Granule-plasma Membrane Fusion Research Articles

VAMP7 knockdown in secretory granules impairs CCL2 secretion in mast cells

Mast cells (MCs) possess numerous potent inflammatory mediators and undergo differential regulation in response to antigen (Ag) stimulation. Among the regulatory systems governing secretory responses, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in facilitating granule-plasma membrane fusion and subsequent secretion. Our previous investigation documented the involvement of vesicle-associated membrane protein 3 (VAMP3) in regulating cytokine secretions in RBL-2H3 cells, a model for MC IgE-mediated responses. In addition to VAMP3, VAMP7 is expressed in MCs, but its functional role remains elusive. The present study seeks to explore VAMP7-specific regulatory mechanisms in MCs, shedding light on one of the mechanisms governing heterogeneous secretory responses in these cells. Murine bone marrow-derived mast cells (BMMCs) were examined to analyze the subcellular distribution of inflammatory mediators, specifically TNFα, CCL2, and histamine, and VAMPs (i.e., VAMP3, VAMP7, and VAMP8). Immunocytochemistry and the transient expression of fluorescent protein-conjugated target proteins were used to discern the distribution of various inflammatory mediators and VAMP7 through confocal laser scanning microscopy. Each inflammatory mediator (TNFα, CCL2, and histamine) was found in secretory granules of different sizes within BMMCs. VAMP7 exhibited a distinct distribution compared to VAMP3 in these granules. Notably, an overlapping distribution was observed between VAMP7 and CCL2, but not between VAMP7 and TNFα or VAMP7 and histamine. This suggests that CCL2 resides within VAMP7-expressing granules and is subject to VAMP7-dependent secretory regulation. Consistently, BMMCs with VAMP7 knockdown showed markedly reduced CCL2 secretion after Ag stimulation. These observations underscore the heterogeneity of MC secretory responses and unveil a novel VAMP7-dependent CCL2 secretion mechanism within MCs. This discovery might pave the way for the development of more precise therapeutic strategies to modulate MC secretion in allergic conditions.

A Critical Role for the IKKβ/Lipid Rafts Axis in Regulating Platelet Exocytosis, Via the Spatial Regulation of SNARE Proteins

Lipid rafts, which are membrane microdomains that are rich in cholesterol and glycosphingolipids, have been implicated in the regulation of intracellular protein trafficking. Furthermore, we have previously shown that SNAREs mediate granule-plasma membrane fusion during exocytosis in platelets, and that IKKβ regulates SNAREs and granule exocytosis. Based on these considerations, we sought to investigate the regulatory relationship between IKKβ and lipid rafts, by employing the PF4-Cre/IKKβflox/flox platelets. We demonstrate that lipid rafts play a critical role in mediating IKKβ-/--dependent regulation of SNARE proteins and SNARE complexes. Specifically, we found that methyl β-cyclodextrin (MβCD, which disrupts lipid rafts) is able to inhibit platelet aggregation, dense and alpha granule secretion, PS exposure and αIIbβ3 integrin activation. We also observed that MβCD is able to inhibit platelet spreading, clot retraction, as well as 7S complex formation. In terms of co-localization, the SNARE protein SNAP-23 was found to associate with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in control platelets, but not in the IKKβ-/- platelets. Furthermore, in resting platelets, plasma membrane SNARE proteins such as syntaxin-11 and SNAP-23 or the granule-associated SNARE vesicle-associated membrane protein VAMP-8 were neither present in the microdomains of control platelets, nor in those from IKKβ-/- mice. Interestingly however, agonist/thrombin stimulation led to the relocation of syntaxin-11 and VAMP-8 into the lipid rafts, and as part of raft-associated phospho-SNAP-23/syntaxin-11/VAMP-8 complexes in control platelets. This seems to be IKKβ dependent, as these associations did not take place in the IKKβ-/- platelets. Taken together, these data reveal a critical role for the IKKβ/lipid rafts axis in regulating platelet exocytosis, via the spatial regulation of SNARE proteins. DisclosuresNo relevant conflicts of interest to declare.

Platelet secretion is kinetically heterogeneous in an agonist-responsive manner

AbstractPlatelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically controlled process? Here we sought to describe the platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (ie, thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules, whereas thrombin rapidly induced the greatest release. Cargo with opposing actions (eg, proangiogenic and antiangiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with 3 classes of release events, differing in their rates. The distribution of cargo into these 3 classes was heterogeneous, suggesting that platelet secretion is a stochastic process potentially controlled by several factors, such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes.

Loss of TRPC1-mediated Ca2+ influx contributes to impaired degranulation in Fyn-deficient mouse bone marrow-derived mast cells

MC degranulation requires the influx of calcium from the extracellular environment. Orai1/STIM1 is essential to MC SOCE, as shown in rat peritoneal MCs, the rat MC lines (RBL-2H3), or in Orai1 null embryo liver-derived, cultured MCs. However, minimal information exists about the role of other calcium channels expressed on these cells. Here, we demonstrate that the nonselective TRPC1 participates in FcεRI-mediated calcium entry in mouse BMMCs. We found that Fyn null MCs, which have an impaired degranulation response, expressed reduced levels of TRPC1, had normal depletion of intracellular calcium stores but an impaired calcium influx, and failed to depolymerize cortical F-actin (a key step for granule-plasma membrane fusion). Partial RNAi silencing of TRPC1 expression in WT MCs (to the level of Fyn null MCs) mimicked the Fyn null defect in calcium influx, cortical F-actin depolymerization, and MC degranulation. Ectopic expression of Fyn or TRPC1 in Fyn null MCs restored calcium responses and cortical F-actin depolymerization and increased MC degranulation. Together with our findings that expression of Orai1 is not altered in Fyn null MCs, our findings suggest that TRPC1 participates in calcium influx and other key events required for MC degranulation. This demonstrates that in addition to a role described previously for Orai1 in promoting MC degranulation, nonselective cation channels participate in promoting the exocytotic response.

Munc18-2 regulates exocytotic membrane fusion positively interacting with syntaxin-3 in RBL-2H3 cells

Recent studies have revealed that SNARE proteins are involved in exocytotic granular content release in mast cells as well as in neurotransmitter release in neural cells. However, the proteins that regulate the structure and activity of SNARE proteins in mast cells are not well understood. Munc18 is one such regulatory protein that plays a crucial role in neurotransmitter release. In this study, we investigated the role of Munc18 and its mechanism for regulating exocytotic release (degranulation) in rat basophilic leukemia cells (RBL-2H3). We obtained RBL-2H3 cells that express a low level of Munc18-2 and found that degranulation was remarkably inhibited in knockdown cells without any change in the expression level of syntaxins or Ca 2+ mobilization. We also observed the behavior of secretory granules in a single cell, and found no significant changes in their number and distribution in Munc18-2 knockdown cells. Using chimera proteins fused with fluorescent proteins, we demonstrated that Munc18-2 interacted with syntaxin-3, but not with syntaxin-4, in vivo. Interestingly, this interaction occurred not only on plasma membrane but also on secretory granules, suggesting that Munc18-2 may regulate granule–granule fusion as well as granule–plasma membrane fusion. These observations suggest that Munc18-2 together with syntaxin-3 regulate degranulation positively during the process of membrane fusion between secretory granules and plasma membrane, rather than during processes that regulate the number or behavior of secretory granules.

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.

Ternary SNARE Complexes Are Enriched in Lipid Rafts during Mast Cell Exocytosis

Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule-plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP-23 co-localized with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule-associated SNARE vesicle-associated membrane protein (VAMP)-2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP-2 in these rafts were present in SNARE complexes containing SNAP-23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP-23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP-23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP-2 present in rafts increased twofold, and these proteins were now present in raft-associated phospho-SNAP-23/syntaxin 4/VAMP-2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.

Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells

The inositol lipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] is involved in a myriad of cellular processes, including the regulation of exocytosis and endocytosis. In this paper, we address the role of PtdIns(4,5)P2 in compound exocytosis from rat peritoneal mast cells. This process involves granule-plasma membrane fusion as well as homotypic granule membrane fusion and occurs without any immediate compensatory endocytosis. Using a novel quantitative immunofluorescence technique, we report that plasma membrane PtdIns(4,5)P2 becomes transiently depleted upon activation of exocytosis, and is not detected on the membranes of fusing granules. Depletion is caused by phospholipase C activity, and is mandatory for exocytosis. Although phospholipase C is required for Ca2+ release from internal stores, the majority of the requirement for PtdIns(4,5)P2 hydrolysis occurs downstream of Ca2+ signalling - as shown in permeabilised cells, where the inositol (1,4,5)-trisphosphate-Ca2+ pathway is bypassed. Neither generation of the PtdIns(4,5)P2 metabolite, diacylglycerol (DAG) or simple removal and/or sequestration of PtdIns(4,5)P2 are sufficient for exocytosis to occur. However, treatment of permeabilised cells with DAG induces a small potentiation of exocytosis, indicating that it may be required. We propose that a cycle of PtdIns(4,5)P2 synthesis and breakdown is crucial for exocytosis to occur in mast cells, and may have a more general role in all professional secretory cells.

FcεRI-mediated mast cell degranulation requires calcium-independent microtubule-dependent translocation of granules to the plasma membrane

The aggregation of high affinity IgE receptors (Fcɛ receptor I [FcɛRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcɛRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcɛRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcɛRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule–plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

Differential Regulation of Exocytotic Fusion and Granule-Granule Fusion in Eosinophils by Ca2+ and GTP Analogs

Dynamics of degranulation was studied in horse eosinophils by patch clamp capacitance measurements. Degranulation was stimulated by intracellular application of calcium, and GTPgammaS or guanosine 5'-(beta,gamma-imido)triphosphate at different concentrations via the patch pipette. Degranulation was quantified by measuring the delay time between the beginning of intracellular perfusion and the first exocytotic event, determining the distribution of time intervals between fusion events and the capacitance step size distributions under the different conditions. The degranulation dynamics could be well reproduced using a computer model assuming three independent rate constants for granule-plasma membrane fusion, granule fusion with already exocytosed granules, and intracellular granule-granule fusion. The rate of granule-plasma membrane fusion is sensitive to both, the GTP analog and [Ca2+]i. The rate of granule-exocytosed granule fusion is sensitive to [Ca2+]i but insensitive to the GTP analogs, and the rate of granule-to-granule fusion is sensitive to the GTP analog but insensitive to [Ca2+]i. Granule fusions with the three different target compartments thus involve different regulatory mechanisms.

Phospholipase A2-Mediated Fusion of Neutrophil-Derived Membranes Is Augmented by Phosphatidic Acid

Phosphatidic acid (PA), the product of phospholipase D (PLD) metabolism, is not only an important second messenger in neutrophil signal transduction but PA generation increases membrane fusogenicity. Following neutrophil stimulation, PA formation can be detected in azurophil, specific, and plasma membrane vesicle subcellular fractions, suggesting a potential role for PA formation in granule–plasma membrane fusion. Neutrophil stimulation also activates phospholipase A2 (PLA2) and the release of arachidonic acid. In vitro fusion of plasma membrane vesicles and specific granules with complex liposomes were dependent on PLA2 (<10 μM Ca2+) while the presence of PA in the liposomes augmented the effects of PLA2. Azurophil granules were extremely resistant to fusion (no fusion at 12 mM Ca2+ even in the presence of PLA2). However, in the presence of both PA and PLA2 fusion could be detected at <5 μM Ca2+, suggesting a direct role for phospholipid metabolism in neutrophil degranulation.

Identification of SNAREs Involved in Regulated Exocytosis in the Pancreatic Acinar Cell

The molecular basis of exocytotic membrane fusion in the pancreatic acinar cell was investigated using an in vitro assay that measures both zymogen granule-plasma membrane fusion and granule-granule fusion. These two fusion events were differentially sensitive to Ca(2+), suggesting that they are controlled by different Ca(2+)-sensing mechanisms. Botulinum neurotoxin C (BoNT/C) treatment of the plasma membranes caused cleavage of syntaxin 2, the apical isoform of this Q-SNARE, but did not affect syntaxin 4, the basolateral isoform. BoNT/C also cleaved syntaxin 3, the zymogen granule isoform. BoNT/C treatment of plasma membranes abolished granule-plasma membrane fusion, whereas toxin treatment of the granules reduced granule-plasma membrane fusion and abolished granule-granule fusion. Tetanus toxin cleaved granule-associated synaptobrevin 2 but caused only a small reduction in both granule-plasma membrane fusion and granule-granule fusion. Our results indicate that syntaxin 2 is the isoform that mediates fusion between zymogen granules and the apical plasma membrane of the acinar cell. Syntaxin 3 mediates granule-granule fusion, which might be involved in compound exocytosis. In contrast, the major R-SNARE on the zymogen granule remains to be identified.

Exocytotic events in eosinophils and mast cells

Exocytotic events in eosinophils and mast cells

Adhesion-Dependent Degranulation of Neutrophils Requires the Src Family Kinases Fgr and Hck

Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-alpha and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck-/- fgr-/- PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-alpha but responded to PMA as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck-/- fgr-/- PMN. Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr-/- or hck-/- PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck-/- fgr-/- or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca2+ but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmacological control of adhesion-dependent degranulation in the inflammatory site.

Identification of glyceraldehyde-3-phosphate dehydrogenase as a Ca2+-dependent fusogen in human neutrophil cytosol.

The membrane fusion events observed during neutrophil degranulation are important aspects of the immunoregulatory system. In an attempt to understand the regulation of granule-plasma membrane fusion, we have begun characterizing human neutrophil cytosol for fusion activity, finding that 50% of the fusogenic activity could be attributed to members of the annexin family of proteins. The major non-annexin fusion activity (25% of the total cytosolic activity) was enriched by ion exchange chromatography after depletion of annexins by Ca2+-dependent phospholipid affinity chromatography. The fusion activity co-purified with a 10,14-kDa dimer identified as leukocyte L1 (which was non-fusogenic), along with an approximately 36-kDa protein. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by amino-terminal sequencing, and the fusion activity was verified using commercially available GAPDH. GAPDH may play an important role in degranulation because it is as potent as annexin I on a mass basis and may constitute up to 25% of the total cytosolic fusion activity of the neutrophil.

Calcium-calmodulin-stimulated phosphorylation of rat parotid secretion granule proteins

In studies designed to determine the mechanism by which Ca ++ and calmodulin stimulate the fusion of parotid secretion granules with plasma membrane vesicles, the hypothesis tested was that Ca ++ and calmodulin act by stimulating protein phosphorylation. It was earlier found that Ca ++ and calmodulin, but neither alone, stimulated the phosphorylation of four secretion granule proteins with molecular masses of 64, 58, 55 and 31 kDa, and decreased the degree of phosphorylation of a 36-kDa protein. Further studies have shown that in the presence of an optimal concentration of calmodulin (2.4 μM), half-maximal activation of phosphorylation of the four proteins occurred at approx. 8 μM Ca ++, and at a maximally effective Ca ++ concentration (10 −4M), half-maximal stimulation occurred at calmodulin concentrations between 0.13 and 1.1 μM for the different proteins. The studies now described also demonstrate that the need for calmodulin for stimulating the phosphorylation, but not the dephosphorylation, is specific; two other Ca ++-binding proteins, parvalbumin and troponin, could not replace calmodulin in stimulating phosphorylation of the four secretion granule proteins, but either one could substitute for calmodulin in stimulating dephosphorylation of the 36-kDa protein. Additionally, the phosphorylated proteins appear to be located on the granule surface. When secretion granules were subjected to mild treatment with a concentration of trypsin that did not lyse the granules, the 31-, 36−, 55−, 58− and 64-kDa proteins were no longer observed. In the presence of optimal concentrations of Ca ++ and calmodulin, a dose-dependent inhibition of the phosphorylation of the various proteins by two calmodulin antagonists, trifluoperazine and calmidazolium, was observed; 50% inhibition of phosphorylation of the different proteins was obtained at approx. 20–40 μM trifluoperazine and at about 2.5–3.0 μM calmidazolium. Inhibition of the dephosphorylation of the 36-kDa protein required greater concentrations of trifluoperazine and calmidazolium; 128 μM and 50 μM, respectively. These results are consistent with the hypothesis that the phosphorylation of one or more of the 31−, 55−, 58− and 64-kDa Proteins, but not the dephosphorylation of the 36-kDa protein, may be involved in the action of Ca ++ and calmodulin in secretion granule-plasma membrane fusion.

Proteins on exocytic vesicles mediate calcium-triggered fusion.

In many exocytic systems, micromolar concentrations of intracellular Ca2+ trigger fusion. We find that aggregates of secretory granules isolated from sea urchin eggs fuse together when perfused with greater than or equal to 10 microM free Ca2+. Mixing of membrane components was demonstrated by transfer of fluorescent lipophilic dye, and melding of granule contents was seen with differential interference microscopy. A technique based upon light scattering was developed to conveniently detect fusion. Two protein modifiers, trypsin and N-ethylmaleimide, inhibit granule-granule fusion at concentrations similar to those that inhibit granule-plasma membrane fusion. We suggest that molecular machinery sufficient for Ca(2+)-triggered fusion resides on secretory granules as purified and that at least some of these essential components are proteinaceous.

Cortical granule matrix disassembly during exocytosis in sea urchin eggs

Cortical granule exocytosis in sea urchins was studied using hyperosmotic and polymer-containing seawater to halt granule matrix dispersal. Addition of Na 2SO 4-containing seawater (2.5 osmole/kg) to Strongylocentrotus purpuratus eggs 10 to 40 sec after insemination resulted in arrest of the exocytic wave during propagation. EM examination of these eggs revealed that matrix disassembly occurred in distinct stages. In the earliest stage, granule-plasma membrane fusion had occurred, but the matrix remained completely intact. This early stage was observed in hyperosmotic media, either ionic or nonionic, suggesting that matrix hydration is required for disassembly and exocytic pore widening, but not for membrane fusion. Subsequent stages, in which partially disassembled matrices remained within ω-configured pockets, were captured by activating eggs in 30% dextran in seawater. Stability of these intermediates stages required the presence of Ca 2+ and Mg 2+; in the absence of divalent cations the matrices completely disassembled and the exocytic pockets flattened. Divalent cations appeared to prevent fragmentation of the matrix lamellae. Late stages of matrix disassembly, in which the lamellae fragmented and formed small particles, were inhibited by media of high ionic strength. Hyperosmolality alone, provided by sucrose, was unable to halt these late stages suggesting that water availability does not play an important role once a critical point in matrix dispersal has been reached.

Reconstitution of single calcium channels from secretory granules of rat adenohypophysis in planar lipid membranes

Free cytosolic Ca 2+ ([Ca 2+] i) has been demonstrated to play a crucial role in the prolactin secretory pathway. It regulates events as apparently diverse as prolactin gene transcription and the fusion of secretory granules with the plasma membrane. It is therefore important to understand the mechanisms which regulate the level of [Ca 2+] i. Because prolactin secretory granule membranes have been shown to contain large amounts of Ca 2+ and there is evidence that this calcium can be released independently of the granule hormone content, we have investigated the possibility that prolactin secretory granule membranes contain Ca 2+ channels. When purified prolactin secretory granules were fused with an artificial phospholipid bilayer, we found a Ca 2+ channel with a linear current-voltage relationship (conductance ~ 45 pS) in symmetrical 50 mM CaCl 2 solutions. Said channel had an open probability that was weakly dependent on the transmembrane potential, and a very good selectivity for calcium over chloride ions. The channel opened and closed very rapidly, when the majority of events lasting well below 25 ms. This channel could be important for the provision of high [Ca 2+] i levels necessary for granule-plasma membrane fusion and could also be involved in the modulation of Ca 2+ fluxes across the plasma membrane after the exocytotic release of prolactin.

Hyperosmolality inhibits exocytosis in sea urchin eggs by formation of a granule-free zone and arrest of pore widening

Hyperosmolality is known to inhibit membrane fusion during exocytosis. In this study cortical granule exocytosis in sea urchin eggs is used as a model system to determine at what step this inhibition occurs. Strongylocentrotus purpuratus eggs were incubated in hyperosmotic seawater (Na2SO4, sucrose or sodium HEPES used as osmoticants), the eggs activated with 20 microM A23187 to trigger exocytosis, and then quick frozen or chemically fixed for electron microscopy. Thin sections and freeze-fracture replicas show that at high osmolality (2.31 osmol/kg), there is a decrease in cortical granule size, a 90% reduction in granule-plasma membrane fusion, and formation of a granule-free zone between the plasma membrane and cortical granules. This zone averages 0.64 micron in thickness and prevents the majority of granules from docking at the plasma membrane. The remaining granules (approximately 10%) exhibit early stages of fusion which appear to have been stabilized; the matrix of these granules remains intact. We conclude that exocytosis is blocked by two separate mechanisms. First, the granule-free zone prevents granule-plasma membrane contact required for fusion. Second, in cases where fusion does occur, opening of the pocket and dispersal of the granule contents are slowed in hyperosmotic media.