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

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 Munc13-4 enhances in vitro soluble NSF attachment protein receptor (SNARE)-dependent, proteoliposome fusion in a Ca²⁺ - and phosphatidylserine (PS)-dependent manner that was independent of SNARE concentrations. Munc13-4-SNARE interactions, under the conditions used, were minimal in the absence or presence of Ca²⁺ . However, Munc13-4 was able to bind and cluster liposomes harbouring PS in response to Ca²⁺ . Interestingly, Ca²⁺ -dependent liposome binding/clustering and enhancement of proteoliposome fusion required both Munc13-4 C2 domains, but only the Ca²⁺ -liganding aspartate residues of the C2B domain. Analytical ultracentrifugation (AUC) measurements indicated that, in solution, Munc13-4 was a monomeric prolate ellipsoid with dimensions consistent with a molecule that could bridge two fusing membranes. To address the potential role of Munc13-4 as a tethering protein in platelets, we examined mepacrinestained, dense granule mobility and secretion in platelets from wild-type and Munc13-4 null (Unc13d^Jinx ) mice. In the absence of Munc13-4, dense granules were highly mobile in both resting and stimulated platelets, and stimulation-dependent granule release was absent. These observations suggest that dense granules are stably docked in resting platelets awaiting stimulation and that Munc13-4 plays a vesicle-stabilizing or tethering role in resting platelets and also in activated platelets in response to Ca²⁺ . In summary, we show that Munc13-4 conveys Ca²⁺ sensitivity to platelet SNARE-mediated membrane fusion and reveal a potential mechanism by which Munc13-4 bridges and stabilizes apposing membranes destined for fusion.