title: Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion.
abstract: The Munc13 family of exocytosis regulators has multiple Ca(2+)-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 Ca(2+)- 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 Ca(2+). However, Munc13-4 was able to bind and cluster liposomes harbouring PS in response to Ca(2+). Interestingly, Ca(2+)-dependent liposome binding/clustering and enhancement of proteoliposome fusion required both Munc13-4 C2 domains, but only the Ca(2+)-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 (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 Ca(2+). In summary, we show that Munc13-4 conveys Ca(2+) sensitivity to platelet SNARE-mediated membrane fusion and reveal a potential mechanism by which Munc13-4 bridges and stabilizes apposing membranes destined for fusion.
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Chicka
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
authors.forename: Qiansheng
authors.initials: Q
authors.lastname: Ren
authors.affiliation: Department of Anesthesia, Cincinnati Children's Hospital and Medical Center, Cincinnati, OH 45229, U.S.A.
authors.forename: David
authors.initials: D
authors.lastname: Richards
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
authors.forename: Lance M
authors.initials: LM
authors.lastname: Hellman
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
authors.forename: Jinchao
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Fried
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, U.S.A. whitehe@uky.edu.
authors.forename: Sidney W
authors.initials: SW
authors.lastname: Whiteheart
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000502:physiology* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism; D002459:Cell Fusion; D005089:Exocytosis; D006801:Humans; D008081:Liposomes; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018345:Mice, Knockout; D009154:Mutation; D010718:Phosphatidylserines / Q000378:metabolism; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000502:physiology / Q000648:ultrastructure
chemical_list: D008081:Liposomes; D008565:Membrane Proteins; D010718:Phosphatidylserines; D050600:SNARE Proteins; C507969:UNC13D protein, human; C520225:Unc13d protein, mouse; D002118:Calcium
keywords: C2 domains; Munc13-4; exocytosis; membrane fusion; platelets