Previous studies indicated that CAPS (calcium-dependent activator protein for secretion) functions as an essential component for the Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells. However, recent mouse knock-out studies suggested an alternative role in the vesicular uptake or storage of catecholamines. To genetically assess the functional role of CAPS, we characterized the sole Caenorhabditis elegans CAPS ortholog UNC-31 (uncoordinated family member) and determined its role in dense-core vesicle-mediated peptide secretion and in synaptic vesicle recycling. Novel assays for dense-core vesicle exocytosis were developed by expressing a prepro-atrial natriuretic factor-green fluorescent protein fusion protein in C. elegans. unc-31 mutants exhibited reduced peptide release in vivo and lacked evoked peptide release in cultured neurons. In contrast, cultured neurons from unc-31 mutants exhibited normal stimulated synaptic vesicle recycling measured by FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide] dye uptake. Conversely, UNC-13, which exhibits sequence homology to CAPS/UNC-31, was found to be essential for synaptic vesicle but not dense-core vesicle exocytosis. These findings indicate that CAPS/UNC-31 function is not restricted to catecholaminergic vesicles but is generally required for and specific to dense-core vesicle exocytosis. Our results suggest that CAPS/UNC-31 and UNC-13 serve parallel and dedicated roles in dense-core vesicle and synaptic vesicle exocytosis, respectively, in the C. elegans nervous system.

Cancer cells secrete copious amounts of exosomes, and elevated intracellular Ca²⁺ is critical for tumor progression and metastasis, but the underlying cellular mechanisms are unknown. Munc13-4 is a Ca²⁺-dependent SNAP receptor– and Rab-binding protein required for Ca²⁺-dependent membrane fusion. Here we show that acute elevation of Ca²⁺ in cancer cells stimulated a fivefold increase in CD63⁺, CD9⁺, and ALIX⁺ exosome release that was eliminated by Munc13-4 knockdown and not restored by Ca²⁺ binding–deficient Munc13-4 mutants. Direct imaging of CD63-pHluorin exosome release confirmed its Munc13-4 dependence. Depletion of Munc13-4 in highly aggressive breast carcinoma MDA-MB-231 cells reduced the size of CD63⁺ multivesicular bodies (MVBs), indicating a role for Munc13-4 in MVB maturation. Munc13-4 used a Rab11-dependent trafficking pathway to generate MVBs competent for exosome release. Membrane type 1 matrix metalloproteinase trafficking to MVBs by a Rab11-dependent pathway was also Munc13-4 dependent, and Munc13-4 depletion reduced extracellular matrix degradation. These studies identify a novel Ca²⁺- and Munc13-4-dependent pathway that underlies increased exosome release by cancer cells.

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P2) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P2 as ∼6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P2 soluble NSF attachment protein receptor (SNARE)–dependent liposome fusion is inhibited. Inhibition by PI 4,5-P2 likely results from its intrinsic positive curvature–promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P2, suggesting that syntaxin sequesters PI 4,5-P2 to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P2, we test a PI 4,5-P2–binding priming factor required for vesicle exocytosis. Ca2+-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P2 dependent. These results indicate that PI 4,5-P2 regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca²⁺ is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca²⁺ also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca²⁺-dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca²⁺-dependent SNARE binding or in Ca²⁺-dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of different-sized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca²⁺-dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca²⁺-dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca²⁺-dependent membrane insertion and SNARE binding to drive fusion pore expansion.