Munc13 proteins control regulated exocytosis in mast cells

Abstract

Mast cells (MCs) are involved in host defenses against pathogens and inflammation. Stimulated MCs release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 (mammalian homolog of uncoordinated gene 13) proteins play essential roles in regulated exocytosis. Here, we found that MCs express Munc13-2 and -4, and we studied their roles using global and conditional knock-out (KO) mice. In a model of systemic anaphylaxis, we found no difference between WT and Munc13-2 KO mice, but global and MC-specific Munc13-4 KO mice developed less hypothermia. This protection correlated with lower plasma histamine levels and with histological evidence of defective MC degranulation but not with changes in MC development, distribution, numbers, or morphology. assays revealed that the defective response in Munc13-4-deficient MCs was limited to regulated exocytosis, leaving other MC secretory effector responses intact. Single cell capacitance measurements in MCs from mouse mutants differing in Munc13-4 expression levels in their MCs revealed that as levels of Munc13-4 decrease, the rate of exocytosis declines first, and then the total amount of exocytosis decreases. A requirement for Munc13-2 in MC exocytosis was revealed only in the absence of Munc13-4. Electrophysiology and EM studies uncovered that the number of multigranular compound events ( granule-to-granule homotypic fusion) was severely reduced in the absence of Munc13-4. We conclude that although Munc13-2 plays a minor role, Munc13-4 is essential for regulated exocytosis in MCs, and that this MC effector response is required for a full anaphylactic response.

Syntaxin 3, but not syntaxin 4, is required for mast cell–regulated exocytosis, where it plays a primary role mediating compound exocytosis

“…This defect affected exclusively the secretion of granule contents and not the secretion of cytokines or eicosanoids. Unlike what we have seen in the absence of Munc13-4 (20) and Munc18-2 (22), elimination of Stx3 in MCs did not affect the anaphylactic response.…”

“…We crossed Stx3 F/F and Stx4 F/F mice with B6.C-Tg(CMV-cre)1Cgn/J mice to generate germ line (global or constitutive) deletants (Ϫallele) or with Tg(Cma1-cre) ARoer mice to remove expression only in MCs (⌬ allele). We have shown the efficacy of this strategy before (20).…”

“…We have described that in mature MCs, regulated exocytosis depends on synaptotagmin-2 (19), Munc13-2 and -4 (20), and Munc18-2 (21,22). About the Stxs involved in this process, it has been accepted for more than a decade (23,24) that Stx4 is an essential participant in MC exocytosis, based on multiple studies on RBL-2H3 cells (25)(26)(27)(28)(29), rat peritoneal MCs (30), and human intestine-derived MCs (31).…”

“…sity of their granules secondary to hydration and loss of granule contents (47,48), and we quantified this change as gain in electron lucency (20). Compared with unstimulated controls, stimulated Stx3 ϩ/ϩ MCs have a significant increase in electron lucency of their granules (right shift), whereas the signal from stimulated Stx3 ⌬/⌬ MCs was intermediate between those two groups ( Fig.…”

Munc18-2, but not Munc18-1 or Munc18-3, regulates platelet exocytosis, hemostasis, and thrombosis

“…4, B-D). Assuming that most granules are almost spherical, the simultaneous stability of these two stereology values indicates that there are no changes in the number or size of granules (26,27).…”