THE MOLECULAR MECHANISMS UNDERLYING

EXOCYTOSIS IN SEA URCHIN EGGS.

Julia Catherine Avery

Department of Physiology

University College

University of London.

A Thesis submitted for the degree of Doctor of Philosophy

in the University of London.

March 1996. ProQuest Number: 10017484

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT.

Exocytosis is a ubiquitous cellular mechanism for the export of secretory products and the insertion of proteins and lipid into the plasma membrane. The sea urchin egg is an ideal system in which to study exocytosis. Fertilization in the sea urchin egg is characterized by a transient increase in intracellular calcium. This triggers the exocytosis of cortical secretory granules which lie immediately beneath the plasma membrane of unfertilized eggs, producing a structure called the fertilization envelope. Exocytosis can also be studied directly; the exocytotic apparatus, the egg cortex, can be isolated and responds to the physiological trigger - Ca^^ - in vitro.

It has been suggested that the molecular mechanisms underlying exocytosis are evolutionarily conserved. I have used western blotting to identify whether homologues of proteins implicated in exocytosis in other secretory systems are present in sea urchin eggs. Using this approach, I have identified homologues of the synaptic proteins synaptobrevin and rabSA, three members of the annexin family of Ca^Vphospholipid- binding proteins and a calcineurin-like protein in sea urchin eggs.

Using immunocytochemical confocal microscopy, I have localized the synaptobrevin homologue to the cortical granule membrane. I show that tetanus toxin light chain (TeTx LC), which inhibits neurotransmitter release by specifically cleaving synaptobrevin, cleaves the sea urchin synaptobrevin homologue in vitro. I demonstrate that preincubation of isolated cortices with TeTx LC causes a time-dependent inhibition of Ca^^-stimulated exocytosis which correlates with cleavage of the synaptobrevin homologue. These results suggest that a tetanus toxin-sensitive synaptobrevin homologue is required for exocytosis in sea urchin eggs. The Ca^Vcalmodulin-dependent phosphoprotein phosphatase calcineurin has been implicated in exocytosis in Paramecium. In sea urchin eggs, irreversible protein phosphorylation and antibodies to calmodulin block exocytosis in vitro. I have investigated the role played by calcineurin in exocytosis. Neither specific inhibitors nor functionally inhibitory antibodies to calcineurin affect calcium-stimulated exocytosis in vivo or in vitro. These results suggest that calcineurin is not involved in exocytosis in sea urchin eggs. Table of Contents.

Chapter 1. Introduction.

1. Exocytosis - A Universal Secretory Mechanism. 13

1.1 INTRODUCTION. 13 (i) The Exocytotic Fusion Pore. 14 (ii) Biophysical Aspects of Exocytosis. 15 1.2 THE ROLE OF CALCIUM IN THE CONTROL OF EXOCYTOSIS. 15 (i) Intracellular Targets for Calcium in Exocytosis. 16 (a) Calmodulin. 17

(b) Annexins and 14-3-3 Proteins. 18 (c) Synaptotagmin. 20 (d) The Cortical Cytoskeleton. 22 1.3 THE MODULATION OF CALCIUM SENSITIVITY. 24 (i) Protein Kinase Modulation of Exocytosis. 24 (ii) G-protein Control of Exocytosis. 26 1.4 THE ROLE OF RAB PROTEINS IN EXOCYTOSIS. 28 1.5 THE REQUIREMENT FOR ATP IN EXOCYTOSIS. 30 (i) The Effects of ATP on Exocytosis in Mast Cells. 31 (ii) Exocytosis and Protein Dephosphorylation in Paramecium. 32 (iii) The Identification of Priming F actors in Regulated Exocytosis. 3 3 (iv) Evidence that the Polyphosphoinositides are Necessaiy for Exocytosis. 35

2. The Exocytotic Mechanism in Synaptic Transmission. 36

2.1 THE MOLECULAR MECHANISM OF ACTION OF CLOSTRIDIAL NEUROTOXINS. 36 2.2 THE PUTATIVE EXOCYTOTIC FUSION COMPLEX. 37 2.3 HOMOLOGUES OF THE SYNAPTIC FUSION PROTEINS FUNCTION IN NON-NEURONAL EXOCYTOSIS. 41 2.4 THE MOLECULAR MECHANISMS UNDERLYING MEMBRANE FUSION. 43 2.5 ALTERNATIVE MODELS OF MEMBRANE FUSION. 45

3. Exocytosis in the Sea Urchin Egg. 46

3.1 SEA URCHIN EGG ACTIVATION AT FERTILIZATION. 46 3.2 CORTICAL GRANULE EXOCYTOSIS IN THE SEA URCHIN EGG. 47 3.3 SEA URCHIN EGG EXOCYTOSIS - A MODEL SYSTEM. 48 3.4 CALCIUM IS THE SOLE REQUIREMENT FOR TRIGGERING EXOCYTOSIS. 51 (i) The Effects of Protein Kinases and Inhibitors of Cytoskeletal Function. 52 (ii) GTP-Binding Proteins and Exocytosis. 52 3.5 CALCIUM TARGETS IN SEA URCHIN EGG EXOCYTOSIS. 53 (i) Calcium-Binding Proteins. 53 (ii) Calcium-Stimulated Phospholipases. 54 3.6 THE EFFECT OF ATP ON EXOCYTOSIS IN SEA URCHIN EGGS. 55 (i) Is ATP Required for an Energy-Dependent Step During Exocytosis? 56 (ii) The Efkct of Irreversible Protein Phosphorylation on Exocytosis. 56 (iii) The Role of the Polyphosphoinositides in Exocytosis. 57 3.7 PROTEINS ARE REQUIRED FOR REGULATING THE EXOCYTOTIC REACTION IN VITRO. 58 3.8 THE EFFECT OF CLOSTRIDIAL NEUROTOXINS ON CORTICAL GRANULE EXOCYTOSIS. 59

4. Experimental Approach. 61

Chapter 2. Materials and Methods.

1. GAMETE HANDLING. 62 2. MICROINJECTION TECHNIQUES. 63 3. TECHNIQUES USED TO INVESTIGATE CORTICAL GRANULE EXOCYTOSIS IN VITRO. 64 (i) Preparation of Cortices. 64 (ii) Preparation of Calcium-Containing Solutions. 65 (iii) Measuring the Extent of Exocytosis in Cortices. 65

4. POLYACRYLAMIDE GEL ELECTROPHORESIS. 6 6

(i) Preparation of Protein Samples. 6 6

(ii) Protein Assay of Samples. 6 8

(iii) Running of Gels. 6 8 5. WESTERN BLOTTING OF SDS-PAGE GELS. 69 (i) Transfer of Proteins. 69 (ii) Immunodetection. 69

6 . IMMUNOCYTOCHEMISTRY OF CORTICES. 70 (i) Fixing Egg Cortices. 70 (ii) hnmunocytochemistry. 71 (iii) Fluorescence Staining of Cortices. 71 (iv) Confocal Microscopy. 72 7. THE USE OF ISOTOPES TO RADIOLABEL INOSITOL PHOSPHOLIPIDS. 73 (i) Treatment of Eggs and Sample Preparation. 73 (ii) Analysis of Inositol Phospholipids. 74

8 . ASSAY OF CALCINEURIN ACTIVITY. 74

Chapter 3. Looking for Proteins Involved in Exocytosis in Sea Urchin Eggs.

1. INTRODUCTION. 75 2. EXPRESSION OF A SYNAPTOBREVIN HOMOLOGUE IN SEA URCHIN EGGS. 76 3. A RAB PROTEIN IS ASSOCIATED WITH CORTICAL GRANULES. 77 4. LOOKING FOR HOMOLOGUES OF SYNAPTOTAGMIN IN SEA URCHIN EGGS. 81 5. IS THE ALPHA-LATROTOXIN RECEPTOR PRESENT IN SEA URCHIN EGGS? 84

6 . THE IDENTIFICATION AND SUBCELLULAR LOCALIZATION OF ANNEXINS. 85

7. EXPRESSION OF CALCINEURIN IN SEA URCHIN EGGS. 89

8 . CONCLUSION. 90

Chapter 4. A Tetanus Toxin-Sensitive Synaptobrevin Homologue is Required for Exocytosis in Sea Urchin Eggs.

1. INTRODUCTION. 93 2. THE SUBCELLULAR LOCALIZATION OF THE SYNAPTOBREVIN HOMOLOGUE. 94 (i) Immunoblot Analysis of Subcellular Protein Fractions. 94 (ii) Immunocytochemical Staining of Egg Cortices. 96 3. CLEAVAGE OF THE SYNAPTOBREVIN HOMOLOGUE BY TETANUS TOXIN LIGHT CHAIN. 99 (i) Cleavage of the Synaptobrevin Homologue In Vitro. 99 (ii) Incubation of Cortices with Tetanus Toxin Light Chain. 100 (iii) Incubation of Cortices with Tetanus Toxin Light Chain at 37 °C. 104 4. TETANUS TOXIN LIGHT CHAIN INHIBITS CORTICAL GRANULE

EXOCYTOSIS IN VITRO. 104 5. THE SEA URCHIN EGG SYNAPTOBREVIN HOMOLOGUE INTERACTS

WITH A RECOMBINANT SYNTAXIN FRAGMENT IN VITRO. 106

6 . CONCLUSION. 109

Chapter 5. The Role of Calcineurin in Cortical Granule Exocytosis.

1. INTRODUCTION. 110 2. THE EFFECT OF A MLCK PEPTIDE ON EXOCYTOSIS. 111 3. THE EFFECT OF CALCINEURIN INHIBITORS ON EXOCYTOSIS. 117 (i) An Antibody to Calcineurin has No EfiFect on Exocytosis. 117 (ii) The Effect of a Peptide Corresponding to the Autoinhibitory Domain of Calcineurin. 119 (iii) The Effect of Deltamethrin on Exocytosis. 121 4. THE EFFECT OF OKADAIC ACID ON THE PHOSPHATASE ACTIVITY OF SEA URCHIN EGG CYTOSOL. 125 5. CONCLUSION. 127

Chapter 6. The Inositol Phospholipids and Exocytosis.

1. INTRODUCTION. 129 2. THE INFLUENCE OF ATP ON EXOCYTOSIS IN VITRO. 130 3. MEASURING THE LEVELS OF THE INOSITOL PHOSPHOLIPIDS IN THE EGG CORTEX. 135 (i) The Exocytosis Response After a Thirty Hour Incubation. 13 5 (ii) Measuring the Levels of the Inositol Phospholipids After Incubation in the Presenee or Absence of ATP. 137 4. THE EFFECT OF NEOMYCIN ON EXOCYTOSIS. 140 5. CONCLUSION. 144

Chapter 7. Discussion.

1. EXOCYTOSIS - A UNIVERSAL SECRETORY MECHANISM. 145 2. THE IDENTIFICATION OF POTENTIAL SECRETORY PROTEINS IN SEA URCHIN EGGS. 146 (i) Investigating Whether Homologues of Neuronal Secretory Proteins are Expressed in Sea Urchin Eggs. 146 (a) The Identification of a Synaptobrevin Homologue in Sea Urchin Eggs. 146 (b) Testing for the Expression of SNAP-25 and Syntaxin in Sea Urchin Eggs. 148 (c) Testing for the Expression of Synaptotagmin and the Alpha-Latrotoxin Receptor in Sea Urchin Eggs. 149 (ii) The Identification and Subcellular Localization of a Rab3 A Homologue. 150 (iii) The Identification of Annexins in Sea Urchin Eggs. 152 (iv) The Identification of Calcineurin in Sea Urchin Eggs. 154 3. THE SEA URCHIN EGG SYNAPTOBREVIN HOMOLOGUE IS REQUIRED FOR EXOCYTOSIS. 155

(i) Tetanus Toxin Light Chain Cleaves the Synaptobrevin Homologue In Vitro. 156 (ii) Tetanus Toxin Light Chain Inhibits Cortical Granule Exocytosis. 156 (a) Is the Sea Urchin Egg Synaptobrevin Homologue Required at a Stage Prior to Fusion? 158

(b) Two Release Mechanisms in Sea Urchin Eggs? 159 (iii) The Evolutionary Conservation of the Exocytotic Fusion Complex. 160 4. THE ROLE OF CALCINEURIN IN EXOCYTOSIS. 161 (i) Exocytosis in Paramecium. 161 (ii) The Effect of a Calmodulin Antagonist on Exocytosis. 162 (iii) The Effects of Calcineurin Antagonists on Exocytosis. 163 (iv) The Role of Phosphatases in Exocytosis. 164 5. THE INOSITOL PHOSPHOLIPIDS AND EXOCYTOSIS. 165 (i) The ATP Requirement of Exocytosis in Sea Urchin Eggs. 166 (ii) The Level of Exocytosis In Vitro Correlates with the Level of Cortical Phosphatidylinositol 4,5-Bisphosphate. 167

6 . SUMMARY. 169

Acknowledgements. 171

References. 172

Some Abbreviations which Appear in the Text. 207 List of Tables.

Chapter 1.

1.1 Events initiated at fertilization. 47

Chapter 3.

3.1 Sequence of the 1D 12 antigenic peptide and the corresponding region within synaptotagmin from different species. 82

Chapter 4.

4.1 The effect of IeTx LC on exocytosis in vitro at 16 °C. 102

Chapter 5.

5.1 Sequence and some properties of the calmodulin-binding domains of calcineurin,

MLCK and the MLCK 7 9 7 .8 1 3 peptide. 113

5.2 The effect of the MLCK7 9 7 _g, 3 peptide on exocytosis in vivo. 115

5.3 The effect of the MLCK7 9 7 .g , 3 peptide on exocytosis in vitro. 117 5.4 The effect of an antibody to calcineurin on exocytosis in vivo. 118 5.5 The effect of an antibody to calcineurin on exocytosis in vitro. 119 5.6 Characteristics of the CaN inhibitory peptide. 120 5.7 The effect of the CaN inhibitory peptide on exocytosis in vivo. 120 5.8 The effect of deltamethrin on exocytosis in vivo. 124

5.9 The effect of deltamethrin on exocytosis in vitro. 124

Chapter 6.

6 .1 Distribution of inositol label incorporated into cortical inositol phospholipids prepared from unfertilized sea urchin eggs. 138 Chapter 7.

7.1 Calcium-dependence of annexin binding to membranes or liposomes. 153

10 List of Figures.

Chapter 1.

1.1 Model showing the currently known members of the synaptic fiision machine and their susceptibility to clostridial neurotoxins. 40 1.2 Fertilization envelope elevation. 49 1.3 Cortical granule exocytosis in vitro. 50

Chapter 2.

2.1 Structure of 1,1 '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC,g). 71

Chapter 3.

3.1 Expression of a synaptobrevin homologue. 78 3.2 Expression and subcellular localization of rab3A. 80 3.3 Is synaptotagmin expressed in sea urchin eggs? 83

3.4 Is the a -latrotoxin receptor expressed in sea urchin eggs? 8 6

3.5 Expression of annexins in sea urchin eggs. 8 8 3.6 Expression of calcineurin in sea urchin eggs. 91

Chapter 4.

4.1 The subcellular localization of the 17.5 kD synaptobrevin homologue. 95 4.2 Immunocytochemical staining to show the distribution of the synaptobrevin homologue within the egg cortex. 97 4.3 Tetanus toxin light chain specifically cleaves the 17.5 kD synaptobrevin

homologue in vitro. 1 0 1 4.4 The effect of temperature on exocytosis/■« v//ro. 103 4.5 Tetanus toxin light chain inhibits cortical granule exocytosis when applied to isolated cortices. 105 4.6 Sea urchin egg synaptobrevin interacts with a soluble recombinant syntaxin fragment cloned from a bovine adrenal medullary cDNA library. 107

11 Chapter 5.

5.1 The effect of the MLCKygy.g ] 3 peptide on caleineurin activity. 114 5.2 The effect of a calcineurin inhibitory peptide on calcineurin activity. 121 5.3 Structure of deltamethrin. 122 5.4 The effect of deltamethrin on calcineurin activity. 123 5.5 Characterization of the phosphatase activity in sea urchin egg cytosol. 126

Chapter 6.

6.1 Loss of calcium-sensitivity with time following cortex isolation. 131

6.2 Comparison of exocytosis in vitro after incubation in either the presence or absence of ATP. 133 6.3 The influence of ATP on exocytosis in vitro. 134 6.4 Comparison of the exocytotic response of cortices prepared from eggs immediately after and thirty hours after shedding. 136 6.5 Autoradiochi'omatograph of inositol-labelled cortical lipid extracts. 139 6.6 Changes in polyphosphoinositide levels in the presence and absence of ATP. 141 6.7 The effect of neomycin on exocytosis in vitro. 143

12 Chapter 1. Introduction.

1. Exocytosis - A Universal Secretory Mechanism.

1.1 INTRODUCTION.

Exocytosis is a ubiquitous cellular mechanism for the export of secretory products from cells and for the insertion of proteins and lipid into the plasma membrane. All cases of secretory exocytosis involve the fiision of prepackaged secretory granules with the plasma membrane and the release of the secretory granule contents into the extracellular space. A general feature of all exocytotic events is that fiision and the release of the granule contents occurs without the release of cytoplasmic constituents. Douglas (1968) demonstrated that the proportion of catecholamines to other secretory substances in the perfusate of stimulated adrenal chromaffin glands was very similar to that found within chromaffin granules, suggesting that exocytosis involved the expulsion of discretely packaged intracellular compartments. The quantal nature of exocytosis was most elegantly demonstrated by measuring capacitance changes in patch-clamped secretory cells. Discrete changes in membrane capacitance, indicating changes in cell surface area, were demonstrated upon stimulation of single chromaffin cells (Neher and Marty, 1982). These capacitance changes were due to the fusion of individual secretory granules. Exocytosis is therefore a means of releasing material from cells in a highly specific fashion whilst conserving the biochemical integrity of the cell.

Exocytosis can be either constitutive or triggered. In constitutive exocytosis, vesicles exocytose approximately as soon as they reach the cell membrane (Kelly, 1985). In triggered or regulated exocytosis, secretory granules are stimulated to fijse with the plasma membrane in response to particular stimuli. In some cells, a transient increase in the intracellular free calcium ion concentration ([Ca^^]J, typically from a resting level of approximately 100 nM to a stimulated level approaching micromolar, is sufficient to

13 trigger exocytosis (Knight and Kesteven, 1983; Rorsman et al., 1984; Kao and Schneider, 1985). In other cells, such as mast cells, calcium is merely a cofactor (Howell et al., 1987; Neher, 1988) and in others, exocytosis can be stimulated in the absence of a rise in [Ca^^]; (Fernandez 1984; Barrowman cr a/., 1986).

(i) The Exocytotic Fusion Pore.

Quick-freeze electron microscopy showed that an early event in the exocytotic fusion of a secretory granule was the formation of a fiision pore, an aqueous channel that connected the lumen of a secretory granule with the extracellular space (Chandler and Heuser, 1980; Ornberg and Reese, 1981; Schmidt et a l, 1983). Conductance measurements, using the patch-clamp technique, revealed the presence of early fusion pores that were much smaller than those observed in electron micrographs (pore diameter of 1-2 nm as compared to 10-20 nm) (Aimers, 1990; Spruce et a l, 1990). The molecular structure of these fusion pores was proposed to resemble that of an ion channel (Aimers, 1990; Aimers and Tse, 1990; Spruce etal, 1990; Lollike etal, 1995), a model which was consistent with the experimental observation that early fusion pores opened abruptly with a well-defined conductance, that subsequently either expanded or closed again (Spruce etal, 1990). Furthermore, the conductance of exocytotic fusion pores was measured to be within the same order of magnitude as the conductances of many ion channels (Lollike eta l, 1995). The final irreversible expansion of the fusion pore was proposed to occur when lipid molecules interspersed between the protein subunits that made up the pore (Aimers, 1990). An alternative model proposed that the fusion pore was completely lipidic, with pore opening induced by tension in the secretory granule membrane, generated by a protein scaffold (Monck et al, 1991; Nanavati et a l, 1992). More recently, Curran et al. (1993) proposed that the initial fusion pore was composed of a lipid/protein complex. Clearly, consideration of these models will be useful when determining the molecular mechanisms underlying the fusion event.

14 (ii) Biophysical Aspects of Exocytosis.

Many of the ideas about how exocytosis may be regulated and its underlying mechanisms have been determined using biophysical approaches, that is, using artificial lipid bilayers to study the changes in membrane properties that occur upon fusion, and the factors that induce it. Artificial bilayers made from biological lipids do not readily fuse with each other under the conditions found in most living cells (Rand, 1981). Both electrostatic repulsion and more importantly at low separation (20-30 A), hydrostatic forces, must be overcome in order for opposing membranes to fuse (Cowley et a l, 1979). Fusion of bilayers has been suggested to involve phase transitions. Changes from lamellar to inverted-hexagonal phase may encourage the fusion of bilayers (Siegel, 1987) and the tendency for bilayers to undergo such transitions is dependent on biological lipids such as cholesterol (Cullis et al., 1978), cholesterol esters (Tilcock et al., 1984), diacylglycerol (Das and Rand, 1985, 1986) and lysophospholipids (Poole e ta l, 1970). In an artificial system, the fusion of phospholipid vesicles to phospholipid bilayers required high (millimolar) concentrations of calcium (Cohen et a l, 1980). However, when a Ca^^- binding protein was incorporated into the planar membrane, fusion was elicited at more physiologically relevant calcium concentrations (Zimmerberg et a l, 1980). These results suggested that biological fusion cannot be explained solely in terms of lipid interactions and that proteins must be involved in the control of exocytosis (for review see Zimmerberg, 1987).

1.2 THE ROLE OF CALCHJM IN THE CONTROL OF EXOCYTOSIS.

It was established more than twenty years ago that the secretion of many neurotransmitters, hormones and enzymes is dependent upon the presence of extracellular calcium (Douglas, 1968; Katz, 1969). Based on these studies, it was proposed that in response to a stimulus, calcium entered cells from the extracellular fluid as a result of an increased membrane permeability and acted at an intracellular site to trigger exocytosis (Douglas, 1968; Katz, 1969). An increase in the level of the intracellular calcium ion

15 concentration ([Ca^^]j) was therefore proposed to be the trigger for secretion. This proposal was supported by studies using radioactive tracers (Douglas and Poisner, 1962) and Ca^^-selective ionophores (Gomperts, 1984).

Studies using permeabilized cells allowed the direct demonstration that exocytosis was triggered by an increase in [Ca^^J^. Exocytosis was triggered by introducing micromolar levels of calcium directly into the cytosol of electropermeabilized adrenal chromaffin cells (Baker and Knight, 1981; Knight and Baker, 1982) and other secretory cells (for review see Knight and Scrutton, 1986a). The use of Ca^^-sensitive fluorescent dyes or photoproteins enabled the demonstration that in a number of different cell types, secretion was associated with a transient increase in [Ca^^Jj. This was demonstrated in both cell populations (Cheek and Burgoyne, 1985; O'Sullivan and Burgoyne, 1989; Kim and Westhead, 1989; Stauderman and Pruss, 1989) and in single cells (Cobbold et al., 1987; O'Sullivan et al, 1989; Cheek et a l, 1989). Both theoretical calculations in nerve terminals (Smith and Augustine, 1988) and patch-clamp experiments in adrenal chromaffin cells (Neher and Augustine, 1992; Augustine and Neher, 1992) indicated that the [Ca^^]j existing within a few nanometres of the exocytotic sites lay within the range 10-100 pM. It was therefore concluded that in these cells, only the influx of external calcium delivered calcium in sufficient amounts to the subplasmalemmal exocytotic sites to trigger exocytosis (Neher and Augustine, 1992; Augustine and Neher, 1992). In other cells, such as sea urchin eggs (Whitaker and Steinhardt, 1982) and Paramecium (Knoll et a l, 1991; Erxleben and Plattner, 1994), the release of calcium from intracellular stores is sufficient to trigger exocytosis.

(i) Intracellular Targets for Calcium in Exocytosis.

Although it is clear that calcium triggers exocytosis in most secretory cells, the Ca^^-receptor has not yet been identified. Exocytosis from different cell types was shown to be mediated by Ca^^-binding proteins with different affinities, suggesting the involvement of distinct Ca^^-binding proteins, or alternatively, related members of a protein family (for review see Burgoyne and Morgan, 1995). There are several candidates

16 for the Ca^^-receptor of exocytosis and the evidence supporting an involvement of each of these in exocytosis will be discussed.

(a) Calmodulin.

Calmodulin (CaM) is a ubiquitous Ca^^-binding protein which has been shown to be involved in a large number of Ca^^-dependent cellular processes (Cheung, 1980). It was indirectly implicated in exocytosis because several CaM antagonists were shown to inhibit secretion in a variety of cells. Trifluoperazine, a CaM antagonist, inhibited secretion in pancreatic p-cells (Gagliardino et a l, 1980; Henquin, 1981), mast cells (Douglas and Nemeth, 1982) and in hamster insulinoma cells (Schubart et a l, 1980). In adrenal chromaffin cells, trifluoperazine inhibited Ca^^-evoked catecholamine secretion (Baker and Knight, 1981; Brooks and Treml, 1983, 1984). This inhibition was shown to occur at the level of secretory granule-plasma membrane fusion (Burgoyne et a l, 1982), distal to the rise in [Ca^^]j (Kenigsberg et al, 1982). In platelets, the CaM antagonist, W- 7, inhibited thrombin-induced serotonin secretion and simultaneously inhibited Ca^^- dependent myosin light chain kinase activity, an enzyme activity known to be under the control of CaM (Nishikawa et a l, 1980). In another study, however, W-7, used at a similar concentration, was shown to have no effect on thrombin-induced secretion (Sanchez et a l, 1983). The results obtained using these CaM antagonist have been questioned, since at high concentrations they were shown to cause release due to a non­ specific hydrophobic effect on the plasma membrane (Seeman and Weinstein, 1966).

More convincing evidence to support a role for CaM in exocytosis was obtained using antibodies directed against CaM. In adrenal chromaffin cells, microinjection of antibodies against CaM specifically inhibited Ca^^-stimulated secretion (Kenigsberg and Trifaro, 1985). In both sea urchin eggs and in Paramecium, antibodies against CaM inhibited Ca^^-stimulated exocytosis in vitro, when applied directly to isolated cell cortices (Steinhardt and Alderton, 1982; Momayezi et a l, 1987). Addition of exogenous CaM to digitonin-permeabilized adrenal chromaffin cells enhanced Ca^^-stimulated exocytosis (Okabe eta l, 1992) but had no effect on secretion in saponin-permeabilized cells (Brooks

17 and Treml, 1983).

The precise role that CaM plays during exocytosis is unknown, however, it was recently demonstrated that CaM acted in the Ca^^-dependent triggering stage of exocytosis in permeabilized adrenal chromaffin cells (Chamberlain et a l, 1995). Strong candidates for the target of CaM in exocytosis include synaptotagmin and the Ca^^/CaM- dependent phosphatase calcineurin. The evidence supporting a role for these proteins in exocytosis will be discussed in later sections.

(b) Annexins and 14-3-3 Proteins.

The annexins are a major group of Ca^^-binding proteins which are characterized by their ability to associate reversibly with phospholipids in a Ca^^-dependent manner. Annexins consist of two regions: the tail, which contains phosphorylation sites and the core, which contains binding sites for calcium, phospholipids and cytoskeletal elements. It is these properties which make the annexins ideal candidates for involvement in exocytosis (for review see Geisow et a l, 1987; Burgoyne and Geisow, 1989; Creutz, 1992). Interest in the involvement of annexins in membrane-membrane interactions originally stemmed from studies on annexin VII (synexin), which was shown to promote the Ca^Vdependent fusion of chromaffin granules in vitro, in the presence of arachidonic acid or other c/\y-unsaturated fatty acids (Creutz, 1981). This property was shared by several other members of the annexin family (Creutz et a l, 1987; Drust and Creutz, 1988). However, only annexin II (calpactin I) promoted Ca^^-dependent aggregation and fatty acid-dependent fusion at physiologically relevant Ca^^ concentrations (Burgoyne, 1988; Drust and Creutz, 1988).

A direct involvement of annexin II in Ca^Vdependent exocytosis was demonstrated by its ability to retard exocytotic run-down in permeabilized adrenal chromaffin cells (Ali eta l, 1989; Burgoyne and Morgan, 1990; Sarafian etal, 1991). This effect was inhibited by both an affinity-purified antibody to annexin II and a synthetic peptide based on the most conserved region of the annexins (Ali et al, 1989). Since annexin II promoted the

18 aggregation and fusion of secretory vesicles in vitro, it was postulated that it might stimulate fusion between secretory vesicles and the plasma membrane in an analogous manner. Indeed, ultrastructural studies showed annexin II to form cross-links between chromaffin granules and the plasma membrane in stimulated adrenal chromaffin cells (Nakata et al, 1990). The observation that release of arachidonic acid accompanied Ca^^- dependent exocytosis in both intact and permeabilized bovine adrenal chromaffin cells supported the idea that fusion in vivo was analogous to fusion in vitro (Frye and Holz, 1984). However, it was demonstrated that arachidonic acid was not required for Ca^^- dependent exocytosis in digitonin-permeabilized adrenal chromaffin cells (Morgan and Burgoyne, 1990b). It is not yet clear whether annexin II functions only in Ca^^-dependent cross-linking of the granules to the plasma membrane or is also responsible for membrane fusion.

Annexins have also been implicated in exocytosis in mammary epithelial cells (Handel etal, 1991; Burgoyne eta l, 1991) and in Madin-Darby canine kidney (MDCK) cells (Fiedler et a l, 1995). In mouse mammary epithelial cells, annexin II (calpactin I) became concentrated at the apical (secretory) membrane, upon cell differentiation, suggesting that it might participate in protein and possibly lipid secretion from these cells (Handel et al., 1991; Burgoyne et a l, 1991). In permeabilized MDCK cells, antibodies against annexin XEHb inhibited vesicular transport to the apical plasma membrane but did not affect transport to the basolateral plasma membrane in permeabilized MDCK cells (Fiedler et a l, 1995). This result suggested that two different transport mechanisms occurred within the same cell.

Exocytosis was stimulated in a Ca^^-dependent manner in permeabilized adrenal chromaffin cells by two other cytosolic proteins, Exol and Exo2 (Morgan and Burgoyne, 1992b). Exol was shown to belong to the 14-3-3 family of proteins, which possess a conserved domain very similar to the conserved C-terminus of the annexins (Toker et al., 1990). A synthetic peptide corresponding to this conserved domain partially inhibited Ca^^-dependent exocytosis in permeabilized adrenal chromaffin cells, leading to the suggestion that this domain may be involved in protein-protein interactions necessary for

19 exocytosis (Roth e ta l, 1993). Recently, 14-3-3 proteins were shown to be involved in the ATP- and Ca^^-dependent priming step, rather than the Ca^^-mediated triggering step, in exocytosis in permeabilized adrenal chromaffin cells (Chamberlain et a l, 1995).

(c) Synaptotagmin,

Synaptotagmin is an integral membrane protein of synaptic vesicles (Matthew et al., 1981; Perin et a l, 1990) and both the synaptic-like microvesicles and chromaffin granules of adrenal chromaffin cells (Tugal et a l, 1991; Perin et a l, 1991; Walch- Solimena et a l, 1993). It binds calmodulin (Fournier and Trifaro, 1988; Trifaro et al, 1989) and contains two copies of a domain (C2) known to be involved in Ca^^-dependent membrane interactions (Perin et al, 1990). In vitro, synaptotagmin was shown to bind via its first C2 domain to both phospholipids (Brose et a l, 1992; Davletov and Siidhof, 1993; Chapman and Jahn, 1994) and to syntaxin (Li et a l, 1995) in a Ca^^-dependent manner. It was also shown to undergo a Ca^^-dependent conformational change in vitro (Davletov and Siidhof, 1994). These properties make synaptotagmin an ideal candidate for the calcium sensor of regulated exocytosis. Synaptotagmin was shown to interact in vitro with N-type calcium channels via its interaction with syntaxin (Bennett et a l, 1992; Yoshida et a l, 1992; Leveque et a l, 1994). Synaptotagmin was also shown to interact directly with presynaptic membrane neurexins via its carboxyl terminus (Petrenko et al, 1991; Hata et a l, 1993a), an association that was proposed to play a role in synaptic vesicle docking and the regulation of neurotransmitter release (Petrenko et a l, 1991; Hata et a l, 1993a). Sollner et al (1993b) reported that synaptotagmin formed a stable complex in vitro with syntaxin, synaptobrevin and SNAP-25, the three core proteins of a putative fusion complex (Sollner et al, 1993a, b). These authors proposed a model in which synaptotagmin functioned as a 'fiision clamp', preventing the binding of the general fusion proteins NSF and a-SNAP, in the absence of a signal for exocytosis. Interestingly, calcium did not effect the exchange of synaptotagmin for a-SNAP, therefore this model failed to account for a Ca^^-dependent ftmction of synaptotagmin in exocytosis.

Investigations into the precise function of synaptotagmin in exocytosis have

20 produced conflicting and often contradictory results. Synaptotagmin I- and Il-deficient mutant PCI2 cells were able to secrete catecholamines in a Ca^^-dependent manner, suggesting that synaptotagmin was not essential for catecholamine secretion (Shoji-Kasai etal, 1992). However, it was subsequently shown that novel isoforms of synaptotagmin, synaptotagmin III and IV, were abundantly expressed in PCI2 cells and may therefore compensate for the lack of synaptotagmins I and II (Mizuta et a l, 1994; Hilbush and Morgan, 1994). Antibodies against synaptotagmin inhibited secretion when microinjected into PCI2 cells (Elferink et a l,1993) and soluble peptides containing the C2 repeat domain blocked neurotransmission when microinjected into the squid giant synapse (Bommert etal, 1993). These results suggested that synaptotagmin was essential for the exocytosis of both synaptic vesicles and chromaffin granules.

Genetic studies in which thesynaptotagmin geneI was disrupted were conducted in Drosophila (DiAntonio et al, 1993; Littleton et a l, 1993), C elegans (Nonet et al, 1993) and mouse (Geppert et al, 1994a). These studies showed that continued, but greatly reduced, evoked transmission occurred in synaptotagmin I mutants, suggesting that synaptotagmin I was not essential for, but played an important regulatory role in, neurotransmission (for review see Littleton and Bellen, 1995). The residual Ca^- dependent release seen in these synaptotagmin mutantsI suggested the presence of other isoforms of synaptotagmin, which might also function in Ca^^-dependent exocytosis. Alternatively, it is possible that synaptotagmin acts in concert with another Ca^^-receptor protein to yield release. If the putative co-receptor was a related protein, for example rabphilin, which also contains a C2 repeat motif (Shirataki et a l, 1993), this hypothesis would explain why synaptotagmin mutationsI produced a reduction in transmission but were not able to completely eliminate it, whereas C2 domain peptides completely blocked secretion. A role for synaptotagmin as a negative regulator of exocytosis was suggested, based on the observation that spontaneous secretion was enhanced in synaptotagmin- deficient Drosophila (Littleton et a l, 1993, 1994).

21 (d) The Cortical Cytoskeleton.

In many secretory systems, secretory granule transport is an essential factor in stimulated exocytosis. The secretory granules are embedded in a cytoskeletal network and must reach the plasma membrane in order for fusion to occur. For example, in adrenal chromaffin cells, most of the secretory granules were shown to be excluded from the cell periphery and appeared to be trapped within a cytoskeletal mesh (Burgoyne et al., 1982; Kondo eta l, 1982) and in pancreatic P-cells, insulin-containing granules were held away from the plasma membrane by a cortical cytoskeletal lattice (Orci et al., 1972).

Evidence suggests that the cortical actin cytoskeleton plays a role in the control of exocytosis by acting as a barrier to secretory granule movement and that disassembly or reorganization of the cortical cytoskeleton is necessary, but not sufficient, for a full secretory response (for review see Burgoyne and Cheek, 1987; Aunis and Bader, 1988; Burgoyne, 1991). One of the earliest events shown to occur following activation of chromaffin cells and elevation of [Ca^^Jj was the rapid, transient disassembly of the cortical actin network (Cheek and Burgoyne, 1986; Sontag a/., 1988; Vitale g/ a/., 1991, 1995). Cortical filamentous actin (F-actin) disassembly was shown to occur upon stimulation in several other secretory cell types, including mast cells (Koffer et al., 1990), parotid acinar cells (Perrin et a l, 1992), pancreatic acinar cells (Jungermann et al., 1995) and neutrophils (Lew et al., 1986).

A variety of stimuli that activated exocytosis were shown to promote actin reorganization (for review see Cheek and Burgoyne, 1992; Trifaro and Vitale, 1993). For example, cytochalasin, which causes disruption of F-actin networks, lowered the Ca^^- requirement of secretion in neutrophils (Lew etal., 1986) and enhanced glucose-induced insulin secretion from pancreatic p-cells (Orci et a l, 1972). In digitonin-permeabilized adrenal chromaffin cells, cytochalasin promoted secretion, whereas phalloidin, an actin filament stabilizer, prevented secretion (Lelkes etal, 1986). These compounds, however, did not affect exocytosis in electrically-permeabilized adrenal chromaffin cells (Knight and Baker, 1982). The reorganization of actin appeared to involve both Ca^^-dependent and

22 Ca^'-independent mechanisms (Burgoyne el ai, 1989). It was proposed that cyclic AMP, which inhibited both nicotine-induced actin disassembly and secretion, might function during the normal events in chromaffin cells to terminate the secretory response (Cheek and Burgoyne, 1987).

Actin-binding proteins may be important regulators of secretion in cells where granule transport is a precursor to fusion. In adrenal chromaffin cells, antibodies against a-fodrin, a protein thought to link actin filaments to the plasma membrane, partially inhibited Ca^-induced catecholamine release (Perrin et a/., 1987). In adrenal medullary extracts, the cross-linking of actin filaments by caldesmon, a Ca^-sensitive actin-binding protein which is present on adrenal chromaffin granules, was inhibited by micromolar levels of calcium (Burgoyne et al., 1986). Scindrin, a Ca^-dependent F-actin-severing protein, redistributed in a Ca^-dependent manner upon stimulation in adrenal chromaffin cells (Vitale et ai, 1991; Zhang et ai, 1995). This event concurred with cortical F-actin disassembly (Vitale et ai, 1991; Del Castillo et ai, 1992). Both these processes might be expected to enhance secretion upon elevation of [Ca^’]j. Exogenous gelsolin, an F- actin-severing protein, induced microfilament disassembly and as a consequence, enhanced secretion in permeabilized mast cells (Borovikov et ai, 1995). Gelsolin has also been proposed to promote disassembly of the F-actin network upon calcium influx in the presynaptic nerve terminal (Miyamoto, 1995). In isolated nerve terminals, synaptic vesicles which were not pre-docked at the plasma membrane, were cross-linked within the peripheral cytoskeletal network by the extrinsic vesicle protein synapsin I (Siidhof et ai, 1989b). Synaptic vesicle availability at the plasma membrane was thought to be mediated via phosphorylation of synapsin I (Elinas et al., 1985; Nichols et ai, 1990; Benfenati et cf/., 1992; Ceccaldi g/o/., 1995).

In view of these findings, modulation of the cytoskeleton may be important factor in mediating the exocytotic response. It is likely that there is regulation at the level of the interactions of secretory granules with the cytoskeleton as well as regulation at the level of membrane fusion itself. Since in adrenal chromaffin cells, stimulation of actin disassembly in the absence of a rise in [Ca^'Jj, did not result in extensive exocytosis

23 (Burgoyne et a l, 1989), it is unlikely that removal of the cytoskeletal barrier is itself sufficient to allow exocytosis to proceed.

1.3 THE MODULATION OF CALCIUM SENSITIVITY.

Having discussed some of the intracellular receptors for Ca^^ which might play a role in exocytosis, the mechanism by which the Ca^^-sensitivity of secretion might be modulated will be discussed. It is clear that in some cases, exocytosis can proceed in the absence of elevated [Ca^^Jj (Fernandez et a l, 1984; Rink et a l, 1983). In particular, the modulatory roles of protein kinases and GTP-binding proteins in the control of exocytosis will be discussed.

(i) Protein Kinase Modulation of Exocytosis.

In a wide variety of cells, activation of the Ca^^- and phospholipid-dependent protein kinase C (PKC) was shown to potentiate exocytosis. The responses generally fell into one of three categories: a modest increase in Ca^^-afFinity, a large increase in Ca^"^- affmity, or an increase in the extent of release with little alteration in the affinity for calcium.

In both permeabilized adrenal chromafiBn cells and permeabilized pancreatic acinar cells, activation of PKC, using the phorbol ester TP A, produced a small potentiation of Ca^^-stimulated secretion by increasing the Ca^^-aflSnity of the process (Knight and Baker, 1983; Pocotte et a l, 1985; Brocklehurst and Pollard, 1985; O'Sullivan and Jamieson, 1992). Conversely, inhibition of PKC activity partially inhibited Ca^^-stimulated exocytosis both in adrenal chromafiBn cells (Burgoyne et al, 1988; Morgan and Burgoyne, 1990b; Vitale eta l, 1992) and in pancreatic acinar cells (Verme et a l, 1989; Edeveen et a l, 1990). The addition of exogenous PKC to exocytotically run-down permeabilized adrenal chromafiBn cells, which lose endogenous PKC in the absence of phorbol esters (Terbush and Holz, 1986), enhanced Ca^^-stimulated exocytosis in a dose-dependent

24 manner (Morgan and Burgoyne, 1992a). Taken together, these results suggested that in adrenal chromaffin cells, PKC played an important stimulatory role in exocytosis.

By measuring [Ca^^]; in platelets using the indicator dye quin2. Rink et al. (1983) demonstrated that thrombin-induced 5-HT secretion occurred in the absence of elevated [Ca^^j. Exogenously added 1,2-diacylglycerol (DAG), or TP A, also stimulated exocytosis at basal calcium levels (Rink et al., 1983). From these results it was concluded that secretion could occur, independent of changes in [Ca^^]j, through the modulatory effects of protein kinase C (Rink et al., 1983). Studies using electropermeabilized platelets indicated that thrombin, DAG (one of the products of thrombin stimulation - Haslam and Davidson, 1984a), and TPA increased the Ca^^-sensitivity of 5-HT secretion (Haslam and Davidson, 1984a; Knight and Scrutton, 1984; Knight et al., 1984). It was therefore concluded that in permeabilized platelets, thrombin enhanced Ca^^-induced serotonin secretion by increasing the level of DAG and hence activation of PKC (Knight et al., 1984). In contrast, thrombin increased the extent of Ca^^-induced secretion of the lysosomal enzyme p -A-acetylglucosaminidase from permeabilized platelets, without affecting the Ca^^-sensitivity (Knight etal, 1984; Knight and Scrutton, 1984). In isolated rat pancreatic islets, TPA increased both the Ca^^-affinity and extent of Ca^^-stimulated insulin secretion (Jones et al., 1985; Tamagawa et al., 1985).

There is also evidence which suggest that other protein kinases may play a modulatory role in the control of exocytosis. In both pancreatic islets (Tamagawa et al., 1985), and in RINm5F cells (Wollheim et al., 1984), forskolin, an activator of adenylate cyclase and thus, indirectly, cAMP-dependent protein kinase (PKA), stimulated insulin release. Again, at least in RINm5F cells, this effect seems to be through the modulation of Ca^^-sensitivity since no change in [Ca^^Jj was measured when cells secreted insulin in response to forskolin (Wollheim et al., 1984). In both permeabilized parotid acini (Takuma and Icida, 1991) and permeabilized pituitary cells (Macrae et al., 1990), cAMP elicited Ca^^-independent secretion. In permeabilized pancreatic acini, cAMP enhanced Ca^^-dependent secretion and it was shown that this effect was mediated by PKA (O'Sullivan and Jamieson, 1992). In permeabilized adrenal chromaffin cells, Exo2, which

25 retarded the loss of secretory response in exocytotically run-down cells (Morgan and Burgoyne, 1992b), was identified as the catalytic subunit of PKA (Morgan et al., 1993), suggesting that PKA played a role in the control of exocytosis in these cells. In platelets, both cAMP and cGMP-dependent protein kinases may modulate secretion: cGMP was shown to increase, and cAMP decrease, the Ca^^-sensitivity of thrombin-stimulated 5-HT release from permeabilized platelets (Knight and Scrutton, 1984).

These data from a variety of different secretory cells indicate that the protein kinases play an important modulatory role in exocytosis. Either the Ca^^-sensitivity or the extent of exocytosis are affected, depending upon the cell type and the experimental conditions used.

(ii) G-Protein Control of Exocytosis,

Although calcium is the major signal for the triggering of regulated exocytosis, stimulatory effects of GTP analogues on exocytosis have been demonstrated in many cell types. As stated previously, the provision of 1,2-diacylglycerol (DAG) and the activation of protein kinase C (PKC) enhanced the Ca^^-affinity of exocytosis in electropermeabilized platelets (Knight and Scrutton, 1984). This effect was mimicked by GTPyS, a thio- analogue of GTP (Haslam and Davidson, 1984b; Knight and Scrutton, 1986b). Since DAG is produced by the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C (PEG), which is under the control of G-proteins in platelets (Haslam and Davidson, 1984a), it was proposed that GTPyS mediated its effects through the stimulation of PEG, with consequent liberation of DAG and activation of PKG (Knight and Scrutton, 1986b).

In permeabilized neutrophils, GTPyS stimulated secretion in the absence of calcium (less than 2 x 10'^^ M) (Barrowman et al., 1986). Since the activation of PEG does not occur under these condition (Cockcroft, 1986), the effect of GTPyS was unlikely to be mediated via protein kinase G. In support of this proposal, PMA inhibited Ga^- induced exocytosis in these cells (Barrowman et al, 1986), indicating that GTPyS was

26 not stimulating exocytosis through the production of DAG, since, if this were the case, it would be expected that the modulatory effects of both PMA and GTPyS on exocytosis would be the same. GDPpS inhibited both GTPyS- and Ca^^-stimulated exocytosis in permeabilized neutrophils. These results suggested that the two stimuli acted through a common pathway and that GTPyS mediated its effect at a late stage in this pathway, perhaps by a direct effect on the exocytotic apparatus (Barrowman et al., 1986).

In mast cells, the combination of calcium plus guanine nucleotide are the essential effectors to elicit an exocytotic response (Howell et al., 1987). In permeabilized mast cells, neomycin, an aminoglycoside antibiotic which prevents the hydrolysis of polyphosphoinositides by PLC, inhibited the generation of InsP^ and DAG but did not prevent the stimulation of exocytosis by GTPyS and calcium (Cockcroft et al., 1987). This result again suggested that GTPyS mediated its effect directly on the exocytotic apparatus. The GTP-binding protein (G-protein) that stimulated exocytosis was termed Ge (Gomperts, 1986; Gomperts etal, 1986; Cockcroft et a l, 1987) and in mast cells, Gg was proposed to be a heterotrimeric G-protein (Lillie and Gomperts, 1992; Aridor et al., 1993).

In adrenal chromaffin cells, GTP analogues have been reported to exert both inhibitory and stimulatory effects on exocytosis. In permeabilized cells, GTPyS was reported to stimulate Ca^^-independent catecholamine secretion (Bittner et al., 1986; Morgan and Burgoyne, 1990a), to enhance Ca^^-dependent secretion (Bader et al., 1989; Burgoyne eta l, 1989) and to inhibit Ca^^-dependent secretion (Knight and Baker, 1985). All three responses were elicited in one study using streptolysin 0-permeabilized chromaffin cell (Ahnert-Hllger et a l, 1992). These seemingly contradictory results were reconciled by the proposal that in adrenal chromaffin cells, exocytosis is controlled by multiple G-proteins which can be differentially activated by GTP analogues under various conditions (Burgoyne et a l, 1994). In intact adrenal chromaffin cells, GDPpS inhibited the Ca^^-independent exocytosis induced by XTP, an analogue of GTP, but had no effect on Ca^^-induced exocytosis (Burgoyne and Handel, 1994), indicating that calcium and GTP analogues acted on parallel but distinct pathways in these cells.

27 Evidence from a variety of cells indicates that a GTP-binding protein is intimately involved in the regulation of exocytotic membrane fusion. The nature of this G-protein and whether it is the same in different secretory cell types has not been established. However, since GTPyS almost always stimulates regulated exocytosis (for review see Gomperts, 1990a,b), Gg is likely to be a heterotrimeric G-protein. The other class of GTP-binding proteins, the low molecular weight (20-30 kD) monomeric G-proteins have also been implicated in exocytosis. The role of this class of GTP-binding proteins in exocytosis will be discussed in the following section.

It now appears that, although calcium is often a trigger for exocytosis, other regulatory processes such as protein phosphorylation, or the actions of G-proteins, can control the sensitivities of exocytotic systems to calcium.

1.4 THE ROLE OF RAB PROTEINS IN EXOCYTOSIS.

Low molecular weight GTP-binding proteins of the rab family are strong candidates for regulators of membrane traffic, including exocytosis. This family of G- protein are thought to cycle between a soluble and membrane-associated state, acting as molecular 'switches' which control the docking/fusion of transport vesicles with their acceptor membrane (for review see Balch, 1990; Goud and McCaffrey, 1991). The differential subcellular localization of different rab proteins has led to the suggestion that each is responsible for controlling a specific membrane fusion event. In particular, members of the rabS subfamily of G-proteins have been implicated in the fusion of secretory vesicles with the plasma membrane, both in constitutive and in regulated secretion (for review see Lledo et a l, 1994).

Secretory vesicles from several cell types have rab3 proteins bound to their cytoplasmic surfaces (for review see Fischer von Mollard et a l, 1994). In neurons, rab3 A was found specifically on the membranes of synaptic vesicles (Fischer von Mollard et al, 1991). Furthermore, rab3A dissociated from these vesicles during Ca^^-stimulated

28 neurotransmitter release in synaptosomes (Fischer von Mollard et al., 1991), making it an ideal candidate for a regulator of exocytosis. However, in the frog neuromuscular synapse, rab3 A was translocated to the plasma membrane upon massive stimulation of exocytosis, suggesting that dissociation occurred at a step distal to fusion and before vesicles were newly reformed by endocytosis (Matteoli et al., 1991). The role of rab3 A in Ca^^-stimulated exocytosis therefore remained unclear. The use of synthetic peptides homologous to the predicted effector domain of rab3 suggested that rab3 proteins played a stimulatory role in exocytosis (Padfield et al., 1992; Senshyn et al., 1992; Oberhauser et a l, 1992; Olszewski et al., 1994; Edwardson e ta l, 1993). However, the specificity of these peptides was subsequently challenged, since the stimulatory effect persisted when the amino acid sequence was substantially modified (MacLean et a l, 1993).

Experiments with mutant rab3A proteins or with antisense oligonucleotides directed to the rabSB mRNA clearly established a role for rab3 proteins in regulated exocytosis. Mutant rab3A proteins, defective in either GTP hydrolysis or in guanine nucleotide binding, inhibited Ca^^-stimulated exocytosis in both bovine adrenal chromaffin cells and PCI2 cells (Johannes etal, 1994), suggesting that rab3A was a regulatory factor that prevented exocytosis from occurring until secretion was triggered. In anterior pituitary cells, antisense oligonucleotides against rabSB mRNA specifically blocked expression of rab3B and inhibited Ca^^-stimulated exocytosis (Lledo et a l, 1993). These results suggested that in these cells, rab3B might play a more active role in regulating exocytosis.

Other experiments have suggested that rab proteins are not essential for Ca^- stimulated exocytosis but play a modulatory role in mediating the secretory response. In adrenal chromaffin cells, antisense oligonucleotides directed to rahSA mRNA led to an increasing potential to respond to repetitive stimulations, suggesting that rab3 A may be involved in adaptive processes such as response habituation (Johannes et a l, 1994). An opposite effect was seen in mice in which the rab3A gene was deleted by homologous recombination, i.e. synaptic depression following short trains of repetitive stimuli was significantly increased (Geppert et al, 1994b). These authors suggested that rab3A was

29 required for the recruitment of synaptic vesicles to release sites during repetitive stimuli. Interestingly, this hypothesis was supported by the observation that in yeast, a rab protein promoted the assembly of the putative fusion complex (Sogaard et al., 1994) which has been proposed to mediate all intracellular hasion events, including exocytosis (Sollner et a l, 1993a, b).

In summary, several experiments have suggested that rab proteins are essential for exocytosis, whilst others have suggested that they merely modulate the exocytotic response. It remains to be established precisely how rab proteins regulate the fusion machinery. The synaptic vesicle protein rabphilin 3 A was shown to interact with rab3A in a GTP-dependent manner, leading to the suggestion that it might be the effector of rab function (Shirataki et a l, 1993). Rabphilin 3A was shown to contain an N-terminal rab3A-binding region and a C-terminal domain that contains two copies of an internal repeat that are homologous to the C2 domains of synaptotagmin (Yamaguchi et al, 1993). In support of the hypothesis that rabphilin 3A may be an effector of rab3A function, recent experiments showed rabphilin 3 A to be a positive regulator of secretion {Chung et a l, 1995).

1.5 THE REQUIREMENT FOR ATP IN EXOCYTOSIS.

MgATP is required to support exocytosis in most secretory systems. Studies on exocytosis from a variety of permeabilized cell systems established that MgATP was necessary for optimal secretion from a number of cell types including chromaffin cells (Baker and Knight, 1981; Knight and Baker, 1982; Dunn and Holz, 1983), platelets (Knight and Scrutton, 1980), pituitary tumour cells (Running and Martin, 1986) and insulin-secreting tumour cells (Vallar et al, 1987). The classic work of Baker and Knight (1981) established that Ca^^-dependent catecholamine release from permeabilized adrenal chromaffin cells was absolutely dependent upon exogenous MgATP. However, this assertion was challenged by Holz et a l (1989), who showed that there were two components of exocytosis from permeabilized adrenal chromaffin cells; an initial fast wave

30 of exocytosis which, although primed by MgATP, did not require exogenous MgATP to proceed; and a second, slower phase which required exogenous MgATP for fusion to occur. These authors suggested that in intact cells, the secretory apparatus was primed by intracellular ATP, so that, immediately upon permeabilization, there was a component of secretion which was independent of exogenous ATP. This MgATP-independent component of secretion was labile and rapidly lost with time after permeabilization (Holz et al., 1989). An important conclusion of their work was that MgATP acted before calcium in the secretory pathway, priming the secretory apparatus in preparation for Ca^^- stimulated exocytosis. A similar conclusion was also reached in mast cells (Howell et at., 1988) and 'mParamecium (Vilmart-Seuwen etal., 1986).

The biochemical reactions underlying the ATP-dependent priming step of exocytosis remain to be elucidated. In experiments on permeabilized PC 12 cells. Hay and Martin (1992) showed that MgATP-dependent priming was inhibited by a broad range of protein kinase inhibitors, suggesting that priming involved a phosphorylation reaction. More recently, the phosphoinositide kinases have been assigned a role in exocytotic priming (Hay et al., 1995). In permeabilized adrenal chromaffin cells, ATP-dependent priming, which also requires the presence of calcium (Bittner and Holz, 1992), was stimulated by a-SNAP and 14-3-3 proteins (Chamberlain et al., 1995).

(i) The Effects of ATP on Exocytosis in Mast Cells.

ATP is not required for exocytosis from permeabilized mast cells: in metabolically inhibited cells, exocytosis was stimulated by the combination of GTPyS and Ca^^ (Howell et al., 1987). ATP, although not essential, increased the Ca^^-sensitivity of exocytosis, by reactions which were catalysed by protein kinase C (Cockcroft et al., 1987; Howell et al., 1989). The onset of exocytosis from permeabilized mast cells was retarded by ATP (Tatham and Gomperts, 1989). Since delays preceding exocytosis were induced by ATP, but not by its non-hydrolysable analogue AppNHp, it was suggested that the effect was most likely due to maintained phosphorylation of an inhibitory regulator protein. This result suggested that dephosphorylation of an unknown regulator protein may comprise

31 a step in the exocytotic pathway. However, since exocytosis occurred when ATP was excluded, it is clear that this phosphorylation reaction did not constitute an essential step in the mechanism of exocytosis.

The idea that inhibitory phosphoproteins can prevent Ca^^-stimulated exocytosis has been illustrated in other secretory systems. In both adrenal chromaffin cells (Brooks et al., 1984; Brooks and Brooks, 1985) and in sea urchin eggs (Whalley et al., 1991), Ca^^-stimulated exocytosis was blocked by ATPyS, a thio-analogue of ATP. ATPyS can be used as a phosphoryl donor by protein kinases but the resulting thiophosphoproteins are not readily dephosphorylated by phosphoprotein phosphatases (Gratecos and Fischer, 1984). These data suggest that phosphorylation primes the secretory apparatus for secretion whilst release depends upon dephosphorylation of specific cell proteins. However, the quest for a significant phosphorylation or dephosphorylation associated with exocytosis has proved elusive. In a variety of secretory cells, including adrenal chromaffin cells (Côté et al., 1986; Lee and Holz, 1986), platelets (Knight e ta l, 1984), pancreatic acini (Burnham and Williams, 1982) and parotid cells (Spearman et al., 1984) there is no single phosphorylation event which is uniquely associated with exocytosis, although there are a number of possible candidates.

(ii) Exocytosis and Protein Dephosphorylation inParamecium.

In Paramecium, there is a well-documented change in the phosphorylation state of a single protein which correlates well with secretory activity. In Paramecium, exocytosis was shown to involve the rapid (< 1 s) dephosphorylation of a 65 kD membrane-bound phosphoprotein (Gilligan and Satir, 1982; Zieseniss and Plattner, 1985). The transient dephosphorylation of this 65 kD phosphoprotein strictly paralleled the actual amount of exocytosed organelles. In non-discharge mutant strains of Paramecium, which did not undergo exocytosis, dephosphorylation of the 65 kD protein was not observed. These data strongly suggest that in Paramecium, dephosphorylation of a 65 kD phosphoprotein is involved in triggering the exocytotic response.

32 The Paramecium is similar to the sea urchin egg, in that the secretory granules (trichocysts) are docked at the plasma membrane and can be triggered to undergo exocytosis solely by an increase in [Ca^^], (Knoll etal., 1991). The plasma membrane and attached trichocysts can also be isolated and respond to calcium in vitro. As is the case in the sea urchin egg, these fragments have been termed cortices (Vilmart-Seuwen et at., 1986).

In Paramecium, both calmodulin and a calcineurin-like protein were localized to the preformed exocytotic sites (Momayezi et al., 1986, 1987), therefore the effects of adding exogenous phosphatases and antibodies to calmodulin (CaM) and calcineurin (CaN) on exocytosis were investigated. Injection into intact cells or application to isolated cortices, of alkaline phosphatase or a Ca^-CaM-CaN complex induced exocytosis and a parallel dephosphorylation of the 65 kD phosphoprotein, whereas inhibitors of phosphatase activity and anti-CaN antibodies blocked these events (Momayezi et al., 1987). The microinjection of Ca^-CaM-CaN into non-discharge mutant strains did not cause exocytosis. Since both CaM and a CaN-like protein were localized to the secretory sites'm Paramecium, it was suggested that exocytosis might be mediated by a Ca^-CaM- CaN complex acting on the 65 kD phosphoprotein (Momayezi et al., 1987).

In summary, a specific protein dephosphorylation has been proposed to be part of the mechanism that triggers exocytosis in Paramecium. Evidence suggests that, in vivo, this dephosphorylation event is likely to be mediated by a calcineurin-like phosphoprotein phosphatase. Because of the similarities between Paramecium and sea urchin eggs, it seem highly possible that exocytosis in these two systems might proceed by a similar mechanism. Experiments in which this possibility was investigated are described in Chapter 5.

(Hi) The Identification of Priming Factors in Regulated Exocytosis.

Regulated secretion consists of sequential priming and triggering steps which depend on ATP and calcium, respectively. Experiments in permeabilized PC 12 cells

33 established that distinct cytosolic factors were responsible for mediating each of these steps in exocytosis (Hay and Martin, 1992). Three factors were identified that reconstituted the ATP-dependent priming of Ca^^-activated exocytosis from permeabilized PC 12 cells. Two of the priming factors were isolated and identified as mammalian phosphatidylinositol transfer protein (PtdlnsTP) (Hay and Martin, 1993) and phosphatidylinositol-4-phosphate 5-kinase (PtdInsP5K) (Hay et al., 1995). PtdlnsTP was originally identified by its ability to transfer pho sphatidylino sitol (Ptdlns) and to a lesser extent, phosphatidylcholine between membrane bilayers in vitro (for review see Cleves et al., 1991). Since phospholipid transfer by PtdlnsTP does not require ATP, its proposed function in ATP-dependent priming was to provide substrate to the recipient membrane for further phosphorylation by the phosphoinositide kinases (Hay et al., 1995). This hypothesis was supported by the observation that priming by PtdlnsTP and PtdInsf5K was accompanied by an increase in the formation of phosphatidylinositol 4,5-bisphosphate (Ptdlnsfj hi permeabilized PCI2 cells (Hay et al., 1995). Taken together, these results suggest that synthesis ofPtdIns ? 2 might be one of the biochemical reaction underlying the priming of exocytosis.

Recent reports have indicated that PtdlnsTP is an essential component of the polyphosphoinositide synthesis machinery (Cunningham et al., 1995; Kauffmann-Zeh et al., 1995). Its proposed hmction was to bind to and present Ptdlns to phosphatidylinositol 4-kinase (for review see Liscovitch and Cantly, 1995). Since this activity would also lead to the production of PtdInsP 2 , it could equally explain the role of PtdlnsTP in ATP-dependent priming.

In summary, PtdlnsTP functions in the ATP-dependent priming of Ca^^-activated secretion either by transferring Ptdlns to, or activating Ptdlns in, a membrane for phosphorylation by ATP-dependent phosphoinositide kinases. The identification of both PtdlnsTP and PtdInsP5K as ATP-dependent priming factors in Ca^^-activated secretion, suggests that lipid kinase-mediated phosphorylation is an important basis for ATP use in the exocytotic pathway.

34 (iv) Evidence that the Polyphosphoinositides are Necessary for Exocytosis.

What is the precise role of the polyphosphoinositides in regulated exocytosis? In many secretory cells, the coupling of the initial stimulus to secretion involves the activation of phospholipase C (PLC), which hydrolyses phosphatidylinositol 4,5- bisphosphate (PtdlnsPj to form the key intracellular signalling molecules inositol 1,4,5- trisphosphate (InsPg) and 1,2-diacylglycerol (DAG). InsPj produces an increase in the intracellular calcium concentration ([Ca^^]J by releasing calcium from intracellular stores, whereas DAG activates protein kinase C (PKC). Calcium and PKC are then thought to act in concert to modulate secretion (for review see Nishizuka, 1986). Increasing evidence, however, suggests that the polyphosphoinositides also play a non-signalling role in Ca^^-regulated exocytosis.

Eberhard et al. (1990) showed a strict correlation between the levels of the polyphosphoinositides and Ca^^-stimulated exocytosis in permeabilized adrenal chromaffin cells. When the levels of phosphatidylinositol 4-phosphate (PtdlnsPj and Ptdlns / * 2 were decreased, either by treatment with an exogenous PLC or by removal of ATP, secretion was concomitantly decreased (Eberhard et a l, 1990). Conversely, preincubation of permeabilized adrenal chromaffin cells with neomycin, in the absence of ATP, to maintain the levels of the polyphosphoinositides, increased the level of subsequent Ca^^-stimulated secretion (Eberhard et al., 1990). Neither InsPj nor DAG, the products of PtdlnsPj hydrolysis, reversed the inhibition induced by the exogenous PLC (Eberhard et al., 1990). This suggested a role for the polyphosphoinositides in secretion independent of their being substrates for the generation of these signalling molecules. Similarly, in permeabilized PC12 cells, exogenous PLC inhibited Ca^^-activated secretion (Hay et al., 1995).

In summary, ATP has been shown to act before calcium in regulated secretion. This ordering of events is a very important consideration when evaluating proposed models of exocytotic membrane fusion. The biochemical reactions underlying ATP- dependent priming of Ca^^-activated exocytosis are unknown, however, several proteins which show a stimulatory effect in this process have been identified.

35 2. The Exocytotic Mechanism in Synaptic Transmission.

An understanding of the molecular mechanisms underlying exocytotic membrane fusion has been greatly advanced by studies on the regulated release of neurotransmitter at the synapse between neurons. In the resting nerve terminal, synaptic vesicles that contain neurotransmitters are docked at "active zones". When an action potential arrives, the presynaptic plasma membrane depolarizes and voltage-gated Ca^^ channels become activated. The influx of extracellular Ca^^ triggers membrane fusion with a delay time of only 200-300 ps, indicating that the proteins involved in membrane fusion must exist in a preassembled and activated state (for review see Augustine et al., 1987). The synapse therefore provides an excellent model for studying the molecular mechanisms underlying membrane fusion.

2.1 THE MOLECULAR MECHANISM OF ACTION OF CLOSTRIDIAL NEUROTOXINS.

Neurotransmitter release is potently blocked by a group of neurotoxins produced by the anaerobic bacteria Clostridium tetani and Clostridium hotulinum. C.tetani produce a single toxin species, tetanus toxin (TeTx), whereas strains of C. hotulinum synthesize at least seven serologically distinct neurotoxins, designated hotulinum neurotoxin (BoNT)/A, B, Cl, D, E, F and G. The neurotoxins are synthesized as single-chain polypeptides of about 150 kD. They are subsequently activated by proteolysis at a single site, resulting in the generation of di-chain toxins in which the toxigenic light (L) chains (50 kD) remain linked to the heavy (H) chains (100 kD) via disulphide bonds. The H chain mediates selective binding to neurons and has been proposed to form a proteinaceous channel that allows the L chain to escape into the cytoplasm (Schmid et al., 1993). The free L chains of the neurotoxins cause a long-lasting inhibition of exocytosis in virtually all neurons examined. The block affects membrane fusion itself since other aspects of nerve terminal function such as membrane potential, voltage-gated Ca^^ currents as well as the

36 morphology of intracellular structures, remain unchanged (for review see Simpson, 1989; Niemann, 1991; Niemann a/., 1994).

A major breakthrough in the study of neurotransmission came when the molecular mechanism of clostridial neurotoxin action was elucidated. Both BoNT/B and TeTx were shown to be zinc-dependent endopeptidases that selectively cleaved the synaptic vesicle protein synaptobrevin/VAMP (vesicle-associated membrane protein), at the same single site (Schiavo et a l, 1992; Link et a l, 1992). Other botulinal neurotoxins were subsequently shown to have a similar mode of action:- BoNT/C, which had no effect on synaptobrevin, was shown to be a zinc-dependent endopeptidase specific for the presynaptic plasma membrane protein syntaxin (Blasi et a l, 1993a). Similarly, both the A and E serotypes of BoNT were shown to be zinc-dependent endopeptidases which specifically cleaved the presynaptic plasma membrane protein, SNAP-25 (synaptosomal- associated protein of MW 25 kD) (Blasi e ta l, 1993b; Schiavo e ta l, 1993; Binz et al, 1994), although the inhibition of neurotransmitter release by these toxins was not 100% (Blasi et a l, 1993b).

The identification of synaptobrevin, syntaxin and SNAP-25 as probably the only molecular targets of the clostridial neurotoxins implies that each of these proteins is required for exocytosis of synaptic vesicles.

2.2 THE PUTATIVE EXOCYTOTIC FUSION COMPLEX.

The second major breakthrough in the study of neurotransmission was the identification of synaptobrevin, syntaxin and SNAP-25 as the membrane receptors (SNAREs: SNAP receptors) for the general fusion proteins NSF (V-ethylmaleimide- sensitive factor) and a/p/y-SNAPs (soluble NSF attachment proteins) (Sollner et al, 1993a). NSF and SNAPs were originally identified as soluble factors which were able to restore intercistemal Golgi transport in a cell-fi’ee system in which intracellular membrane trafficking had been blocked by A-ethylmaleimide treatment (for review see Rothman and

37 Orci, 1992). NSF, SNAPs and SNAREs were isolated as a 20S complex from detergent extracts of mammalian brain (Sollner et a l, 1993a). Furthermore, the three SNARE proteins, together with synaptotagmin, were shown to form a stable 7S particle in vitro, in the absence of NSF and SNAPs (Sollner et al., 1993b). The series of complexes formed by these proteins in vitro were proposed to represent the in situ protein associations responsible for vesicle docking, activation and fusion (Sollner et al., 1993b). According to this model, the 7S complex represented synaptic vesicles docked at presynaptic release sites. Binding of a-SNAP to the complex displaced synaptotagmin and allowed the binding of NSF. The complex disassembled in vitro upon ATP hydrolysis by NSF, an event which was proposed to represent the changes in protein-protein interactions underlying membrane fusion (Sollner et al., 1993a, b). As discussed later, this model is not consistent with other observations regarding Ca^^-stimulated exocytosis and alternative models have been proposed.

The discovery of a link between the processes of constitutive membrane trafficking and regulated exocytosis led to the 'SNARE' hypothesis which proposed that the exocytotic machinery at the synapse was a specialized version of a ubiquitous fusion machinery which functioned in all eukaryotic fusion events (for review see Rothman, 1994). According to the SNARE' hypothesis, an integral membrane protein resident on the transport vesicle (v-SNARE) pairs with its receptor on the target membrane (t- SNARE). Each intracellular fusion event has its own specific v-SNAREs and t-SNAREs whilst NSF and SNAPs operate in all of these steps. The specificity of vesicular targeting is guaranteed since each v-SNARE only interacts with its matching t-SNARE, ensuring that the transport vesicle fuses with its correct acceptor compartment. In support of this hypothesis, multiple isoforms of syntaxin (Bennett et al., 1993) and synaptobrevin (McMahon et al., 1993) have been identified with various intracellular distributions and binding specificities (Calakos etal., 1994).

The use of a constitutively operating mechanism in regulated exocytosis implies the existence of "clamps" that prevent fusion, until receival of an appropriate signal. According to the 'SNARE' hypothesis, synaptotagmin acts as a "fusion clamp" in synaptic

38 transmission (Sollner et al., 1993b). Other proteins that also interact with the SNARE proteins include synaptophysin, a synaptic vesicle transmembrane protein, which binds to synaptobrevin in a complex that excludes syntaxin and SNAP-25 (Bennett et a l, 1992; Edelmann et a l, 1995; El Far et al, 1995) and nSec 1 p/mSec 1 p/Munc-18, which binds syntaxin with high affinity and prevents the binding of synaptobrevin and SNAP-25 (Pevsner a/., 1994; Hata^^a/., 1993b; Garcia^/a/., 1995; Hodel et a l, 1994; Tellam etal, 1995). These protein-protein interactions were proposed to regulate the availabilty of synaptobrevin and syntaxin, respectively, for assembly into the core fiision complex (Edelmann et a l, 1995; El Far et al, 1995; Pevsner et a l, 1994; Tellam et a l, 1995). Small GTP-binding proteins of the rab subfamily were also shown to interact with the SNARE complex (Horikawa etal, 1993; Sogaard etal, 1994) and were proposed to play a role its assembly (Sogaard et a l, 1994; Geppert et a l, 1994b). Figure 1.1, taken from Niemann et a l (1994), shows the currently known members of the putative synaptic fusion machine and their susceptibility to clostridial neurotoxins.

39 T eT x, BoNT/B,D,F,G B oN T /A ,E

Synaptotagmin Neurexin

Munc-18 SNAP-25, Channel

Syntaxin/HPC-1 B oN T /C I Synaptobrevin/VAMP

Synaptobrevin/VAMP

Syntaxin/HPC-1 Munc-18. SNAP-25

ISF lAP

VM ATP ADP PM

Figure 1.1 Model showing the currently known members of the synaptic fusion machine and their susceptibility to clostridial neurotoxins.

Synaptobrevin, syntaxin and SNAiP-25 form the core complex that interacts with the complex of NSF and a-, p- and Y-SNAP. Synaptotagmin interacts with this complex in a mutually exclusive manner with a-SNAP, In addition, synatotagmm binds to neurexins. Syntaxin, in addition to being a component of the complex, interacts with synaptotagmin and presynaptic Ca^^ channels and with Munc-18/rbSecl. VM, vesicle membrane; PM, plasma membrane. Figure taken from Niemann et ai, 1994.

40 2.3 HOMOLOGUES OF THE SYNAPTIC FUSION PROTEINS FUNCTION IN NON-NEURONAL EXOCYTOSIS.

In yeast, ten genes have been identified whose products are structurally related to the three SNARE proteins (Aalto et al., 1993; Protopopov et al., 1993; Dascheret al., 1991; Gerst etal, 1992; Brennwald e ta l, 1994). The idea that synaptobrevin, syntaxin and SNAP-25 are essential for membrane fusion was strongly supported by in vivo and in vitro studies which showed that several of these gene products were required for the docking and/or fiision of transport vesicles with their acceptor compartments at different stages of the constitutive secretory pathway, including exocytosis (for review see Bennett and Scheller, 1993; Ferro-Novick and Jahn, 1994; Rothman and Warren, 1994). NSF and a-SNAP are also highly conserved in evolution and correspond to the yeast proteins Seel 8 and Sec 17, respectively (Wilson et a l, 1989; Griff et a l, 1992).

A synaptobrevin homologue was identified in Drosophila (Siidhof et a l, 1989a) and was shown to be involved in neurotransmitter release (Sweeny et a l, 1995). Cellubrevin, a non-neuronal homologue of synaptobrevins 1 and 11, was shown to be expressed in virtually all non-neuronal tissues examined (McMahon et a l, 1993). Cleavage of cellubrevin by the light chain of tetanus toxin (TeTx LC) did not affect endosome-endosome fusion in a cell-free assay (Link et a l, 1993) but did impair the exocytosis of endosome-derived, constitutively recycling vesicles in streptolysin-0- permeabilized CHO cells (Galli et a l, 1994). These results indicate that cellubrevin is involved in the exocytotic limb of the membrane cycle (Galli et a l, 1994). The residual exocytotic activity seen after the complete proteolysis of cellubrevin (Galli et a l, 1994) could be explained by the existence of TeTx-insensitive synaptobrevin homologues which can also function in this membrane fusion step. In rat kidney epithelial cells, TeTx cleavage of a synaptobrevin homologue inhibited the ftision of light endosomes in vitro (Jo et al, 1995). These authors suggested that the differences between these results and those of Link et al. (1993) might be due to differences arising because light endosomes are derived from a specialized endosomal compartment rather than from early endosomes of the constitutive pathway.

41 Homologues of synaptobrevin, syntaxin and SNAP-25 have been identified in pancreatic p-cells (Jacobsson etal., 1994), adrenal chromaffin cells (Hodel et a l, 1994; Roth and Burgoyne, 1994) and in the related clonal neuroendocrine cell line PC 12 (Oho et al., 1995). The three SNARE proteins were isolated in a 'SNARE' complex from adrenal chromaffin cells (Roth and Burgoyne, 1994) and from PCI2 cells (Oho et al., 1995). Ca^^-dependent catecholamine secretion in digitonin-permeabilized adrenal chromaffin cells was stimulated by the introduction of exogenous a-SNAP (Morgan and Burgoyne, 1995) and was inhibited in intact cells by TeTx cleavage of cellubrevin and synaptobrevin H, which were localized to the chromaffin granule membrane (Hohne-Zell et al., 1994; Foran et al., 1995; Co ville et a l, 1992). It was not clear whether both synaptobrevin II and cellubrevin were essential for Ca^^-induced exocytosis or whether one of them sufficed (Foran et al., 1995). In PC12 cells, TeTx LC inhibited Ca^- stimulated catecholamine secretion (Ahnert-Hilger and Weller, 1993). Cellubrevin was co-localized with synaptobrevin I and II on the membranes of both synaptic-like microvesicles and dense-core granules in PC 12 cells and all three proteins were shown to interact with the same neuronal syntaxin I and SNAP-25 isoforms (Chilcote et al., 1995). Co-localization of cellubrevin and synaptobrevin II on synaptic-like microvesicles and dense-core granules appears not to be unique since it was also observed in pancreatic p- cells (Jacobssong/a/., 1994; Regazzi e ta l, 1995). Furthermore, Ca^^-stimulated insulin secretion was inhibited in permeabilized cells by pretreatment with TeTx or BoNT/B (Regazzi et a l, 1995). As in adrenal chromaffin cells, it was not clear whether both synaptobrevin II and cellubrevin were essential for Ca^^-dependent exocytosis. Although synaptobrevin immunoreactivity was not detected on dense core granules in neurons (Baumert et a l, 1989), taken together, these results suggest that the mechanism controlling the exocytosis of large dense core vesicles is similar to that controlling the exocytosis of small synaptic vesicles.

Both a synaptobrevin II homologue and a cellubrevin homologue were shown to be associated with GLUT4-containing vesicles in adipocytes and both proteins were translocated to the plasma membrane, together with GLUT4, in response to insulin (Cain etal, 1992; Volchuka/., 1995). Synaptobrevin II and cellubrevin were also identified

42 in rat skeletal muscle and in a muscle cell line, however, their function in these cells is unknown (Volchuk a/., 1994).

2.4 THE MOLECULAR MECHANISMS UNDERLYING MEMBRANE FUSION.

An understanding of precisely how cleavage of the 'SNARE' proteins by clostridial neurotoxins leads to an inhibition of neurotransmitter release should help to elucidate the precise nature and sequence of the molecular interactions underlying membrane fiision. TeTx-toxified squid giant nerve terminals contained more docked synaptic vesicles than non-toxified synapses (Hunt et al., 1994), suggesting that the membrane-anchored synaptobrevin fragment obtained upon cleavage was sufficient to mediate vesicle docking but not fusion. Synaptobrevin, syntaxin and SNAP-25 were reported to form a stable stoichiometric complex in vitro that was resistant to sodium dodecyl sulphate (SDS) and also present in brain (Hayashi et al., 1994). This complex was shown to form a high affinity binding site for a-SNAP (Hayashi et al., 1994; McMahon and Siidhof, 1995). Some of the clostridial neurotoxins inhibited the conversion of a ternary complex into an SDS-resistant complex and it was suggested that this conversion played a pivotal role in making synaptic vesicles competent for exocytosis (Hayashi et al., 1994). The N-terminal synaptobrevin fragment generated by BoNT/F cleavage, but not that generated by TeTx cleavage, impaired the disassembly of the reconstituted fusion complex in vitro (Hayashi et al., 1995). Cleavage of SNAP-25 by BoNT/A inhibited neither the assembly or disassembly of the reconstituted fusion complex in vitro (Hayashi et al., 1995; Otto et al, 1995). These results suggested that some of the clostridial neurotoxins might inhibit neurotransmission by inhibiting the disassembly of the fusion complex. Alternatively, inhibition might be mediated by toxin cleavage products at a step between complex disassembly and membrane fusion itself (Hayashi et al., 1995; Otto e ta l, 1995).

It was reported that significant amounts of both syntaxin I and SNAP-25 recycled

43 through synaptic vesicles (Walch-Solimena et a l, 1995). Furthermore, the vesicular pool of syntaxin I was preferentially cleaved by BoNT/Cl, suggesting that the protein underwent changes in its conformation and/or interactions with other proteins during the cycle of exo- and endo-cytosis. Interestingly, NSF and a-SNAP bound directly to isolated syntaxin, inducing a conformational change in the molecule which reduced its afiSnity for a-SNAP, synaptobrevin and SNAP-25 (Hanson eta l, 1995). It was proposed that the disassembly of the 20S fusion complex was mediated by this conformational change in syntaxin (Hanson et a l, 1995). In support of this hypothesis, neither the NSF/SNAP-induced conformational change nor the dissociation of the 20S fusion complex in vitro was observed using an N-terminally truncated form of syntaxin (Hanson et a l, 1995; Hayashiet a l, 1995). These observations also suggested that the amino- terminal part of the molecule played an essential role in its function.

As stated earlier, synaptophysin and nSeclp/mSeclp/Munc-18 were proposed to regulate the assembly of the core fusion complex. Mutations in the Seel homologues in the nervous systems of C.elegans and D.melanogaster led to defective neurotransmitter secretion (Hosono et al, 1992; Harrison et a l, 1994). Conversely, overexpression of synaptophysin enhanced neurotransmitter release at Xenopus neuromuscular synapses (Alder et a l, 1995). These jesults demonstrate that Seclp and synaptophysin are involved in the control of exocytotic membrane fusion. Syntaxin 1/SNAP-25 complexes were found to be present in non-synaptic areas of neuronal plasma membranes (Garcia et al, 1995). Furthermore, these complexes were dissociated by binding a-SNAP/NSF. It was suggested that this dissociation may represent a regulatory mechanism, resulting in the removal of potential vesicle attachment and fiision sites from areas outside the active zones (Hayashi gr a/., 1995).

The minimum domains essential for the interactions between synaptobrevin, syntaxin and SNAP-25 were identified and were shown to be regions with a high propensity to form a-helical coiled coils (Inoue et a l, 1992; Chapman et a l, 1994; Hayashi et a l, 1994). The three SNARE proteins were therefore suggested to interact by forming a heterotrimeric coiled-coil (Hayashi et a l, 1994; Chapman et a/., 1994).

44 Coiled-coils are formed by two or more right-handed a-helices wound around each other into a tight compact structure with a left-handed super-helical twist. This type of intermolecular interaction has already been linked to membrane fusion (Carr and Kim, 1993; Bullough et al., 1994; Yu et al., 1994): the influenza virus hemagglutinin (HA) forms homotrimers that mediate binding to the cell surface, internalization of the virus particle and fusion with cellular membranes. It was therefore suggested that the three neuronal SNAREs may mediate membrane fusion via a similar mechanism (Chapman et al., 1994b; Hayashi a/., 1994).

2.5 ALTERNATIVE MODELS OF MEMBRANE FUSION.

There are two major discrepancies in the molecular model of membrane fusion proposed by Sollner et al. (1993b). First, events in the model may occur too slowly to account for the rapid kinetics of Ca^^-regulated exocytosis in the synapse, which indicate a time delay of only about 100 ps between calcium accumulation and neurotransmitter exocytosis (Augustine etal., 1987). Second, ATP hydrolysis by NSF occurs at a late step immediately preceding membrane fusion but in many different cell types, there is no evidence which suggests that ATP hydrolysis is required for the triggering step of exocytosis (Bittner and Holz, 1992; Neher and Zucker, 1993). These discrepancies were reconciled by an alternative model proposed by O'Connor et al. (1994), in which SNAP/NSF binding and ATP hydrolysis occur at a step preceding calcium influx. In this model, NSF and SNAPs are required to energize docked vesicles into a so-called prefusion ("primed") state, which is reached upon ATP hydrolysis and dissociation of NSF and a-SNAP. Synaptotagmin then associates with this metastable release complex in a conformation that can be rapidly regulated by calcium to trigger membrane fusion. This model is more easily reconcilable with the observation that ATP acts before calcium in regulated exocytosis (Holz et al., 1989). In support of this alternative model, Chamberlain et al. (1995) showed, using stage-specific assays in permeabilized adrenal chromafiSn cells, that a-SNAP acted in an early ATP-requiring priming step but was not active in Ca^^-dependent exocytotic triggering.

45 Although the 'SNARE' hypothesis is proposed to cover all targeted fusion events during secretion and endocytosis, there is evidence to support the existence of an NSF/a- SNAP-independent exocytotic mechanism. In permeabilized Madin-Darby canine kidney (MDCK) cells, transport from the trans-GoXgi network to the basolateral plasma membrane was inhibited by antibodies against NSF and stimulated by a-SNAP (Ikonen et al., 1995). In contrast, transport to the apical cell surface was not affected by either of these treatments. Furthermore, apical transport was insensitive to tetanus and hotulinum neurotoxins, which inhibited basolateral transport (Ikonen et al., 1995). These results indicated that NSF and a-SNAP fimctioned specifically in vesicular transport to the basolateral plasma membrane but were not involved in transport to the apical plasma membrane (Ikonen et al., 1995). This result points towards the existence of at least one NSF-independent fusion pathway in the cell. Furthermore, this alternative pathway was shown to involve a member of the annexin family of Ca^^-dependent phospholipid-binding proteins (Fiedler etal., 1995).

The 'SNARE' hypothesis has provided a useful tool in elucidating the molecular mechanisms underlying exocytotic membrane fusion. One of its main conclusions is that a ubiquitous fusion machinery functions in all eukaryotic fusion events. However, the apparent lack of involvement of NSF and a-SNAP in apical transport in MDCK cells indicates that the whole picture is not yet known.

3. Exocytosis in the Sea Urchin Egg.

3.1 SEA URCHIN EGG ACTIVATION AT FERTILIZATION.

When a sperm fertilizes a sea urchin egg, a whole array of events occur which change it from being a quiescent to an actively-dividing cell. Table 1.1, taken from Whitaker and Steinhardt (1982), shows the events which occur in the egg during the first few minutes following fertilization.

46 Table 1.1 Events Initiated at Fertilization.

Na dependent action potential release of Ca^^ from intracellular stores cortical granule exocytosis changes in the cytoskeleton activation of NAD kinase increased levels of reduced nicotinamide nucleotides cytoplasmic alkalinisation activation of mitochondria and increased oxygen uptake lipid peroxidation increased tyrosine kinase activity

The activation of all of the events triggered by gamete interactions at fertilization is caused by changes in the concentrations of two ions; a transient increase in intracellular Ca^^ concentration ([Ca^^]J and a permanent decrease in intracellular concentration ([H^Ji) (Whitaker and Steinhardt, 1982). These ionic changes at fertilization are linked to changes in the phosphoinositide messengers inositol 1,4,5-trisphosphate (InsPj) and 1,2-diacylglycerol (DAG), which are generated through the action of the enzyme phospholipase C on the plasma membrane phospholipid, phosphatidylinositol 4,5- bisphosphate. InsPj causes Ca^^ release from intracellular stores while DAG causes efflux by stimulating protein kinase C and the Na^/H^ antiport (Whitaker, 1989; Swann and Whitaker, 1990). That changes in the levels of [Ca^^]j and [H^]j alone are sufficient to cause all of the other events associated with egg activation constitutes the ionic hypothesis of egg activation (Whitaker and Steinhardt, 1982).

3.2 CORTICAL GRANULE EXOCYTOSIS IN THE SEA URCHIN EGG.

The work in this thesis concerns one of the events triggered at fertilization, namely, cortical granule exocytosis. Exocytosis in sea urchin eggs is an example of triggered exocytosis, the trigger being, at fertilization, a sperm-induced rise in the intracellular free calcium ion concentration ([Ca^^]j) from a resting level of around lOOnM

47 to several micromolar (Steinhardt et al., 1977; Baker and Whitaker, 1978; Baker et al, 1980). The increase in [Ca^^J^ begins at the point of sperm entry and propagates in a wave-like fashion across the egg (Whitaker and Steinhardt, 1982; Eisen et al, 1984; Swann and Whitaker, 1986), causing the exocytosis of secretory cortical granules which lie immediately beneath the plasma membrane of unfertilized eggs (Jaffe, 1983; Swann and Whitaker, 1986). Cortical granule exocytosis results in the formation of a fertilization envelope around the egg, which is a physical barrier to prevent further sperm from reaching the egg and is thus an effective barrier to polyspermy (Kay and Shapiro, 1985). The prevention of polyspermy is essential in allowing normal embryonic development to progress. Figure 1.2 shows a sea urchin egg before and after fertilization.

3.3 SEA URCHIN EGG EXOCYTOSIS - A MODEL SYSTEM.

The sea urchin egg forms an ideal model system in which to study exocytosis both in vivo and in vitro. Eggs are easily obtainable in large quantities and can be fertilized simply by the mixing of the male and female gametes. The eggs are amenable to microinjection, allowing the introduction of substances into the egg cytoplasm. An in vitro system can be obtained by shearing eggs attached to polylysine-coated surfaces using a buffer similar to the internal milieu of the cell (Vacquier, 1975). When the cytoplasmic contents of the cell are washed away, the egg cortex, complete with its array of cortical secretory granules, is left attached to the surface via the vitelline layer (an extracellular glycocalyx). Using this isolated system it was shown that, in a medium similar in ionic composition to that of ooplasm, exocytosis was triggered simply by the addition of buffers containing micromolar levels of calcium (Vacquier, 1975; Baker and Whitaker, 1978; Moy et al., 1983). This exocytosis is also very easy to visualize. The cortical granules have a diameter of about 1 pm and when they fuse with the plasma membrane they lose their integrity and seem to disappear, leaving a flattened dome in their place. Cortical granule exocytosis is shown in figure 1.3.

48 Figure 1.2 Fertilization envelope elevation.

The upper panel shows an unfertilized sea urchin egg. The lower panel shows an egg, 5 minutes after insemination. The transparent halo surrounding the egg is the fertilization envelope which is formed by the calcium-stimulated exocytosis of the cortical secretory granules which lie immediately beneath the plasma membrane (Moser, 1939; Schuel etal, 1972).

49 Figure 1.3 Cortical granule exocytosis in vitro.

The figure shows the effect of elevated calcium ion concentration on isolated cortices. The top panel shows a cortical fragment isolated in a medium containing 10 mM EGTA. The lower panel shows the same fragment 3 minutes after the addition of 5.9 pM Ca^^. The addition of calcium has caused the disappearance of many of the cortical granules. This is due to exocytosis.

50 The reconstitution of exocytosis in vitro, between plasma membranes and isolated cortical granules, is also possible (Crabb and Jackson, 1985). Cortical granules, mechanically dislodged from cortical lawn preparations with a jet of isolation buffer, produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached to polylysine-coated microscope slides (Crabb and Jackson, 1985). When freshly prepared cortical granules were incubated with plasma membrane lawns, the granules reassociated with the cytoplasmic face of the plasma membrane and underwent exocytosis when calcium was added (Crabb & Jackson, 1985). Using an immunofluorescence-based vectorial transport assay, it was shown that the Ca^^- stimulated release of cortical vesicle contents in cortical lawns and reconstituted lawns was the in vitro equivalent of exocytosis (Crabb et al., 1987).

Thein vitro systems permit the study of the effect of compounds on the exocytotic reaction, both when applied to the isolated cortex and by treatment of the individual components of the exocytotic machinery. Using these systems, it should be possible to investigate the individual contributions made by each component to exocytosis.

3.4 CALCIUM IS THE SOLE REQUIREMENT FOR TRIGGERING EXOCYTOSIS.

A sperm-induced increase in [Ca^^Jj is the sole requirement for triggering cortical granule exocytosis in sea urchin eggs (Whitaker and Steinhardt, 1982, 1985). In concordance with this, exocytosis was triggered by treating eggs with the calcium ionophore A23187, which releases calcium from intracellular stores (Steinhardt and Epel, 1974; Zucker et al, 1978) and by microinjecting the Ca^^-releasing messenger, InsPj, into eggs (Whitaker and Irvine, 1984; Swann and Whitaker, 1986). Conversely, when the calcium chelator, EGTA, was microinjected into eggs prior to fertilization, the sperm- induced rise in [Ca^^j was inhibited and polyspermie eggs were produced, since formation of the fertilization envelope was prevented (Zucker and Steinhardt, 1978; Swann and Whitaker, 1986; Swann et a l, 1992). Experiments in which fertilization and

51 parthenogenic activation were performed in Ca^^-free seawater confirmed that the increase in [Ca^^]j was due to release from intracellular stores since, under these conditions, egg activation and fertilization envelope elevation still occurred (Steinhardt and Epel, 1974; Schmidt et al., 1982; Crossley et al., 1988). In summary, it has been shown that [Ca^^]j increases at fertilization, that cortical granule exocytosis can be caused by the addition of Ca^^ ionophore to unfertilized eggs and that blocking an increase in [Ca^^Jj inhibits exocytosis. Although calcium has been shown to be essential for stimulating exocytosis in sea urchin eggs, its precise site of action has not been elucidated.

(i) The Effects of Protein Kinases and Inhibitors of Cytoskeletal Function.

As discussed in section 1.2(i), modulation of cytoskeletal elements and secretory granule transport have been shown to be important factors in the regulation of exocytosis in many different secretory systems. In sea urchin eggs, however, the secretory cortical granules are already firmly attached to the plasma membrane, therefore no granule transport step is required. In concordance with this, drugs that interfere with the normal functions of microfilaments and microtubules did not affect exocytosis in sea urchin eggs, eitherin vivo or in vitro (Byrd and Perry, 1980; Whitaker and Baker, 1983). In addition, it was shown that protein kinase C activators such as PMA or DiCg and cyclic nucleotides, did not affect exocytosis directly, neither did they modulate the sensitivity towards calcium (Lau et al, 1986). It can be concluded then that in the sea urchin egg there is no regulation of exocytosis at the level of cytoskeletal elements.

(ii) GTP-Binding Proteins and Exocytosis.

Both cholera toxin (CTX) and GTPyS, a thio-analogue of GXP, stimulated cortical granule exocytosis when microinjected into sea urchin eggs (Turner et al., 1986, 1987; Swann et al., 1987; Crossley et al., 1991). Since both of these agents activate GTP-binding proteins, it was proposed that they stimulated exocytosis by causing an increase in [Ca^^]j through the activation of phospholipase C (PLC) and generation of inositol 1,4,5-trisphosphate (InsPj) (Turner a/., 1986, 1987; Swann 1987). In

52 support of this hypothesis, Crossley et al. (1991) demonstrated that microinjection of GTPyS into eggs stimulated exocytosis and generated a [Ca^^Jj transient identical in magnitude and duration to the fertilization [Ca^^j, transient. It was postulated that sperm may activate eggs in an analogous manner via a G-protein linked sperm receptor, activation of which would stimulate phospholipase C (Turner et al., 1986, 1987) . However, microinjection of GDPpS inhibited cortical granule exocytosis at fertilization without inhibiting the sperm-induced increase in [Ca^^Jj (Crossley et al., 1991), suggesting that sperm did not activate eggs via a G-protein linked receptor. This conclusion was further supported by the observation that microinjection of the soluble fraction of a sperm homogenate into unfertilized eggs caused the elevation of the fertilization envelope (Dale et al., 1985).

The inhibition of exocytosis at fertilization by GDPpS, distal to the sperm-induced increase in [Ca^^Jj, suggested that GDPpS was exerting its effect directly on the exocytotic machinery (Crossley et al., 1991). This observation provides evidence to support the existence of a G-protein which is directly involved in the mechanism of cortical granule exocytosis in sea urchin eggs. The involvement of G-proteins in exocytosis in other systems has been well documented and the G-protein involved has been termed G^ (Gomperts, 1986). Whether the Gg in sea urchin eggs is the same as the Gg that functions during exocytosis in other systems remains to be established.

3.5 CALCIUM TARGETS IN SEA URCHIN EGG EXOCYTOSIS.

(i) Calcium-Binding Proteins.

There is evidence to suggest that calmodulin (CaM) is important in the control of exocytosis in sea urchin eggs. The CaM antagonist trifluoperazine, inhibited cortical granule exocytosis both in vivo and in vitro (Baker and Whitaker, 1979; Whitaker and Baker, 1983). However, as discussed earlier and as pointed out by these authors, the effect of trifluoperazine on exocytosis might be due to its detergent-like effect on the cell

53 membrane. A more direct relationship between CaM and exocytosis was shown using an antibody directed against CaM (Steinhardt and Alderton, 1982). In this study, a complete block of exocytosis with calcium concentrations up to 1 mM was achieved in vitro after long incubations with the antibody. Using immunofluorescence, the CaM was shown to be localized on the plasma membrane, beneath the layer of cortical granules and occurred at the site of plasma membrane/cortical granule attachment (Steinhardt and Alderton, 1982). This study provided strong evidence for CaM regulating exocytosis in the sea urchin egg.

Other Ca^^-binding proteins which were also identified in sea urchin eggs include members of the annexin family of Ca^^-dependent phospholipid-binding proteins (Shen et al, 1994), a calcineurin-like protein (Iwasa and Ishiguro, 1986) and an unidentified 15 kD cytoplasmic protein (Hosoya et a l, 1986). The involvement of these proteins in exocytosis, however, was not investigated.

(ii) Calcium-Stimulated Phospholipases.

In sea urchin eggs, InsP^ and DAG are generated at fertilization through the hydrolysis ofPtdIns ? 2 by phospholipase C (PLC) (Ciapa and Whitaker, 1986). PLC was shown to be activated in vitro at identical calcium concentrations as those required to stimulate exocytosis (Whitaker and Aitchison, 1985). Neomycin, an aminoglycoside antibiotic, which inhibits the hydrolysis of PtdInsP 2 by PLC, inhibited Ca^^-stimulated exocytosis in vitro (Whitaker and Aitchison, 1985). Since DAG was shown to be fusogenic, it was suggested that the effect of neomycin may have been due to the inhibition of production of DAG. Alternatively, it is possible that the effect of neomycin on exocytosis may have been due to its effect on the surface potential of the membranes (McLaughlin and Whitaker, 1988). Since 1,2-dioctanoylglycerol (an analogue of DAG) did not stimulate exocytosis in vitro (Swann and Whitaker, 1985; Shen and Burgart, 1986; Lau et a l, 1986), it is unlikely that DAG stimulates exocytosis or modulates the Ca^^- sensitivity of exocytosis through the activation of protein kinase C.

54 There is some evidence to suggest that phospholipase A 2 (PLA2 ) may play a role in cortical granule exocytosis (Schuel, 1978). PLA 2 cleaves phospholipids to produce arachidonate and a lysophosphoglyceride. Both arachidonate (Creutz, 1981; Drust and Creutz, 1988) and lysophospholipids (Poole et al., 1970) have been shown to be fusogenic. Ca^^-dependent PLA 2 activity was identified in sea urchin egg homogenates

(Ferguson and Shen, 1984) and melittin, a PLA 2 activator triggered cortical granule exocytosis (Shimadae/a/., 1982). Conversely, quinacrine, an inhibitor of PLA 2 , blocked exocytosis but not sperm incorporation in intact eggs (Ferguson and Shen, 1984). These results suggested that the calcium transient at fertilization might activate PLA 2 which would then produce fusogenic lysophosphoglycerides. However, quinacrine is also considered to be a calmodulin antagonist (Weiss et al., 1982), therefore its effect on exocytosis may not have been due to a specific effect on PLA 2 The role of PLA 2 in sea urchin egg exocytosis remains unclear.

3.6 THE EFFECT OF ATP ON EXOCYTOSIS IN SEA URCHIN EGGS.

When sea urchin eggs were incubated with the metabolic poison cyanide, there was a decrease in intracellular ATP and a concomitant decrease in fertilization envelope elevation in response to both sperm and ionophore (Baker and Whitaker, 1978). This data suggested that a supply of ATP was required in order to maintain the cortical response to calcium. Cortices isolated in the absence of ATP slowly lost their ability to exocytose in response to micromolar levels of calcium (Baker & Whitaker, 1978; Sasaki & Epel, 1983) and after prolonged exposure in ATP-free media, readmission of ATP to cortices restored the high Ca^^-affmity of the exocytotic reaction, but failed to restore Ca^^- sensitivity to refractory granules (Baker and Whitaker, 1978). From these results it was concluded that although a supply of free ATP was not necessary for exocytosis to occur, ATP, or something derived from it, was required either to maintain the exocytotic apparatus primed, ready to undergo exocytosis, or to confer Ca^^-sensitivity upon it. The requirement for ATP can be explained in a number of ways, and the evidence in support of each explanation will be presented.

55 (i) Is ATP Required for an Energy-Dependent Step During Exocytosis?

Baker and Whitaker (1978) postulated that exocytosis might involve the release of calcium from an intracellular store which only remained filled in the presence of ATP. Thus, in the absence of this store, sperm or ionophore would not be able to raise [Ca^^Jj to threshold levels for triggering exocytosis. In support of this hypothesis, it was shown that isolated cortices from unfertilized eggs sequestered calcium in an ATP-dependent manner and cortical regions preloaded with in the presence of ATP, dramatically increased their rate of Ca^^ efflux upon addition of the Ca^^ ionophore A23187 (Oberdorf et al., 1986; Payan et at., 1986). This pool of calcium is most likely sequestered in the portion of the eggs endoplasmic reticulum that remains associated with the cortical region during its isolation (Sardet, 1984; Chandler, 1984).

It was shown that microinjection of adenosine 5'-0-3-thiotriphosphate (ATPyS), a thio-analogue of ATP, into eggs completely blocked cortical granule exocytosis in response to fertilizing sperm (Whalley et al., 1991). ATPyS can be used as a phosphoryl donor by protein kinases but the resulting thiophosphoproteins are not readily dephosphorylated by phosphoprotein phosphatases (Gratecos and Fischer, 1974).

Microinjection of the non-hydrolysable analogues of ATP, AppNHp and AppCH 2 p, which are not kinase substrates, had no effect on exocytosis upon subsequent fertilization (Whalley et al., 1991). These results suggested that ATPyS was not exerting its effect by competing with normal hydrolysable cellular ATP since, if this was the case, it would be predicted that both AppNHp and AppCH 2 p would have a similar effect. These results therefore argue against ATP being required for an energy-requiring step in the exocytotic reaction.

(ii) The Effect of Irreversible Protein Phosphorylation on Exocytosis.

Microinjection of ATPyS into eggs completely blocked cortical granule exocytosis in response to fertilizing sperm, distal to the increase in [Ca^^jj (Whalley et al., 1991). Cortices isolated from ATPyS-treated eggs failed to undergo exocytosis in response to

56 calcium, however, ATPyS did not affect exocytosis when applied directly to isolated cortices. These results indicated that the inhibitory effect was mediated by proteins associated with the egg cortex and that inhibition required the action of a cytosolic protein kinase which was lost when cortices were prepared (Whalley et al., 1991). Okadaic acid, an inhibitor of phosphoprotein phosphatases (Bialojan & Takai, 1988; Haystead et al., 1989), also inhibited cortical granule exocytosis at fertilization in a dose-dependent fashion, without affecting calcium mobilisation (Whalley et al., 1991). From these results it was concluded that an inhibitory phosphoprotein associated with the egg cortex obstructed Ca^^-stimulated exocytosis in sea urchin eggs. Two cortical proteins of 33 kD and 27 kD were strongly labelled using [^^S]ATPyS and it was suggested that these proteins might mediate the inhibitory effect of ATPyS (Whalley et al., 1991).

(iii) The Role of the Polyphosphoinositides in Exocytosis.

Eberhard et al. (1990) demonstrated that in permeabilized adrenal chromaffin cells, the polyphosphoinositides, phosphatidylinositol 4-phosphate (PtdlnsP) and phosphatidylinositol 4,5-bisphosphate (Ptdlnsf 2 ) were required for Ca^^-stimulated exocytosis, independent of their being substrates for phospholipase C and the generation of InsPg and DAG. When the levels of PtdlnsP and PtdInsT *2 were decreased by the addition of exogenous phospholipase C, or by removing ATP, Ca^^-dependent exocytosis concurrently decreased (These results are discussed in detail in section 1.5). On the basis of these results, it can be argued that in sea urchin eggs, the decrease in the secretory response seen in the absence of ATP may be due to a decrease in the levels of PtdlnsP and

PtdInsP 2 in the egg cortex. In unfertilized eggs, both phosphatidylinositol 4-phosphate 5-kinase (PtdInsP5K) and phosphatidylinositol 4-kinase (PtdIns4K) activities were found associated with cortical membranes (Oberdorf et al., 1989). The subcellular localization of these kinases provides further support for the proposed role of ATP in exocytosis. It follows that in the absence of ATP, PtdInsP5K and PtdIns4K would be unable to maintain the levels of the polyphosphoinositides in the cortex, which would decrease due to the action of endogenous phosphatases. (Phosphatase activity has been localized to the cortices of unfertilized sea urchin eggs - Iwasa and Ishiguro, 1986).

57 In summary, it is possible that ATP is required to maintain the levels of the polyphosphoinositides in the egg cortex prior to Ca^^-stimulated exocytosis. Experiments in which this hypothesis was investigated are presented in Chapter 6.

3.7 PROTEINS ARE REQUIRED FOR REGULATING THE EXOCYTOTIC REACTION IN VITRO,

Although specific proteins have been implicated to be involved in cortical granule exocytosis, other experiments have shown that, as yet unidentified, proteins control the Ca^^- and ATP-sensitivity, and also the rate of the exocytotic reaction. Mild treatment of cortices with either trypsin or the sulfhydryl-modifying reagent A^ethylmaleimide (NEM), inhibited exocytosis in vitro by increasing the threshold free calcium concentration required to trigger exocytosis (Haggerty and Jackson, 1983; Jackson et a l, 1985; Whalley and Sokoloff, 1994). These results suggested that proteins were responsible for modulating the Ca^^-sensitivity of exocytosis. Indeed, Sasaki (1984), demonstrated that eggs of Hemicentrotus pulcherrimus contained a KCl-extractable protein of approximately 100 kD that was required for conferring Ca^^-sensitivity to the exocytotic reaction. Removal of the protein was attributed to the chaotropic effect of chloride ions. After extraction, the original level of Ca^^-sensitivity was restored by adding back the protein to the cortex in a medium closely assimilating the internal milieu of the cell (intracellular medium-IM) (Sasaki, 1984). The 100 kD protein was not identified, however, a protein which had a similar effect on the isolated cortex was found in extracts of murine brain (Sasaki, 1992). These results suggest that the mechanism conferring Ca^^- sensitivity to the exocytotic reaction may be highly conserved throughout evolution.

Cortices isolated in EM exhibited the same Ca^^-sensitivity of exocytosis as that of intact eggs (Baker and Whitaker, 1978). Furthermore, the Ca^^-sensitivity of exocytosis both in vivo and in vitro was maintained by the presence of ATP (Baker and Whitaker, 1978; Sasaki and Epel, 1983). The sensitivity to ATP in vitro was lost when cortices were isolated in KCl-containing medium (Sasaki & Epel, 1983). These data suggested

58 that the cortices isolated in IM best reflected the in vivo situation. The rate of discharge of cortices isolated in the KCl-containing medium in response to calcium was much faster than that of cortices isolated in IM and there were significant differences in morphology and protein composition of the two types of preparation (more proteins present in the cortices prepared in IM) (Sasaki and Epel, 1983). From these results it was concluded that proteins, which were extracted from cortices using high salt buffers, controlled the rate of the exocytotic reaction and also conferred ATP-sensitivity upon it. The identification of these proteins will greatly aid the elucidation of the exocytotic mechanism.

3.8 THE EFFECT OF CLOSTRIDIAL NEUROTOXINS ON CORTICAL GRANULE EXOCYTOSIS.

Clostridial neurotoxins are a group of zinc-dependent endopeptidases that potently inhibit Ca^^-stimulated neurotransmitter release by specifically cleaving one of three synaptic proteins, namely synaptobrevin/VAMP (vesicle-associated membrane protein), syntaxin and SNAP-25 (synaptosomal-associated protein of MW 25 kD). Clostridial neurotoxins have been shown to inhibit exocytosis in a number of different cell types (Galli et al, 1994; Hohne-Zell et a l, 1994; Regazzi et a l, 1995), implying that the basic mechanism of membrane fusion is highly conserved throughout evolution.

In unfertilized sea urchin eggs, cortical granule exocytosis and membrane resealing in response to micropuncture were unaffected by treatment with botulinum neurotoxins A and B (Steinhardt et al, 1994). In fertilized eggs, however, membrane resealing was inhibited by prior microinjection of either of these toxins (Steinhardt et a l, 1994). From these results it was concluded that homologues of SNAP-25 and synaptobrevin, which are cleaved by botulinum neurotoxins A and B respectively, participate in membrane resealing in fertilized eggs. Sea urchin eggs have been shown to require calcium to reseal (Swezey and Epel, 1989) and membrane resealing was proposed to occur by a mechanism similar to vesicular exocytosis (Steinhardt et a l, 1994). It was demonstrated that synaptobrevin

59 and SNAP-25 were not susceptible to cleavage by TeTx or BoNT/A, respectively, when assembled into the 7S heterotrimeric complex which was proposed to mediate vesicle docking at the plasma membrane (Hayashi et a l, 1994; Pellegrini et a l, 1994). It was therefore suggested that the lack of effect of the neurotoxins in unfertilized eggs was because membrane resealing utilized cortical granules already docked at the plasma membrane (Steinhardt et al, 1994). It follows that in fertilized eggs, where membrane resealing involves the transport of vesicles to and the docking at the plasma membrane, the putative homologues of synaptobrevin and SNAP-25 are susceptible to cleavage before assembly of a 7S heterotrimeric complex. In contrast to these findings, results presented in Chapter 4 demonstrate the involvement of a synaptobrevin homologue in cortical granule exocytosis in unfertilized sea urchin eggs.

60 4. Experimental Approach.

The work in this thesis consists of three parts. In the first part, I used immunoblotting to investigate whether homologues of proteins implicated in exocytosis in other secretory systems were present in sea urchin eggs. Using this approach, I show the presence of the synaptic proteins synaptobrevin and rab3A, three members of the annexin family of Ca^Vphospholipid-binding proteins and a calcineurin-like protein in sea urchin eggs.

The second part of the thesis is concerned with the role played by two of these proteins in exocytosis. I used tetanus toxin light chain (TeTx LC) to investigate whether the sea urchin synaptobrevin homologue was essential for cortical granule exocytosis. TeTx LC is a zinc-dependent endopeptidase which potently blocks neurotransmitter release by specifically cleaving synaptobrevin (Schiavo et al., 1992). I show that TeTx LC specifically cleaves the sea urchin synaptobrevin homologue in vitro. I demonstrate that preincubation of isolated cortices with TeTx LC causes a time-dependent inhibition of Ca^^-stimulated exocytosis which correlates with cleavage of the synaptobrevin homologue. I also show that the synaptobrevin homologue interacts with a recombinant syntaxin fragment cloned from a bovine adrenal medullary cDNA library. I also investigated the possible role played by the Ca^Vcalmodulin-dependent phosphoprotein phosphatase, calcineurin, in controlling exocytosis. Using specific inhibitors and functionally inhibitory antibodies, I show that calcineurin is not involved in exocytosis in sea urchin eggs.

The final part of the thesis concentrates on the role played by the polyphosphoinositides in exocytosis. In many secretory systems, the exocytotic response decreases in the absence of ATP and it has been suggested that this decrease may be due, in part, to a decrease in the levels of the polyphosphoinositides. In order to measure levels of the polyphosphoinositides in the egg cortex, I radiolabelled eggs using myo-2-Ÿ^]- inositol. I correlate the decrease in the exocytotic response in vitro due to the absence of ATP with a decrease in the level of polyphosphoinositides in the egg cortex.

61 Chapter 2. Materials and Methods.

1. GAMETE HANDLING.

Sea urchins of the species Lytechinus pictus (Pacific Biomarine Laboratories Inc., Venice, CA., USA and Marinus Inc., Long Beach, C A , USA) were held in thermostatted tanks containing natural sea water at 15 - 16 °C in order to keep them continually gravid. They were fed fortnightly on seaweed (Marine Biological Station, Millport, Scotland). Eggs were obtained by the intracoelomic injection of female urchins with 100 pi 0.5 M KCl and were collected by upending injected urchins in a beaker of artificial seawater (ASW; 435 mM NaCl, 40 mM MgCl^, 15 mM MgSO^, 11 mM CaCl^, 10 mM KCl, 2.5 mM NaHCOj, 1 mM ethylenediaminetetra-acetic acid (EOTA), pH 8.0). Male urchins were shed in a similar manner but were placed right side up in a small volume of ASW. Sperm was collected "dry" by pipetting directly from the gonadopores. Sperm was kept undiluted at 4 °C and used within 2 days of collection. Fertilizations were performed by diluting this concentrated sperm 10 000 times, which produced typical sperm concentrations of 0.5 -1 xlO^ sperm/ml. The viability of the eggs from each female was tested at the beginning of experiments by fertilizing a sample of eggs. Only batches of eggs in which fertilization was greater than 95 % were used. Eggs were nominally used within 8 hours of shedding.

Eggs are shed from urchins in a jelly-like substance that protects the eggs and which was only removed immediately prior to egg use. The jelly was removed by multiple passage of the eggs through a Nitex mesh (mesh size -80 pm). The eggs were allowed to settle and were washed several times before use in order to remove the egg jelly from the ASW. Occasionally eggs were washed into solutions other than ASW. This was done by spinning egg suspensions in a hand centrifuge and resuspending in the appropriate medium. Eggs were routinely used as soon after dejellying as possible in order to retain their capability of fertilization.

62 2. MICROINJECTION TECHNIQUES.

The injection of solutions into eggs was performed on glass slides or coverslips mounted on the stage of an inverted microscope (Leitz Diavert, Leitz Instruments, Luton, U.K.). The glass was coated with a solution of polylysine at 0.01 mg/ml for 30 seconds and this was then rinsed with distilled water and allowed to dry. Using this concentration of polylysine ensured that eggs stuck down firmly but did not become flattened, which makes microinjection difficult. A rectangular perspex bath was then mounted onto the coverslip using high vacuum silica grease (Dow Coming) which ensured the whole apparatus was watertight. An egg suspension was applied to the completed bath and after settling, the eggs were washed. Eggs were viewed using bright field optics with lOx or 40x objectives. Microinjections were always performed with the eggs viewed at 40x. Glass micropipettes were made from capillary glass with an internal diameter of 1.5 mm (type 15OF, Clark Electromedical, Pangbourne, U.K.) using a Palmer Bioscience Microelectrode Puller (Bioscience, Sheamess, U.K.). Micropipettes were backfilled with the solution to be injected, a process facilitated by an internal glass filament in the capillaries that draws the solution to the pipette tip by capillarity. Typically 0.1 to 1 pi of solution was used. All injections were performed using high pressure pulses applied to the back of the solution. This was done by holding the pipettes in an electrode holder (Clark Electromedical, Pangbourne, U.K.), to which a length of teflon tubing was attached. This, in turn, was attached to a gas cylinder via a solenoid valve (British Fluidics, Bishops Stortford, U.K.) which allowed pulses of pressure to force solutions from the tips of the micropipettes. The whole assembly was held in a hydraulic micromanipulator (Narishige Instruments, Japan) which was attached to the microscope stage. Whilst the pipette assembly was outside the egg, continuous pulsing at 1 Hz was used to prevent contamination of the tip contents by sea water.

To inject an egg, the micropipette was moved so that it just touched the egg surface and was moved into the egg such that it caused a large deformation. In order to penetrate the egg, the top of the pipette holder was tapped very gently. Once the desired volume of solution had been injected, the pulsing was stopped and the micropipette

63 withdrawn carefully. The volume of injection was estimated by observing the cytoplasmic displacement following each pulse, and its size was estimated with reference to an eyepiece graticule. Injections expressed as a percentage of total egg volume were then converted to a final cytoplasmic concentration given the pipette concentration and the relationship : Volume = 4/3 n Radius^. Eggs which showed any cytolysis following microinjection were rejected from the experiment.

3. TECHNIQUES USED TO INVESTIGATE CORTICAL GRANULE EXOCYTOSIS IN VITRO.

(i) Preparation of Cortices.

The cortical preparation, which was first described by Vacquier (1975), has proved to be a very useful preparation for the study of exocytosis in sea urchin eggs. The cortical preparation is a means by which plasma membrane fragments with the cortical granules attached are isolated and remain active to their physiological trigger. Isolated cortices respond to the same signal in vitro as that which causes exocytosis in the intact cell, that is, a rise in the intracellular free calcium concentration ([Ca^^jJ. The cortical preparation is also useful because exocytosis in vitro can be monitored using conventional light microscopy. Cortices respond to Ca^^ at 1-3 pM when kept in a medium similar in composition to that of ooplasm (Baker and Whitaker, 1978) and this [Ca^^] is similar to the peak concentration occurring at fertilization (Steinhardt et al., 1977; Swann and Whitaker, 1986).

To prepare cortices, a slurry of dejellied eggs was applied to a #1 coverslip pretreated for 1 minute with 0.1 mg/ml polylysine and was allowed to settle for 2 minutes. Any unattached eggs were rinsed away using ASW to leave a confluent layer of eggs attached to the coverslip. Sea water was removed and the eggs were rinsed gently with intracellular medium (IM: 220 mM potassium glutamate, 500 mM glycine, 10 mM NaCl,

5 mM MgCl 2 , 2.5 mM ATP, 10 mM ethylene glycol-bis( p -aminoethylether)#,#-

64 tetraacetic acid (EGTA), pH 6.7). Eggs were sheared by pipetting vigorously with a jet of IM. Cortices remained bound to the coverslip and the cytosol was removed by 3-5 further washes with 0.5 ml IM (for experiments using TeTx LC, IM contained 2 mM EGTA). Cortices were generally used as soon as they had been prepared unless incubations with particular chemicals were required. A perfusion cell was constructed on a glass slide by pressing strips of PTFE tape onto the edges of the slide. The coverslip was inverted and mounted onto the PTFE supports using silica grease, to complete the cell. These perfusion cells typically held a volume of 20 pi. In order to achieve rapid perfiasion of solutions across the cortical field, solutions were added to the cell at one end and drawn over the cortices using capillarity, by placing a strip of filter paper at the opposite end of the cell. Cortices were examined using phase contrast microscopy at lOOOx magnification on a Leitz Optiphot microscope (Leitz Instruments, Luton, U.K.).

(ii) Preparation of Calcium-Containing Solutions.

Calcium-EGTA buffers were used in experiments on exocytosis in isolated cortices and were prepared as described in Baker et al. (1980). IM contained 10 mM EGTA and various total calcium concentrations. The free calcium concentrations were calculated using the constants of Martell and Sillen (1964). The calcium-EGTA ratios and free calcium were 0.607, 1.9 pM; 0.756, 4.0 pM; 0.838, 5.9 pM; 0.942, 17.0 pM. The pH of calcium-EGTA buffers, which was pH 6.7, was routinely checked before and immediately after each experiment.

(iii) Measuring the Extent of Exocytosis in Cortices.

The extent of exocytosis in cortical lawns was determined using either one of two methods. In the first method, the cortical lawn was illuminated using dark-field optics so that the light passing through the specimen was related to the degree of light scattering. As cortical granules fused with the plasma membrane they lost their retractility and light scattering ability. Thus, the extent of exocytosis was inversely proportional to light

65 scattering. This was determined quantitatively by attaching a photodiode to the camera port of the microscope and sending the output directly to a potentiometric chart recorder. For each experiment, maximum exocytosis was determined by perfusing with a 10 mM Ca^^ solution. The effects of other solutions were expressed in terms of the decrease in light scattering compared to that when a solution containing 10 mM Ca^^ was added. In the second method, cortices were viewed at lOOOx magnification using phase contrast optics. Individual cortical granules can be seen using these optics and appear as dark spheres against a light background. Experiments were recorded by attaching a camera to the microscope, which in turn was attached to a video recorder. Images were stored on Sony Videos tapes. The extent of exocytosis was calculated by counting the number of cortical granules, in a given area, that underwent exocytosis on addition of a Ca^- containing buffer.

4. POLYACRYLAMIDE GEL ELECTROPHORESIS.

(i) Preparation of Protein Samples.

All buffers used in the preparation of protein samples contained 1 pg/ml leupeptin, I pg/ml pepstatin and 0.1 mMPMSF. To prepare whole egg lysates, cytosolic and whole egg membrane protein fi-actions, packed dejellied eggs (0.2 ml) were resuspended in 5 ml IM at 4 °C. The suspension was gently homogenised by 3-5 strokes of a tight-fitting stainless-steel Dounce-type homogeniser. An aliquot of the homogenate was taken to give the whole egg lysate. The remainder of the homogenate was centrifuged at lOOOx g for 10 minutes to pellet nuclei and intact cells. The resulting supernatant was centrifuged at 100 OOOx g for 1 hour at 4 °C in a Sorvall ultracentrifuge. The supernatant, containing soluble cytosolic proteins, was carefully removed and the pellet was resuspended in 2 ml 1 % Triton-X-100.

To prepare a cortical protein fi-action, cortical lawns were prepared on glass Petri dishes as described in section 3(i) and were removed from the dishes by dissolution in 0.5

66 ml 1 % (v/v) Triton-X-100. Plasma membrane and cortical granule protein fractions were prepared from dishes of cortical lawns which were exhaustively washed until completely free of cytoplasmic organelles. Washed cortices were examined using dark field optics and were not used for the preparation of cortical granules until the washing solution was free of cytoplasmic particles. IM was drained from these cortical lawns and was replaced with 1 ml KEA buffer (450 mM KCL, 5 mM EGTA, 50 mMNH4CI, pH 9.1). Cortical granules were removed by shearing with a more powerful jet of KEA which removed more than 95 % of the cortical granules but left the plasma membrane fragments adhering to the slides. This shearing was achieved by forcing 1 ml of KEA from a disposable syringe. Removal of the granules was accompanied by a clearing of the translucent cortical lawn and this proved to be a convenient measure of the extent of granule detachment. This process has previously been shown not to tear away fragments of plasma membrane together with cortical granules (Whalley, 1990). Cortical granules from at least 6 dishes were prepared in 1 ml KEA. Once the cortical granules had been removed, the denuded plasma membrane lawns were detached from dishes by dissolution in 0.5 ml 1 % (v/v) Triton-X-100.

At this stage, aliquots from each protein sample were taken for use in protein determination. Proteins were concentrated from samples by the addition of 3 volumes of chloroform/methanol (2:1). The precipitated protein was washed twice with distilled water before dissolving in a minimum volume of SDS-PAGE sample buffer (2 % sodium dodecyl sulphate (SDS), 10 % glycerol, 80 mM Tris-HCl, pH 6.8 and 5 % 2- mercaptoethanol). Sample buffer also contained bromophenol blue (1 % of a 0.2 % stock in ethanol), which allowed the running of the gel to be followed. Samples were briefly sonicated to ensure solubilisation using a 4710 series ultrasonic homogeniser (Cole Parmer Instrument Co. Chicago, USA), before storage at -20°C.

To prepare a rat brain membrane protein fraction, three brains were washed 3 times in homogenisation buffer (130 mM NaCl, 2 mM EGTA, 50 mM PIPES, pH 6.8) to remove any blood. The brains were then chopped and homogenised in 5 ml buffer by 4-5 strokes in a stainless-steel Dounce-type homogeniser. The homogenate was centrifuged

67 at lOOOx g for 10 minutes at 4 °C and the resulting pellet, containing nuclei and intact cells, was discarded. The supernatant was then centrifuged at 100 OOOx g for 1 hour at 4 °C. The supernatant from this centrifugation was discarded and the pellet, containing membrane proteins, was resuspended in 1 ml buffer. Proteins were then precipitated from this suspension and redissolved in SDS-PAGE sample buffer as described above.

(ii) Protein Assay of Samples.

Protein from the aliquots taken, was precipitated using chloroform/methanol extraction and redissolved in 1 M NaCl. The protein concentrations in these samples were determined using the BCA (bicinchoninic acid) protein assay protocol (Pierce, Illinois, USA). Briefly, aliquots of the protein samples were added to test tubes and enough 1 M NaCl added to make a final volume of 1 ml. To each test tube 1 ml of Working Reagent (Pierce) was added and mixed well. Tubes were then incubated at 60 °C for 1 hour. After incubation, tubes were cooled to room temperature and the absorbance at 562 nm for each tube was measured. The protein concentration was calculated by comparison with an Bovine Serum Albumin (BSA) standard.

(iii) Running of Gels.

Protein samples were analysed using SDS polyacrylamide gel electrophoresis, as described by Laemmli (1970). Briefly, slab gels of 0.75 mm thickness were used with an acrylamide/bisacrylamide ratio of 29:1. The final acrylamide concentration in the running gel was 12 % and in the stacking gel was 4 %. Typically, 10 pg of protein dissolved in SDS-PAGE sample buffer was loaded per lane. The running buffer consisted of 0.38 M glycine, 0.05 M Tris and 0.1 % SDS. Electrophoresis was performed using the Bio-Rad Mini-PROTEAN n Cell system. Samples were run into the stacking gel using 100 V and this voltage was increased to 185 V (approx. 25 mA) as soon as the sample had entered the running gel. Electrophoresis was stopped when the running front was within 0.25 cm of the end of the gel. Gels were then either fixed and the proteins visualized using colloidal coomassie, or used in Western blotting. For staining, gels were fixed for 1 hour

68 in 25 % trichloroacetic acid (TCA). Proteins were visualized by incubating gels in a solution of coomassie blue G250 in 20 % methanol, 2 % phosphoric acid and 10 % ammonium sulphate for 20 hours with agitation. Gels were destained in a solution containing 25 % methanol and 7 % acetic acid for several hours and were dried at 80 °C using a Bio-Rad 543 gel dryer. In order to estimate the molecular weights (MW) of separated proteins, a lane of molecular weight standards (Bio-Rad Laboratories, CA, USA) was also run per gel. For gels to be Western blotted, pre-stained standards were used (Bio-Rad Laboratories, CA, USA).

5. WESTERN BLOTTING OF SDS-PAGE GELS.

(i) Transfer of Proteins.

Polyvinylidene difluoride transfer membrane (PVDF, Millipore Corp.,MA, USA), which was cut to the dimensions of the gel, was wetted by a brief immersion in methanol. The membrane was then soaked in Bjerrum and Schafer-Nielsen transfer buffer with SDS (48 mM Tris, 39 mM glycine, 20 % methanol, 1.3 mM SDS (0.0375 %), pH 9.2) for 15 minutes at 4 °C prior to transfer. Eight sheets of filter paper, which were also cut to the dimensions of the gel, were completely saturated by soaking in transfer buffer at 4 °C. Following electrophoresis, the gel was equilibrated in transfer buffer for 10 minutes. A gel/membrane sandwich was then assembled into a Bio-Rad Trans Blot SD Cell in the following order: (i) saturated filter paper on the platinum anode; (ii) pre-wetted transfer membrane; (iii) rinsed gel; (iv) saturated filter paper. The cathode was placed firmly onto the stack to ensure complete electrical contact. After securing the safety cover, proteins were electophoretically transferred onto the PVDF membrane at a constant voltage of 5 V for 45 minutes.

(ii) Immunodetection

After protein transfer, unoccupied binding sites on the membrane were blocked

69 by overnight incubation in PBS (137 mM NaCl, 2.7 mM KCl, 7.0 mM Na^HPO^, 1.25 mM NaH^PO^, 1.5 mM KHPOJ containing 0.01 % Tween 20, 5 % milk protein and 1.5 % BSA at 4 ®C. Incubations with primary antibodies were performed in PBS containing 0.01 % Tween 20, 5% normal goat serum (Sigma) and 1.5 % BSA (Sigma) with agitation for 16 hours at 4 °C and for 1 hour at Room Temperature. Incubations with horseradish- peroxidase-conjugated secondary antibodies (Pierce) were performed in the same buffer for 1 hour at Room Temperature. After washing extensively in PBS containing 0.01 % Tween 20, immunoreactive proteins were visualized using the enhanced chemiluminescence system (Amersham).

6. IMMUNOCYTOCHEMISTRY OF CORTICES.

(i) Fixing Egg Cortices.

Before immunostaining, most biological tissues must be fixed. Fixation makes cells permeable to the stains and cross-links their macromolecules so that they are stabilized and locked in position. Some of the earliest fixation procedures involved a brief immersion in acids or organic solvents such as methanol, while current procedures usually include exposure of the cell to reactive aldehydes, particularly formaldehyde and glutaraldehyde. The latter compounds form covalent bonds with the free amino groups of proteins and thereby link adjacent molecules together.

Cortices, which were prepared on #1 coverslips as described in section 3(i), were fixed by immersing for 30 minutes in cortex isolation buffer (0.6 M mannitol, 2.5 mM

MgCl 2 , 20 mM EGTA, 50 mM HEPES, 50 mM PIPES, pH 6.8) containing 3 % formaldehyde and 0.5 % glutaraldehyde. To stop the fixation process, the coverslips were incubated in aldehyde quench solution (75 mM KCl, 2 mM MgCl 2 , !0 mM EGTA, 150 mM glycine, 320 mM sucrose, 25 mM PIPES, pH 6.8) for 1 hour at 4 °C. Cortices were rehydrated by 5 x 10 minute incubations in PBS. At this stage fixed cortices can be dried completely and stored at - 70 °C until required.

70 (ii) Immunocytochemistry.

Fixed cortices attached to #1 coverslips were rehydrated by placing in a humidity chamber for 10 - 15 minutes. The humidity chamber consisted of a container lined with filter paper saturated with water. The rehydrated cortices were then incubated with primary antibody in blocking buffer (1 % BSA, 2 % normal goat serum, 0.1 % Triton-X- 100, in PBS, pH 7.5) at approximately lOOx the concentration used for Western blotting. The cortices were incubated with primary antibody for 16 hours at 4 °C and for 1 hour at Room Temperature. Cortices were washed 5 x 10 minutes in PBS before incubation with fluorescein-conjugated anti-mouse IgG (Pierce) in blocking buffer for 1 hour in the dark, at Room Temperature. After washing the coverslips 5x10 minutes in PBS and twice in distilled water, the coverslips were inverted and mounted in glycerol on glass slides. The edges of the coverslips were sealed using clear varnish.

(iii) Fluorescence Staining of Cortices.

1,1 -dioctadecyl-3,3,3 ',3 '-tetramethylindocarbocyanine perchlorate (DilC^g) was purchased fi’om Molecular Probes (Ohio, USA). DilCjg has a fluorescent head group with two long hydrocarbon chains (Figure 2.1).

CHzCH-CH

CIO

CH, CH,

Figure 2.1 Structure of 1,1 '-dioctadecyl-3,3,3',3'-tetramethyl­ indocarbocyanine perchlorate (DilCig)

71 The hydrocarbon chains intercalate into membrane bilayers; once within a bilayer leaflet, Dil remains in the leaflet but diffuses freely within that leaflet (Honig and Hume, 1986). Dil suspensions stain membranes by random collisions of the Dil aggregates. Although Dil suspensions do not stain endoplasmic reticulum (ER) membranes specifically, the ER is stromgly stained because the widespread ER membranes can be hit in many places and because the continuity of the ER allows dye spreading.

Cortices were stained according to the method of Terasaki et al. {\99\). A 7.5 pg/ml Dil "suspension" was made by diluting 3 pi stock solution (2.5 mg/ml in ethanol, protected from light at room temperature) into 1 ml of intracellular medium (IM). After mixing, the suspension was used within a few minutes as Dil aggregates collide with each other to form fewer and larger aggregates which stain fewer cortical compartments with greater intensity. Cortices were stained for 1 minute, washed 5 x 3 minutes in IM and then mounted in IM in perfusion chambers, as described in section 3(i). Non-specific fluorescence due to staining of the back of the coverslip, was reduced by wiping with ethanol.

(iv) Confocal Microscopy.

Immuno stained and fluorescence-stained cortices were observed using a laser scanning confocal microscope (model 122050, Leica Lasertechnik, Heidelberg, Germany). Observations were made using a Leica 40x/1.3 oil immersion objective lens. The laser light source was an argon laser with a wavelength of 488 nm. The excitation light was separated fi*om the emission light via a dichroic mirror (510 nm cutoff used with excitation of 488 nm) and the emission light passed to the photomultiplier. The software of the confocal was based on a Leica 'confocal laser scanning microscopy' (CLSM, Leica Lasertechnik) system run by a Motorola VME147 computer. Images were stored on Ricoh ROD-5064F optical memory disks.

72 7. THE USE OF ISOTOPES TO RADIOLABEL INOSITOL PHOSPHOLIPIDS.

(i) Treatment of Eggs and Sample Preparation.

A^o-2-[^H]-inositol was used to investigate whether the decrease in secretory response of cortices incubated in the absence of ATP could be explained in terms of the levels of the polyphosphoinositides. Afyo-2-[^H]-inositol is taken up into the eggs and is incorporated into the polyphosphoinositide phospholipids. Isotopic equilibrium in Lytechinus pictus eggs has been shown to be attained after 30 hours incubation (Ciapa et al, 1992). The advantage of using labelled inositol rather than orthophosphate is that the label is incorporated solely into the metabolic pool under consideration.

Jellied eggs were incubated for 30 hours at 16 °C with 10 pCi/ml of myo-2-^H]- inositol (NEN DuPont) as a 20 % (v/v) suspension in 5 ml ASW containing 0.01 % of the antibiotic sulfadiazine. Eggs were then rinsed 4 times by décantation in cold ASW until the external solution was essentially free of labelled inositol, before dejellying. Dejellied eggs were washed 3 times in ASW, followed by washing and resuspension 4-5 times in IM. Cortices were prepared in IM on glass Petri dishes coated with 1 mg/ml polylysine and were incubated in either the presence or absence of 2.5 mM ATP. After incubation, cortices were detached using 0.5 ml 1 M NaOH. This solution was then transferred into an eppendorf containing 0.5 ml 25 % TCA at 4 ”C, mixed thoroughly and stored on ice for 30 minutes. After centrifugation, the resulting TCA pellet was washed twice with deionised water and extracted with chloroform/methanol/11 N HCl (100/200/5 v/v) for 30 minutes at 4 °C. 100 pg of carrier polyphosphoinositides (Sigma) was also added at this stage. 400 pi of a mixture of chloroform/water (1:1) was then added to give two distinct phases. After centrifugation, the lipid-containing chloroform phase was isolated and stored in a glass vial under N 2 at -20 °C.

73 (ii) Analysis of Inositol Phospholipids.

pH]inositol-labelled polyphosphoinositides were dried completely under vacuum before resuspension in 50 pi chloroform. Samples were loaded onto heat-activated, oxalate-impregnated silica gel 60 thin-layer chromatography plates (Merck, Darmstadt) and were separated by chromatography in chloroform/methanol/acetone/acetic acid/water (40/13/15/12/7 v/v), according to the method of Allan and Cockroft (1983). All lipids were identified using co-migration with commercial standards (Sigma) as the criterion and were visualized by brief staining with I 2 vapour. Lipids were recovered from the substrate and added to scintillation vials containing 200 pi methanol and 300 pi liquid scintillant. After vortexing, samples were counted in a Hewlett-Packard Tri-Carb 1500 scintillation counter.

8. ASSAY OF CALCINEURIN ACTIVITY.

Calcineurin activity was measured using p-nitrophenylphosphate (PNPP) as substrate, according to the method of Pallen and Wang (1983). All enzyme assays were performed in micro-titer assay plates in triplicate. Each well contained 0.16 pM calcineurin (Upstate Biotechnology Incorp.) in 50 mM Tris-HCl, pH 7.0, 2.5 mM NiCl 2 , 0.25 mg/ml BSA (Sigma), 0.1 mM Ca% 0.38 pM calmodulin (Calbiochem) and a varying quantity of the inhibitor to be analysed. The volume of the assay mixture was made up to 80 pi using 50 mM Tris-HCl, pH 7.0. In all sets of assays a blank, containing no calcineurin and a control, containing no inhibitor were also prepared. The assay plate was incubated for 15 minutes at 30 °C. After incubation, the plate was returned to room temperature and 120 pi of substrate (1.5 mg/ml PNPP in 50 mM HCl, pH 7.0) was added. The substrate mixture was freshly prepared before each assay. After 5 minutes the reaction was stopped by adding 20 pi of 13 % KHPO4. The absorbance of samples was measured at 405 nm against a blank using a micro-titer plate reader. The extent of the phosphatase reaction is proportional to the absorbance of the assay mixture.

74 Chapter 3. Looking for Proteins Involved in Exocytosis in Sea Urchin Eggs.

1. INTRODUCTION.

There is much evidence to support the idea that exocytosis is mediated by proteins in sea urchin eggs. As far back as 1975, Vacquier reported that trypsin treatment of isolated cortices caused a gradual decrease in plasma membrane-cortical granule attachment. Tryptic digestion of either isolated plasma membranes or cortical granules inhibited the interactions between these two components on subsequent reconstitution (Whalley, 1990). These data show that the docking of cortical granules is mediated by proteins on both the granules and the plasma membrane. Haggerty and Jackson (1983) reported that alkylation of sulphydryl-groups in cortices, using A-ethylmaleimide (NEM), decreased the Ca^^-sensitivity of exocytosis in vitro and that prolonged treatment with NEM completely blocked exocytosis. The exocytotic activity of NEM-inactivated cortices was restored by brief proteolytic digestion (Jackson et al., 1985). Sasaki (1984) reported that exposure of cortices to chaotropic anions decreased the Ca^^-sensitivity of exocytosis due to dissociation of specific proteins. One dissociated protein could be added back to cortices to restore the original level of Ca^^-sensitivity (Sasaki, 1984) and a protein which had a similar effect on isolated cortices was found in murine brain (Sasaki, 1992). Together, these results suggest that one or more proteins confer micromolar Ca^- sensitivity to exocytosis in sea urchin eggs and that functionally related components occur in different species.

The idea that the mechanism of vesicular membrane fusion is evolutionarily conserved was supported by the observation that chromaffin granule membranes underwent exocytosis in response to a rise in [Ca^^], when microinjected into Xenopus laevis eggs (Scheuner and Holz, 1994). Further support for this hypothesis came from studies on synaptic vesicle exocytosis. Several proteins required for synaptic vesicle

75 exocytosis have been identified and their homologues in yeast were shown to fimction in membrane fiasion events along the constitutive secretory pathway (for review see Bennett and Scheller, 1993; Rothman, 1994; Ferro-Novick and Jahn, 1994). Based on these findings, it was postulated that the mechanism of regulated secretion evolved from the constitutive secretory pathway of lower eukaryotes. In sea urchin eggs, reconstitution of the cortical granules of Lytechinus variegatus with the plasma membranes of Lytechinus pictus resulted in the formation of reconstituted heterologous cortical lawns which underwent exocytosis on the addition of calcium (Whalley, 1990). This result indicates that the mechanism controlling exocytosis is conserved, at least between these two species.

Since the mechanism of exocytosis appears to be evolutionarily conserved, it seemed pertinent to look for sea urchin homologues of proteins shown to function in exocytosis in other secretory systems. In this chapter I present the results of immunoblotting experiments designed to detect potential exocytotic proteins in sea urchin eggs.

2. EXPRESSION OF A SYNAPTOBREVIN HOMOLOGUE IN SEA URCHIN EGGS.

The synaptic proteins synaptobrevin, syntaxin and SNAP-25 form a heterotrimeric complex which has been proposed to function in all intracellular fusion events in eukaryotic cells (Sollner et al., 1993a,b). Steinhardt et a/.(1994) reported that both botulinum neurotoxins A and B inhibited membrane resealing after puncture in fertilized sea urchin eggs, suggesting that membrane resealing occurs by an exocytotic process similar to neurotransmitter release. Since the target proteins of these neurotoxins are SNAP-25 and synaptobrevin, respectively, this result suggested that homologues of these proteins exist in sea urchin eggs.

I immunoblotted sea urchin egg proteins using a monoclonal antibody. Cl 10.1,

76 raised against rat brain synaptobrevin (Baumert et al., 1989) (kind gift of Dr. R. Jahn). The antibody recognized a protein of approximately 18.5 kD in rat brain membranes, which was assigned as synaptobrevin (Figure 3.1). In sea urchin egg subcellular protein fractions, the antibody recognized a protein of approximately 17.5 kD in the whole egg lysate, cortical fraction and whole egg membrane fraction. No immunogenic proteins were present in the cytosolic fraction.

These results suggest that sea urchin eggs contain a synaptobrevin homologue which is membrane-associated and present in the egg cortex. This result is consistent with the fact that synaptobrevin is an integral membrane protein of secretory vesicles (Baumert et al., 1989). The putative synaptobrevin homologue detected in membranous compartments throughout the egg may indicate the presence of a synaptobrevin isoform which functions in vesicular fusion events along the constitutive secretory pathway. In Chapter 4, I describe experiments in which I investigated whether the synaptobrevin homologue localized to the cortex is involved in exocytosis.

I also immunoblotted sea urchin egg proteins using a monoclonal antibody against chick brain syntaxin (kind gift of Dr. M. Takahashi) and a polyclonal antibody against SNAP-25 (kind gift of Dr. S. Catsicas). No sea urchin egg proteins were recognized by either antibody.

3. A RAB PROTEIN IS ASSOCIATED WITH CORTICAL GRANULES.

There is evidence to support the involvement of GTP-binding proteins in exocytosis in sea urchin eggs. Prior microinjection of the G-protein antagonist, GDP p S, blocked cortical granule exocytosis upon insemination in sea urchin eggs (Turner et al., 1986; Crossley et al, 1991). Crossley et al. (1991) demonstrated that this inhibition was caused by a direct effect on cortical granule exocytosis and not by an effect on the signal transduction pathway generating an increase in [Ca^^Jj. This discovery suggested the presence of an exocytotic G-protein (G^), located in the egg cortex. In support of this

77 kD

- 3 2 .5

- 2 7 .5

- 18.5

-6.5

Figure 3.1 Expression of a synaptobrevin homologue.

Sea urchin egg subcellular protein fractions (50 p^g protein per lane) and rat brain membrane proteins (50 pg) were resolved by SDS-PAGE and transferred to a P VDF membrane by Western blotting. Immunoreactive proteins were detected using a monoclonal antibody (Cl 10.1) against rat brain synaptobrevin and were visualized using enhanced chemiluminescence. The positions of molecular-mass standards are shown on the right.

78 hypothesis, the egg plasma membrane was shown to contain proteins that were ribosylated by cholera and pertussis toxins, a property characteristic of the heterotrimeric class of GTP-binding proteins (Turner et al, 1987).

Evidence from other secretory systems has shown the involvement of ras-like small GTP-binding proteins in vesicular fusion events. Rab3A, a small GTP-binding protein highly concentrated on synaptic vesicles, was proposed to play a role in synaptic vesicle exocytosis (Fischer von Mollard et al., 1991; Matteoli et a l, 1991). Rab3 A, or related proteins, were shown to be involved in Ca^^-stimulated exocytosis in other secretory systems (Lledo et al., 1993; Johannes et al., 1994). Since it is possible that the effect of GDPpS on exocytosis in sea urchin eggs is mediated by a member of this class of GTP- binding protein, I looked for the presence of homologues of rab3A in these cells.

I immunoblotted different egg subcellular protein fractions using monoclonal antibody Cl 42.1 (kind gift of Dr. R. Jahn), which was raised against recombinant bovine rab3A (Matteoli e/ a/., 1991). A strongly immunoreactive protein of approximately 25 kD was detected in the sea urchin egg cortical fraction which migrated with the same mobility on gel electrophoresis as rat brain rab3A (Figure 3.2a). When cortical proteins were divided into cortical granule and plasma membrane proteins, the immunoreactive protein was localized to the cortical granule fraction. Another immunoreactive protein, of slightly lower molecular weight (approx. 20 kD), was also detected in the cortical granule fraction. This may be another isoform of rab3. The lack of detection of the 25 kD immunoreactive protein in the whole egg lysate is most likely due to the fact that in this complex protein sample, the protein was present in a quantity too small for detection.

Fischer von Mollard et al. (1991) reported that the dissociation of rab3A from synaptic vesicle membranes specifically correlated with Ca^^-stimulated exocytosis in isolated synaptosomes. These authors suggested that the cycling of rab3A between membrane-bound and free states was a mechanism by which it acted as a molecular "switch", directing synaptic vesicles during exocytosis. Matteoli et al. (1991), however, showed that rab3 A was translocated to the plasma membrane during exocytosis and

79 (a) / A a> o « rf^ xO-- _0 O' O ^

-8 0 -49.5

-32.5

-6.5

(b)

-49.5

-32.5

- 18.5

Figure 3.2 Expression and subcellular localization of rab3A.

(a) Sea urchin egg subcellular protein hactions (10 gg protein per lane) were resolved by SDS-PAGE and transferred to a PVDF membrane by Western blotting. Immunoreactive proteins were detected using a monoclonal antibody against rat brain rab3A (Cl 42.1) and were visualized using enhanced chemiluminescence. (b) Cortical proteins were prepared either before (-) or after (+) exocytosis was stimulated by addition of 17 pM Ca^^. Samples (10 pg protein per lane) were resolved by SDS-PAGE, transfeiTcd to a PVDF membrane and immunoblotted with mcAb Cl 42.1. The positions of moleculai-mass standards are shown on the right.

80 dissociated ifrom the membrane at some time after fusion. In order to determine the fate of the sea urchin homologue of rab3 A upon Ca^^-stimulated exocytosis, I immunoblotted cortical proteins prepared both before and immediately after exocytosis was stimulated by the addition of 10 mM Ca^\ The rab3A homologue remained membrane-associated both before and after exocytosis occurred (Figure 3.2b). This result, which is consistent with the findings of Matteoli et al. (1991), indicates that rab3A does not dissociate from membranes upon Ca^^-stimulated exocytosis in sea urchin eggs but is translocated to the plasma membrane.

4. LOOKING FOR HOMOLOGUES OF SYNAPTOTAGMIN IN SEA URCHIN EGGS.

Synaptotagmin is a 65 kD transmembrane protein of synaptic vesicles that has been proposed to be the Ca^^-sensor of regulated exocytosis (Perin et al., 1990, 1991). Synaptotagmin binds both negatively-charged phospholipids (Brose et a l, 1992) and calmodulin in a Ca^^-dependent manner (Fournier and Trifaro, 1988; Trifaro et al., 1989). Calmodulin has been implicated in exocytosis in sea urchin eggs and was localized on the plasma membrane, at the site of plasma membrane/cortical granule attachment in unfertilized eggs (Steinhardt and Alderton, 1982). Synaptotagmin therefore possesses several properties which make it an ideal candidate for involvement in cortical granule exocytosis in sea urchin eggs.

1 immunoblotted sea urchin egg subcellular protein fractions using a monoclonal antibody, ID 12, which was raised against chick brain synaptotagmin (Takahashi et al., 1991) and a polyclonal antibody which was raised against a synthetic peptide corresponding to a conserved region within the first C2 domain of synaptotagmin (Both antibodies were a kind gift from Dr. M. Takahashi). The sequence of the immunogenic peptide and its homology to the corresponding region within the first C2 domain of synaptotagmin from different species are shown in Table 3.1.

81 Table 3.1 Sequence of the ID 12 antigenic peptide and the corresponding region within synaptotagmin from different species.

Peptide RRLKKRKTSI KKNTLN

Human KRLKKKKTTI KKNTLN Rat-1 KRLKKKKTTI KKNTLN Ommata-A KRLKKKKTTI KKNTLN Rat-2 KRLKKKKTTVKKKTLN Ommata-B KRLKKKKTTVKKNTLN Drosophila KRLKKKKTS VKKCTLN Squid K R I KKKKTTVKKCTLN C. elegans KRLKKKKTSI KKCTLN Ommata-C RRLKKRKTSI KKNTLN

Conserved residues are shown in bold.

Figure 3.3 shows the results of immunoblotting sea urchin egg subcellular protein fractions using the anti-peptide antibody. In the control lane, which was loaded with rat brain membrane proteins, the antibody recognized the 65 kD protein, synaptotagmin, and also a 39 kD protein. The 39 kD protein is probably a proteolytic fragment of synaptotagmin, since a 39 kD proteolytic fragment of synaptotagmin, which was generated by cleavage at a hypersensitive site, has previously been reported (Perin et al., 1991). No corresponding proteins with a similar molecular weight were detected in any of the sea urchin egg protein fractions. The antibody recognized immunoreactive proteins of approximately 78, 48 and 26 kD in the whole egg lysate. The 48 and 26 kD species could either be degradation products of the 78 kD protein, or other proteins that contain C2 domains. The 78 and 26 kD proteins were also present in the cytosolic fraction, although in very small quantities. The lack of detection of the 78 kD protein in either the cortical or cytosolic fractions, implies that it is associated with intracellular membranes. The 78 kD protein possibly represents a sea urchin homologue of protein kinase C (PKC). PKC isozymes contain a C2 motif and have a molecular weight of approximately 76 kD (Nishizuka, 1992). A PKC activity has been identified and partially characterized in sea urchin eggs, although this activity was localized to the cytosol (Shen and Ricke, 1989). A protein of approximately 49 kD was detected in the cytosolic fraction. No immunoreactive proteins were detected using monoclonal antibody ID 12 (results not

82 kD

- 8 0

-49.5

-32.5

-18.5

Figure 3.3 Is synaptotagmin expressed in sea urchin eggs?

Sea urchin egg subcellular protein fractions (10 pg protein per lane) were resolved by SDS-PAGE and transferred to a PVDF membrane by Western blotting. Immunoreactive proteins were detected using a polyclonal antibody against a conserved region of chick brain synaptotagmin and were visualized using enhanced chemiluminescence. The positions of molecular-mass standards are shown on the right.

83 shown).

These results show no evidence to support the presence of a synaptotagmin homologue in sea urchin eggs. Several proteins containing C2 domains were detected, one of which, a 78 kD protein, may be a PKC family member.

5. IS THE ALPHA-LATROTOXIN RECEPTOR PRESENT IN SEA URCHIN EGGS?

Alpha latrotoxin (a-LTX) is a potent neurotoxin, which is the major constituent of black widow spider venom. a-LTX acts presynaptically in vertebrates at nanomolar concentrations, where it induces a massive quantal release of most, perhaps all, types of neurotransmitter (Scheer et al, 1986; Meldolesi et a l, 1983). A receptor for a-LTX has been purified fi"om brain (Scheer and Meldolesi, 1985; Petrenko et a l, 1990) and was the first member of a novel class of neuronal cell surface proteins now known as the neurexins (Ushkaiyov et al, 1992). The receptor is composed of four subunits; two proteins of 200 kD and 160 kD which both bound a-LTX with high affinity and two others of 79 kD and 43 kD that did not bind toxin at the nanomolar concentrations at which a-LTX is active. The latter two subunits, P and y respectively, are peripheral membrane proteins, extractable in high salt buffer (Surkova, 1990). Although the effects of a-LTX are restricted to tissues and cells of neuronal origin (Finkelstein, et a l, 1976), homologues of the P- and y-subunits have been shown to be present in a-LTX-insensitive cells (Petrenko e ta l, 1993).

The effects of a-LTX on sea urchin eggs have been investigated. Falugi and Prestipino (1989), reported that treatment of unfertilized sea urchin eggs with nanomolar amounts of a-LTX neither triggered cortical granule exocytosis, nor prevented the elevation of the fertilization envelope upon sperm-egg fusion. Treatment of fertilized eggs with a-LTX, however, blocked egg development and caused noticeable alterations in cell surface topography. The authors explained these results by proposing that either a-LTX

84 receptors were absent in unfertilized eggs, or that the vitelline layer did not allow the toxin to reach the plasma membrane. In contrast, in fertilized eggs, the cortical reaction exposed or activated a-LTX receptors, thus allowing the toxin to display its specific activity.

I immunoblotted sea urchin egg proteins using a polyclonal antiserum (ISSl) to the 79 kD p protein of the a-LTX receptor purified from bovine brain (kind gift of Dr. I. Surkova). In the control lane, the 79 kD p protein, which was purified from bovine brain, and a characteristic degradation product of 62 kD, are strongly labelled by the antibody (Figure 3.4). In the sea urchin egg protein fractions, immunogenic proteins migrating to the same positions as the p protein and the 62 kD degradation product were detected in the whole egg lysate. In the cortical fraction, no immunogenic proteins were detected. Comparison with an identical blot incubated with pre-immune serum indicated that these bands were specifically labelled by the antiserum. Thus, a 79 kD protein, which is recognized by an antiserum to the p protein of the a-LTX receptor, is present in unfertilized sea urchin eggs but was not detected in the egg cortex. If the 79 kD protein is a homologue of the p protein of the a-LTX receptor, then these results imply that the lack of effect of a-LTX on unfertilized eggs (Falugi and Prestipino, 1989) was due to the absence of receptor proteins at the cell surface.

6. THE H)ENTIFICATION AND SUBCELLULAR LOCALIZATION OF ANNEXEVS.

The annexins are a major group of Ca^^-dependent phospholipid-binding proteins with unknown function (Drust and Creutz, 1988; Creutz, 1992), however evidence suggests a role for annexin II in exocytosis. Annexin II was shown to retard exocytotic run-down in permeabilized adrenal chromaffin cells (Ali et al., 1989; Burgoyne and Morgan, 1990; Sarafian et a l, 1991) and promoted the aggregation of chromaffin granules in vitro (Drust and Creutz, 1988). Ultrastructural studies showed annexin II to be concentrated at exocytotic sites in stimulated chromaffin cells (Nakata et a l, 1990).

85 -205

-116 -97.4

Figure 3.4 Is the a-latrotoxin receptor expressed in sea urchin eggs?

Samples of sea urchin egg proteins (10 pg protein per lane) were resolved by SDS-PAGE and transfened to a PVDF membrane by Western blotting. Immunoreactive proteins were detected using an antiseiaim to the P subunit of the a-latrotoxin receptor purified from bovine brain and were visualized using enhanced chemiluminescence. The positions of molecular-mass standards are shown on the right.

86 I looked for the presence of annexins in sea urchin eggs by immunoblotting using polyclonal antisera raised against mammalian annexins (Moss et a l, 1988; Putter et al, 1993). Figure 3.5a shows that the presence of immunoreactive proteins in whole cell lysates of approximately 34 kD, 35 kD and 68 kD was revealed by detection with antisera to human annexins I, V and VI respectively. Several other immunoreactive proteins were also detected with the antisera. In the results shown in Figure 3.5a, the antiserum to annexin I identified a major band at 34 kD and a minor band at approximately 80 kD. The antiserum to annexin V recognized minor bands at 35 kD and 55 kD and a more prominent band at 68 kD. The 68 kD protein detected with this antiserum is probably due to the antiserum cross-reacting with the 68 kD protein detected with the antiserum to annexin VI. The antiserum to annexin VI revealed a major band at 68 kD and a series of slightly smaller bands that may represent degradation products. Given that only three annexin antisera were tested in this study and all identified proteins in sea urchin eggs, it seems likely that other members of the annexin family are also present in these cells.

Using immunoblotting, I also determined the subcellular localization of the putative sea urchin annexins. Both the 34 kD and 35 kD proteins were predominantly localized to the cytosol. The localization of the 68 kD annexin varied from season to season and with different batches of sea urchins. In the experiment shown in figure 3.5b, it was concentrated in the cortical fraction, with only a small amount present in the cytoplasmic fraction. This finding is surprising in view of the fact that annexins require Ca^^ to bind to lipids and the sea urchin egg protein fractions were prepared in the presence of the Ca^^ chelator EGTA. However, annexin VI has previously been reported to be predominantly localized to the plasma membrane fraction of adrenal medullary cells, in the absence of Ca^^ (Drust and Creutz, 1991). Thus, it appears that in both sea urchin eggs and adrenal medullary cells, a 67-68 kD annexin binds to membranes in a Ca^^- independent manner.

In summary, I detected the presence of at least three annexin homologues in sea urchin eggs. Two of these homologues were localized to the cytosol, whereas one was primarily localized to the cortex. Only the 67 kD annexin could be directly involved in

87 (a) (b)

Annexin

I V VI kD kD

- 116.5

- 80

-49.5

-32.5 -49.5

-32.5

-27.5

Figure 3.5 Expression of annexins in sea urchin eggs.

(a) Samples of sea urchin egg whole egg lysate (20 pg protein per lane) were resolved by SDS-PAGE and transferred to Immobilon P by Western blotting. Immunoreative proteins were detected using antisera to human annexins I, V and VI as indicated and the products visualized using either chemiluminescence (annexins I and V) or Western Blue (annexin VI). (b) Samples of cortical and cytosolic proteins (20 pg protein per lane) were immunoblotted using the anti serum to human annexin VI and were visualized using Western Blue. The immunoreactive protein of approximately 67 kD is localized to the cortical protein fraction. The positions of molecular-mass standards are shown on the right.

88 cortical granule exocytosis in vitro, since it was the only annexin which was found associated with the cortex after isolation in the presence of EGTA. (Cortices isolated in the presence of EGTA undergo exocytosis on the subsequent addition of calcium). The mode of action of annexins in exocytosis, however, remains to be determined.

7. EXPRESSION OF CALCINEURIN IN SEA URCHIN EGGS.

In a number of different secretory systems, dephosphorylation of phosphoproteins in response to exocytotic triggering has been observed (Burnham and Williams, 1982; Gilligan and Satir, 1982; Spearman et al., 1984; Côté et al., 1986). Paramecium, a strict correlation was found between the degree of dephosphorylation of a 65 kD cortical phosphoprotein and the extent of Ca^^-stimulated exocytosis (Zieseniss and Plattner, 1985). This dephosphorylation step involved the activity of the Ca^Vcalmodulin(CaM)- dependent phosphoprotein phosphatase calcineurin (CaN), which was localized to the cell cortex (Momayezi et al., 1987). In unfertilized Strongylocentrotus intermedins eggs, a Ca^7CaM-dependent phosphoprotein phosphatase was identified which consisted of two subunits of 17 kD and 55 kD (Iwasa and Ishiguro, 1986). Peptide mapping showed the 17 kD subunit to be homologous to the 19 kD subunit (CaN B) of porcine CaN. The 55 kD subunit, however, showed less homology to the 61 kD subunit (CaN A) of porcine CaN. It was not satisfactorily determined as to whether the CaN-like phosphatase was present in isolated cortices (Iwasa and Ishiguro, 1986).

I used a polyclonal antibody against bovine brain calcineurin (kind gift of Dr. C. Klee) to determine the presence and subcellular localization of calcineurin in Lytechinus pictus eggs. Figure 3.6 shows the results of immunoblotting egg subcellular protein fractions and purified bovine brain calcineurin using this antibody. In the lane loaded with purified bovine brain calcineurin, proteins of approximately 61 kD and 16 kD were strongly immunostained. These can be assigned as CaN A and CaN B, respectively. Three other proteins of approximately 63 kD, 50 kD and 48 kD were also recognized by the antibody. A protein of approximately 62 kD was immunostained in all three sea urchin

89 egg protein fractions, although the degree of staining in the cortical fraction was much less than that seen in the cytosolic fraction and the whole egg lysate, which suggests that the protein is much less abundant in the cortex than in the cytosol. A protein which migrated with the same mobility as the 16 kD CaN B was also detected in the cytosolic fraction and the whole egg lysate but was not detected in the cortical fraction. The 62 kD and 16 kD proteins detected in the sea urchin egg protein fractions can be assigned as homologues of CaN A and CaN B, respectively. In brain extracts, CaN A is always found associated with CaN B in a Ca^^-independent manner (Klee et a l, 1988). The 55 kD and 17 kD subunits of the sea urchin egg CaN-like protein detected by Iwasa and Ishiguro (1986) were also bound to each other in a Ca^^-independent manner (Iwasa and Mohri, 1983). Therefore, it seems reasonable to assume that a homologue of CaN B is also present in the egg cortex but is present in too small a quantity for detection.

In summary, I detected the presence of a calcineurin-like protein in sea urchin eggs in both the cytosol, and to a lesser extent, associated with the cortex. In Paramecium, a CaN-like phosphatase localized to the cell cortex was implicated in exocytosis (Momayezi et al., 1987). It is possible that the cortex-associated CaN-like protein detected in sea urchin eggs is involved in exocytosis in an analogous manner. In Chapter 5 ,1 describe experiments in which I investigated the role played by CaN in cortical granule exocytosis.

8. CONCLUSION.

This chapter describes the occurrence in sea urchin eggs of several proteins which may be involved in exocytosis. I identified homologues of the synaptic proteins synaptobrevin and rab3 A in sea urchin eggs. Although immunoreactivity and molecular weight are not proof of identity, the specificity of the antibodies used for detection and the subcellular localization of the proteins suggests that they are true homologues of the synaptic proteins. I showed that both proteins are present in the egg cortex and that rab3 A is specifically localized to the cortical granules. The proteins are therefore in an appropriate location for involvement in cortical granule exocytosis. Synaptobrevin is one

90 'y

y /y / kD

- 66 m

- 45

- 29

Figure 3.6 Expression of calcineurin in sea urchin eggs.

Egg subcellular protein fractions (10 pg protein per lane) and 0.1 pg ealeineurin purified from bovine brain (Sigma) were resolved by SDS-PAGE and transferred to a PVDF membrane by Western blotting. Immunoreactive proteins were detected using a polyclonal antibody to bovine brain calcineurin and were visualized using Western blue. The positions of molecular-mass standards are shown on the right.

91 of the three proteins that form the core of a putative fusion complex proposed to function in all eukaryotic fusion events (Sollner etal., 1993a). Sogaard et al. (1994), reported that a rab protein is required for the assembly of this fiision complex. The fact that two elements of the synaptic vesicle targeting and docking complex have been found in sea urchin eggs suggests a similar exocytotic mechanism in these two systems. Although the presence of homologues of the other putative fusion proteins was not detected in sea urchin eggs by immunoblotting, evidence from other sources supports their existence (Steinhardt et al., 1994; Model - personal communication). Synaptotagmin, a protein which also interacts with the putative fusion complex (Sollner et al., 1993b), was not detected in sea urchin eggs.

I showed the presence in sea urchin eggs of a 79 kD protein immunologically related to the 79 kD p protein of the a-LTX receptor. The protein was detected in whole cell lysates of unfertilized sea urchin eggs but was not detected in the egg cortex. Since a-LTX has no effect when applied to unfertilized sea urchin eggs (Falugi and Prestipino, 1989), it is unlikely that an a-LTX receptor is involved in cortical granule exocytosis.

I identified the presence in sea urchin eggs of three annexins, one of which is in an appropriate subcellular localization for involvement in cortical granule exocytosis in vitro. I also identified the presence of a calcineurin-like protein in sea urchin eggs. It is possible that either, or both of these proteins may be involved in cortical granule exocytosis.

92 Chapter 4. A Tetanus Toxin-Sensitive Synaptobrevin Homologue is Required for Exocytosis in Sea Urchin Eggs.

1. INTRODUCTION

Synaptobrevin is an 18 kD integral membrane protein of synaptic vesicles (Baumert et al, 1989). It was first implicated in the process of neurotransmission because it was identified as the sole substrate for cleavage by tetanus toxin, a potent neurotoxin (Schiavo et a l, 1992). Tetanus toxin was shown to inhibit the release of neurotransmitters from presynaptic nerve endings. The mature toxin is a heterodimer consisting of a heavy chain linked via a disulphide bond to a light chain. The heavy chain mediates cell entry by binding selectively to target neurons. Once internalized, the light chain mediates its toxic effect by acting as a zinc-dependent endopeptidase specific for synaptobrevin (Schiavo et a l, 1992; Link et al, 1992). Homologues of synaptobrevin have been identified in Drosophila (Südhof et a l, 1989a) and in an enriched synaptic vesicle fraction isolated fi*om Torpedo electric organ (Trimble et a l, 1988). Although synaptobrevin is predominantly expressed in the nervous system, non-neuronal homologues of synaptobrevin have also been identified. A protein immunologically related to synaptobrevin was identified in adipocytes, where it was associated with vesicles responsible for the intracellular sequestration of the glucose transporter GLUT4 (Cain et al, 1992). In the yeast, Saccharomyces cerevisiae, two genes, SNCJ and SNC2, which encode homologues of vertebrate synaptobrevins, were shown to be required late in the constitutive secretory pathway (Protopopov etal, 1993). Other yeast proteins that share structural homology with Sncl and the synaptobrevin/VAMP family include Sly2/Sec22 (Dascher et a l, 1991; Newman et a l, 1992), Betl and Bosl (Newman et a l, 1992). These proteins were shown to be required for the transport of endoplasmic reticulum- derived vesicles to the Golgi. Cellubrevin, a non-neuronal homologue of synaptobrevin, was found to be ubiquitously expressed and was also cleaved by tetanus toxin light chain

93 in vitro (McMahon et al., 1993).

The evolutionary conservation of synaptobrevin suggests that it plays a central role in membrane fusion events, including exocytosis. Since intracellular membrane fusion events in all eukaryotes appear to involve similar mechanisms using identical or related components, it is possible that cortical granule exocytosis in sea urchin eggs may also employ homologues of these proteins. In this chapter I describe experiments in which I determined the subcellular localization of the sea urchin homologue of synaptobrevin, using immunoblotting of egg subcellular protein fractions and immunocytochemistry of fixed cortices. I describe experiments in which I used tetanus toxin light chain to investigate whether the synaptobrevin homologue is involved in cortical granule exocytosis. Finally, I show the results of experiments in which I demonstrated the interaction between the sea urchin synaptobrevin homologue and a recombinant syntaxin fragment cloned from a bovine adrenal medullary cDNA library.

2. THE SUBCELLULAR LOCALIZATION OF THE SYNAPTOBREVIN HOMOLOGUE.

(i) Immunoblot Analysis of Subcellular Protein Fractions.

I investigated the intracellular distribution of the sea urchin synaptobrevin homologue by immunoblotting various egg subcellular protein fractions. If there are similarities between vertebrate and invertebrate exocytotic systems, one would expect to find homologous proteins at homologous locations in order to mediate general exocytotic functions. Accordingly, if the sea urchin synaptobrevin homologue performs an analogous role to its neuronal counterpart, then it would be expected to be an integral membrane protein of cortical secretory vesicles.

Figure 4.1 shows the results of immunoblotting with monoclonal antibody Cl 10.1, which recognizes multiple synaptobrevin isoforms (kind gift of Dr. R. Jahn). The

94 - 18.9 kD

Figure. 4.1 The subcellular localization of the 17.5 kD synaptobrevin homologue.

Egg subcellular protein fractions (50 pg protein per lane) were resolved by SDS-PAGE and transferred to a PVDF membrane by Western blotting. The blot was labelled for synaptobrevin using monoclonal antibody Cl 10.1 and visualized using enhanced chemiluminescence. The position of a molecular-mass standard is shown on the right.

95 synaptobrevin homologue is present in both intracellular membranes and in the egg cortex. When cortical proteins are further subdivided into plasma membrane proteins and cortical granule-associated proteins, it is clear that the protein is specifically localized to the cortical granule fraction and none is present in the plasma membrane fraction. This localization is analogous to that of neuronal synaptobrevin which follows the distribution of small synaptic vesicles during subcellular fi*actionation (Baumert et a l, 1989). The presence of the protein in the cytosolic fraction is difficult to explain as synaptobrevin is an integral membrane protein. However, I performed this Western blot at least six times and detected the immunoreactive protein in the cytosolic fraction only in this experiment. I therefore concluded that its presence is probably due to contamination of the protein fraction with egg membranes during preparation.

(ii) Immunocytochemical Staining of Egg Cortices.

I used immunofluorescence confocal microscopy to further clarify the distribution of the synaptobrevin homologue within the cortical region. I fixed egg cortices and after rehydration in aldehyde quench solution, cortices were incubated with the monoclonal antibody Cl 10 . 1 in blocking buffer. The cortices were then stained with a fluorescein- conjugated secondary antibody and visualized using confocal microscopy. Figure 4.2a shows that immunostaining results in a "honeycomb-like" pattern of staining which is indicative of staining of the cortical granule membranes. No staining of granule contents is observed, which is expected since synaptobrevin is an integral membrane protein.

For comparison, I stained living cortices with the dicarbocyanine, DilCjg, which stains cortical endoplasmic reticulum (ER) (Henson e ta l, 1989; Terasaki etal., 1991). Dil has a fluorescent head group with two long hydrocarbon chains and Dil suspensions stain membranes by random collisions of the Dil aggregates. The hydrocarbon chains intercalate into membrane bilayers. Once within a bilayer leaflet, Dil remains in the leaflet but drffiases fi'eely within that leaflet (Honig and Hume, 1986). Although Dil suspensions do not stain ER membranes specifically, the ER is stained because it is widespread and because the continuity of the ER allows dye spreading.

96 Figure 4.2 Immunocytochemical staining to show the distribution of the synaptobrevin homologue within the egg cortex..

Fixed egg cortices were immunostained using monoclonal antibody Cl 10.1 and were visualized using a fluorescein-conjugated secondary antibody and confocal microscopy: (a) horizontal section; (b) vertical section. For comparison, unfixed cortices were stained with DiIC,g, to show endoplasmic reticulum staining, (c) horizontal section; (d) vertical section. Bar, 10 pm.

97 98 Figure 4.2c shows the results of staining unfixed cortices with a 75 |ig/ml Dil suspension. An extensive reticular network is stained by this process which is characteristic of endoplasmic reticulum. Figure 4.2d shows a zx scan through cortices which were stained using a 1.5 mg/ml Dil suspension. At this higher dye concentration, the probability of dye aggregates colliding with membranous structures is greatly increased. Nevertheless, only a few cortical granules were stained. Three stained granules are visible in the centre of the figure and these clearly show the specific membrane staining. The other granules which were stained are out of the plane of the scan and are visible as circles of bright staining. Staining of the plasma membrane is also evident. The fluorescence due to ER staining is probably masked by the signal from the plasma membrane. Figure 4.2b shows an xz scan through the immunostained fixed cortices of Figure 4.2a. The cortical granule membrane is clearly immunostained, whereas the plasma membrane is unstained. A comparison of these two images indicates that the monoclonal antibody Cl 1 0 . 1 specifically stains the cortical granule membrane.

3. CLEAVAGE OF THE SYNAPTOBREVIN HOMOLOGUE BY TETANUS TOXIN LIGHT CHAIN.

(i) Cleavage of the Synaptobrevin HomologueIn Vitro.

Because tetanus toxin is specific for members of the synaptobrevin protein family, structural similarities between synaptobrevin and the sea urchin egg 17.5 kD protein would be demonstrated if they were both recognized by tetanus toxin. Both synaptobrevin II and its cellular homologue, cellubrevin, were shown to be cleaved by tetanus toxin light chain (TeTx LC) in vitro at 37 °C (Schiavo et al., 1992; Link et at., 1992; McMahon et al., 1993). In order to determine whether TeTx LC cleaves the sea urchin synaptobrevin homologue in vitro, I prepared a whole egg membrane fraction in IM and incubated it with 270 nM TeTx LC at 37 °C. After incubation at 15, 30, 60 and

1 2 0 minutes, protein aliquots were withdrawn, concentrated by chloroform/methanol extraction and dissolved in SDS-PAGE sample buffer. Samples were separated on a 12

99 % polyacrylamide gel (10 pg/lane) and electrophoretically transferred onto a PVDF membrane. Figure 4.3a clearly shows that the sea urchin synaptobrevin homologue was cleaved by TeTx LC under these conditions. As judged by immunoblot analysis, most of the synaptobrevin molecule was digested within the first 15 minutes of incubation and complete digestion was obtained afl;er 30 minutes (Figure 4.3a - upper panel). As a control, the same blot was incubated with monoclonal antibody Cl 42.1, which recognizes the cortical granule protein rab3 A. The level of rab3 A was not affected by toxin treatment even after 1 2 0 minutes, as determined by immunoblot analysis, indicating that the cleavage of the synaptobrevin homologue was specific. In a parallel experiment in which no toxin was added, the level of the synaptobrevin homologue remained unchanged (Figure 4.3a - lower panel). The cleavage products of toxin digestion were not detected in this polyacrylamide gel/blot system, although two fragments of 6 - 8 and 11-13 kD, resulting from cleavage at a site similar to the one determined in rat and Aplysia synaptobrevins (Schiavo et al., 1992), should be recovered. These results clearly show that the sea urchin egg synaptobrevin homologue is specifically cleaved in vitro by tetanus toxin light chain.

(ii) Incubation of Cortices with Tetanus Toxin Light Chain.

In order to demonstrate the involvement of the synaptobrevin homologue in cortical granule exocytosis, it was necessary to determine whether the toxin would cleave the protein when applied directly to isolated cortices. This was important in view of the findings by some groups that synaptobrevin was not susceptible to cleavage by tetanus toxin when assembled into the 7S heterotrimeric complex that was postulated to mediate vesicle docking at the plasma membrane (Hayashi et a l, 1994; Pellegrini et al., 1994). Sea urchin eggs fijnction optimally at 16 °C, their normal physiological temperature. I incubated isolated cortices for up to 2 hours in the presence o f270 nM TeTx LC at 16 °C before stimulating exocytosis by the addition of 17 pM Ca^^. At this temperature, incubation with TeTx LC had no effect on the exocytotic reaction in vitro (Table 4.1). In both toxin and control incubations, the cortical response to calcium diminished over time. This rundown effect has been reported previously (Moy a/., 1983). After parallel incubations, cortical proteins were dissolved in SDS-PAGE sample buffer and analysed

100 (a)

Time (min.) 0 15 30 60 120

+ TeTx LC

- TeTx LC

(b)

Synaptobrevin

RabSA

Figure 4.3 Tetanus toxin light chain specifically cleaves the 17.5 kD synaptobrevin homologue in vitro.

(a) A membrane protein fraction was incubated either in the presence {upper panel), or absence {lower panel), of 270 nM TeTx LC at 37 °C. Samples were taken at the times indicated. Proteins (10 pg/lane) were resolved by SDS-PAGE and immimoblotted using monoclonal antibody Cl 10.1. (b) Cortices were incubated either in the presence (+), or absence (-), of270 nM TeTx LC for 15 min. at 37 °C. Cortical proteins were then dissolved in SDS-PAGE sample buffer, separated by SDS-PAGE (20 pg protein per lane) and transferred to a PVDF membrane by Western blotting. The blot was cut horizontally between rab3 A and synaptobrevin and each part was immunostained using the respective antibody. Synaptobrevin was completely proteolysed by TeTx LC {middle panel). In contrast, no breakdown of rab3 A was observed {lower panel).

101 by polyacrylamide gel electrophoresis and immunoblotting. Immunoblotting with monoclonal antibody Cl 10.1 revealed that under these incubation conditions, the synaptobrevin homologue was not cleaved by TeTx LC.

Table 4.1 The effect of TeTx LC on exocytosis in vitro at 16 °C

% Exocytosis Incubation (min.) Control + TeTx LC

30 99.6 +/- 0.3 95.3 +/-3.3 60 81.3 +/- 12.0 92.3 +/- 7.7 90 61.3 +/- 17.7 . 61.3+/-11.4

1 2 0 48.0+/-23.0 38.7+/-21.5

Egg cortices were incubated with 270 nM tetanus toxin light chain at 16 °C for the times indicated. After incubation, the cortices were rinsed briefly in IM and then analysed using phase contrast microscopy. Exocytosis was stimulated by the addition of 5.9 pM Ca^^ and was measured 3 minutes after Ca^^ addition. Mean +/- s.e.m. are shown, n=4.

As the sea urchin synaptobrevin homologue was cleaved by incubating isolated membrane proteins with TeTx LC at 37 °C, I decided to test whether it was possible to cleave the protein by incubating isolated cortices with TeTx LC at 37 °C. Firstly, I looked to see if the cortices were still viable under these conditions. The results presented in Figure 4.4 show that cortices underwent exocytosis after incubation at 37 °C, although the extent of the response was diminished in comparison to that of controls. This effect was probably due to some dénaturation of sea urchin egg proteins which are involved in mediating the exocytotic response. The rate of many enzyme-catalysed reactions are approximately doubled if the temperature is increased by 10 °C (Scheve, 1984). If this is the case for tetanus toxin, then a rise in temperature from 16 °C to 37 °C would approximately quadruple the reaction rate. Therefore, although 37 °C is non- physiological, I decided to conduct experiments at this temperature.

102 1 0 0

16 X

80 -

(g 60 - w I 40 -

20 -

0 5 15 Incubation time (min.)

Figure 4 .4 The effect of temperature on exocytosis in vitro.

Ca^^-stimulated exocytosis in vitro was measmed after incubation at either 16 °C or 37 °C, for 5 or 15 minutes. After incubation, cortices were stimulated to undergo exocytosis at 16 °C by the addition of 5.9 ^iM Ca^". Cortices were viewed using phase contrast microscopy and exocytosis was measuied 1 minute after Ca^^ addition. The results were obtained using two different urchins on different days. Mean +/- s.e.m. are shown, n= 6 .

103 (iii) Incubation of Cortices with TeTx LC at 37 “C.

Cortices prepared in IM were incubated with 270 nM TeTx LC for 15 minutes at 37 °C. Proteins were then dissolved in Triton X-100 and precipitated by chloroform/methanol extraction. Analysis of cortical proteins by immunoblotting with monoclonal antibody Cl 10.1. showed that the synaptobrevin homologue was specifically digested by incubation of cortices with TeTx LC (Figure 4.3b - upper panel). The TeTx LC was selective for the synaptobrevin homologue since another cortical granule- associated protein, rab3A, was not affected by the toxin (Figure 4.3b - lower panel). The results indicate that complete digestion occurred after 15 minutes. This experiment was repeated three times and each time, complete digestion of the synaptobrevin homologue was obtained after 15 minutes incubation.

In summary, the results show that at 37 °C, TeTx LC cleaves the sea urchin synaptobrevin homologue, indicating structural similarities with both neuronal synaptobrevin II and cellubrevin. The results also indicate that the synaptobrevin homologue is accessible for cleavage by TeTx LC, even when granules are docked at the plasma membrane.

4. TETANUS TOXIN LIGHT CHAIN INHIBITS CORTICAL GRANULE EXOCYTOSIS IN VITRO.

Schiavo et a l (1993) correlated the decrease in exocytotic response in Aplysia neurons with cleavage of synaptobrevin. Since I have shown that a sea urchin homologue of synaptobrevin is cleaved by TeTx LC, I determined the effect of this cleavage on cortical granule exocytosis. I conducted the same experiments as those described in section 2(iii), except for the isolation of cortical proteins. Instead, cortices were rinsed briefly in IM and analysed using phase contrast microscopy. Exocytosis was stimulated by addition of 5.9 pM free Ca^^ and was measured one minute after calcium addition. Exocytosis was scored as the proportion of cortical granules that fused with the plasma

104 1 0 0 Control + 270 nM TeTx LC

o< 0

g LU

5 15 Time of incubation with TeTx LC (min.)

Figure 4.5 Tetanus toxin light chain inhibits cortical granule exocytosis when applied to isolated cortices.

Egg cortices were preincubated at 37 °C for 5 or 15 minutes, either in the presence or absence of 270 nM tetanus toxin light chain. Cortices were viewed using phase contrast microscopy. Exocytosis was stimulated by the addition of 5.9 pM free Ca^^ and was measuied 1 mmute after Ca^^ addition. Mean +/- s.e.m. are shown, n= 6 .

105 membrane. Figure 4.5 indicates that cortices incubated at 37 °C in the absence of TeTx LC, underwent 64 % and 55 % exocytosis, after incubation for 5 and 15 minutes, respectively. These values are comparable to those obtained in Figure 4.4 (64 % and 52 %, respectively). Incubation with 270 nM TeTx LC caused a time-dependent inhibition of exocytosis. Exocytosis was inhibited by 43 % after 5 minutes and 63 % after 15 minutes (Figure 4.5). These results indicate that TeTx LC inhibits cortical granule exocytosis in sea urchin eggs.

5. THE SEA URCHIN SYNAPTOBREVIN HOMOLOGUE INTERACTS WITH A RECOMBINANT SYNTAXIN FRAGMENT IN VITRO,

Synaptobrevin, syntaxin and SNAP-25 have been shown to interact in vitro to form both binary and ternary complexes (Sollner g/ a/., 1993; Hayashi a/., 1994). The regions within each molecule that are responsible for these interactions have been identified and were shown to be regions which have a high propensity to form a-helices (Hayashieta l, 1994). Hodel et al. (1994) produced a soluble, biotinylated syntaxin lA fragment (biotinylated (his) ^syntaxirf) cloned from a bovine adrenal medullary cDNA library. The fragment contains the region of the molecule shown to interact with both synaptobrevin and SNAP-25 (Hayashi et al., 1994). In immunoblots of different subcellular fractions from both mouse brain and bovine adrenal medulla, (his)^syntaxirf interacted specifically with SNAP-25 in the plasma membrane fractions of both species. No interactions with proteins of a purified bovine adrenal chromaflSn granule fraction were detected (Schafer - personal communication). Interacting proteins were detected using peroxidase-coupled avidin and visualized using enhanced chemiluminescence. This assay can be used to identify proteins that may be components of, or interact with, the heterotrimeric fusion complex.

I used this overlay assay, firstly to determine whether the sea urchin egg synaptobrevin homologue will interact with (his)^syntaxirf and secondly, to look for the presence of homologues of SNAP-25 in sea urchin eggs. No homologues of either

106 /

y y kD

-26.1

- 18.9

-7.3

Figure 4.6 Sea urchin egg synaptobrevin interacts with a soluble recombinant syntaxin fragment cloned from a bovine adrenal medullay cDNA library.

Egg subcellular protein fractions (50 pg protein per lane) were resolved by SDS-PAGE and transfen ed to a PVDF membrane by Western blotting. The blot was incubated with a soluble, biotinylated, recombinant s>nta\in Ifagment cloned from a bovine adrenal medullary cDNA libraiy. Interacting proteins were detected by incubation with peroxidase-coupled avidin and were visualized using enhaneed ehemiluminescence.

107 SNAP-25 or syntaxin were identified by immunoblot analysis (see Chapter 3).

Sea urchin egg subcellular protein fi-actions were separated by SDS-PAGE and electrophoretically transferred to a PVDF membrane. After blocking, the membrane was incubated with 500 ng/ml (his)^syntaxirf dissolved in blocking buffer. Interacting proteins were detected by incubation with peroxidase-coupled avidin and visualized using enhanced chemiluminescence. As shown in Figure 4.6, the (his)^syntaxirf fragment interacted with a protein of approximately 17.5 kD in the cortical granule fraction. This corresponds in molecular weight to the protein recognized by the monoclonal antibody to synaptobrevin. Cl 10.1, in Figure 4.1. The (his)^syntaxirf fvdLgmQni also interacted with a protein of a slightly lower molecular weight, approximately 16.5 kD, in the cytosolic fraction. This interaction may either be non-specific or may be due to the presence of an, as yet unidentified, constituent of the membrane fusion complex. One candidate for such a protein is a member of the complexin family. Complexins are hydrophilic proteins that have been shown to interact with syntaxin (McMahon et ai, 1995). Interestingly, the (his)^syntaxirf fragment did not interact with the synaptobrevin homologue present in the whole egg membrane fraction which was recognized by monoclonal antibody Cl 10.1 in Figure 4.1. No bands were visible in the plasma membrane fraction, the fraction in which homologues of SNAP-25 would be predicted to be localized. Subsequent work has shown the (his)^syntaxirf fragment to interact with a protein of approximately 25 kD in the plasma membrane fraction of sea urchin eggs which may be a homologue of SNAP-25 (A. Hodel - personal communication).

The interaction of a synaptobrevin homologue from sea urchin eggs with a syntaxin fragment cloned from a bovine adrenal medullary cDNA library supports the hypothesis that the molecular machinery underlying membrane fusion in exocytosis is evolutionarily conserved.

108 6. CONCLUSION.

In this chapter I showed that the sea urchin homologue of synaptobrevin is present in membranous compartments throughout the egg, including the egg cortex. Using immunocytochemical confocal microscopy I showed that, within the egg cortex, the protein is localized to the cortical granule membrane.

I showed that TeTx LC specifically cleaves the synaptobrevin homologue in vitro. The cleavage site of the light chain remains to be established since no cleavage products were detected by immunoblotting. TeTx LC also cleaves the synaptobrevin homologue when applied directly to isolated cortices. Under the same incubation conditions, I showed that TeTx LC causes an inhibition of Ca^^-stimulated exocytosis. Taken together, the results show that the inhibition of cortical granule exocytosis in sea urchin eggs by TeTx LC is associated with a specific degradation of cortical synaptobrevin. One discrepancy in the results is that although immunoblot analysis indicates complete proteolysis of the sea urchin synaptobrevin homologue after 15 minutes incubation of cortices with TeTx LC, cortical granule exocytosis is only decreased by 63 % under the same incubation conditions. The residual exocytotic activity may be due to the existence of an exocytotic mechanism that is independent of synaptobrevin function.

Finally, I showed that the sea urchin egg synaptobrevin homologue interacts with a recombinant syntaxin 1A fragment in vitro. This observation supports the hypothesis that the membrane fusion machinery has been structurally conserved during evolution. This observation also suggests that, although no sea urchin egg homologues of syntaxin and SNAP-25 have been identified, the fusion machinery functioning in cortical granule exocytosis may contain functionally related proteins.

109 Chapter 5. The Role of Calcineurin in Cortical Granule Exocytosis.

1. INTRODUCTION.

In sea urchin eggs, irreversible phosphorylation of proteins associated with the egg cortex, using adenosine-5'-0-(3-thiotriphosphate) (ATPyS), inhibited exocytosis (Whalley et al., 1991). ATPyS can be used as a phosphoryl donor by protein kinases but the resulting thiophosphoproteins are not readily dephosphorylated by phosphoprotein phosphatases (Gratecos and Fischer, 1974). It was therefore proposed that in sea urchin eggs, specific dephosphorylation of one or more phosphoproteins associated with the egg cortex was required for exocytosis (Whalley et at., 1991). Protein dephosphorylation has been proposed to be an exocytotic trigger in other secretory systems (Brooks et al., 1984; Brooks and Brooks, 1985; Gilligan and Satir, 1982; Zieseniss and Plattner, 1985; Momayezi et al., 1987; Tatham and Gomperts, 1989). In Paramecium, the rapid dephosphorylation of a 65 kD cortical phosphoprotein was shown to be required for exocytosis (Gilligan and Satir, 1982; Zieseniss and Plattner, 1985). This dephosphorylation was mediated by a Ca^Vcalmodulin-dependent, calcineurin-like phosphatase associated with the cell cortex (Momayezi et al., 1987). In both Paramecium and sea urchin eggs, calmodulin and calcineurin have been localized to the cell cortex (Momayezi et al., 1986, 1987; Steinhardt and Alderton, 1982; Chapter 3). Thus, it is possible that exocytosis in sea urchin eggs proceeds in a manner analogous to exocytosis in Paramecium.

Calcineurin is probably the best candidate for a phosphatase involved in exocytosis in sea urchin eggs, since there is evidence to show an involvement of calmodulin in exocytosis in this system. An antibody against calmodulin, which was localized to the cytoplasmic face of the plasma membrane, inhibited exocytosis in vitro (Steinhardt and Alderton, 1982) and the phenothiazine drug trifluoperazine, which is a calmodulin

110 antagonist, inhibited exocytosis both in vivo and in vitro (Baker and Whitaker, 1979; Whitaker and Baker, 1983). Although these authors suggested that the effect of trifluoperazine might be due to a non-specific hydrophobic effect on the cell membrane, trifluoperazine specifically inhibited the activity of purified bovine brain calcineurin at concentrations comparable to those that inhibited exocytosis in vitro (Stewart et al., 1983; Whitaker and Baker, 1983). The only evidence which argued against an involvement of calcineurin in exocytosis was the observation that okadaic acid inhibited exocytosis in vivo at micromolar concentrations (Whalley et al., 1991). Okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A and a much less potent inhibitor of calcineurin (Cohen et al, 1990). However, it is possible that okadaic acid mediated its effect on upstream regulatory elements since it had no effect on exocytosis in vitro.

In this chapter I describe experiments in which I investigated the involvement of calcineurin in exocytosis in sea urchin eggs. Firstly, I describe experiments in which I used a peptide antagonist of calmodulin to confirm the involvement of calmodulin in exocytosis. Secondly, I present the results of experiments in which I determined the effect of specific inhibitors of calcineurin on exocytosis, both in vivo and in vitro. Finally, I show results in which I characterised the phosphatase activity in sea urchin egg cytosol, which I correlated with previous findings from this laboratory.

Cortices isolated in intracellular medium exhibited variation in Ca^^-sensitivity from season to season, as reported previously by Sasaki and Epel (1983). The concentration of calcium used to trigger exocytosis therefore varied between experiments and was chosen to give approximately 1 0 0 % exocytosis in the controls.

2. THE EFFECT OF A MLCK PEPTH)E ON EXOCYTOSIS.

A peptide antagonist of calmodulin, the myosin light chain kinase (MLCK) peptide (Torok and Trentham, 1994), was used to examine whether a calmodulin-dependent enzyme is involved in exocytosis in sea urchin eggs. The MLCK 7 9 7 . 8 1 3 peptide is a peptide

111 sequence (residues 797-813) derived from the calmodulin binding site of MLCK (Kemp et al., 1987). It binds to calmodulin in a Ca^^-dependent manner and acts as a specific inhibitor of Ca^Vcalmodulin-dependent signalling (Kemp et a l, 1987). The end groups of the peptide are blocked by acétylation of the N-terminus and amidation of the C- terminus (Torok and Trentham, 1994). These modifications enable the peptide to bind calmodulin very tightly: its equilibrium dissociation constant (K J for calmodulin was measured to be 0.005 nM in vitro (Torok and Trentham, 1994). The residues responsible for hydrophobic interactions with calmodulin are Trp*®° and Leu*^^ (Torok and Whitaker, 1994). Proteins can bind calmodulin in several different ways (Torok and Whitaker, 1994). A comparison of the calmodulin-binding domains of MLCK and calcineurin indicated that the two sites were approximately 81 % identical, suggesting that the two proteins bound calmodulin in a similar manner (Kincaid et al., 1988). A comparison of the amino acid sequences of the calmodulin-binding domains of calcineurin, MLCK and the MLCK7 9 7 . 8 1 3 peptide are shown in Table 5 .1. The sequences are taken fi-om Kemp et al. (1987), Kincaid et al. (1988) and Torok and Trentham (1994). Because of this similarity, the MLCK peptide might be expected to antagonize calcineurin-mediated reactions more specifically than the more general calmodulin antagonists used previously.

In order to determine the effect of the MLCK 7 9 7 . 8 1 3 peptide on calcineurin activity I analysed its effect on the activity of purified bovine brain calcineurin, using /?- nitrophenylphosphate as substrate. Unfortunately, it was not possible to detect calcineurin activity in sea urchin egg homogenates using this colorimetric method, due to the presence of calmodulin- and calcium-independent phosphatases (results not shown). Thus, the effect of the MLCK7 9 7 . 8 1 3 peptide on calcineurin activity could not be determined. However, since structural similarities have been demonstrated between calcineurin A from different species (Hubbard and Klee, 1989) and since the antibody that recognized sea urchin egg calcineurin also recognized bovine brain calcineurin, indicating structural similarities between these two proteins, the effect of the peptide on the activity of bovine brain calcineurin should qualitatively reflect its effect on the activity of sea urchin egg calcineurin.

112 Table 5.1 Sequence and properties of the calmodulin-binding domains of calcineurin, MLCK and the MLCK 7 9 7 . 8 1 3 peptide.

Protein/peptide Sequence Kj for calmodulin (nM)

Calcineurin ARKE- - VI RNKI R AI G K MA 0.1

MLCK ARRKWQKTGHAVRAIGRLS 0.11-0.24

MLCK7 9 7 . 8 1 3 peptide NAc - R R K WQ KTGHAVRAI GRL - CONH2 0.005

A comparison of the amino acid sequences of the calmodulin binding domains of calcineurin, MLCK and the MLCK 7 9 7 .8 1 3 peptide. Hydrophobic residues responsible for the interaction with calmodulin are shown in bold. The equilibrium dissociation constant (K J values for calmodulin binding are also shown. The sequences and Kj values are taken from Kemp et al. (1987), Kincaid et a l (1988) and Torok and Trentham (1994). 100

> I c 60 3 (Ü I 40 (Q ^ 20

0 2 4 6 8 10 [MLCK797.^g3 peptide] (pM)

Figure 5.1 The effect of the MLCK7 9 7 . 8 1 3 peptide on calcineurin activity.

The phosphatase activity of purified bovine brain calcineurin was assayed using 0.16 pM calcineurin in 50 mM Tris-HCl, p H 7.0,2.5 mMNiClj, 0.25 mg/ml BSA in the presence of 0.1 mM Ca^^, 0.38 pM calmodulin and the indicated concentration ofMLCKyg?^,] peptide. 3.4 raM PNPP was used as substrate. All assays were performed in microtiter assay plates in triplicate and phosphatase activity was monitored by measuring the absorbance at 405 nm. Mean +/- s.e.m. are shown, n=3.

114 The MLCK7 9 7_gi 3 peptide potently inhibited the activity of purified bovine brain calcineurin, with an half maximal inhibitory concentration (I.C.50) value of approximately 1 pM (Figure 5.1). Complete inhibition of calcineurin activity was obtained using 5 pM

MLCK7 9 7 . 8 1 3 peptide.

Since in Xenopus oocytes, the cytosolic calmodulin concentration was shown to be minimally 34 pM (Cartaud et al., 1980), I microinjected the MLCK 7 9 7.gi 3 peptide (50 mM in 125 mM KCl, 0.25 M EGTA, 5 mM PIPES, pH 6.7) to a final cytoplasmic concentration of 100 pM in order to ensure that sufficient peptide was injected to inhibit calcineurin activity in vivo. After microinjection, the eggs were left for 15-20 minutes and were examined for exocytosis, as judged by elevation of the fertilization envelope. This concentration of the MLCK 7 9 7_gi 3 peptide did not cause exocytosis in any of 19 eggs. When the eggs were subsequently inseminated, all raised complete fertilization envelopes (Table 5.2).

Table 5.2 The effect of the MLCK7 9 7.gi 3 peptide on exocytosis in vivo.

Treatment Number of eggs % Exocytosis microinjected

MLCK797_gj3 19 100

Control 19 100

The MLCKygy^ij peptide was injected to give a final cytoplasmic concentration of 100 pM. Eggs were fertilized 15 minutes after injection. Exocytosis was scored by counting the number of eggs which raised complete fertilization envelopes. Control eggs were injected with vehicle only.

The identification of calmodulin in cortices which were isolated in the presence of 10 mM EGTA (Steinhardt and Alderton, 1982) suggests that calmodulin is interacting in

115 a Ca^^-independent manner with a cortical protein. There have been many reports of Ca^^- independent binding of calmodulin (Noland et al., 1985; Olson et al., 1985; Aitken et al., 1988; Hernandez et al., 1994). The Ca^Mndependent interaction of proteins with calmodulin was proposed to play a role in calmodulin compartmentalisation (Hernandez et al., 1994). It can be hypothesised that upon the addition of calcium to cortices, the Ca^VCaM complex could be displaced from its original binding site by binding to calcineurin. This proposal is supported by the fact that the affinity of calcineurin for the Ca^VCaM complex is very high (K^ = 0.2 x 10^ M'^), and it has been reported to displace most other calmodulin-binding proteins from calmodulin (Klee and Krinks, 1978). The inhibitory activity of the antibody against calmodulin on exocytosis, found by Steinhardt and Alderton (1982), could be explained by supposing that the binding of antibody prevented the interaction of calmodulin with its target, i.e. calcineurin, upon calcium addition.

I incubated cortices with 100 pM MLCK 7 9 7 . 8 1 3 peptide for 45 minutes at 16 °C, after which exocytosis was stimulated using 5.9 pM Ca^^. As a control, cortices were incubated with a MLCK control peptide, in which the two hydrophobic residues involved in calmodulin binding were replaced by glutamate residues. Glutamate substitution was chosen to further prevent binding of the peptide to calmodulin due to charge repulsion. The MLCK control peptide was measured to have a K^ for calmodulin of 11.8 pM and did not significantly inhibit calmodulin-dependent myosin light chain kinase activity in vitro (Torok and Trentham, 1994). Preincubation with the MLCK 7 9 7 . 8 1 3 peptide inhibited Ca^Lgtimulated exocytosis in vitro by approximately 30 % (Table 5.3). Exocytosis was measured to be 74% as compared to 96 % after incubation with the MLCK control peptide and 98 % after incubation with buffer only.

116 Table 5.3 The effect of the peptide on exocytosisin vitro.

Treatment % Exocytosis

MLCK7 9 7 . 8 1 3 73.88 +/- 3.55

MLCK control peptide 95.88 +/- 2.10

Control 98.00+/-0.71

Cortices were incubated with 100 ^iM MLCKyg 7 _g, 3 or MLCK control peptide in IM, pH6.7, for 45 minutes at 16 °C. Control cortices were incubated in IM only. Exocytosis was stimulated by addition of buffer containing 5.9 pM free Ca^^. Cortices were viewed using phase contrast microscopy and

exocytosis was measured 1 minute after Ca^^ addition. Mean +/- s.e.m are shown, n = 8 for peptide

incubations and n - 6 for incubation with buffer only.

In summary, the MLCK7 9 7 . 8 1 3 peptide, a peptide antagonist of calmodulin, had no effect on exocytosis in vivo but inhibited exocytosis in vitro by approximately 30 %.

3. THE EFFECT OF CALCINEURIN INHIBITORS ON EXOCYTOSIS.

(i) An Antibody to Calcineurin has No Effect on Exocytosis.

In Chapter 3 ,1 showed that a polyclonal antibody against bovine brain calcineurin (CaN Ab) (Momayezi et al, 1987) recognized two proteins of approximately 16 kD and 61 kD in sea urchin eggs. These proteins correspond in molecular weight to the A and B subunits of bovine brain calcineurin, respectively. The immunoreactive proteins were detected in both cytosolic and cortical protein fractions. The same antibody that was used in immunoblot analysis also recognized a calcineurin-like protein in Paramecium (Momayezi et a l, 1987). This antibody inhibited trichocyst release in response to an exocytotic trigger when microinjected into Paramecium and also inhibited exocytosis in vitro when applied to the isolated cortices of these cells (Momayezi et a l, 1987). I

117 investigated the effect of this polyclonal CaN Ab on exocytosis in sea urchin eggs.

The polyclonal CaN Ab inhibited the activity of bovine brain calcineurin in vitro at a concentration of 30 nM (C. Klee, personal communication). I microinjected the polyclonal CaN Ab (5 mg/ml in 125 mM KCl, 250 mM EGTA, 5 mM PIPES, pH 6.7) into sea urchin eggs to give a final cytoplasmic concentration of 25 pg/ml (170 nM). This concentration is approximately six times the concentration that inhibited the phosphatase activity of purified bovine brain calcineurin in vitro. After incubation for 15 minutes, eggs were inseminated and the number of eggs that raised fertilization envelopes was counted. The CaN Ab had no effect on exocytosis in vivo. Out of 20 eggs injected with the antibody, all raised complete fertilization envelopes (Table 5.4).

Table 5.4 The effect of an antibody to calcineurin on exocytosis in vivo.

Treatment Number of eggs % Exocytosis microinjected

Calcineurin Ab 20 100

Control 20 100

The antibody against calcineurin was injected to give a final cytoplasmic concentration of 25 pg/ml. Eggs were fertilized 15 minutes after injection. Exocytosis was scored by counting the number of eggs which raised complete fertilization envelopes. Control eggs were injected with vehicle only.

The effect of the antibody on exocytosis in vitro was then investigated. Cortices were isolated and then incubated with the antibody at a final concentration of 50 pg/ml (340 nM). After 30 minutes, the cortices were rinsed and exocytosis was stimulated by addition of 17 pM Ca^^. The antibody had no inhibitory effect on Ca^^-stimulated exocytosis in vitro. Exocytosis was 97.99 % as compared to 91.68 % in control incubations (Table 5.5).

118 Table 5.5 The effect o f an antibody to calcineurin on exocytosisin vitro.

Treatment % Exocytosis

CaNAb 97.99+/- 2 . 0

Control 91.68+/-5.4

Cortices were incubated with 50 pg/ml CaN Ab for 30 minutes at 16 °C. Control cortices wer incubated in IM only. Cortices were viewed using dark field microscopy. Exocytosis was stimulated by the addition of 17 pM Ca^^ and was measured by measuring the degree of light scattering. Mean +/- s.e.m. are shown, n-5.

In summary, the antibody against calcineurin had no effect on exocytosis either in vivo or in vitro.

(ii) The Effect of a Peptide Corresponding to the Autoinhibitory Domain of Calcineurin.

Calcineurin A, which contains the active site of the enzyme also contains an autoinhibitory domain which interacts with the active site and prevents phosphatase activity in the absence of Ca^Vcalmodulin (Hashimoto et al., 1990). In calcineurin, the autoinhibitory domain is 43 residues COOH-terminal of the putative CaM-binding domain (Hashimoto et al., 1990). In the absence of bound Ca^VCaM, it prevents the binding of ATP and protein substrates (Perrino et al., 1995). Binding of Ca^VCaM to calcineurin A and also binding of Ca^^ to calcineurin B induces a conformational change which displaces the autoinhibitory domain from the active site, thus relieving its inhibition of calcineurin activity (Perrino et al., 1995). A synthetic peptide corresponding to the autoinhibitory domain inhibited the phosphatase activity of purified bovine brain calcineurin in vitro (Hashimoto et al., 1990). I investigated the effect of this autoinhibitory peptide on exocytosis in sea urchin eggs. Since both sea urchin egg calcineurin and bovine brain calcineurin are similarly recognized by the same polyclonal antibody and as stated earlier.

119 structural similarities have been demonstrated between calcineurin A from different species, it is likely that the sea urchin egg calcineurin also contains an autoinhibitory region and would be affected by the inhibitory peptide. The sequence of the inhibitory peptide is shown in Table 5.6 and its effect on the phosphatase activity of purified bovine brain calcineurin is shown in Figure 5.2.

Table 5.6 Characteristics of the CaN inhibitory peptide.

Sequence of the CaN inhibitory peptide I C . 5 0 (pM)

ITSFEEAKGLDRINERMPPRRDAMP 15

The sequence is taken Jfrom Hashimoto et al. (1990). The I.C.5 0 value was calculated from the results shown in Figure 5.3, which shows the inhibition of the phosphatase activity of purified bovine brain calcineurin.

I microinjected the CaN inhibitory peptide (50 mM in 125 mM KCl, 0.25 M EGTA, 5 mM PIPES, pH 6.7) to give a final cytoplasmic concentration of 100 pM. After incubation for 15 minutes the eggs were inseminated and the number of eggs that raised complete fertilization envelopes was counted. Injection of the peptide had no effect on exocytosis in vivo. All of the eggs injected raised complete fertilization envelopes (Table 5.7). The effect of the peptide on exocytosis in vitro was not tested.

Table 5.7 The effect of the CaN inhibitory peptide on exocytosis in vivo.

Treatment Number of eggs % Exocytosis microinjected

CaN inhibitory peptide 20 1 0 0

Control 18 100

The CaN inhibitory peptide was injected to give a final cytoplasmic concentration of 100 ^iM. Eggs were fertilized 15 minutes after injection. Exocytosis was scored by counting the number of eggs which raised complete fertilization envelopes. Control eggs were injected with vehicle only.

120 100

6 0 3 0> I 4 0 (Ü ^ 20

0 2 5 5 0 75 100

[CaN Peptide] (pM)

Figure 5.2 The effect of a calcineurin inhibitory peptide on calcineurin activity.

The phosphatase activity of purified bovine brain calcineurin was assayed using 0.16 p.M purified bovine brain CaN in 50 mM Tris-HCl, pH 7.0, 2.5 mM NiClj, 0.25 mg/ml BSA in the presence of 0.1 mM Ca^"’, 0.38 p.M calmodulin and the indicated concentration of inhibitory peptide. 3.4 mM PNPP was used as substrate. All assays were performed in micro-titer assay plates in triplicate and phosphatase activity was monitored by measuring the absorbance at 405 nm. Mean +/- s.e.m. are shown, n=3.

121 (iii) The Effect of Deltamethrin on Exocytosis.

Deltamethrin is a type II pyrethroid insecticide which was shown to be a potent inhibitor of calcineurin activity (Enan and Matsumura, 1992). In intact synaptosomes, 0.1 pM deltamethrin caused an increase in the level of phosphoproteins due to an inhibition of calcineurin activity (Enan and Matsumura, 1992). I assayed the effect of deltamethrin on the phosphatase activity of purified bovine brain calcineurin. The structure of deltamethrin is shown in Figure 5.3 and its effect on the activity of purified bovine brain calcineurin is shown in Figure 5.4. The I.C . 5 0 value for deltamethrin was calculated to be 0.9 pM. This value is higher than that calculated by Enan and Matsumura (1992). The discrepancy between these two values could be due to differences in the assays used to measure phosphatase activity or could be due to the different sources used to supply calcineurin.

H3C CH3 COOCH

Figure 5.3 Structure of deltamethrin.

I microinjected deltamethrin (10 mM in 0.5 M KCl, 1 mM EGTA, 20 mM PIPES, pH 6.7) into sea urchin eggs to give a final cytoplasmic concentration of 20 pM. After incubation for 15-20 minutes, the eggs were inseminated and the number of eggs that raised complete fertilization envelopes was counted. All of the eggs microinjected with deltamethrin raised complete fertilization envelopes, indicating that deltamethrin had no effect on exocytosis in vivo (Table 5.8).

122 100

8 0

î 6 0 4 c 3 Q) C 4 0 Ü o(0 nP 20

-9 -8 -7 -G -5 Ig^Q [Deltamethrin] (M)

Figure 5.4 The efifect of deltamethrin on calcineurin activity.

The phosphatase activity of purified bovine brain calcineurin was assayed using 0.16 ^iM calcineurin in 50 mM Tris-HCl, pH 7.0,2.5 mM NiCl^, 0.25 mg/ml BSA in the presence of 0.1 mM Ca^^, 0.38 |iM calmodulin and the indicated concentration of deltamethrin. 3.4 mM PNPP was used as substrate. All assays were performed in microtiter assay plates in triplicate and phosphatase activity was monitored by measuring the absorbance at 405 nm. Mean +/- s.e.m. are shown, n=3.

123 Table 5.8 The effect o f deltamethrin on exocytosisin vivo.

Treatment Number of eggs % Exocytosis microinjected

Deltamethrin 15 100

Control 15 100

Deltamethrin was injected to give a final cytoplasmic concentration of 20 p.M. Eggs were fertilized 15 minutes after injection. Exocytosis was scored by counting the number of eggs which raised complete fertilization envelopes. Control eggs were injected with vehicle only.

The efifect of deltamethrin on exocytosis in vitro was also investigated. Cortices were isolated in intracellular medium and were incubated for 20 minutes with 10 |iM deltamethrin. Cortices were viewed using phase contrast microscopy. Exocytosis was stimulated by the addition of 17 pM Ca^^ and was measured 1 minute after calcium addition. Deltamethrin had no effect on Ca^^-stimulated exocytosis in vitro (Table 5.9). The extent of exocytosis was 99 % as compared to 95 % in control experiments, in which no deltamethrin was added.

Table 5.9 The efifect of deltamethrin on exocytosis in vitro.

Treatment % Exocytosis

Deltamethrin 99.00+/- 1.0

Control 95.00 +/- 5.0

Cortices were incubated with 20 pM deltamethrin for 20 minutes at 16 °C. Control cortices were incubated in IM only. Cortices were viewed using phase contrast microscopy. Exocytosis was stimulated by the addition of 17 |iM Ca^^ and was measured 1 minute after Ca^^ addition. Mean +/- s.e.m are shown, n=5.

124 In summary, deltamethrin, which is a potent inhibitor of calcineurin, had no effect on exocytosis either in vivo or in vitro.

4. THE EFFECT OF OKADAIC ACID ON THE PHOSPHATASE ACTIVITY OF SEA URCHIN EGG CYTOSOL.

It was previously shown that okadaic acid inhibited exocytosis when microinjected into sea urchin eggs but had no effect on exocytosis in vitro (Whalley et a l, 1991). Okadaic acid must therefore be inhibiting a regulatory step that is not present when cortices are isolated in vitro and is therefore unlikely to be an essential step in the mechanism of exocytotic membrane fusion. However, since this step appears to be part of the cascade of reactions leading to exocytosis in vivo, I characterised the phosphatase activity of sea urchin egg cytosol in order to determine whether this effect was mediated through a cytosolic phosphatase. The effect is unlikely to be mediated via calcineurin, since okadaic acid does not inhibit the activity of this enzyme in the concentration range used to inhibit exocytosis (Cohen et a l, 1990). Okadaic acid is, however, is a potent inhibitor of both protein phosphatases 1 and 2 A in this concentration range (Cohen et al, 1990).

I measured the phosphatase activity of an egg cytosolic fraction, using p- nitrophenylphosphate (PNPP) as substrate. Samples were incubated in either the presence of 10 mM Ca^^ or 0.1 mM EGTA, with or without 500 nM okadaic acid. When microinjected into sea urchin eggs, 500 nM okadaic acid inhibited exocytosis by approximately 70 % (Whalley et a/.,1991). 500 nM okadaic acid inhibited the Ca^^- dependent phosphatase activity of sea urchin egg cytosol in vitro by only 7.5 %, whereas it inhibited the Ca^^-independent phosphatase activity by approximately 69 % (Figure 5.5). Ca^^-dependent phosphatase activity accounted for 27 % of the total phosphatase activity and Ca^^-independent phosphatase activity accounted for 73 % of the total.

125 0.4 -

0.3

8 C CD f 0.2 - O CO <

0.1 -

0.0 10 mM CaCL

0.1 mM EGTA + 500 nM Okadaic acid +

Figure 5.5 Characterization of the phosphatase activity in sea urchin egg cytosol.

The phosphatase activity of sea urchin egg cytosol was assayed using 10 pg of cytosolic protein in 50 mM Tns-HCl, pH 7.0,2.5 mM NiCf, 0.25 mg/ml BSA, 0.38 pM calmodulin in either the presence of 10 mM Ca^^ or 0.1 mM EGTA, with or without 500 nM okadaic acid. 3.4 mM PNPP was used as substrate. All assays were perfonned in microtiter assay plates in triplicate and phosphatase activity was monitored by measuring the absorbance at 405 nm. Mean +/- s.e.m. are shown, n-3.

126 In summary, 500 nM okadaic acid inhibited the Ca^^-independent phosphatase activity of sea urchin egg cytosol by an extent that correlated with the amount by which it inhibited exocytosis in vivo. It had very little effect, however, on the Ca^^-dependent phosphatase activity of sea urchin egg cytosol.

5. CONCLUSION.

In this chapter, I showed evidence against the involvement of a Ca^^-dependent, calcineurin-like phosphoprotein phosphatase in exocytosis in sea urchin eggs. An autoinhibitory antibody, which was shown to inhibit exocytosis in Paramecium (Momayezi et al., 1987), had no effect on exocytosis either in vivo or in vitro in sea urchin eggs. Similarly, peptide inhibitors of calcineurin had no effect on exocytosis. These results suggest that exocytosis in sea urchin eggs does not proceed in an manner analogous to that of Paramecium.

I showed that a calmodulin antagonist, the MLCK 7 9 7 . 8 1 3 peptide, inhibited exocytosis in vitro. This result supports the findings of Steinhardt and Alderton (1982) and Baker and Whitaker (1979), who showed that calmodulin was involved in exocytosis in sea urchin eggs. The fact that the MLCK7 9 7 . 8 1 3 peptide only inhibited exocytosis by approximately 30 % could be explained by a high affinity for calmodulin of a putative calmodulin-binding protein in the egg cortex and hence only a small displacement by the

MLCK7 9 7 . 8 1 3 peptide. The inhibitory effect was specific, since a control peptide, with a similar sequence but a low affinity for calmodulin, had no effect on exocytosis. The lack of effect of the peptide on exocytosis in vivo could be explained by the high level of calmodulin in eggs, which would prevent the MLCK 7 9 7 . 8 1 3 peptide from antagonizing all calmodulin-dependent reactions.

Finally, I showed that okadaic acid inhibited a cytosolic Ca^^-independent phosphatase activity in the egg cytosol, at concentrations comparable to those that inhibited exocytosis in vivo (Whalleyet al., 1991). This result suggests that okadaic acid

127 may mediate its effect on exocytosis in vivo, by inhibiting the activity of a Ca^^- independent cytosolic phosphatase. This hypothesis would also explain why okadaic acid had no effect on exocytosis in vitro (Whalley et a l, 1991). It can be postulated that if protein dephosphorylation is an important step in the regulation of exocytosis in vivo, this reaction is most likely to be mediated by either protein phosphatase 1 or protein phosphatase 2A.

128 Chapter 6. The Inositol Phospholipids and Exocytosis.

1. INTRODUCTION.

The requirement for ATP to maintain a competent secretory apparatus has been clearly demonstrated, both in sea urchin eggs (Baker and Whitaker, 1978; Moy et a/., 1983; Sasaki and Epel, 1983) and in many other secretory systems (Howell et a l, 1989; Wagner and Vu, 1989; Holz et a l, 1989; Vilmart-Seuwen et a l, 1986). The precise site and mechanism of action of ATP, however, remain to be elucidated. It has been proposed that ATP may be required to maintain the exocytotic apparatus in a particular protein phosphorylation state. More recently, ATP has been proposed to be required to maintain the levels of the polyphosphoinositides prior to Ca^^-stimulated fusion (Eberhard et al, 1990). A tight correlation between polyphosphoinositide levels and secretion was demonstrated in permeabilized adrenal chromaffin cells and the requirement for the polyphosphoinositides in Ca^^-stimulated secretion was shown to be independent of their being substrates for the generation of InsP^ and DAG (Eberhard et a l, 1990). The proposal that the polyphosphoinositides are required for exocytosis was supported by the identification of phosphatidylinositol transfer protein (PtdlnsTP) and pho sphatidylino sitol - 4-phosphate 5-kinase (PtdlnsPSK) as priming factors in Ca^^-stimulated secretion (Hay et a l, 1993; 1995). Indeed, exocytotic priming by PtdlnsTP and PtdlnsPSK in permeabilized PC 12 cells was accompanied by an increase in Ptdlns ? 2 formation (Hay et a l, 1995). These results suggest that the polyphosphoinositides are required for Ca^^- stimulated secretion and that the phosphoinositide kinases may, at least in part, be responsible for the ATP requirement in exocytotic priming.

In sea urchin eggs, incubation of isolated cortices in the absence of ATP resulted in a decrease in exocytotic response (Baker and Whitaker, 1978; Moy et a l, 1985; Sasaki and Epel, 1983). Since both phosphatidylinositol 4-kinase (PtdIns4K) and PtdInsf5K

129 activities were identified in the cortical region of unfertilized sea urchin eggs (Oberdorf et al, 1989), the decrease in exocytotic response due to the absence of ATP might be due to a decrease in the levels of the polyphosphoinositides in the egg cortex.

In this chapter I describe experiments in which I investigated whether the loss of exocytotic activity in vitro in the absence of ATP was due to a decrease in the levels of the polyphosphoinositides in the egg cortex. Firstly, I present results in which I characterised the effect of ATP on Ca^^-stimulated exocytosis in vitro. Secondly, I present results in which I determined the effects of incubation in the presence or absence of ATP on the levels of polyphosphoinositides in the egg cortex. Finally, I describe experiments in which I investigated whether neomycin, an aminoglycoside antibiotic which prevents the hydrolysis of polyphosphoinositides, prevented the decrease in exocytotic response in vitro due to the absence of ATP.

2. THE INFLUENCE OF ATP ON EXOCYTOSIS IN VITRO.

In order to determine the ATP requirement of exocytosis in vitro, isolated cortices were incubated for various times either with or without ATP. After incubation, exocytosis was stimulated by the addition of 5.9 pM free Ca^^ and the extent of exocytosis was measured. When exocytosis was stimulated immediately after cortex preparation, the level of exocytosis was the same for cortices prepared in either the presence or absence of ATP and was approximately 80 % (Figure 6.1). The Ca^^-sensitivity of exocytosis rapidly declined in a time-dependent manner following the preparation of cortices, in both the presence and absence of ATP. Afl;er a 15 minute incubation, the level of exocytosis had decreased to approximately 78 % for cortices incubated in the presence of ATP and 35 % for cortices incubated in its absence. The level of exocytosis continued to decline with time and after 60 minutes had fallen to approximately 40 % and 17 % for cortices incubated in the presence and absence of ATP, respectively. This loss of Ca^^-sensitivity with time after cortex preparation was previously described by Moy et a l (1983) and was termed "aging". These authors reported that inclusion of ATP in the incubation buffer

130 100 n

8 0 -

g 60 -

IS uj 4 0 -

20 -

0 15 3 0 4 5 60

Incubation time (min.)

Figure 6.1 Loss of calcium-sensitivity with time following cortex isolation.

Isolated cortices were incubated either with ( • ) or without (O) 2.5 mM ATP in IM, pH 6.7 at 16 °C. At the times indicated, exocytosis was stimulated by the addition of 5.9 pM Ca^^. Cortices were viewed using phase contrast microscopy and exocytosis was measured 3 minutes after Ca^^ addition by counting the proportion of granules that had undergone fusion. The results are taken from two independent experiments. Mean and s.e.m. are shown, n = 4.

131 retarded the rate of loss of Ca^^-sensitivity, an effect which is apparent in the results presented in Figure 6.1. The results presented in Figure 6.1 are consistent with those of Moy et al. (1983), who showed that after a 60 minute incubation, exocytosis decreased to approximately 40 % and 9 % in the presence and absence of ATP, respectively. Phase contrast micrographs of cortices, before exocytosis was stimulated and after addition of calcium, are shown in Figure 6.2. The increase in the number of refractory granules for cortices incubated in the absence of ATP is apparent. The maintenance of the exocytotic response in vitro by ATP has also been reported by other groups (Baker and Whitaker, 1978; Sasaki and Epel, 1985). In contrast to my results, Sasaki and Epel (1985) reported no loss of Ca^^-sensitivity in cortices incubated for up to 12 hours in the presence of ATP. This group also reported that the Ca^^-sensitivity of isolated cortices varied from season to season, therefore this observation may explain the discrepancy between the results.

In another set of experiments, the kinetics of the exocytotic reaction were studied. Addition of 5.9 pM calcium immediately after cortex preparation resulted in 63 % and 52 % exocytosis in the first 30 seconds for cortices isolated in the presence or absence of ATP respectively (Figure 6.3). Cortices that had been incubated for 60 minutes in the presence of ATP underwent 37 % exocytosis in the first 30 seconds, whereas cortices incubated for 60 minutes in the absence of ATP, underwent 9 % exocytosis in the first 30 seconds. These results indicate that inclusion of ATP in the incubation buffer affected the initial rate of secretion. When exocytosis was stimulated immediately after cortex preparation, the rate of exocytosis in the first minute following Ca^^ addition was 65 %/minute and 60%/minute for cortices prepared in the presence and absence of ATP respectively. This rate dropped to 37 %/minute after incubation for 60 minutes in the presence of ATP and to 11.8 %/minute for cortices incubated for 60 minutes in the absence of ATP.

In summary, both the initial rate and the Ca^^-sensitivity of exocytosis in vitro decreased with time, following cortex isolation. Inclusion of ATP in the incubation buffer partially inhibited the rate of loss of Ca^^-sensitivity, thus maintaining the extent of exocytosis triggered after prolonged incubation times.

132 Figure 6.2 Comparison of exocytosis in vitro after incubation in either the presence or absence of ATP.

Cortices were isolated and incubated for 60 minutes in IM, in either the presence (a,b) or absence (c,d) of 2.5 mM ATP. Coifiees were viewed using phase contrast microscopy and images were taken before (a,c), and 3 minutes after exocytosis was stimulated by the addition of 5.9 pM (b,d). Bar = 10 pm.

133 80

6 0

(0 I 4 0

20

0

1

Time after addition of Ca^^ (min.)

Figure 6.3 The influence of ATP on exocytosis in vitro.

Isolated cortices were incubated in IM, pH 6.7 in either the presence ( • ■ ) , or absence (O D ) of 2.5 mM ATP for 5 minutes (O # ) or 60 minutes (■ □ ) at 16 °C. After incubation, exocytosis was stimulated by addition of 5.9 pM Ca^\ Cortices were viewed using phase contrast microscopy and exocytosis was scored 3 minutes after Ca^^ addition by counting the proportion of granules that had undergone fusion. Mean and s.e.m. are shown, n = 4.

134 3. MEASURING THE LEVELS OF THE INOSITOL PHOSPHOLIPIDS IN THE EGG CORTEX.

In order to measure the levels of the inositol phospholipids in the egg cortex, eggs were radiolabelled to isotopic equilibrium using myo-2-[^H]-inositol, according to the method of Ciapa et al (1992). After labelling to isotopic equilibrium, subsequent measurements of the levels of the different polyphosphoinositides should reflect alterations in the chemical amounts of the polyphosphoinositides, if it is assumed that the entire pool of inositol phospholipids is exchangeable. This is important if the levels of the different polyphosphoinositides are to be correlated with levels of exocytosis.

(i) The Exocytotic Response After a Thirty Hour Incubation.

Labelling of eggs to isotopic equilibrium involved a thirty hour incubation period. It was previously noted that this long-term labelling of eggs resulted in lowered rates of sperm-induced fertilization (Ciapa et al, 1992). It was therefore important to ensure that the exocytotic response of cortices prepared from eggs after a thirty hour incubation was identical to that of cortices prepared from eggs immediately after shedding. The exocytotic response of cortices prepared from eggs taken from the same urchin was therefore measured immediately after the eggs were shed and after a thirty hour incubation in ASW containing 0.001 % sufadiazine at 16 °C, the conditions used for labelling eggs to isotopic equilibrium. Identical experiments were conducted on the two samples of eggs.

Isolated cortices were incubated at 16 °C either with, or without ATP, after which time exocytosis was stimulated by the addition of 5.9 \iM free Ca^^. The exocytotic responses of cortices prepared from the two different samples of eggs were comparable under all incubation conditions (Figure 6.4). When exocytosis was stimulated immediately after cortex preparation, the exocytotic response was the same for cortices prepared in the presence or absence of ATP and was approximately 80 %. This was true for cortices prepared from eggs immediately after shedding and for cortices prepared from eggs that

135 100 (a)- + ATP -ATP

w to (b). I I

0 60 Incubation time (min.)

Figure 6.4 Comparison of the exocytotic response of cortices prepared from eggs immediately after and thirty hours after shedding.

Eggs shed from a smgle urchin were either used immediately (a), or incubated for 30 hours in ASW containing 0.001 % sulfadiazine at 16 °C (b). Cortices were prepaied and incubated for the tunes indicated either with, or without, 2.5 mM ATP. Cortices were viewed using phase contrast microscopy. Exocytosis was stimulated by the addition of 5.9 pM Ca^^ and was measured 3 minutes after Ca^^ addition. The results are taken from two difterent experiments. Mean and s.e.m. are shown, n = 4.

136 were incubated for 30 hours. The phenomenon of "aging" was apparent in both samples of eggs and both samples "aged" at the same rate and to the same extent. The exocytotic response of cortices after incubation for 60 minutes in the presence of ATP was approximately 50 % for both samples of eggs. Similarly, after incubation for 60 minutes in the absence of ATP, the exocytotic response was 28 % for cortices prepared from both samples of eggs. The results presented in Figure 6.4 are from independent experiments conducted on different urchins on different days. These results indicate that the exocytotic response of cortices is not affected by the time eggs are kept before cortex preparation.

(ii) Measuring the Levels of the Inositol Phospholipids After Incubation in the Presence or Absence of ATP.

A 20 % suspension of freshly shed eggs was labelled to isotopic equilibrium using /w>^o-2-pH]-inositol, according to the method of Ciapa et al. (1992). Briefly, jellied eggs were incubated for thirty hours at 16 °C with 10 pCi/ml of /Myo-2--inositol as a 20 % (v/v) suspension in 5 ml ASW containing 0.01 % of the antibiotic sulfadiazine. After labelling, eggs were rinsed four times by décantation in cold ASW until the external solution was essentially free of labelled inositol. Eggs were then dejellied, washed and resuspended in intracellular medium. Cortices were prepared from the eggs and lipids extracted using acidified chloroform/methanol, according to the method of Allan and Cockcroft (1983). After separation using thin layer chromatography, the amount of each inositol phospholipid was determined using scintillation counting. The relative proportion of each inositol phospholipid was compared. The relative proportion of phosphatidylinositol (Ptdlns), phosphatidylinositol 4-phosphate (PtdlnsPj and phosphatidylinositol 4,5-bisphosphate (Ptdlns? 2 ) in the egg cortex was 73.5:15.5:10.9 (Table 6.1).

137 Table 6.1 Distribution of [^HJinositol label incorporated into cortical inositol phospholipids prepared from unfertilized sea urchin eggs.

Inositol phospholipid % of total ino sitol phospholipids

Ptdlnsf^ 10.9+/- 1 . 0 2

PtdlnsP 15.5 +/- 0.50

Ptdlns 73.5+/- 1.50

A 20 % suspension of freshly shed eggs was labelled to isotopic equilibrium using myo-l- [^H]-inositol, according to the method of Ciapa et al. (1992). Cortices were prepared from labelled eggs and lipids were extracted using acidified chloroform/methanol, according to the method of Allan and Cockcroft (1983). After separation using thin layer chromatography, the radioactivity in each of the inositol phospholipids was measured using scintillation counting. Mean and s.e.m. are shown, n=3.

In another set of experiments, cortices prepared from eggs labelled to isotopic equilibrium were incubated either in the presence or absence of ATP for 60 minutes. Lipids were extracted and separated using thin layer chromatography (TLC). An autoradiochromatograph of the TLC plate was made before the radioactivity in each of the inositol phospholipids was measured using scintillation counting.

The autoradiochromatograph clearly shows that the amount of PtdlnsPj in the cortex decreased after incubation in the absence of ATP (Figure 6.5). Using scintillation counting, the amounts of Ptdlns, PtdlnsP and Ptdlnsp 2 were determined. The relative proportion of Ptdlns;PtdlnsP:PtdlnsP 2 was calculated for each incubation condition. At time 0, the ratio Ptdlns:PtdlnsP:PtdlnsP 2 was 82.4:11.4:6.2 for cortices prepared in the presence of ATP and 82.9:9.2:7.9 for cortices prepared in the absence of ATP. After a

60 minute incubation in the presence of ATP, the ratio Ptdlns:PtdlnsP:Ptdlns ? 2 was 87.9:5.0:7.1 and in the absence of ATP, was 90.4:6.1:3.6. The results show that after a

60 minute incubation in the absence of ATP, the relative proportion ofPtdlns ? 2 decreased by approximately 54 %, whereas in the presence of ATP, it actually increased by

138 + ATP -ATP

Ptdlns

PtdlnsP i i » MÊi

PtdlnsP.

Origin

0 60 0 60 Time (min.)

Figure 6.5 Autoradiochromatograph of pH]inositol-labelled cortical lipid extracts.

Lipids were extracted from cortices after incubation for 0 or 60 minutes, in either the presence or absence of 2.5 mM ATP. The thin layer chromatogi aph shows the pattern of label in lipids exti acted from the isolated egg cortex. The positions of the inositol phospholipids was detennined by eompai ison with a lane of unlabelled standaids. Radiolabel is lost from the PtdlnsPj spot after incuabation for 60 minutes in the absence of ATP.

139 approximately 13 %. In both the presence and absence of ATP, the proportion of PtdlnsP decreased.

The results from three independent experiments, performed on different batches of eggs, are presented in Figure 6.6. As determined by changes in counts per minute

(C.P.M.), the amount of Ptdlns ? 2 decreased by approximately 50 % and the amount of PtdlnsP decreased by approximately 40 %, after incubation of cortices in the absence of ATP. In contrast, there was very little change in the amounts of these two inositol phospholipids after incubation in the presence of ATP. The total amount of pH]-labelled inositol phospholipid varied considerably between experiments. This variation may have been due to differences in the amount of lipid recovered from each experiment, therefore, the relative proportion of each inositol phospholipid was compared. Using this method of comparison, the proportion of Ptdlnsp 2 decreased by 35 % after incubation in the absence of ATP and by 19 % after incubation with ATP. The proportion of PtdlnsP decreased by approximately 23 % in both cases.

In summary, after incubation in the absence of ATP, the amount of Ptdlns ? 2 in the egg cortex decreased, although the amount by which it decreased varied from experiment to experiment. In all experiments, the amount of PtdlnsP in the egg cortex decreased after incubation in both the presence and absence of ATP.

4. THE EFFECT OF NEOMYCIN ON EXOCYTOSIS.

Neomycin is an aminoglycoside antibiotic that prevents the hydrolysis of polyphosphoinositides by binding to their polar headgroups (Schibeci and Schacht, 1977; Schacht, 1978; Downes and Michell, 1981). In permeabilized adrenal chromaffin cells, preincubation with neomycin prevented the loss of secretory response due to the absence of ATP, by maintaining the levels of the polyphosphoinositides (Eberhard et a l, 1990). In sea urchin eggs, incubation of isolated cortices with neomycin both inhibited exocytosis and prevented the hydrolysis of the polyphosphoinositides with the same concentration

140 (a). + ATP (b). - ATP

0. o qT* ccn 2 o_

CL U

CL ccn g

CL O cc/l

60 0 60

Incubation time (min.)

Figure 6.6 Changes in polyphosphoinositide levels in the presence and absence of ATP

[^I Ijlnositoi-prelabelled eggs were incubated in either the presence (a), or absence (b), of 2.5 mM ATP for the times indicated at 16 "C. After incubation, lipids were extracted and were separated using thin layer chromatogiaphy. The levels of the diiferent inositol phospholipids were measured using scintillation counting and are expressed as counts per minute (C.P.M.). Mean and s.e.m. are shown, n=3.

14 dependence (Whitaker and Aitchison, 1985; Whitaker, 1987; McLaughlin and Whitaker, 1988). However, the effect of preincubation with neomycin prior to Ca^^-stimulated exocytosis in its absence was not investigated. I preincubated cortices with neomycin in the absence of ATP and investigated whether this would prevent the decrease in the secretory response normally seen in the absence of ATP. If maintenance of the secretory response was observed, then it would provide further evidence to support the proposal that maintenance of polyphosphoinositide levels is one of the ATP-requiring steps in exocytotic priming.

Isolated cortices were incubated with different concentrations of neomycin in the absence of ATP. After incubation, cortices were washed extensively in order to remove the neomycin. This was important because neomycin was shown to directly inhibit exocytosis in vitro in sea urchin eggs, when present in both the incubation buffer and in the calcium-containing buffer used to stimulate exocytosis (Whitaker and Aitchison, 1985). It was therefore important to ensure that the exocytotic response in vitro was not affected by the presence of residual neomycin. The inclusion of neomycin in the incubation bufter did not prevent the loss of secretory response due to the absence of ATP (Figure 6.7). On the contrary, it inhibited exocytosis in a dose-dependent manner. In these experiments, exocytosis was stimulated by the addition of 17 pM Ca^^, which resulted in 87 % exocytosis when added immediately after cortex isolation and 61 % exocytosis after a 60 minute incubation in the absence of ATP. Inclusion of 10 pM neomycin in the incubation buffer had no effect on exocytosis, however, at higher concentrations, exocytosis was inhibited in a dose-dependent manner, with 200 pM neomycin inhibiting exocytosis by approximately 80 %.

The best explanation for these observations is that neomycin was not removed from the cortices by extensive washing. This finding is in contrast to the experiments of Eberhard et al. (1990) on permeabilized adrenal chromaffin cells. Similar concentrations of neomycin were used in both sets of experiments. The dose-dependency of inhibition is apparent at the low concentrations of neomycin used, i.e. in the micromolar range, as compared to the concentration which inhibited exocytosis completely, 10 mM, in the

142 100

80

20

50 100 150 200

[Neomycin] (jjM)

Figure 6 .7 The effect of neomycin on exocytosis in vitro.

Cortices were isolated in intracellular medium (IM), in the absence of ATP and were incubated for 1 hour with the indicated concentration of neomycin. After incubation, cortices were washed extensively using IM before exocytosis was stimulated by the addition of 17 ^iM Ca^^. Cortices were viewed using phase contrast microscopy and exocytosis was measured 3 minutes after Ca^^ addition. Mean and s.e.m. are shown, n = 3.

143 experiments of Whitaker and Aitchison (1985).

In summary, it was not possible to use neomycin to determine whether maintenance of the polyphophoinositides explained the requirement for ATP to maintain the exocytotic response in vitro. The results suggested that it was not possible to entirely remove neomycin from cortices after incubation. The results, however, do support the observation of Whitaker and Aitchison (1985), who showed that polyphosphoinositide hydrolysis was required for exocytosis in vitro in sea urchin eggs.

5. CONCLUSION.

In this chapter, I showed that the Ca^^-sensitivity and the extent of exocytosis in vitro decreased with time following cortex isolation. I showed that the inclusion of ATP in the incubation buffer retarded the rate of loss of Ca^^ sensitivity, thus maintaining the extent of exocytosis triggered after prolonged incubation times. I showed that the decrease in exocytotic response due to the absence of ATP was paralleled by a decrease in the amount ofPtdIns ? 2 in the egg cortex. In the absence of ATP, both the decrease in exocytotic response and the decrease in the level ofPtdIns ? 2 were approximately doubled, as compared to the decreases seen after incubation in the presence of ATP. The decrease in the amount of PtdlnsP was approximately the same in both the presence and absence of ATP.

In summary, I correlated the exocytotic response seen in vitro with the amount of

PtdInsP 2 in the egg cortex. My results support the hypothesis that the ATP requirement of exocytosis may in part, be due to a requirement by the phosphoinositide kinases.

144 Chapter 7. Discussion.

1. EXOCYTOSIS - A UNIVERSAL SECRETORY MECHANISM.

Exocytosis is a ubiquitous cellular mechanism for the export of secretory products and the insertion of proteins and lipid into the plasma membrane. Exocytosis in sea urchin eggs is an example of triggered exocytosis, the trigger being, at fertilization, a sperm- induced rise in the intracellular free calcium ion concentration ([Ca^^]J from a resting level of around 100 nM to several micromolar (Steinhardt et a l, 1977; Baker and Whitaker, 1978; Baker et al., 1980). A large body of experimental evidence has shown that the fundamental mechanisms of exocytosis are similar in all eukaryotic cells (for review see Bennett and Scheller, 1993; Ferro-Novick and Jahn, 1994; Rothman and Warren, 1994). Several key proteins, namely synaptobrevin, syntaxin and SNAP-25, have been proposed to function in all intracellular membrane fusion events, including exocytosis (Sollner et al., 1993a,b). However, it should be noted that there are examples of membrane fusion events which occur independently of these three proteins, for example, annexin II and annexin VII were shown to promote the Ca^^-dependent fusion of chromaffin granules in vitro, in the presence of arachidonic acid (Creutz, 1981; Burgoyne, 1988; Drust and Creutz, 1988). Another protein, calcineurin, has been implicated in exocytosis 'm Paramecium (Momayezi et al., 1987), a cellular system very similar to sea urchin eggs.

There were two underlying themes in this thesis. The first was to identify the presence in sea urchin eggs of proteins implicated in exocytosis in other secretory systems. The second was to investigate whether exocytosis in sea urchin eggs involved these proteins and proceeded in a manner analogous to exocytosis in other secretory systems.

145 2. THE IDENTIFICATION OF POTENTIAL SECRETORY PROTEINS IN SEA URCHIN EGGS.

Sea urchin egg proteins were immunoblotted using antibodies raised against proteins implicated in exocytosis in other secretory systems. This was the first approach that I used in order to identify the presence of putative secretory proteins in sea urchin eggs. The functions of these proteins in other secretory systems may then provide insights into their possible roles in exocytosis in the sea urchin egg. The results of immunoblotting, which are presented in Chapter 3, indicate that homologues of several such proteins are expressed in sea urchin eggs.

(i) Investigating Whether Homologues of Neuronal Secretory Proteins are Expressed in Sea Urchin Eggs.

The three synaptic proteins synaptobrevinA^AMP (vesicle-associated membrane protein), syntaxin and SNAP-25 (synaptosomal-associated protein of MW 25 kD) form the constitutive core of a putative fusion complex proposed to function in all eukaryotic fusion events, including exocytosis (Sollner et al., 1993a,b). Since homologues of synaptobrevin, syntaxin and SNAP-25 function in exocytosis in a large number of secretory cell types, I investigated whether homologues of these three proteins were expressed in sea urchin eggs.

(a) The Identification of a Synaptobrevin Homologue in Sea Urchin Eggs.

I immunoblotted sea urchin egg proteins using a monoclonal antibody raised against rat brain synaptobrevin (Baumert et al, 1989). An immunogenic protein of approximately 17.5 kD was detected in the cortical fraction of sea urchin eggs. The 17.5 kD protein was also detected in an intracellular membrane fraction but was not detected in the cytosolic fraction. No other proteins were detected using the antibody, illustrating its high specificity. Results presented in Chapter 4 show that, within the egg cortex, the 17.5 kD immunogenic protein was localized to the cortical granule fraction, as determined

146 using immunoblot analysis. Immunostaining of fixed cortices using the monoclonal antibody revealed that the protein was specifically localized to the cortical granule membrane. Staining was uniform throughout the cortex, suggesting that the 17.5 kD protein was evenly distributed throughout the granule population.

On the basis of immunoreactivity, molecular weight and subcellular localization, the 17.5 kD protein can be assigned as a synaptobrevin homologue. Within the cortex, its localization to the cortical granule membrane is consistent with the localization of synaptobrevin in neurons, where it is concentrated on the synaptic vesicle membrane (Baumert et al., 1989). The sea urchin egg synaptobrevin homologue is thus in an appropriate location for involvement in cortical granule exocytosis. The identification of the 17.5 kD synaptobrevin homologue in an intracellular membrane fraction suggests an involvement in one or more steps along the constitutive secretory pathway. However, the precise localization of the protein within intracellular membrane compartments was not determined. It was not possible to determine whether the different subcellular localizations of synaptobrevin were due to the presence of two different isoforms, since the monoclonal antibody used recognizes the conserved cytoplasmic domain of synaptobrevins (R. Jahn, personal communication) and has been reported to recognize multiple synaptobrevin isoforms (Südhof et al, 1989). Homologues of synaptobrevin have been shown to fimction in the constitutive secretory pathway in yeast and in the exocytosis of endosome-derived, constitutively recycling vesicles in permeabilized Chinese hamster ovary (CHO) cells (Protopopov et a l, 1993; Galli et a l, 1994). One anomaly is the possible involvement of the same protein in both constitutive and regulated secretion. According to the 'SNARE' hypothesis, the specificity of intracellular membrane fusion events is determined by each having its own specific isoforms o f SNARE' proteins in the vesicle membrane (v-SNARE) and target membrane (t-SNARE), which only interact with each other (Sollner etal, 1993a, b). However, this hypothesis was disputed by Chilcote et a l (1995) who showed that cellubrevin was co-localized with synaptobrevin I and II on the dense-core granule membrane in PCI2 cells and demonstrated that all three proteins interacted with the same neuronal syntaxin I and SNAP-25 isoforms. All three proteins were implicated in Ca^^-stimulated exocytosis

147 (Chilcote et al., 1995). It therefore appears that the specificity of membrane-membrane interactions may not be determined by the 'SNARE' proteins alone and that different isoforms function in the same membrane fusion event. The results obtained in sea urchin eggs suggest that the converse may also be true, i.e. that the same isoforms may function in different membrane fusion steps in both the constitutive and regulated secretory pathways. However, results which are discussed in section 3(iii) provide evidence to suggest that the two subcellular localizations are due to the existence of two different synaptobrevin isoforms. The identification of a synaptobrevin homologue localized to the cortical granule membrane suggests that exocytosis in sea urchin eggs might proceed in a manner analogous to synaptic vesicle exocytosis.

(b) Testing for the Expression of SNAP-25 and Syntaxin in Sea Urchin Eggs.

I also investigated whether homologues of SNAP-25 and syntaxin, the other components of the heterotrimeric fusion complex, were expressed in sea urchin eggs. Sea urchin egg proteins were immunoblotted using a monoclonal antibody against chick brain syntaxin and a polyclonal antibody against rat brain SNAP-25. No sea urchin egg proteins were detected using either antibody. The lack of detection of immunogenic proteins using these antibodies does not necessarily mean that homologues of SNAP-25 and syntaxin are not present in sea urchin eggs. The lack of detection could be due to the fact that the epitopes recognized by the antibodies are not present in the sea urchin egg protein isoforms. This would be especially true when using a monoclonal antibody. A similar lack of detection of syntaxin and SNAP-25 was observed in adipocytes and skeletal muscle in which synaptobrevin was detected (Cain et al., 1992; Volchuk et al., 1994). The existence of a syntaxin homologue in sea urchin eggs has been suggested in experiments by Bi et al. (1995), who showed that under certain conditions, cortical granule exocytosis in unfertilized eggs could be inhibited by botulinum neurotoxin Cl (BoNT/Cl). BoNT/Cl has been shown to inhibit neurotransmitter release by specifically cleaving neuronal syntaxin (Blasi etal., 1993a).

148 (c) Testing for the Expression of Synaptotagmin and the Alpha-Latrotoxin Receptor in Sea Urchin Eggs.

Synaptotagmin, which is an integral membrane protein of synaptic vesicles, is a strong candidate for the Ca^^-sensor of regulated exocytosis (for review see Littleton and Bellen, 1995). Synaptotagmin was reported to form a stable complex in vitro with syntaxin, synaptobrevin and SNAP-25, the three core proteins of the putative fusion complex (Sollner et al., 1993a,b). It was also shown to interact directly with the alpha- latrotoxin receptor, which is present in the presynaptic plasma membrane (Petrenko et al, 1991), an association that was proposed to play a role in synaptic vesicle docking and the regulation of neurotransmitter release (Petrenko et al., 1991).

Sea urchin egg proteins were immunoblotted using a polyclonal antibody against a synthetic peptide corresponding to a conserved region within the first C2 domain of synaptotagmin. Several immunogenic proteins were detected using the antibody, including proteins of 78 kD and 26 kD proteins which were detected in the cytosolic fraction. These proteins and a protein of 48 kD were also detected in the whole cell lysate. None of the immunogenic proteins, however, were found in the cortical fraction or were of a similar molecular weight as synaptotagmin. C2 domains are thought to confer Ca^^-dependent phospholipid-binding properties to proteins (Nishizuki, 1992). They occur in several different proteins, such as protein kinase C, phospholipase A2 and rabphilin 3 A. Any one of these proteins could have been detected using this antibody, for example, the 78 kD immunogenic protein is of a suitable molecular weight and in an appropriate subcellular localization to be assigned as an isoform of protein kinase C. The identification of a rabphilin 3 A homologue would have been particularly interesting in view of the identification of a rab3A homologue in sea urchin eggs. However, no immunogenic proteins were in an appropriate subcellular localization for assignment as a rabphilin 3 A homologue. In summary, using immunoblot analysis, no synaptotagmin homologues were identified in sea urchin eggs.

Sea urchin egg proteins were immunoblotted using a polyclonal anti serum against

149 the P protein of the alpha-latrotoxin (a-LTX) receptor, purified from bovine brain. An immunogenic protein which migrated with the same electrophoretic mobility as purified bovine brain p protein was detected in the whole egg lysate. A characteristic 62 kD degradation product was also detected. No immunogenic proteins were detected in the cortical fraction. On the basis of immunoreactivity and electrophoretic mobility, it is possible that the sea urchin egg 79 kD protein is a homologue of the mammalian p protein of the a-LTX receptor. However, the lack of detection of the protein in the cortical fraction implies that it is not involved in cortical granule exocytosis. Falugi and Prestipino (1989) showed that treatment of unfertilized eggs with a-LTX neither triggered cortical granule exocytosis, nor prevented the elevation of the fertilization envelope upon sperm- egg fusion. These observations are consistent with the lack of detection of immunogenic proteins in the cortical fraction using the anti-P protein antiserum. In contrast, treatment of fertilized eggs with a-LTX blocked egg development and caused noticeable alterations in cell surface topography (Falugi and Prestipino, 1989). Perhaps translocation of the putative a-LTX receptor from an unknown intracellular storage site to the cell surface is induced at fertilization in a manner analogous to the translocation of GLUT4 glucose transporters to the cell surface in muscle and fat cells induced by insulin (Volchuk et al., 1995). The physiological relevance of this, however, is unknown. In summary, no immunogenic proteins corresponding to the p protein of the a-LTX receptor were detected in the cortical fraction of unfertilized sea urchin eggs. This finding is consistent with earlier findings that a-LTX had no effect on unfertilized eggs (Falugi and Prestipino, 1989).

(ii) The Identification and Subcellular Localization of a Rab3A Homologue.

Rab proteins, which are members of the class of small monomeric GTP-binding proteins, have been proposed to cycle between a soluble and membrane-associated state, acting as molecular 'switches' which control the docking or fusion of transport vesicles with their target membrane (for review see Balch, 1990; Goud and McCaffrey, 1991). Rab3 proteins represent a small subfamily of rab proteins that are exclusively localized to secretory vesicles (Fischer von Mollard et a l, 1994). Rab3 A, which has been localized

150 to synaptic vesicles, has been implicated in the control of neurotransmitter release (Fischer von Mollard etal, 1991; Matteoli et al., 1991). Other rab3 proteins, which are localized to the secretory organelles of other secretory cell types, have also been implicated in the regulation of Ca^^-stimulated exocytosis (for review see Lledo et al., 1994).

I immunoblotted sea urchin egg proteins using a monoclonal antibody which recognizes bovine brain rab3A (Matteoli et al., 1991). An immunogenic protein of approximately 25 kD, which migrated with the same mobility on gel electrophoresis as rat brain rab3 A, was detected. By immunoblotting different egg subcellular protein fractions using the monoclonal antibody, the 25 kD immunogenic protein was shown to be specifically localized to the cortical granule fraction. This localization is consistent with the association of neuronal rab3A with synaptic vesicles (Fischer von Mollard et al., 1991). Taken together, the immunoreactivity of the 25 kD sea urchin egg protein, its subcellular localization and the fact that it migrated with the same mobility on gel electrophoresis as rat brain rab3A suggest that it is a true homologue of rab3A. As revealed using immunoblot analysis, the protein also behaved in a manner analogous to neuronal rab3 A during Ca^^-stimulated exocytosis, i.e. it was translocated to the plasma membrane upon stimulation (Matteoli etal., 1991). Rab3A was shown to dissociate from membranes at a stage distal to fusion and before vesicles were newly reformed by endocytosis (Matteoli etal, 1991). Thus, in sea urchin eggs it is possible that the rab3A homologue dissociates from membranes at a stage following exocytosis.

There is evidence to support the existence of a GTP-binding protein (G^) which is directly involved in cortical granule exocytosis in sea urchin eggs (Crossley et al, 1991). However, it is not clear whether Gg is a heterotrimeric GTP-binding protein or a small monomeric GTP-binding protein. The specific localization of the sea urchin egg rab3 A homologue to cortical secretory granules suggests that it performs an analogous role to its counterparts in other secretory cell types. Elucidation of the function of rab3 proteins in these secretory cells should therefore reveal the function the rab3 A homologue in exocytosis in sea urchin eggs.

151 (ii:) The Identification of Annexins in Sea Urchin Eggs.

The annexins are a family of Ca^^-dependent phospholipid-binding proteins. All members of the annexin family contain four or eight repeats of a highly conserved 70 amino acid motif which is the region of the protein responsible for both Ca^^- and lipid- binding. It has been proposed that members of the annexin family may function in exocytosis by initiating contact and fusion between the secretory vesicle membrane and the plasma membrane (for review see Geisow et al., 1987; Burgoyne and Geisow, 1989; Creutz, 1992).

Using immunoblotting, I detected the presence of three members of the annexin family in sea urchin eggs. Immunogenic proteins of approximately 34 kD, 35 kD and 68 kD were detected using antisera against human annexins I, V and VI, respectively. The MWs of human annexins I, V and VI are 34 kD, 35 kD and 68 kD, respectively (Moss et al., 1988; Putter et al., 1993; Owens et al., 1984). Therefore, on the basis of immunoreactivity and molecular weight, the sea urchin egg proteins may be homologues of their respective mammalian counterparts. In support of this proposal, the sea urchin egg 68 kD annexin migrated with the same mobility on gel electrophoresis as purified human placental annexin VI (Shen et al., 1994). However, peptide sequence analysis of an N-terminal fi^agment from the 34 kD annexin indicated that it was a novel member of the annexin super-gene family (Shen et al., 1994).

I investigated the subcellular distribution of the sea urchin egg annexins by immunoblotting different subcellular protein fi*actions using the antisera. When subcellular protein fi-actions were prepared in buffer containing 10 mM EGTA, the 34 kD and 35 kD annexins were localized to the cytosolic fraction. The 68 kD annexin was localized primarily to the cortical fraction, with a small amount present in the cytosolic fraction. It must therefore be localized to the cortex in a Ca^^-independent manner. Thus, under the same experimental conditions, the different annexins had different subcellular distributions in sea urchin eggs. The subcellular distribution of the 34 kD, 35 kD and 68 kD annexins corresponds to the distribution of annexins I, IV and VI, respectively, in

152 adrenal chromaffin cells (Drust and Creutz, 1991).

Is it possible that any of the sea urchin egg annexins fimction in cortical granule exocytosis? Most members of the annexin family are capable of promoting the Ca^^- dependent aggregation of chromaffin granules in vitro (Creutz et al., 1987). Table 7.1, taken from Burgoyne (1988), shows the calcium concentrations at which members of the annexin family bind to membranes or liposomes.

Table 7.1 Calcium-dependence of annexin binding to membranes or liposomes.

Protein Concentration of Ca^^ for Ref. half-maximal binding (pM)

p70* (annexin VI) 2.6 1 lipocortifr (annexin I) 4S, 20 2 endonexin I* (annexin IV) 5.5 3 endonexin 11^ (annexin V) 40 4 calpactin^ (annexin II)

* Binding assayed with chromaffm granule membranes; ^ Binding assayed with phosphatidyl serine liposomes; + Binding assayed with phosphatidylethanolamine liposomes; ^ Data for phosphorylated form; II A large component of binding was detected at low calcium levels, with a second calcium- dependent component. References: 1, Geisow & Burgoyne, 1982; 2, Schlaepfer & Haigler, 1987; 3, Siidhof e/a/., 1984; 4, Schlaepferet a t, 1987; 5, Powell & Glenney, 1987.

As illustrated in Table 7.1, several of the annexins bind to membranes or liposomes at the calcium concentrations shown to trigger cortical granule exocytosis in sea urchin eggs, i.e. 3-5 pM (Steinhardt et al., 1977; Baker and Whitaker, 1978). It is therefore conceivable that members of the annexin family might play a role in exocytosis in these cells. It is not known, however, whether the granule aggregating activity of the annexins represents their function in vivo.

From the subcellular distribution of the sea urchin egg annexins it can be concluded that the 34 kD and 35 kD annexins are not essential for exocytosis in vitro.

153 Cortices isolated in the presence of 10 mM EGTA undergo exocytosis on the subsequent addition of micromolar levels of calcium (Baker and Whitaker, 1978). Under such isolation conditions the 34 kD and 35 kD annexins would be removed in the cytosol. The 68 kD annexin, a possible homologue of human annexin VI, was localized primarily to the cortical fraction and is thus in a suitable location for involvement in exocytosis both in vivo and in vitro. Its possible role may be to promote contact between the cortical granules and the plasma membrane, in an manner analogous to that proposed for annexin II in adrenal chromaffin cells (Nakata et al., 1990). However, it should be noted that investigations into the function of annexin VI in adrenal chromaffin cells indicated that it inhibited the aggregation of chromaffin granules in vitro by annexin VII (synexin) and annexin II (calpactin) (Creutz et al., 1987).

In summary, three members of the annexin family of Ca^^-dependent lipid-binding proteins were identified in sea urchin eggs. The subcellular distribution of these proteins suggests that only the 68 kD annexin might play a role in exocytosis in vitro. However, the demonstration that a molecule is at the correct site to be involved in a process does not on its own demonstrate that the molecule under study is involved in that process. It is possible that any of the three annexins might function in exocytosis in vivo. One of the most successfirl methods used for identifying cytosolic factors involved in exocytosis has been the reconstitution of exocytosis in exocytotically run-down permeabilized cells. Indeed, the involvement of annexin II in exocytosis in adrenal chromaffin cells was demonstrated using this approach (Ali eta l, 1989; Burgoyne and Morgan, 1990; Sarafian et al., 1991). Since sea urchin eggs gradually lose their exocytotic response following electropermeabilization, due to the loss of cytosolic proteins (Swezey and Epel, 1989), reconstitution using cytosolic protein fractions could be used to investigate whether any of the three annexins are involved in cortical granule exocytosis in these cells.

(iv) The Identification of Calcineurin in Sea UrchinEggs.

In Paramecium, a Ca^Vcalmodulin-dependent calcineurin-like phosphatase localized to the cell cortex was implicated in exocytosis (Momayezi et al., 1987). There

154 are many similarities between exocytosis in Paramecium and exocytosis in sea urchin eggs, suggesting that calcineurin (CaN) may be involved in sea urchin egg exocytosis in a manner analogous to that in Paramecium. I therefore investigated the presence and subcellular localization of CaN in sea urchin eggs.

I immunoblotted sea urchin egg proteins using a polyclonal antibody raised against bovine brain CaN (Momayezi et al, 1987). The antibody recognises both the A and B subunits of bovine brain CaN. In sea urchin eggs the antibody recognized two immunogenic proteins of approximately 62 kD and 16 kD. The 62 kD protein was detected in the whole egg lysate, cytosolic fraction and to a lesser extent, in the cortical fraction. The 16 kD protein, which migrated with the same mobility on gel electrophoresis as purified bovine brain CaN B, was detected only in the whole egg lysate and cytosolic fraction and was not detected in the cortical fraction. On the basis of immunoreactivity, electrophoretic mobility and subunit structure, the 62 kD and 16 kD proteins can be assigned as homologues of CaN A and CaN B, respectively. Since in brain extracts, CaN A is always found associated with CaN B in a Ca^^-independent manner (Klee etal., 1988), it is reasonable to assume that a homologue of CaN B is also present in the egg cortex but in too small a quantity for detection.

The identification of a cortical CaN-like protein raises the possibility that exocytosis in sea urchin eggs proceeds in a manner analogous to that proposed for exocytosis in Paramedw/w (Momayezi et al., 1987). The results of experiments in which I investigated this possibility are discussed in section 4.

3. THE SEA URCHIN EGG SYNAPTOBREVEV HOMOLOGUE IS REQUIRED FOR EXOCYTOSIS.

The identification of a 17.5 kD synaptobrevin homologue localized to the cortical granule membrane suggested that exocytosis in the sea urchin egg might proceed in a manner analogous to the mechanism proposed for synaptic vesicle exocytosis (Sollner et

155 al, 1993a,b). The observation that exocytosis in sea urchin eggs is inhibited by treatment with the sulphydryl-modifying agent, A^-ethylmaleimide (NEM) (Haggerty and Jackson, 1983; Jackson etal, 1985; Whalley and Sokolofif, 1994), provides circumstantial evidence to support this hypothesis. According to the model proposed by Sollner et a l (1993a,b), ATP hydrolysis by the NEM-sensitive factor (NSF) constitutes a key step in the series of reactions leading to membrane fusion. The light chain of tetanus toxin (TeTx LC), which is a Zn^^-dependent endopeptidase, potently inhibits neurotransmitter release by specifically cleaving the synaptic vesicle protein synaptobrevin (Schiavo et a l, 1992; Link et a l, 1992). TeTx LC was therefore a useful tool to investigate the function of the synaptobrevin homologue in exocytosis.

(i) Tetanus Toxin Light Chain Cleaves the Synaptobrevin HomologueIn Vitro,

A whole egg membrane fraction was incubated with TeTx LC in vitro at 37 °C. Under these conditions, TeTx LC cleaved the 17.5 kD synaptobrevin homologue. Its effect was extremely rapid - the majority of the synaptobrevin homologue was digested within the first 15 minutes of incubation and cleavage was complete by 30 minutes, as determined by immunoblot analysis. The effect of TeTx LC appeared to be specific for synaptobrevin, since another cortical vesicle protein, rab3 A, was unaffected by the toxin. These results are consistent with those of Schiavo et al (1992) and Link et a l (1992) who showed that TeTx LC specifically cleaved neuronal synaptobrevin II. The susceptibility of the 17.5 kD protein to cleavage by TeTx LC confirms that it truly is a homologue of synaptobrevin.

(ii) Tetanus Toxin Light Chain Inhibits Cortical Granule Exocytosis.

In order to investigate the involvement of the synaptobrevin homologue in cortical granule exocytosis, cortices were incubated with TeTx LC in vitro. TeTx LC cleaved the cortical synaptobrevin homologue when applied to isolated cortices. Complete digestion was obtained after 15 minutes, as determined using immunoblot analysis. This result may seem surprising in view of the fact that in sea urchin eggs, the cortical granules are all

156 docked at the plasma membrane (Schuel et a l, 1972). In vitro studies have shown that synaptobrevin is not susceptible to TeTx LC cleavage when present, together with syntaxin, SNAP-25 and synaptotagmin, in the 7S complex proposed to mediate secretory vesicle docking at the plasma membrane (Pellegrini et a l, 1994; Hayashiet al., 1994). However, since the experiments were conducted at 37 °C rather than at the physiological temperature of 16 °C, a temperature-induced conformational change may have rendered the protein more susceptible to TeTx LC cleavage. This hypothesis would also explain the results of Steinhardt etal. (1994), who showed that TeTx had no effect on exocytosis when microinjected into unfertilized sea urchin eggs, since their experiments were conducted at 21 °C. This temperature is possibly too low for TeTx LC to gain access to the synaptobrevin cleavage site.

The cleavage of the sea urchin egg synaptobrevin homologue by TeTx LC correlated with a significant inhibition of Ca^^-stimulated exocytosis. The simplest interpretation of these results is that cortical granule exocytosis in sea urchin eggs requires synaptobrevin and proceeds via a mechanism analogous to that of synaptic vesicle exocytosis. There is, however, a discrepancy in the results which is that although after a 15 minute incubation of cortices with TeTx LC, the sea urchin synaptobrevin homologue was completely digested by TeTx LC, as determined by immunoblot analysis, the physiological data showed that exocytosis was only inhibited by approximately 63 %. Similar incomplete inhibition of secretion in spite of massive cleavage of synaptobrevin/cellubrevin has been observed in other systems (Galli et al., 1994; Chilcote et al., 1995). How can these results be explained if the sea urchin synaptobrevin homologue is absolutely required for exocytosis? One explanation for this apparent discrepancy is that a second isoform of synaptobrevin exists in sea urchin eggs which was not detected by the monoclonal antibody Cl 10.1 and which is resistant to cleavage by TeTx LC. This isoform would be able to mediate exocytosis in a proportion of the granules after TeTx treatment. Both a TeTx-sensitive and TeTX-insensitive isoform of synaptobrevin have been identified in rat brain (Schiavo et al., 1992) and in yeast, none of the synaptobrevin homologues are cleaved by TeTx LC (Niemann et a l, 1994). It is feasible, therefore, that a TeTx-insensitive isoform exists in sea urchin eggs. It is unlikely,

157 however, that such an isoform would not be recognized by mcAb Cl 10. 1 since this antibody recognizes the conserved cytoplasmic domain of synaptobrevins (R. Jahn, personal communication) and has been shown to cross-react with multiple synaptobrevin isoforms (Südhof et al., 1989). Several other interpretations of the data need to be considered.

(a) Is the Sea Urchin Egg Synaptobrevin Homologue Required at a Stage Prior to Fusion?

Fusion between two biological membranes has been proposed to occur in three consecutive steps: (i) docking, i.e. establishing a contact between the two membranes; (ii) priming, a process in which the proteins involved in the fusion event are aligned and activated; and (iii) the actual fusion event, during which phospholipids are rearranged and a hydrophilic continuum across the two fusing membranes is generated. (Jahn and Südhof, 1994). If synaptobrevin was required in reactions that energized docked granules into a so-called "primed" state, then TeTx LC could inhibit exocytosis by inhibiting this process. This hypothesis could be used to explain the effects of TeTx LC action when applied to isolated cortices, provided that the population of cortical granules existed in both non­ activated and activated states. The partial inhibition of exocytosis in egg cortices by TeTx LC would be due to the proportion of "activated" granules that had already passed the step in which synaptobrevin acts and were competent for fiision. Cleavage of synaptobrevin by TeTx LC would only inhibit exocytosis of "non-activated" granules. Although this argument supports both the Western blot data and the physiological data, it is hard to reconcile with evidence which implicates synaptobrevin to be involved in the membrane fusion reaction (Schiavo et a l , 1992; Link et a l , 1992; Hunt et al ., 1994). The observation that synaptobrevin contains a region which has a high propensity to form an alpha-helix also suggests that it plays a role in membrane fusion (Chapman et al., 1994; Hayashi et al., 1994). Alpha-helices are found in the viral fiision proteins of influenza virus which mediate binding of the virus particle to cell surfaces, internalization and fusion of the virus particle with cellular membranes (Carr and Kim, 1993; Bullough et a l, 1994; Yu et a l, 1994).

158 (b) Two Release Mechanisms in Sea Urchin Eggs?

Another interpretation of the data is that two exocytotic mechanisms exist in sea urchin eggs, one dependent upon and one independent of synaptobrevin function. Immunocytochemical staining of cortices showed a uniform distribution of the sea urchin synaptobrevin homologue throughout the cortical granule population. This observation suggests that all cortical granules possess the ability to exocytose in a reaction involving the synaptobrevin homologue. It is conceivable, however, that at least a proportion of the granules possess a second, as yet unknown, release mechanism. Anstrom et al. (1988) showed that the population of cortical granules in Strongylocentrotus purpuratus eggs was heterogenous. However, this heterogeneity referred to granule content and as yet, no studies regarding the heterogeneity of the membrane constituents of cortical granules has been conducted. The key question is what are the possible alternative release mechanisms? In Madin-Darby canine kidney (MDCK) cells, the existence of two alternative docking and fusion mechanisms has been demonstrated (Ikonen et al., 1995). In streptolysin O-permeabilized MDCK cells, stimulated transport from the trans-Golgi network (TGN) to the basolateral plasma membrane required the function of NSF and a- SNAP and was also sensitive to TeTx. In contrast, transport from the TGN to the apical plasma membrane proceeded by a mechanism that was independent of NSF and a-SNAP, insensitive to TeTx, but required the function of a novel annexin protein, annexin Xlllb (Ikonen et al., 1995; Fiedler et al., 1995). Transport to the basolateral plasma membrane was independent of annexin function (Fiedler et al., 1995). Thus it appears that two docking and fiision mechanisms exist within the same cell, one involving NSF, a-SNAP and a TeTx-sensitive protein (synaptobrevin?) and the other independent of NSF and a- SNAP but mediated by an annexin. Annexin Xlllb was shown to be specifically associated with apical transport vesicles and also behaved as a membrane protein in phase partitioning in Triton X-114, in the absence of added Ca^^ (Wadinger-Ness et a l, 1990). These observations are particularly interesting in view of the identification of members of the annexin family in sea urchin eggs. The 68 kD sea urchin annexin protein also remained associated with membranes in the absence of Ca^^. Morgan and Burgoyne (1995) have shown the involvement of both a-SNAP and annexins in Ca^^-stimulated exocytosis in

159 adrenal chromaffin cells.

(iii) The Evolutionary Conservation of the Exocytotic Fusion Complex.

The 'SNARE' hypothesis states that the specificity of intracellular membrane fusion events is encoded by the SNAREs (SNAP receptors) (Sollner et a l, 1993a,b). According to the SNARE hypothesis, an integral membrane protein resident on the transport vesicle (v-SNARE:synaptobrevin in nerve terminals) pairs with its receptor on the target membrane (t-SNARE: syntaxin and SNAP-25 in nerve terminals). Each form of intracellular fusion has its own set of specific v-SNAREs and t-SNAREs and the v- SNARE interacts only with its matching t-SNARE, ensuring that the transport vesicle fuses with the correct acceptor compartment.

The sea urchin synaptobrevin homologue which was localized to the cortical granule membrane interacted in vitro with a soluble syntaxin fragment (biotinylated (his)^ syntaxirf) cloned from a bovine adrenal medullary cDNA library. No interaction was detected between the (his) ^syntaxirf fragment and the sea urchin egg synaptobrevin homologue which was localized to intracellular membranes. The (his) ^syntaxirf fragment corresponds to the amino terminal of syntaxin 1A and contains the region of the protein shown to interact with both synaptobrevin and SNAP-25 (Hayashi et al., 1994). The fact that the(his) ^syntaxirf fragment only interacted with the synaptobrevin homologue localized to the cortical granule membrane supports the 'SNARE' hypothesis and suggests the existence of two different isoforms of synaptobrevin in sea urchin eggs. The synaptobrevin homologue localized to the cortical granule membrane appears to be structurally similar to synaptobrevins 1 and 2, since syntaxin 1A was shown to specifically interact with these two neuronal synaptobrevin isoforms (Calakos et a l, 1994). In contrast, the synaptobrevin homologue localized to intracellular membranes appears to be a different synaptobrevin isoform which may interact with another syntaxin isoform. This protein may correspond to cellubrevin, the cellular homologue of synaptobrevin, which was shown to be ubiquitously distributed in a constitutively recycling pathway, the receptor-mediated endocytosis pathway, in CHO cells (McMahon et a l, 1993). In some

160 cells, however, cellubrevin and synaptobrevin 2 were co-localized to the secretory vesicle membrane (Chilcote g/a/., 1995;Regazzi etal., 1995).

The interaction of the (his)^syntaxirf fragment with a cytosolic protein of approximately 16.5 kD may be due to interaction with an unidentified constituent of the exocytotic fusion complex. A good candidate for such a protein would be a member of the complexin family (McMahon et al., 1995). Complexins are a family of evolutionarily conserved, ubiquitous cytosolic proteins of around 18-19 kD which specifically interact with syntaxin in vitro (McMahon et al., 1995). Complexins were proposed to regulate the interaction of a-SNAP with the heterotrimeric fusion complex (McMahon et al, 1995). I

In summary, the specific interaction of the sea urchin egg cortical synaptobrevin homologue with a bovine (his)^syntaxirf fragment supports the idea that the molecular machinery underlying exocytotic membrane fusion is evolutionarily conserved. The data also supports the 'SNARE' hypothesis prediction that the specificity of membrane- membrane interactions is, at least in part, encoded by the SNAREs. In order to further test the validity of the SNARE' hypothesis, it would be interesting to probe sea urchin egg proteins with fi’agments corresponding to other syntaxin isoforms. The lack of interaction of the (his) ^syntaxirf fragment with any plasma membrane proteins suggests that the putative sea urchin egg t-SNARE (SNAP-25?) is structurally divergent from neuronal SNAP-25 and thus unable to interact with the mammalian syntaxin fragment.

4. THE ROLE OF CALCINEURIN IN EXOCYTOSIS.

(i) Exocytosis inParamecium.

Paramecium, exocytosis was shown to involve the rapid dephosphorylation of a 65 kD cortical phosphoprotein (Gilligan and Satir, 1982; Zieseniss and Plattner, 1985). Injection into intact cells or application to isolated cortices of a calcium-calmodulin-

161 calcineurin (Ca^^-CaM-CaN) complex induced exocytosis and a parallel déphosphorylation of the 65 kD phosphoprotein, whereas phosphatase inhibitors and antibodies against CaN blocked these events (Momayezi et al., 1987). Both CaM and a CaN-like protein were localized to the preformed exocytotic Paramecium (Momayezi et al., 1986, 1987). It was therefore suggested that exocytosis was triggered by a Ca^-CaM-CaN complex acting on the 65 kD phosphoprotein (Momayezi et a l, 1987). A similar localization of CaM and a CaN-like protein has been demonstrated in sea urchin eggs (Steinhardt and Alderton, 1982; Chapter 3). In sea urchin eggs, irreversible phosphorylation of proteins associated with the egg cortex inhibited exocytosis (Whalley e ta l, 1991). The trigger for exocytosis in sea urchin eggs is an increase in [Ca^^Ji to approximately 3-5 pM, a concentration that will activate the phosphatase activity of CaN (Stewart et a l, 1983). These observations, when taken together with the identification of CaN associated with the egg cortex, provide strong circumstantial evidence for the involvement of CaN in exocytosis. I investigated the role played by the cortical CaN-like protein in exocytosis in order to determine whether exocytosis in sea urchin eggs proceeds in a manner analogous to that proposed for trichocyst discharge in Paramecium (Momayezi et al, 1987).

(ii) The Effect of a Calmodulin Antagonist on Exocytosis.

The effect of a peptide antagonist of calmodulin, the MLCK 7 9 7 . 8 1 3 (myosin light chain kinase) peptide (Torok and Trentham, 1994), on exocytosis in sea urchin eggs was investigated. The MLCK 7 9 7,gi 3 peptide was chosen because it binds calmodulin in the same manner as calcineurin but with a much higher affinity, i.e. of 0.005 nM as compared to 0.1 nM (Kincaid et al, 1988; Tôrôk and Trentham, 1994).

The MLCK7 9 7 . 8 1 3 peptide had no effect on exocytosis when microinjected into sea urchin eggs to a final cytoplasmic concentration of 100 pM. This lack of effect may have been due to the abundance of calmodulin in the cell, making it impossible for the

MLCK7 9 7 . 8 1 3 peptide to antagonize all CaM-dependent reactions. When isolated cortices were incubated with the MLCK7 9 7 . 8 1 3 peptide at a concentration of 100 pM, Ca^^-

162 stimulated exocytosis was inhibited by approximately 30 %. In contrast, incubation with an MLCK control peptide, which does not bind calmodulin with high affinity, had no effect on exocytosis.

The inhibitory effect of the peptide on exocytosis in vitro suggests the involvement of a CaM-dependent enzyme, possibly calcineurin, in cortical granule exocytosis. The specificity of this effect is indicated by the lack of effect on exocytosis of the control peptide. However, it should be noted that the peptide only partially inhibited exocytosis in vitro. It is possible that the number of calmodulin-binding sites antagonised by the peptide was insufficient for full inhibition of exocytosis. This hypothesis is supported by the results of Steinhardt and Alderton (1982), who demonstrated that a large amount of calmodulin is associated with each cortical granule- plasma membrane complex with only a small fraction of it being necessary for full discharge at 5 pM Ca^^. Alternatively, it is conceivable that the partial inhibition of exocytosis is due to the existence of two alternative fusion mechanisms, one calmodulin- dependent and the other calmodulin-independent. Interestingly, the existence of two different fusion mechanisms was also suggested by the data discussed in section 3(ii).

(iii) The Effects of Calcineurin Antagonists on Exocytosis.

In order to determine whether the effect of the MLCK 7 9 7 . 8 1 3 peptide on exocytosis in vitro was mediated by calcineurin, the effects of specific calcineurin inhibitors on exocytosis were investigated. A polyclonal antibody against bovine brain calcineurin had no effect on cortical granule exocytosis either in vivo or in vitro. The same antibody inhibited exocytosis in Paramecium, both when microinjected and when applied to the isolated cortices of these cells (Momayezi et a l, 1987). A synthetic peptide corresponding to the autoinhibitory domain of calcineurin (Hashimoto et a l, 1990) had no effect on exocytosis when microinjected into sea urchin eggs. Finally, deltamethrin, which is a potent inhibitor of calcineurin activity (Enan and Matsumura, 1992), had no effect on exocytosis either in vivo or in vitro when used at concentrations of 20 pM and 10 pM, respectively. Deltamethrin has been reported to inhibit calcineurin activity at

163 concentrations as low as 0.1 nM (Enan and Matsumura, 1992).

In summary, none of the calcineurin inhibitors used affected exocytosis in sea urchin eggs. The consistently negative results obtained both in vivo and in vitro strongly argue against a role for calcineurin in exocytosis. This, in turn, implies that the effects of calmodulin antagonists on cortical granule exocytosis must be mediated by another, as yet unidentified, calmodulin-binding protein. On the basis of these results, it can be concluded that exocytosis in sea urchin eggs does not proceed in an analogous manner to that proposed for Paramecium (Momayezi et al., 1987).

(iv) The Role of Phosphatases in Exocytosis.

In sea urchin eggs, evidence suggests that an inhibitory phosphoprotein can intervene in the pathway that links an increase in to exocytotic membrane fusion (Whalleyetal, 1991). The only clue as to the identity of the phosphatase that mediates this effect was the observation that okadaic acid inhibited exocytosis in vivo (Whalley et al, 1991). The lack of effect of okadaic acid when applied to isolated cortices (Whalley et a l, 1991) suggests that the activation of a cytosolic phosphatase forms part of the mechanism that controls exocytosis in the intact egg and that this aspect of control is absent when exocytosis is studied in vitro. Since Ca^^-stimulated exocytosis occurs in vitro, activation of the phosphatase is not necessary to trigger exocytotic membrane fusion.

I correlated the inhibitory effect of okadaic acid on exocytosis in vivo with an effect on the Ca^^-independent phosphatase activity of sea urchin egg cytosol. Okadaic acid is a potent inhibitor of both protein phosphatases 1 and 2A (Bialojan and Takai, 1988; Haystead etal, 1989). Both of these phosphatases are cytosolic serine/threonine- specific phosphatases which are active in the absence of divalent cations (Cohen et al, 1989). It can therefore be speculated that either of these phosphatases mediates a dephosphorylation reaction which may comprise a step in the exocytotic pathway in vivo. The use of specific inhibitors which differentiate between these two phosphatases should

164 help elucidate precisely which phosphatase mediates this reaction.

The existence of inhibitory phosphoproteins has been suggested in a variety of secretory systems, including adrenal chromaffin cells (Brooks et al., 1984; Brooks and Brooks, 1985), mast cells (Tatham and Gomperts, 1989) and sea urchin eggs (Whalley etal, 1991). In sea urchin eggs, two cortical phosphoproteins of 33 kD and 27 kD were specifically thiophosphorylated after microinjection of p^S]ATPyS (Whalley et al., 1991). It was suggested, but not proven, that these proteins mediated the inhibitory effect of ATPyS on exocytosis (Whalley et at., 1991). The identification and isolation of inhibitory phosphoproteins and the elucidation of their interactions with the proteins that mediate membrane fusion should help unravel the series of molecular interactions that lead to exocytotic membrane fusion.

5. THE INOSITOL PHOSPHOLIPIDS AND EXOCYTOSIS.

ATP has been proposed to prime the exocytotic apparatus prior to Ca^^-stimulated exocytosis in a variety of secretory systems, including sea urchin eggs (Baker and Whitaker, 1978; Holz et al., 1989; Hay and Martin, 1992). The precise site and mechanism of action of ATP, however, is unknown. In permeabilized adrenal chromaffin cells, there is evidence to suggest that polyphosphoinositides are required for Ca^^- stimulated exocytosis, independent of their being substrates for the generation of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (Eberhard et a l, 1990). The proposal that the polyphosphoinositides are required for exocytosis was supported by the identification of phosphatidylinositol transfer protein (PtdlnsTP) and phosphatidylinositol-4-phosphate 5- kinase (PtdlnsPSK) as priming factors in Ca^^-stimulated exocytosis (Hay et al., 1993,1995). Taken together, these results suggest that the phosphoinositide kinases are, at least in part, responsible for the ATP requirement in exocytotic priming, their function being to maintain the level of PtdInsP 2 -

In sea urchin eggs, both phosphatidylinositol 4-kinase (PtdIns4K) and PtdInsP5K

165 activities were identified in the cortical region of unfertilized sea urchin eggs (Oberdorf etal., 1989). I investigated whether the decrease in exocytotic response in vitro due to the absence of ATP (Baker and Whitaker, 1978; Moy eta l, 1983; Sasaki and Epel, 1983) was due to changes in the levels of the polyphosphoinositides in the egg cortex.

(i) The ATP Requirement of Exocytosis in Sea Urchin Eggs.

Several laboratories have demonstrated that ATP is required to maintain a competent exocytotic apparatus in sea urchin eggs (Baker and Whitaker, 1978; Moy et al., 1983; Sasaki and Epel, 1983). However, the results obtained from the different laboratories varied, for example, Sasaki and Epel (1983) reported no loss of Ca^- sensitivity in cortices incubated for up to 12 hours in the presence of ATP, whereas Moy etal. (1983) reported that the Ca^^-sensitivity of exocytosis in isolated cortices decreased with time, even in the presence of ATP. Since the results obtained from the different laboratories varied, I characterized the ATP requirement of exocytosis in the in vitro system used in my experiments.

Cortices were prepared in intracellular medium and were then incubated in either the presence or absence of 2.5 mM ATP. Exocytosis was triggered by the addition of buffer containing 5.9 pM free Ca^^ and was measured 3 minutes after calcium addition. In agreement with previous work (Whitaker and Baker, 1978; Sasaki and Epel, 1983; Moy et a l, 1983), I demonstrated that there was no requirement for ATP per se during Ca^^-stimulated exocytosis in sea urchin eggs. When exocytosis was stimulated immediately after cortex preparation, the exocytotic response was the same for cortices isolated in either the presence or absence of ATP and was approximately 80 %. When calcium was added to cortices after 15 minutes incubation in the absence of ATP, exocytosis decreased to approximately 35 %. After 60 minutes incubation in the absence of ATP, exocytosis further diminished to 17 %. In contrast, in cortices incubated in the presence of ATP, exocytosis decreased only slightly to approximately 78 % after 15 minutes and to approximately 40 % after 60 minutes.

166 From these results it can be concluded that ATP in the intact egg primed the exocytotic apparatus prior to Ca^^-stimulated exocytosis in the isolated cortex. The ATP- dependent "primed" state was labile and decayed rapidly with time following cortex isolation in the absence of ATP Inclusion of ATP in the incubation medium slowed the rate of loss of Ca^^-sensitivity, thus maintaining the extent of exocytosis triggered after prolonged incubation times. However, even in the presence of ATP, the exocytotic response diminished with time following cortex isolation, an effect which was previously described by Moy et al. (1983).

In sea urchin eggs, proteins which confer Ca^^-sensitivity to exocytosis were extracted from cortices using chaotropic anions (Sasaki and Epel, 1983). One such protein, a 100 kD protein, was also found in extracts of murine brain (Sasaki, 1992). The decrease in exocytosis in the presence of ATP might be due to the gradual dissociation of these exocytosis-relevant proteins from the cortex. This phenomenon is also likely to occur in the absence of ATP. The identification of the 100 kD protein in extracts of murine brain suggests that proteins which confer Ca^^-sensitivity to the exocytotic apparatus are evolutionarily conserved. Since cytosolic proteins are required for exocytosis in a number of other secretory cells (Ali et al., 1989; Koffer and Gomperts, 1989; Sarafian et al., 1991; Hay and Martin, 1992; Walent et al., 1992; Morgan and Burgoyne, 1992b), the loss of such proteins in the sea urchin egg in vitro system may also contribute to the decrease in exocytotic response.

(ii) The Level of ExocytosisIn Vitro Correlates with the Level of Cortical Phosphatidylinositol 4,5-Bisphosphate.

By measuring changes in the levels of pH]-labelled cortical inositol phospholipids, the decrease in exocytotic response due to the absence of ATP was correlated with a concomitant decrease in the level of cortical phosphatidylinositol 4,5-bisphosphate

(PtdInsP 2 ). After incubation of cortices in the absence of ATP, the decrease in both the exocytotic response and the level of cortical PtdInsP 2 were approximately doubled, as compared to the decreases seen after incubation in the presence of ATP. In contrast, the

167 level of PtdlnsP decreased by approximately the same amount in both the presence and absence of ATP. These results are consistent with those of Eberhard et al. (1990) who correlated the levels of polyphosphoinositides with Ca^^-stimulated exocytosis in permeabilized adrenal chromaffin cells. Thus, it appears that PtdInsP 2 is required for Ca^^-stimulated exocytosis in sea urchin eggs.

In order to further correlate changes in the amount of cortical Ptdlnsf 2 with exocytotic competence, the effect of preincubation with neomycin was investigated. Neomycin is an aminoglycoside antibiotic that prevents the hydrolysis of polyphosphoinositides by binding to their polar headgroups (Schibeci and Schacht, 1977; Schacht, 1978; Downes and Michell, 1981). In permeabilized adrenal chromaffin cells, preincubation with neomycin prevented the decrease in secretory response due to the absence of ATP by maintaining the levels of the polyphosphoinositides (Eberhard et al., 1990). Preincubation of isolated sea urchin egg cortices with neomycin had the opposite effect on exocytosis, i.e. it inhibited exocytosis in a dose-dependent manner. Since neomycin was shown to directly inhibit exocytosis in sea urchin eggs in vitro (Whitaker and Aitchison, 1985), the best explanation for these results is that neomycin was not removed from the cortices by extensive washing. It is unclear as to why this was the case in sea urchin eggs and not in adrenal chromaffin cells. Similar concentrations of neomycin were used in both sets of experiments.

In sea urchin eggs, the correlation between exocytotic competence and the level of cortical phosphatidylinositol 4,5-bisphosphate (Ptdlns? 2 ) supports the hypothesis that the phosphoinositide kinases are, at least in part, responsible for the ATP requirement of exocytotic priming. However, since I have shown that a synaptobrevin homologue is required for exocytosis in sea urchin eggs, it is possible that an additional ATP-requiring step mediated by the A-ethylmaleimide-sensitive factor (NSF) may also be involved. According to the model of membrane fiision proposed by O'Connor et al. (1994), ATP hydrolysis by NSF primes the exocytotic apparatus prior to Ca^^-stimulated exocytosis which is mediated by synaptobrevin, syntaxin and SNAP-25.

168 In order to further characterize the role played by the inositol phospholipids in exocytosis in sea urchin eggs, the effect of phosphatidylinositol transfer protein (PtdlnsTP) on exocytosis in vitro could be investigated PtdlnsTP has been proposed to function in exocytotic priming by providing Ptdlns for further phosphorylation to produce

PtdInsP 2 (Hay and Martin, 1993). Since both PtdIns4K and PtdInsP5K activities were localized to the cortex of unfertilized sea urchin eggs (Oberdorf et a l, 1989), it is possible that PtdlnsTP might support exocytosis in vitro in an analogous manner as in permeabilized PC12 cells (Hay and Martin, 1993). The effect of antibodies against

PtdInsP 2 on exocytosis in vitro could also be investigated. Such antibodies inhibited Ca^^- stimulated exocytosis in permeabilized PC 12 cells (Martin et a l, 1995). As yet, the role played by Ptdlnsf 2 in exocytotic membrane fusion is unknown.

6. SUMMARY.

Regulated secretion consists of sequential priming and triggering steps which depend on ATP and calcium, respectively (Holz e ta l, 1989; Hay and Martin, 1992). I have demonstrated that in sea urchin eggs the phosphoinositide kinases are, at least in part, responsible for the ATP requirement of exocytotic priming. I have shown that a homologue of the synaptic protein synaptobrevin is localized to the cortical granule membrane in sea urchin eggs and is required for Ca^^-stimulated exocytosis. In contrast, my results show that the Ca^VCaM-dependent phosphoprotein phosphatase calcineurin is not involved in exocytosis.

My results support the hypothesis that universal biochemical mechanisms underlie all eukaryotic fusion events (Sollner et a l, 1993a,b). However, an exciting prospect which was suggested by my results is the possible existence of more than one exocytotic fusion mechanism within the same cell. The existence of more than one fusion mechanism has already been demonstrated in Madin-Darby canine kidney cells (Ikonen et al, 1995).

The sea urchin egg is a unique cellular system in which to study exocytosis. The

169 main advantage of the sea urchin egg is that exocytosis can be studied in vitro. In addition, secretory granules and plasma membrane can be isolated separately and undergo exocytosis in response to calcium when recombined. The sea urchin egg remains the only exocytotic system that can be reconstituted efficiently in this way. My results indicate that the elucidation of the molecular mechanisms underlying exocytosis in the sea urchin egg will contribute towards the understanding of the general fusion mechanisms occurring in all eukaryotic cells.

170 Acknowledgements.

I would like to thank the Medical Research Council for providing funds for the work presented in this thesis.

I would like to thank Prof. Michael Whitaker for his support during three years of stimulating research. I would like to thank Dr. S. Moss for his support and useful discussions. I also thank Dr. R. Patel for his advise and Dr. A. Hodel for proof-reading my thesis.

Above all I thank my parents, my sisters Rachel and Fiona, and Justin Bharucha for their love and support whilst I was working on this thesis.

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206 Some Abbreviations which Appear in the Text.

AppNHp adenylyl-imidodiphosphate ASW artificial sea water ATP adenosine-5'-triphosphate

ATPyS adenosine-5'-0-(3-thiotriphosphate)

[C a^i intracellular free calcium ion concentration CaM calmodulin DAG 1,2-diacylglycerol

EGTA ethylene glycol-bis(P-aminoethylether)A/7V-tetraacetic acid GDPPS guanosine-5'-0-(2-thiodiphosphate) GTP guanosine-5'-triphosphate GTPyS guanosine-5'-0-(3-thiotriphosphate) IM intracellular medium InsPj inositol 1,4,5-trisphosphate kD kilodalton MW molecular weight Ptdlns phosphatidylinositol PtdlnsP phosphatidylinositol-4-phosphate Ptdln&P; phosphatidylinositol 4,5-bisphosphate PtdlnsTP phosphatidylinositol transfer protein PtdIns4K phosphatidylinositol 4-kinase PtdlnsPSK phosphatidylinositol-4-phosphate 5-kinase PLC phospholipase C PKC protein kinase C s.e.m. standard error of the mean TPA tetradecanoyl phorbol acetate

207