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

Michael C. Chicka, Qiansheng Ren, David Richards, Lance M. Hellman, Jinchao Zhang, Michael G. Fried, Sidney W. Whiteheart

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)627-639
Number of pages13
JournalBiochemical Journal
Volume473
Issue number5
DOIs
StatePublished - Mar 1 2016

Bibliographical note

Funding 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.)].

Publisher Copyright:
©2016 Authors; published by Portland Press Limited.

Keywords

  • C2 domains
  • Exocytosis
  • Membrane fusion
  • Munc13-4
  • Platelets.

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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