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 mepacrine-stained, 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.
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March 2016
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Structure of the alpha1, beta1, gamma1 isoform of AMPK bound to a chlorinated analog of A769662 (‘Cl-A769662’). Alpha colored in purple, beta in blue, gamma in light pink. Cl-A769662 shown in yellow sticks and AMP bound to gamma shown in green sticks. Image kindly provided by Francis Rajamohan (Groton, U.S.A.). - PDF Icon PDF LinkFront Matter
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Research Article|
February 24 2016
Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion
Michael C. Chicka;
Michael C. Chicka
1
*Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
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Qiansheng Ren;
Qiansheng Ren
2
*Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
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David Richards;
David Richards
3
†Department of Anesthesia, Cincinnati Children's Hospital and Medical Center, Cincinnati, OH 45229, U.S.A.
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Lance M. Hellman;
Lance M. Hellman
4
*Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
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Jinchao Zhang;
Jinchao Zhang
*Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
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Michael G. Fried;
Michael G. Fried
*Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
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Sidney W. Whiteheart
Sidney W. Whiteheart
5
*Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
5To whom correspondence should be addressed (email whitehe@uky.edu).
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Publisher: Portland Press Ltd
Received:
October 29 2015
Revision Received:
December 03 2015
Accepted:
December 04 2015
Accepted Manuscript online:
December 04 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 Authors; published by Portland Press Limited
2016
Biochem J (2016) 473 (5): 627–639.
Article history
Received:
October 29 2015
Revision Received:
December 03 2015
Accepted:
December 04 2015
Accepted Manuscript online:
December 04 2015
Citation
Michael C. Chicka, Qiansheng Ren, David Richards, Lance M. Hellman, Jinchao Zhang, Michael G. Fried, Sidney W. Whiteheart; Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion. Biochem J 1 March 2016; 473 (5): 627–639. doi: https://doi.org/10.1042/BJ20151150
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