagglutinin. 352-353 induced fusion in CV-I cells, 359 La Crosse virus fusion activity and. 27N mouse mammary tumor virus fusion activity and. 278 lysosomal. polyanionics and, 247 range required for hemagglutinating virus of Japan-mediated cell-to-cell fusion, 308-309 threshold, influenza virus mutants. 350 Phagosome-lysosome fusion fluorescent lysosomal probes acridine orange. 23 1-236 artifacts, 234-235 sulfonated fluors, 236-238 illusion of inhibited fusion of secondary lysosomes, 236 inhibition, Ca" and. 247-248 intracellular digestion and, 242-244 microbicidal activities and. 244-246 promotion by weak bases, 246 Phagosomes acridine orange transfer to, 229-230, 245 dye transfer to. 240 sulfonated Ruor uptake, 238 Thorotrast transfer to, 229-230 yeast digestion without marker, 242-243 intrusion of lysosomal contents in, 236

379 Phase separation, phospholipids. Ca" and M g " roles. 35 Phenothiazines, inhibition of cortical exocytosis. 65 Phenylalklamines. calcium antagonists, I23 Phorbol esters protein kinase C substrate, 12X sen3itivity of exocytotic systems to. 132 Phosphate anion Class 11, 208 permeability, 207 Phosphatidylinositol. intracellular signaling and, 52 Phosphatidylinositol 4.5-bisphosphate, intracellular signaling and, 52-54 Phosphatidylinositol 4-phosphate. intracellular signaling and, 52 Phospholipase. in sperm-egg fusion. 34 Phospholipase A. effect on myoblast fusion in culture. 98 Phospholipase A*. role in cortical exocytosis. 73 Phospholipase C effect on myoblast fusion in culture. 98 GTP-stimulated, 124 phosphatidylinositol-specific,52-53, 74 role in cortical exocytosis, 73 Phospholipids, phase separation. Ca" and Mg'+ roles. 35 Pinosome fusion, polyanionics and. 248-249 PK cells, X-31 gene introduction. hemagglutinin expression, 344 Plasma membranes cholesterol distribution during exocytosis. 185 and granule membrane constituents, intermixing during fusion, 183 intramembrane particle-free areas, 171 mobility before and during fusion. 183-185 Platelets granule membrane. proton-translocating ATPase, 2 I 1 5-hydroxytryptamine secretion, 214 regulated exocytosis, ATP and CaLf requirements, 149 Pneumovirus FI glycoproteins, hydrophobic segments. 260

INDEX

Point defects, membrane fusion initiation, 179- 180 Poliovirus, cell entry route, 288 Polyanionic substances accumulation as hydrosols in lysosomes, 23 I by primary lysosomes, 242 antagonism with weak bases, 246-247 within lysosomes. affinity for acridine orange, 233 polyanionics hypothesis, history, 22 8-230 in secondary lysosomes, digestion and, 242-244 Polycations, inhibition of cortical exocytosis, 65 Polyerythrocytes, spherical, formation, 266 Polyglutamic acid, acridine orange accumulation in macrophage lysosomes and, 232-233 Pol ykaryons influenza virus mutants, 350 spherical, formation, 266 Polylysine, inhibition of cortical exocytosis, 65 Polymers hydrosol formation in water, 239-240 nonionic water-dispersible, behavior, 239-240 Polymorphonuclear leukocytes, azurophilic granules and secretory vesicles, 142-143 Polymyxin 8 , protein kinase C inhibition, 130 Polyphosphoinositide phosphodiesterase, see Phospholipase C, phosphatidylinositol-specific Polyphosphoinositides, hydrolysis, 147- I48 Polyspermy block saccharide residue changes and, 31 sialic acid changes and, 31 Posttranslational cleavage hemagglutinin precursor, 339, 353-359 virus fusion proteins, 258 Potassium ion, extracellular, sperm-egg fusion and, 29-30 Precursor cells fusion proteins, 258-259

hemagglutinin myogenic, 89-90 Prefusion events, sperm-egg fusion, 26 Probenecid inhibition of anion transport in erythrocyte membranes, 208 5-hydroxytryptamine secretion, 214 parathyroid hormone secret ion, 2 14-2 I 5 veratridine-induced secretion in chromaffin cells, 212 Procrustean bed interpretation, 23 I , 236, 239 Pronase effect on sperm-egg fusion, 3 1-32 inhibition of cortical exocytosis, 65 Protease sensitivity, hemagglutinin mutants, 347-350 Proteinase inhibitors, sperm-egg fusion and, 32 Protein kinase C activators, effect on exocytosis, 150 Ca2+ requirement, 129-130 exocytosis and, 129-130 hydrolysis of polyphosphoinositides and, I49 inhibitors, 130- I32 myoblast fusion and, 104 plasma membrane association. 133I34 substrate for phorbol esters, 128 Protein phosphorylation in exocytosis, 126-127 exocytosis modulation and, 153 in membrane fusion, 96, 104 Proteoliasin, in sea urchin cortical vesicles, 50 Proteol ysis in membrane fusion, 103 in myoblast fusion, 104-105 Protonation acidic residues in hydrophobic segments, 28 1 carboxyl groups, 282-284 Proton gradient across chromaffin granule membrane, 205 cultured chromaffin cells, 213 Proton pump, activation, 127-128

INDEX

381

Proton-translocating AI'PdSe bovine anterior pituitary gland. 210 insulin secretory granules, pancreatic islets. 209-210 isolated chromdffin granules, characterization. 204-205 neurosecretory vesicles, 2 10 in platelet granule membrane, 21 1 Protozoa. rapidly frozen, exocytosis, 177-179 Pyridoxal phosphate inhibition of anion transport in erythrocyte membranes. 208 chromaffin granule ATPase, 209 5-hydroxytryptamine secretion. 214 veratridine-induced secretion in chromaffin cells, 212

Q Quick freezing, see Rapid freezing Quin2 Ca" measurement. 149 microinjection, in sea urchin eggs, 51-52 role of Ca?' in stimulus-secretion coupling and, 120

R Rabies virus, pH dependence of viral fusion, 270 Radioimmunoassay s bovine papilloma virus-transformed cells expressing hemagglutinin, 342 surface changes during myoblast fusion. 97 Rapid freezing artifacts, 173-175 cells. exocytosis in, 175-189 Recognition reactions, in myoblast fusion, 91-92.94-95 Recombinant DNA genes encoding influenza virus hemagglutinin. 338 mechanism of membrane fusion and, 360 Regulated secretion, in exocytosis, 46-47 Retroviridae, oncovirus glycoprotein Types B and C, hydrophobic segment, 26 1 Rotary-shadow method, 189

S Saccharide residues, in egg membrane, 31 Suc,c.ho,omyces cerrvisicie. 229 behavior, phagosome-lysosome fusion and, 245 digestion, fusion inhibition and. 242 Sea urchin eggs cell surface complex preparations, 59 cortical lawn preparations. S9 cortical vesicles, 48-51 constituents, 50-51 discharge morphology. 59-62 fertilization envelope, 50 fertilization steps, 48 granule and plasma membrane constituents, intermixing during fusion, 183 hyperosmotic inhibition of exocytosis, 186 plasma membrane glycocalyx. extracellular, 50 vitelline layer, SO rapidly frozen, exocytosis. 177-179 secretory granule swelling. 220 sperm-egg fusion morphology. 4-6 swelling of cortical granules. 185 Secondary structure, influenza virus HA2 amino-terminal segment and, 284 Secretogranin 11, immunocytochemical identification, 143 Secretory vesicles Ca"-activated K' channels, opening, 220 polymorphonuclear leukocytes, 142-143 .sea urchin eggs. 48 triggered exocytosis and, I16 Semliki Forest virus E 1 glycoprotein, 276 free energies for transfer, 280 internal hydrophobic stretch, 259 internal segment, 281-282 molecular weight and function, 258 E2 glycoprotein. 276 molecular weight and function, 258 E3 glycoprotein, 276 molecular weight and function, 258 entry mechanism, 262 envelope fusion in acidic media, 262 with BHK-21 cells, 263

382 genome transfer by fusion with endosomes, 262 low pH-induced hemolytic activity, 275-276 pH dependence of viral fusion, 270 Sendai virus, see Hemagglutinating virus of Japan Serine protease, in sea urchin eggs, 50 SFV virus, see Semliki Forest virus Sialic acid, in egg membrane, 31 Signaling, intracellular G proteins and, 54-55 phosphoinositide cycle and. 52-54 Sindbis virus low pH-induced hemolytic activity, 276 pH dependence of viral fusion, 270 Smooth vesicles, virus uptake, 262, 286 Sonication, hemagglutinating virus of Japan. 265 Species specificity, sperm-egg fusion, 20-24 Spermatozoa acrosome-reacted, particle-free patches, 17 Ca2+ influx, 15 fish sperm head plasma membrane, 9 sturgeon, 9 fusion with egg, see Sperm-egg fusion in Kargagener's syndrome, 18 motility, sperm-egg fusion and, 18-19 plasma membrane freeze-fracture imaging before and after acrosome reaction, 16 intermingling with egg plasma membrane, 19-20 particle-free patches, 34 sperm-egg fusion sites and, 13-17 Sperm-egg fusion antimembrane antibodies and, 32 cytochalasin effects, 33 dithiothreitol effects, 33 effects of extracellular Ca2+,27-28 K ' , 29-30 Mg*+, 29 erythrosine B effects, 33 glycoprotein biosynthesis inhibitors and, 33-34 mechanism, 34-35

INDEX morphology amphibians, 10 birds, I 1 fishes, 8-10 mammals, 11-13 marine invertebrates, 6-8 sea urchins, 4-6 motility of spermatozoa and, 18-19 pH effects, 27 prefusion events, 26 proteinase inhibitors and, 32 proteolytic enzymes and, 31-32 serine proteinase inhibitors and, 32 specific sites egg plasma membrane, 17-18 sperm plasma membrane, 13-17 temperature effects, 26-27 Stearic acids, effect on myoblast fusion. 98 Stimulus-secretion coupling, 47-48 Sulfate anion Class 111, 208 permeability, 207 Sulfolipids, mycobacterial acridine orange accumulation in macrophage lysosomes and, 232-233 acridine orange complexing, 232-233 affinity for acridine orange, 233 phagosome-lysosome fusion and. 229 Sulfonated fluors differential transfer from lysosomes, 24 1-242 and fluoresceinated dextran, differential and sequential delivery, 241-242 labeling of dextran sulfate cells, transfer to phagosomes, 240 labels for macrophage secondary lysosomes, 236-238 Sulforhodamine, 236-237 Suramin acridine orange accumulation in macrophage lysosomes and, 232-233 inhibition of 5-hydroxytryptamine secretion, 214 phagosome-lysosome fusion and, 229 pretreatment effect on acridine orange transfer to phagosomes, 245 SV40-HA vectors, 343

INDEX

Swelling Ca”-induced. 220 cortical granules. IUS sea urchin egg secretory granules. 220 Synapsin I exocytosis and, 117, 143 membrane fusion-fission in exocytosis and. 152 Synaptic vesicles, depletion in endocytosis. I 6 0

T Temperature low. virus-cell interaction at. 312-314 dependence inHuenza virus fusion activity, 269 viral envelope fusion, 264 effects o n myoblast fusion, 100 Temporal dependence, myoblast fusion, 94 Tetanus toxin effect on capacitance. 125 exocytosis, 124 exocytosis inhibition. 153-514 in chromaffin cells, IS4 mechanism. 154 structure and binding. 153-154 Thiocyanate anion permeability, 207. 207 inhibition of Ca”-dependent catecholamine release, 2 16-218 Thorium oxide marker. cross-linking with adsorbed polyanionics, 239 Thorotrast labeling of dextran sulfate cells, nontransfer to phagosomes, 240 polyanion cells, marker traces. 242 transfer to phagosomes. 229-230 Thrombin. stimulation of 5-hydroxytryptarnine secretion. inhibition by anion transport blockers. 214 Transport, of hemagglutinin, 358 Trapping. physical, of colloid,ri I e I eclron microscopic markers. 240 Trichocyst discharge, in P~ircimc~c,irrm, 186- I87 TriHuoperazine inhibition of cortical exocytosis. 65 protein kinase C inhibition, 130

p-TriHuoromet hox yphen ylhydrazone effect on secretion from intact chromaffin cells, 212-213 from permeabilized chromafin cells, 216-217 inhibition of platelet secretion, 214 Trilaminar structure, of membranes, 170- I75 Tripnrrrsres grarlrillti. sperm-egg fusion morphology, 4-6 Trypsin effect on sperm-egg fusion, 32 inhibition of cortical exocytosis. 65 Tunicamycin, inhibition of myoblast fusion, 95

U

UDP-glucose. effect on sperm-egg fusion. 33 Ultrastructure, myoblast, fusion-induced changes, 99-100

V Valinomycin, K -induced lysis, isolated chromaffin granules, 208 Vectorial transport assay. immunofluorescence-based. 62 Vesicular stomatitis virus entry mechanism, 262 envelope fusion and hemolysis with trypsinized erythrocytes, 263 envelope fusion in acidic media, 262 fusion with liposomes. 277 G glycoprotein, 258 free energies for transfer. 280 internal hydrophobic stretch. 259 inlernal segment. 28 1-282 niolecular weight and function. 258 lipids as targets for, 277 low pH-induced hemolysis and cell fusion. 276-277 Vesicular structures, production by aldehyde fixation. 17.5 Vesiculovirus G . glycoproteins, hydrophobic segments, 261 Viral envelope fusion, pH dependence. 262 +

384

INDEX

gl ycoproteins fusion, 262 molecular weight and function, 258 Viral fusion, exocytosis mechanism and, 118-120

Viral glycoproteins, conformation change, 278-279

cross fertilization and, 22 domestic fowl, I 1 fish eggs, 8 mammals, I I in sea urchin egg, 4 Vitelline layer, sea urchin eggs, 50 VSV, see Vesicular stomatitis virus

Viral model, cortical exocytosis and, 72-73

Virus glycoproteins envelope fusion and, 258 posttranslational cleavage of fusion proteins, 258 Virus membrane fusion hydrophobic segment entrance into lipid bilayer, 279-284 free energy of transfer of peptide from aqueous to lipid bilayer phases,

W Western equine encephalitis virus, E glycoproteins, 276 West Nile virus cell entry route, 288 low pH-induced hemolytic activity, 276 pH dependence of viral fusion. 270

Y

279-280

lipid bilayer domain as target, 279 primary sequence and hydrophobicity, 284

hydrophobic segment pH-dependent activity H A 2 N-terminal peptide, pH-dependent activity, 282 H A 2 peptide, 282 succinylated melittin, 282-284 intermediates, 285 mechanism membrane perturbation, 284-285 pH dependence, 285-286 Vitelline envelope amphibian egg, 10 barriers to excess spermatozoa and, 30 cortical granule material effect, 25

Yeasts affinity for acridine orange, 233 digestion by macrophages, 244 in phagosornes without marker, 242-243

marker delivery, phagosome-lysosome fusion and, 248 phagosomes, intrusion of lysosomal contents in, 236 Yellow fever virus low pH-induced hemolytic activity, 276 pH dependence of viral fusion, 270 Z

Zinc ion, effect on sperm-egg fusion, 33