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 (Unc13dJinx ) 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.
|Number of pages||13|
|State||Published - Mar 1 2016|
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health [grant numbers HL56652 and HL082193 (to S.W.W.)], a predoctoral fellowship from the Great Rivers Affiliate of the American Heart Association [grant number 0615238B (to Q.R.)] and a postdoctoral fellowship from the Great Rivers Affiliate of the American Heart Association [grant number 2240048 (to M.C.C.)].
©2016 Authors; published by Portland Press Limited.
- C2 domains
- Membrane fusion
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology