Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion

摘要: The Munc13 family of exocytosis regulators has multiple Ca2+-binding, C2 domains. Here, we probed the mechanism by which Munc13-4 regulates in vitro membrane fusion and platelet exocytosis. We show that enhances soluble NSF attachment protein receptor (SNARE)-dependent, proteoliposome in a Ca2+- phosphatidylserine (PS)-dependent manner was independent SNARE concentrations. Munc13-4–SNARE interactions, under conditions used, were minimal absence or presence Ca2+. However, able to bind cluster liposomes harbouring PS response Interestingly, Ca2+-dependent liposome binding/clustering enhancement required both domains, but only Ca2+-liganding aspartate residues C2B domain. Analytical ultracentrifugation (AUC) measurements indicated that, solution, monomeric prolate ellipsoid with dimensions consistent molecule could bridge two fusing membranes. To address potential role as tethering platelets, examined mepacrine-stained, dense granule mobility secretion platelets from wild-type null ( Unc13dJinx ) mice. In Munc13-4, granules highly mobile resting stimulated stimulation-dependent release absent. These observations suggest are stably docked awaiting stimulation plays vesicle-stabilizing also activated summary, conveys Ca2+ sensitivity SNARE-mediated reveal bridges stabilizes apposing membranes destined for fusion. * AUC, : analytical ultracentrifugation; FRET, fluorescence energy transfer; GPS, grey syndrome; HPS, Hermansky–Pudlak MSD, mean square displacement; munc, mammalian uncoordinated; NBD-PE, N -(7-nitro-2-1,3-benzoxadiazol-4-yl)-1,2-dipalmitoyl phosphatidyl-ethanolamine; PC, phosphatidylcholine; PF4, factor 4; PRP, rich plasma; PS, phosphatidylserine; rhodamine-PE, -(lissamine rhodamine B sulfonyl)-1,2-dipalmitoyl phosphatidylethanolamine; SNAP-23, synaptosomal associated protein-23; SNARE, receptor; Unc, VAMP, vesicle-associated protein; VWF, von Willebrand