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

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 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 harboring 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 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. (246 words)

Gene transfer into hematopoietic stem cells reduces HLH manifestations in a murine model of Munc13-4 deficiency

“…However, it has recently been demonstrated that (1) NK cell cytotoxicity has a distinct immunoregulatory role in HLH disease, 19 and (2) Munc13-4 has a critical function in platelets and neutrophils. [20][21][22][23] By using an LV coding for a Munc13-4/GFP fusion protein, our present study demonstrated that gene transfer into murine HSCs resulted in transgene expression in all the hematopoietic lineages, including NK and myeloid cells. Thus, HSC gene correction (providing multilineage Munc13-4 expression) might be a valuable treatment option for FHL3 in patients.…”

The actin binding protein scinderin acts in PC12 cells to tether dense-core vesicles prior to secretion

“…While it is difficult to distinguish between these possibilities, three lines of argument support a loss of tethering interpretation. Firstly, the shape of the MSD curve in the scinderin knockdown cells has a relatively constant slope, indicative of unconstrained motion, similar to that seen in Munc13-4 knockout platelets (Chicka et al, 2016), although this could also reflect a confounding combination of active transport (positive change in slope) and caging (asymptotic slope). Secondly, in these experiments scinderin knockdown and actin depolymerization have similar effects on vesicle mobility, which is consistent with a comparable mechanism of action, specifically a reduction in actin dependent vesicle tethering.…”

“…Previously, we examined secretory vesicle motion in platelets (Chicka et al, 2016), and we used the same approach to quantify the mobility of secretory vesicles in PC12 cells. Through tracking the visual center of mass of the vesicles over time, we can build up a description of the vesicles displacement over time.…”

“…Number and amplitude of peaks were then compared between conditions. Vesicle tracking was carried out as previously described (Chicka et al, 2016), and based upon centroid tracking of vesicles moving laterally within the TIRF field.…”

How Does Protein Disulfide Isomerase Get Into a Thrombus?

“…15 It is thought to work with the small GTPase, Rab27, to bring granule and plasma membranes together and thus promote Soluble NSF-Attachment Protein Receptor (SNARE) protein-mediated membrane fusion. 16 Does this mean SNAREs are not involved in PDI exteriorization? Perhaps, but there are a number of SNAREs present in platelets, not all of which may require Munc13-4 for function.…”