Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs

At fertilization in sea urchin eggs, elevated cytosolic Ca2+ leads to the exocytosis of 15,000-18,000 1.3-microns-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueous- and lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-microns-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 min. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrin-coated vesicles. When eggs are treated with short wave-length ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures.     

Members of the SNARE hypothesis are associated with cortical granule exocytosis in the sea urchin egg

Cortical granule exocytosis is important for the block to polyspermy at fertilization in the eggs of most vertebrates and many invertebrates. Cortical granules are poised at the cell surface and exocytose in response to sperm stimulation. Following exocytosis, the cortical granule contents modify the extracellular environment of the egg, the major result of which is to block additional sperm binding. Here we show that proteins homologous to members of the SNARE hypothesis—a molecular model designed to explain the trafficking, docking, and exocytosis of vesicles in the secretory compartment—are present in eggs at the right time and place to be involved in the regulation of cortical granule exocytosis. Using polymerase chain reaction (PCR) screens we have found homologues of synaptobrevin/VAMP, syntaxin, synaptotagmin, and rab3. Antibodies generated to fusion proteins or to synthetic peptides encoded by the cloned cDNAs were used in an immunofluorescence assay to show that each of the cognate proteins are present in the cortex of the egg. A synaptobrevin/VAMP homologue appears to be specifically associated with the membrane of cortical granules before fertilization and, following cortical granule exocytosis, is incorporated into the plasma membrane of the zygote. A rab3 homologue is also associated with cortical granules specifically but, following fertilization, the protein reassociates with different, yet undefined, vesicles throughout the cytoplasm of the zygote. Homologues of synaptotagmin and syntaxin are also present at the egg cortex but, in contrast to rab3 and VAMP, appear to be associated with the plasma membrane. Following fertilization, syntaxin and tagmin remain associated with the plasma membrane and are more readily immunolabeled, presumably due to an increased accessibility of the antibodies to the target protein domains. We also show by immunoblotting experiments that the cognate proteins are of the sizes predicted for these homologues. These results suggest that at least some steps in the biology of cortical granules may be mediated by SNARE homologues, and this finding, along with the unique biology of cortical granules, should facilitate examination of specific events of the fertilization reaction and the mechanism of stimulus-dependent exocytosis. Mol. Reprod. Dev. 48:106–118, 1997. © 1997 Wiley-Liss, Inc.     

Synaptotagmin I is involved in the regulation of cortical granule exocytosis in the sea urchin

Cortical granules are stimulus-dependent secretory vesicles found in the egg cortex of most vertebrates and many invertebrates. Upon fertilization, an increase in intracellular calcium levels triggers cortical granules to exocytose enzymes and structural proteins that permanently modify the extracellular surface of the egg to prevent polyspermy. Synaptotagmin is postulated to be a calcium sensor important for stimulus-dependent secretion and to test this hypothesis for cortical granule exocytosis, we identified the ortholog in two sea urchin species that is present selectively on cortical granules. Characterization by RT-PCR, in-situ RNA hybridization, Western blot and immunolocalization shows that synaptotagmin I is expressed in a manner consistent with it having a role during cortical granule secretion. We specifically tested synaptotagmin function during cortical granule exocytosis using a microinjected antibody raised against the entire cytoplasmic domain of sea urchin synaptotagmin I. The results show that synaptotagmin I is essential for normal cortical granule dynamics at fertilization in the sea urchin egg. Identification of this same protein in other developmental stages also shown here will be important for interpreting stimulus-dependent secretory events for signaling throughout embryogenesis.     

Ultrastructural observations of human and mouse oocytes treated with cryopreservatives

The effects of the cryopreservative agents dimethylsulfoxide (DMSO) and propanediol (PROH) on mature human and mature mouse oocytes have been examined with transmission electron microscopy. Treatment of CD-1 mouse oocytes and human preovulatory oocytes in a stepwise manner with either DMSO or PROH up to 1.5 M appears to trigger the exocytosis of 70-80% of the cortical granules in all oocytes. Successive stages in premature dehiscence, including a loss in granule electron density, fusion of the granule-limiting membrane with the oolemma, and extrusion of the cortical granule core into the perivitelline space, have been observed in all human oocytes studied. In addition, all human DMSO- and PROH-treated oocytes exhibited crypt-like invaginations and clusters of endocytic vesicles that subtend the oolemma. The presence of these crypts and pinocytotic vesicles in treated oocytes may suggest a mechanism for the retrieval of cortical granule membrane that is inserted into the original plasmalemma during exocytosis. The paucity of cortical granules in treated mouse and human oocytes as it potentially relates to an impaired ability to elicit the cortical reaction at fertilization is discussed.     

A Nibbling Mechanism for Clathrin-mediated Retrieval of Secretory Granule Membrane after Exocytosis

Clathrin-mediated endocytosis is the major pathway for recycling of granule membrane components after strong stimulation and high exocytotic rates. It resembles “classical” receptor-mediated endocytosis but has a trigger that is unique to secretion, the sudden appearance of the secretory granule membrane in the plasma membrane. The spatial localization, the relationship to individual fusion events, the nature of the cargo, and the timing and nature of the nucleation events are unknown. Furthermore, a size mismatch between chromaffin granules (∼300-nm diameter) and typical clathrin-coated vesicles (∼90 nm) makes it unlikely that clathrin-mediated endocytosis internalizes as a unit the entire fused granule membrane. We have used a combination of total internal reflection fluorescence microscopy of transiently expressed proteins and time-resolved quantitative confocal imaging of endogenous proteins along with a fluid-phase marker to address these issues. We demonstrate that the fused granule membrane remains a distinct entity and serves as a nucleation site for clathrin- and dynamin-mediated endocytosis that internalizes granule membrane components in small increments.     

The biology and dynamics of mammalian cortical granules

Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals.     

A role for myosin 1e in cortical granule exocytosis in Xenopus oocytes

Xenopus oocytes undergo dynamic structural changes during maturation and fertilization. Among these, cortical granule exocytosis and compensatory endocytosis provide effective models to study membrane trafficking. This study documents an important role for myosin 1e in cortical granule exocytosis. Myosin 1e is expressed at the earliest stage that cortical granule exocytosis can be detected in oocytes. Prior to exocytosis, myosin 1e relocates to the surface of cortical granules. Overexpression of myosin 1e augments the kinetics of cortical granule exocytosis, whereas tail-derived fragments of myosin 1e inhibit this secretory event (but not constitutive exocytosis). Finally, intracellular injection of myosin 1e antibody inhibits cortical granule exocytosis. Further experiments identified cysteine string proteins as interacting partners for myosin 1e. As constituents of the membrane of cortical granules, cysteine string proteins are also essential for cortical granule exocytosis. Future investigation of the link between myosin 1e and cysteine string proteins should help to clarify basic mechanisms of regulated exocytosis.     

OOCYTE DIFFERENTIATION IN THE SEA URCHIN, ARBACIA PUNCTULATA, WITH PARTICULAR REFERENCE TO THE ORIGIN OF CORTICAL GRANULES AND THEIR PARTICIPATION IN THE CORTICAL REACTION

This paper presents morphological evidence on the origin of cortical granules in the oocytes of Arbacia punctulata and other echinoderms. During oocyte differentiation, those Golgi complexes associated with the production of cortical granules are composed of numerous saccules with companion vesicles. Each element of the Golgi complex contains a rather dense homogeneous substance. The vesicular component of the Golgi complex is thought to be derived from the saccular member by a pinching-off process. The pinched-off vesicles are viewed as containers of the precursor(s) of the cortical granules. In time, they coalesce and form a mature cortical granule whose content is bounded by a unit membrane. Thus, it is asserted that the Golgi complex is involved in both the synthesis and concentration of precursors utilized in the construction of the cortical granule. Immediately after the egg is activated by the sperm the primary envelope becomes detached from the oolemma, thereby forming what we have called the activation calyx (see Discussion). Subsequent to the elaboration of the activation calyx, the contents of cortical granules are released (cortical reaction) into the perivitelline space. The discharge of the constituents of a cortical granule is accomplished by the union of its encompassing unit membrane, in several places, with the oolemma.     

Structural studies of SNARE complex and its interaction with complexin by molecular dynamics simulation

Since neurotransmitter releasing into the synaptic space delivers electrical signals from presynaptic neural cell to the postsynaptic cell, neurotransmitter secretion must be much orchestrated. Crowded intracellular vesicles involving neurotransmitters present a question of the how secretory vesicles fuse onto the plasma membrane in a fast synchronized fashion. Complexin is one of the most experimentally studied proteins that regulate assembly of fusogenic four‐helix SNARE complex to synchronized neurotransmitter secretion. We used MD simulation to investigate the interaction of complexin with the neural SNARE complex in detail. Our results show that the SNARE complex interacts with the complexin central helix by forming salt bridges and hydrogen bonds. Complexin also can interact with the Q‐SNARE complex instead of synaptobrevin to decrease the Q‐SNARE flexibility. The complexin alpha‐accessory helix and the C‐terminal region of synaptobrevin can interact with the same region of syntaxin. Although the alpha‐accessory helix aids the tight binding of the central helix to the SNARE complex, its proximity with synaptobrevin causes the destabilization of syntaxin and Sn1 helices. This study suggests that the alpha‐accessory helix of complexin can be an inhibiting factor for membrane fusion by competing with synaptobrevin for binding to the Q‐SNARE complex. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 560–570, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini

Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca²⁺-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca²⁺-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca²⁺-sensitive regulatory pathway for zymogen granule exocytosis.     

Characterization, fate, and function of hamster cortical granule components

Little is known about the composition and function of mammalian cortical granules. In this study, lectins were used as tools to: (1) estimate the number and molecular weight of glycoconjugates in hamster cortical granules and show what sugars are associated with each glycoconjugate; (2) identify cortical granule components that remain associated with the oolemma, cortical granule envelope, and/or zona pellucida of fertilized oocytes and preimplantation embryos; and (3) examine the role of cortical granule glycoconjugates in preimplantation embryogenesis. Microscopic examination of unfertilized oocytes revealed that the lectins PNA, DBA, WGA, RCA(120), Con A, and LCA bound to hamster cortical granules. Moreover, LCA and Con A labeled the zona pellucida, cortical granule envelope, and plasma membrane of fertilized and artificially activated oocytes and two and eight cell embryos. Lectin blots of unfertilized oocytes had at least 12 glycoconjugates that were recognized by one or more lectins. Nine of these glycoconjugates are found in the cortical granule envelope and/or are associated with the zona pellucida and plasma membrane following fertilization. In vivo functional studies showed that the binding of Con A to one or more mannosylated cortical granule components inhibited blastomere cleavage in two-cell embryos. Our data show that hamster cortical granules contain approximately 12 glycoconjugates of which nine remain associated extracellularly with the fertilized oocyte after the cortical reaction and that one or more play a role in regulating cleavage divisions.     

Combinatorial SNARE complexes modulate the secretion of cytoplasmic granules in human neutrophils

Mobilization of human neutrophil granules is critical for the innate immune response against infection and for the outburst of inflammation. Human neutrophil-specific and tertiary granules are readily exocytosed upon cell activation, whereas azurophilic granules are mainly mobilized to the phagosome. These cytoplasmic granules appear to be under differential secretory control. In this study, we show that combinatorial soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with vesicle-associated membrane proteins (VAMPs), 23-kDa synaptosome-associated protein (SNAP-23), and syntaxin 4 underlie the differential mobilization of granules in human neutrophils. Specific and tertiary granules contained VAMP-1, VAMP-2, and SNAP-23, whereas the azurophilic granule membranes were enriched in VAMP-1 and VAMP-7. Ultrastructural, coimmunoprecipitation, and functional assays showed that SNARE complexes containing VAMP-1, VAMP-2, and SNAP-23 mediated the rapid exocytosis of specific/tertiary granules, whereas VAMP-1 and VAMP-7 mainly regulated the secretion of azurophilic granules. Plasma membrane syntaxin 4 acted as a general target SNARE for the secretion of the distinct granule populations. These data indicate that at least two SNARE complexes, made up of syntaxin 4/SNAP-23/VAMP-1 and syntaxin 4/SNAP-23/VAMP-2, are involved in the exocytosis of specific and tertiary granules, whereas interactions between syntaxin 4 and VAMP-1/VAMP-7 are involved in the exocytosis of azurophilic granules. Our data indicate that quantitative and qualitative differences in SNARE complex formation lead to the differential mobilization of the distinct cytoplasmic granules in human neutrophils, and a higher capability to form diverse SNARE complexes renders specific/tertiary granules prone to exocytosis.     

‘Porosome’ discovered nearly 20 years ago provides molecular insights into the kiss‐and‐run mechanism of cell secretion

Secretion is a fundamental cellular process in living organisms, from yeast to cells in humans. Since the 1950s, it was believed that secretory vesicles completely merged with the cell plasma membrane during secretion. While this may occur, the observation of partially empty vesicles in cells following secretion suggests the presence of an additional mechanism that allows partial discharge of intra‐vesicular contents during secretion. This proposed mechanism requires the involvement of a plasma membrane structure called ‘porosome’, which serves to prevent the collapse of secretory vesicles, and to transiently fuse with the plasma membrane (Kiss‐and‐run), expel a portion of its contents and disengage. Porosomes are cup‐shaped supramolecular lipoprotein structures at the cell plasma membrane ranging in size from 15 nm in neurons and astrocytes to 100–180 nm in endocrine and exocrine cells. Neuronal porosomes are composed of nearly 40 proteins. In comparison, the 120 nm nuclear pore complex is composed of >500 protein molecules. Elucidation of the porosome structure, its chemical composition and functional reconstitution into artificial lipid membrane, and the molecular assembly of membrane‐associated t‐SNARE and v‐SNARE proteins in a ring or rosette complex resulting in the establishment of membrane continuity to form a fusion pore at the porosome base, has been demonstrated. Additionally, the molecular mechanism of secretory vesicle swelling, and its requirement for intra‐vesicular content release during cell secretion has also been elucidated. Collectively, these observations provide a molecular understanding of cell secretion, resulting in a paradigm shift in our understanding of the secretory process.     

Secretory carrier membrane proteins 31-35 define a common protein composition among secretory carrier membranes.

A novel compositional overlap between membranes of exocrine and endocrine granules, synaptic vesicles, and a liver Golgi fraction has been identified using a monoclonal antibody (SG7C12) raised against parotid secretion granule membranes. This antibody binds secretory carrier membrane proteins with apparent Mr 31,000, 33,000 and 35,000 (designated SCAMPs 31, 33, 35). The proteins are nonglycosylated integral membrane components, and the epitope recognized by SG7C12 is on the cytoplasmic side of the granule membrane. SCAMP 33 is found in all secretory carrier membranes studied so far while SCAMP 35 is found in exocrine and certain endocrine granules and liver Golgi membranes and SCAMP31 only in exocrine granules. They are not related to other similar-sized proteins that have been studied previously in relation to vesicular transport and secretion. Immunocytochemical staining shows that these SCAMPs are highly concentrated in the apical cytoplasm of exocrine cells. Antigens are present not only on exocrine granules and synaptic vesicles but also on other smooth membrane vesicles of exocrine and neural origin as revealed by immunolocalization in subcellular fractions and immunoadsorption to antibody-coated magnetic beads. The wide tissue distribution and localization to secretory carriers and related membranes suggest that SCAMPs 31-35 may be essential components in vesicle-mediated transport/secretion.     

Analysis of sea urchin egg cortical transformation in the absence of cortical granule exocytosis

A burst of endocytosis accompanying microvillar elongation follows cortical granule exocytosis in normal sea urchin development. By 5 min postfertilization the burst is over and a lower level of endocytosis ensues (constitutive phase). To determine whether microvillar elongation and initiation of endocytosis are necessary concommitants of cortical granule exocytosis we utilized Chase's (1967, Ph.D. thesis, University of Washington, Seattle) high-hydrostatic pressure technique to block the latter and then examined developing eggs for endocytosis and microvillar elongation. To accomplish this, eggs were fertilized, after which hydrostatic pressure was quickly raised to 6000-7000 psi at the start of cortical granule exocytosis and maintained for 5 min. Only the cortical granules immediately surrounding the sperm penetration site were secreted (about 3% or less of the egg's total number of cortical granules). Blockage of major cortical granule exocytosis had the following consequences on surface events during first division: (1) The endocytosis burst normally associated with cortical granule exocytosis was effectively eliminated as was early microvillar elongation and elevation. Both occurred to a limited extent around the sperm penetration site which resulted in a highly localized surface transformation. (2) By 20 min after fertilization endocytosis began over the rest of the egg surface in the absence of any further cortical granule exocytosis. (3) Subsequently, during a 30-min period starting midway between fertilization and first cleavage microvilli more than doubled in length and endocytosis levels increased severalfold. These events brought about a complete surface transformation similar to that which normally occurs in early development but in the absence of cortical granule exocytosis. By first cleavage surfaces and cortices of high-pressure-treated and control eggs were nearly indistinguishable except for the presence of cortical granules in cortices of the former. Pressure-treated eggs cleaved normally and developed to larval forms overnight. The period of late surface transformation in high-pressure-treated Strongylocentrotus purpuratus eggs corresponds in timing and some of its characteristics to second phase microvillar elongation observed in normal development in this species and also in S. droebachiensis development. These observations suggest, therefore, that microvillar elongation and endocytosis are necessary membrane remodelling events which must occur for normal development even in the absence of membrane addition from the cortical granules.     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphoniidae) II. Vitellogenesis, follicle cell structure and egg shell formation

By the end of previtellogenesis, the oocytes of Glossiphonia heteroclita gradually protrude into the ovary cavity. As a result they lose contact with the ovary cord (which begins to degenerate) and float freely within the hemocoelomic fluid. The oocyte's ooplasm is rich in numerous well-developed Golgi complexes showing high secretory activity, normal and transforming mitochondria, cisternae of rER and vast amounts of ribosomes. The transforming mitochondria become small lipid droplets as vitellogenesis progresses. The oolemma forms microvilli, numerous coated pits and vesicles occur at the base of the microvilli, and the first yolk spheres appear in the peripheral ooplasm. A mixed mechanism of vitellogenesis is suggested. The eggs are covered by a thin vitelline envelope with microvilli projecting through it. The envelope is formed by the oocyte. The vitelline envelope is produced by exocytosis of vesicles containing two kinds of material, one of which is electron-dense and seems not to participate in envelope formation. The cortical ooplasm of fully grown oocytes contains many cytoskeletal elements (F-actin) and numerous membrane-bound vesicles filled with stratified content. Those vesicles probably are cortical granules. The follicle cells surrounding growing oocytes have the following features: (1) they do not lie on a basal lamina; (2) their plasma membrane folds deeply, forming invaginations which eventually seem to form channels throughout their cytoplasm; (3) the plasma membrane facing the ovary lumen is lined with a layer of dense material; and (4) the plasma membrane facing the oocyte forms thin projections which intermingle with the oocyte microvilli. In late oogenesis, the follicle cells detach from the oocytes and degenerate in the ovary lumen.     

Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs

We investigated the effect of the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate) on the egg morphology of the sea urchin Arbacia lixula. Our study indicates that TPA alters the cortical region of the egg: the pigment granules migrate toward the surface, while cortical granules detach from the plasma membrane. Cortical granule exocytosis did not occur but the endocytosis process was turned on. Prolonged treatment of the eggs by TPA partially inhibits the cortical granule exocytosis normally triggered by fertilization. We discuss the effects of TPA in terms of its interaction with the Ca2+ pool and cytoskeletal structures. In order to discern the respective roles of pHi and protein kinase C activity in endocytosis process activation, we compared the ultrastructural effects of TPA and ammonia. Finally, the role of pigment vesicles in egg metabolism activation is discussed.     

A Rho-signaling pathway mediates cortical granule translocation in the sea urchin oocyte

Cortical granules are secretory vesicles of the egg that play a fundamental role in preventing polyspermy at fertilization. In the sea urchin egg, they localize directly beneath the plasma membrane forming a compact monolayer and, upon fertilization, undergo a Ca(2+)-dependent exocytosis. Cortical granules form during early oogenesis and, during maturation, translocate from the cytosol to the oocyte cortex in a microfilament-mediated process. We tested the hypothesis that these cortical granule dynamics were regulated by Rho, a GTPase of the Ras superfamily. We observed that Rho is synthesized early in oogenesis, mainly in a soluble form. At the end of maturation, however, Rho associates with cortical granules. Inhibition of Rho with the C3 transferase from C. botulinum blocks cortical granule translocation and microfilaments undergo a significant disorganization. A similar effect is observed by GGTI-286, a geranylgeranyl transferase inhibitor, suggesting that the association of Rho with the cortical granules is indispensable for its function. In contrast, the anchorage of the cortical granules in the cortex, as well as their fusion at fertilization, are Rho-independent processes. We conclude that Rho association with the cortical granules is a critical regulatory step in their translocation to the egg cortex.     

Involvement of complexin 2 in docking, locking and unlocking of different SNARE complexes during sperm capacitation and induced acrosomal exocytosis

Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3). By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1) recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2) the assembly of a fusion-driving SNARE complex which executes Ca(2+)-dependent AE, (3) the disassembly of the earlier reported docking SNARE complex, (4) the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca(2+) which are all involved in AE make sperm an ideal model for studying exocytosis.     

Control of local actin assembly by membrane fusion-dependent compartment mixing

Local actin assembly is associated with sites of exocytosis in processes ranging from phagocytosis to compensatory endocytosis. Here, we examine whether the trigger for actin-coat assembly around exocytosing Xenopus egg cortical granules is 'compartment mixing'--the union of the contents of the plasma membrane with that of the secretory granule membrane. Consistent with this model, compartment mixing occurs on cortical granule-plasma membrane fusion and is required for actin assembly. Compartment mixing triggers actin assembly, at least in part, through diacylglycerol (DAG), which incorporates into the cortical granule membranes from the plasma membrane after cortical granule-plasma membrane fusion. DAG, in turn, directs long-term recruitment of protein kinase Cbeta (PKCbeta) to exocytosing cortical granules, where it is required for activation of Cdc42 localized on the cortical granules. The results demonstrate that mixing of two membrane compartments can direct local actin assembly and indicate that this process is harnessed during Xenopus egg cortical granule exocytosis to drive compensatory endocytosis.