Fusion Of Secretory Granules Research Articles

Multifunctional regulation of VAMP3 in exocytic and endocytic pathways of RBL-2H3 cells.

Mast cells (MCs) are inflammatory cells involved in allergic reactions. Crosslinking of the high-affinity receptor for IgE (FcϵRI) with multivalent antigens (Ags) induces secretory responses to release various inflammatory mediators. These responses are largely mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Vesicle-associated membrane protein 3 (VAMP3) is a vesicular-SNARE that interacts with targeted SNARE counterparts, driving the fusion of MC secretory granules with the membrane and affecting subsequent assembly of the plasma membrane. However, the role of VAMP3 in FcϵRI-mediated MC function remains unclear. In this study, we comprehensively examined the role of VAMP3 and the molecular mechanisms underlying VAMP3-mediated MC function upon FcϵRI activation. VAMP3 shRNA transduction considerably decreased VAMP3 expression compared with non-target shRNA-transduced (NT) cells. VAMP3 knockdown (KD) cells were sensitized with an anti-DNP IgE antibody and subsequently stimulated with Ag. The VAMP3 KD cells showed decreased degranulation response upon Ag stimulation. Next, we observed intracellular granule formation using CD63-GFP fluorescence. The VAMP3 KD cells were considerably impaired in their capacity to increase the size of granules when compared to NT cells, suggesting that VAMP3 mediates granule fusion and therefore promotes granule exocytosis in MCs. Analysis of FcϵRI-mediated activation of signaling events (FcϵRI, Lyn, Syk, and intracellular Ca2+ response) revealed that signaling molecule activation was enhanced in VAMP3 KD cells. We also found that FcϵRI expression on the cell surface decreased considerably in VAMP3 KD cells, although the amount of total protein did not vary. VAMP3 KD cells also showed dysregulation of plasma membrane homeostasis, such as endocytosis and lipid raft formation. The difference in the plasma membrane environment in VAMP3 KD cells might affect FcϵRI membrane dynamics and the subsequent signalosome formation. Furthermore, IgE/Ag-mediated secretion of TNF-α and IL-6 is oppositely regulated in the absence of VAMP3, which appears to be attributed to both the activation of FcϵRI and defects in VAMP3-mediated membrane fusion. Taken together, these results suggest that enhanced FcϵRI-mediated signal transduction in VAMP3 KD cells occurs due to the disruption of plasma membrane homeostasis. Hence, a multifunctional regulation of VAMP3 is involved in complex secretory responses in MCs.

The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues.

Exocytosis is a fundamental process in physiology, that ensures communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. We report here a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, we found that Zda is required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen we identified the small GTPase RalA as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane.

ABCA12 regulates insulin secretion from β‐cells

Dysregulation of lipid homeostasis is intimately associated with defects in insulin secretion, a key feature of type 2 diabetes. Here, we explore the role of the putative lipid transporter ABCA12 in regulating insulin secretion from β-cells. Mice with β-cell-specific deletion of Abca12 display impaired glucose-stimulated insulin secretion and eventual islet inflammation and β-cell death. ABCA12's action in the pancreas is independent of changes in the abundance of two other cholesterol transporters, ABCA1 and ABCG1, or of changes in cellular cholesterol or ceramide content. Instead, loss of ABCA12 results in defects in the genesis and fusion of insulin secretory granules and increases in the abundance of lipid rafts at the cell membrane. These changes are associated with dysregulation of the small GTPase CDC42 and with decreased actin polymerisation. Our findings establish a new, pleiotropic role for ABCA12 in regulating pancreatic lipid homeostasis and insulin secretion.

Inhibition of Airway Epithelial Snare/Synaptotagmin Mediated Membrane Fusion by Hydrocarbon-Stapled Peptides

Airway mucin secretion involves membrane fusion of secretory granules that is mediated by SNAREs (syntaxin-3, SNAP-23, VAMP8), synaptotagmin-2, and other proteins. Mucus hypersecretion is a major cause of airway obstruction in the pathophysiology of asthma and cystic fibrosis. Deletion of synaptotagmin-2 protects mice against airway obstruction in a model of allergic asthma indicating the rationality of synaptotagmin-2 as a target for drug design. The so-called primary interface between the neuronal SNARE complex and synaptotagmin-1 is important for neurotransmitter release (Zhou et al., 2015), and it is also conserved for airway epithelial SNAREs and synaptotagmin-2. We designed a series of hydrocarbon-stapled peptides consisting of SNAP-25 fragments that included some of the key residues involved in the primary interface. The most α-helical stapled peptide had an affinity to the synaptotagmin-1 C2B domain that is similar to the affinity between the SNARE complex and the C2B domain. A subset of four stapled peptides, which were α-helical as assessed by circular dichroism, inhibit Ca2+-triggered fusion in a single vesicle fusion assay with reconstituted airway SNAREs and synaptotagmin-2. Inclusion of both Munc13-2 and Munc18-2 in the single vesicle fusion assay, the stapled peptide SP9 strongly suppress the Ca2+-triggered fusion even at the physiological Ca2+ concentrations. Moreover, cell penetrating peptide adjunct stapled peptide can be efficiently delivered into cultured epithelial cells and markedly reduced stimulated mucin secretion. Taken together, hydrocarbon-stapled peptide that interfere with the synaptotagmin-2/SNARE complex primary interface may allow modulation of secretory pathways.

SNARE-Mediated Fusion of Single Chromaffin Granules with Pore-Spanning Membranes

Pore-spanning membranes (PSMs) composed of supported membrane parts as well as freestanding membrane parts are shown to be very versatile to investigate SNARE-mediated fusion on the single-particle level. They provide a planar geometry readily accessible by confocal fluorescence microscopy, which enabled us for the first time, to our knowledge, to investigate the fusion of individual natural secretory granules (i.e., chromaffin granules (CGs)) on the single-particle level by two-color fluorescence microscopy in a time-resolved manner. The t-SNARE acceptor complex ΔN49 was reconstituted into PSMs containing 2 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol-4,5-bisphosphate and Atto488-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and CGs were fluorescently labeled with 2-((1E,3E)-5-((Z)-3,3-dimethyl-1-octadecylindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-1-octadecyl-3H-indol-1-ium perchlorate. We compared the dynamics of docked and hemifused CGs as well as their fusion efficacy and kinetics with the results obtained for synthetic synaptobrevin 2-doped vesicles fusing with PSMs of the same composition. Whereas the synthetic vesicles were fully immobile on supported PSMs, docked as well as hemifused CGs were mobile on both PSM parts, which suggests that this system resembles more closely the natural situation. The fusion process of CGs proceeded through three-dimensional post-lipid-mixing structures, which were readily resolved on the gold-covered pore rims of the PSMs and which are discussed in the context of intermediate states observed in live cells.

18F-FDOPA PET/CT Uptake Parameters Correlate with Catecholamine Secretion in Human Pheochromocytomas

Background: ¹⁸F-FDOPA positron emission tomography/computed tomography (PET/CT) is a sensitive nuclear imaging technology for the diagnosis of pheochromocytomas (PHEO). However, its utility in determining predictive factors for the secretion of catecholamines remains poorly studied. Methods: Thirty-nine histologically confirmed PHEO were included in this retrospective single-center study. Patients underwent ¹⁸F-FDOPA PET/CT before surgery, with an evaluation of several uptake parameters (standardized uptake values [SUV_max and SUV_mean] and the metabolic burden [MB] calculated as follows: MB = SUV_mean × tumor volume) and measurement of plasma and/or urinary metanephrine (MN), normetanephrine (NM), and chromogranin A. Thirty-five patients were screened for germline mutations in the RET, SDHx, and VHL genes. Once resected, primary cultures of 5 PHEO were used for real-time measurement of catecholamine release by carbon fiber amperometry. Results: The MB of the PHEO positively correlated with 24-h urinary excretion of NM (r = 0.64, p < 0.0001), MN (r = 0.49, p = 0.002), combined MN and NM (r = 0.75, p < 0.0001), and eventually plasma free levels of NM (r = 0.55, p = 0.006). In the mutated patients (3 SDHD, 2 SDHB, 3 NF1, 1 VHL, and 3 RET), a similar correlation was observed between MB and 24-h urinary combined MN and NM (r = 0.86, p = 0.0012). For the first time, we demonstrate a positive correlation between the PHEO-to-liver SUV_max ratio and the mean number of secretory granule fusion events of the corresponding PHEO cells revealed by amperometric spikes (p = 0.01). Conclusion: While the ¹⁸F-FDOPA PET/CT MB of PHEO strongly correlates with the concentration of MN, amperometric recordings suggest that ¹⁸F-FDOPA uptake could be enhanced by overactivity of catecholamine exocytosis.

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Imaging FITC-dextran as a Reporter for Regulated Exocytosis.

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Small molecules that inhibit the late stage of Munc13-4–dependent secretory granule exocytosis in mast cells

Ca2+-dependent secretory granule fusion with the plasma membrane is the final step for the exocytic release of inflammatory mediators, neuropeptides, and peptide hormones. Secretory cells use a similar protein machinery at late steps in the regulated secretory pathway, employing protein isoforms from the Rab, Sec1/Munc18, Munc13/CAPS, SNARE, and synaptotagmin protein families. However, no small-molecule inhibitors of secretory granule exocytosis that target these proteins are currently available but could have clinical utility. Here we utilized a high-throughput screen of a 25,000-compound library that identified 129 small-molecule inhibitors of Ca2+-triggered secretory granule exocytosis in RBL-2H3 mast cells. These inhibitors broadly fell into six different chemical classes, and follow-up permeable cell and liposome fusion assays identified the target for one class of these inhibitors. A family of 2-aminobenzothiazoles (termed benzothiazole exocytosis inhibitors or bexins) was found to inhibit mast cell secretory granule fusion by acting on a Ca2+-dependent, C2 domain–containing priming factor, Munc13-4. Our findings further indicated that bexins interfere with Munc13-4–membrane interactions and thereby inhibit Munc13-4–dependent membrane fusion. We conclude that bexins represent a class of specific secretory pathway inhibitors with potential as therapeutic agents.

Kv2.1 clusters on β-cell plasma membrane act as reservoirs that replenish pools of newcomer insulin granule through their interaction with syntaxin-3

The voltage-dependent K+ (Kv) channel Kv2.1 is a major delayed rectifier in many secretory cells, including pancreatic β cells. In addition, Kv2.1 has a direct role in exocytosis at an undefined step, involving SNARE proteins, that is independent of its ion-conducting pore function. Here, we elucidated the precise step in exocytosis. We previously reported that syntaxin-3 (Syn-3) is the key syntaxin that mediates exocytosis of newcomer secretory granules that spend minimal residence time on the plasma membrane before fusion. Using high-resolution total internal reflection fluorescence microscopy, we now show that Kv2.1 forms reservoir clusters on the β-cell plasma membrane and binds Syn-3 via its C-terminal C1b domain, which recruits newcomer insulin secretory granules into this large reservoir. Upon glucose stimulation, secretory granules were released from this reservoir to replenish the pool of newcomer secretory granules for subsequent fusion, occurring just adjacent to the plasma membrane Kv2.1 clusters. C1b deletion blocked the aforementioned Kv2.1-Syn-3-mediated events and reduced fusion of newcomer secretory granules. These insights have therapeutic implications, as Kv2.1 overexpression in type-2 diabetes rat islets restored biphasic insulin secretion.

STX3 represses the stability of the tumor suppressor PTEN to activate the PI3K-Akt-mTOR signaling and promotes the growth of breast cancer cells

Syntaxin 3, also known as STX3, is a protein encoded by the STX3 gene in humans. This protein is one of the fundamental components of the exocytotic machinery required for the docking and fusion of secretory granules with the plasma membrane. The roles of STX3 in human breast cancer remains elusive. Here we report that STX3 acts as an oncogenic protein in human breast cancer. We analyzed the expression of STX3 in 148 patients with breast cancer. The mRNA and protein levels of STX3 are significantly up-regulated in human breast cancer compared with matched adjacent non-cancer tissues. The up-regulation of STX3 is correlated with high disease stage and predicts overall and disease-free survival in patients with breast cancer. Lentivirus-mediated knockdown of STX3 represses in vitro proliferation and colony formation and in vivo growth of breast cancer cells, whereas STX3 overexpression promotes the growth of breast cancer cells in vitro and in vivo. We find that STX3 promotes the proliferation of breast cancer cells by increasing the activation of the Akt-mTOR signaling, and Akt inhibitor Ipatasertib or MK-2206 represses STX3 effects on the growth of breast cancer cells. Further mechanism study shows that STX3 binds to PTEN and increases PTEN ubiquitination and degradation, thus leading to activation of the PI3K-Akt-mTOR signaling. Therefore, STX3 promotes the growth of breast cancer cells by regulating the PTEN-PI3K-Akt-mTOR signaling.

Rab5 is critical for SNAP23 regulated granule-granule fusion during compound exocytosis

Compound exocytosis is considered the most massive mode of exocytosis, during which the membranes of secretory granules (SGs) fuse with each other to form a channel through which the entire contents of their granules is released. The underlying mechanisms of compound exocytosis remain largely unresolved. Here we show that the small GTPase Rab5, a known regulator of endocytosis, is pivotal for compound exocytosis in mast cells. Silencing of Rab5 shifts receptor-triggered secretion from a compound to a full exocytosis mode, in which SGs individually fuse with the plasma membrane. Moreover, we show that Rab5 is essential for FcεRI-triggered association of the SNARE protein SNAP23 with the SGs. Direct evidence is provided for SNAP23 involvement in homotypic SG fusion that occurs in the activated cells. Finally, we show that this fusion event is prevented by inhibition of the IKKβ2 kinase, however, neither a phosphorylation-deficient nor a phosphomimetic mutant of SNAP23 can mediate homotypic SG fusion in triggered cells. Taken together our findings identify Rab5 as a heretofore-unrecognized regulator of compound exocytosis that is essential for SNAP23-mediated granule-granule fusion. Our results also implicate phosphorylation cycles in controlling SNAP23 SNARE function in homotypic SG fusion.

Molecular mechanisms of developmentally programmed crinophagy in Drosophila.

At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface. Here, we show that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. We find that the tethering complex HOPS (homotypic fusion and protein sorting); the small GTPases Rab2, Rab7, and its effector, PLEKHM1; and a SNAP receptor complex consisting of Syntaxin 13, Snap29, and Vamp7 are all required for the fusion of secretory granules with lysosomes. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. Our work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans.

Recent new insights into the role of SNARE and associated proteins in insulin granule exocytosis.

Initial work on the exocytotic machinery of predocked insulin secretory granules (SGs) in pancreatic β-cells mimicked the SNARE hypothesis work in neurons, which includes SM/SNARE complex and associated priming proteins, fusion clamps and Ca2+ sensors. However, β-cell SGs, unlike neuronal synaptic vesicles, exhibit a biphasic secretory response that requires additional distinct features in exocytosis including newcomer SGs that undergo minimal docking time at the plasma membrane (PM) before fusion and multi-SG (compound) fusion. These exocytotic events are mediated by Munc18/SNARE complexes distinct from that which mediates predocked SG fusion. We review some recent insights in SNARE complex assembly and the promiscuity in SM/SNARE complex formation, whereby both contribute to conferring different insulin SG fusion kinetics. Some SNARE and associated proteins play non-fusion roles, including tethering SGs to Ca2+ channels, SG recruitment from cell interior to PM, and inhibitory SNAREs that block the action of profusion SNAREs. We discuss new insights into how sub-PM cytoskeletal mesh gates SG access to the PM and the targeting of SG exocytosis to PM domains in functionally polarized β-cells within intact islets. These recent developments have major implications on devising clever SNARE replacement therapies that could restore the deficient insulin secretion in diabetic islet β-cells.

Molecular Mechanisms of V-SNARE Function in Secretory Granule Exocytosis

Insulin is released by regulated exocytosis and docking of insulin granules at the plasma membrane, followed by the assembly of the secretory machinery is required for insulin release. Newly formed granules require a complex maturation process to become release-ready and the formation of these fusion competent mature secretory granules involves a series of discrete and unique events comprised of granule sorting, maturation, docking, priming, and finally Ca2+-dependent fusion. Soluble NSF attachment receptor (SNARE) proteins are the conserved core machinery required for all intracellular membrane fusion events. While SNAREs specifically control fusion, vesicular SNAREs (v-SNAREs) perform additional roles that are still elusive. In secretory cell types, v-SNAREs are pivotal for targeting vesicles to different locations, mediating both sorting and membrane protein distribution and reductions in v-SNAREs result in defective regulated exocytosis. Using total internal reflection fluorescence (TIRF) microscopy, we studied the role of VAMP8 in secretory granule exocytosis in pancreatic beta cells. We found that VAMP8 associates with early, late and recycling endosome compartment, rather than insulin containing granules. However, VAMP8 traffics and fuses at the plasma membrane specifically with the recycling endosomal compartment. VAMP8 expression compromised secretory granule fusion, whereas VAMP2 expression facilitated granule fusion, delineating a VAMP8-dependent fusion step between recycling endosomes and the plasma membrane. Truncation of the VAMP8 C-terminal region resulted in attenuated exocytosis and a change in secretion kinetics. Moreover, secreting insulin measurements are negatively correlated with increased VAMP8 copy number in human islets. We conclude that VAMP8 vesicles play an important role in granule genesis and sorting of essential membrane proteins regulating insulin secretion. Additionally, the TMD of VAMP8 may be necessary for initiating fusion upon stimulation and appears to have some direct interaction on insulin secretion. This work should fill critical gaps in our understanding of regulated secretion and the treatment of type-2 diabetes.

New Roles of Syntaxin-1A in Insulin Granule Exocytosis and Replenishment

In type-2 diabetes (T2D), severely reduced islet syntaxin-1A (Syn-1A) levels contribute to insulin secretory deficiency. We generated β-cell-specific Syn-1A-KO (Syn-1A-βKO) mice to mimic β-cell Syn-1A deficiency in T2D. Glucose tolerance tests showed that Syn-1A-βKO mice exhibited blood glucose elevation corresponding to reduced blood insulin levels. Perifusion of Syn-1A-βKO islets showed impaired first- and second-phase glucose-stimulated insulin secretion (GSIS) resulting from reduction in readily releasable pool and granule pool refilling. To unequivocally determine the β-cell exocytotic defects caused by Syn-1A deletion, EM and total internal reflection fluorescence microscopy showed that Syn-1A-KO β-cells had a severe reduction in the number of secretory granules (SGs) docked onto the plasma membrane (PM) at rest and reduced SG recruitment to the PM after glucose stimulation, the latter indicating defects in replenishment of releasable pools required to sustain second-phase GSIS. Whereas reduced predocked SG fusion accounted for reduced first-phase GSIS, selective reduction of exocytosis of short-dock (but not no-dock) newcomer SGs accounted for the reduced second-phase GSIS. These Syn-1A actions on newcomer SGs were partly mediated by Syn-1A interactions with newcomer SG VAMP8.

Munc18b Increases Insulin Granule Fusion, Restoring Deficient Insulin Secretion in Type-2 Diabetes Human and Goto-Kakizaki Rat Islets with Improvement in Glucose Homeostasis

Reduced pancreatic islet levels of Munc18a/SNARE complex proteins have been postulated to contribute to the deficient glucose-stimulated insulin secretion (GSIS) in type-2 diabetes (T2D). Whereas much previous work has purported Munc18a/SNARE complex (Syntaxin-1A/VAMP-2/SNAP25) to be primarily involved in predocked secretory granule (SG) fusion, less is known about newcomer SGs that undergo minimal docking time at the plasma membrane before fusion. Newcomer SG fusion has been postulated to involve a distinct SM/SNARE complex (Munc18b/Syntaxin-3/VAMP8/SNAP25), whose levels we find also reduced in islets of T2D humans and T2D Goto-Kakizaki (GK) rats. Munc18b overexpression by adenovirus infection (Ad-Munc18b), by increasing assembly of Munc18b/SNARE complexes, mediated increased fusion of not only newcomer SGs but also predocked SGs in T2D human and GK rat islets, resulting in rescue of the deficient biphasic GSIS.Infusion of Ad-Munc18b into GK rat pancreas led to sustained improvement in glucose homeostasis. However, Munc18b overexpression in normal islets increased only newcomer SG fusion. Therefore, Munc18b could potentially be deployed in human T2D to rescue the deficient GSIS.

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles.

Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell-like RBL-2H3 cells provide direct evidence that Munc13-4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells.

HID-1 is required for homotypic fusion of immature secretory granules during maturation.

Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules.

Opposing roles for SNAP23 in secretion in exocrine and endocrine pancreatic cells.

The membrane fusion of secretory granules with plasma membranes is crucial for the exocytosis of hormones and enzymes. Secretion disorders can cause various diseases such as diabetes or pancreatitis. Synaptosomal-associated protein 23 (SNAP23), a soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) molecule, is essential for secretory granule fusion in several cell lines. However, the in vivo functions of SNAP23 in endocrine and exocrine tissues remain unclear. In this study, we show opposing roles for SNAP23 in secretion in pancreatic exocrine and endocrine cells. The loss of SNAP23 in the exocrine and endocrine pancreas resulted in decreased and increased fusion of granules to the plasma membrane after stimulation, respectively. Furthermore, we identified a low molecular weight compound, MF286, that binds specifically to SNAP23 and promotes insulin secretion in mice. Our results demonstrate opposing roles for SNAP23 in the secretion mechanisms of the endocrine and exocrine pancreas and reveal that the SNAP23-binding compound MF286 may be a promising drug for diabetes treatment.