Phorbolester-activated Munc13-1 and ubMunc13-2 exert opposing effects on dense-core vesicle secretion

Abstract

Munc13 proteins are priming factors for SNARE-dependent exocytosis, which are activated by diacylglycerol (DAG)-binding to their C1-domain. Several Munc13 paralogs exist, but their differential roles are not well understood. We studied the interdependence of phorbolesters (DAG mimics) with Munc13-1 and ubMunc13-2 in mouse adrenal chromaffin cells. Although expression of either Munc13-1 or ubMunc13-2 stimulated secretion, phorbolester was only stimulatory for secretion when ubMunc13-2 expression dominated, but inhibitory when Munc13-1 dominated. Accordingly, phorbolester stimulated secretion in wildtype cells, or cells overexpressing ubMunc13-2, but inhibited secretion in Munc13-2/Unc13b knockout (KO) cells or in cells overexpressing Munc13-1. Phorbolester was more stimulatory in the Munc13-1/Unc13a KO than in WT littermates, showing that endogenous Munc13-1 limits the effects of phorbolester. Imaging showed that ubMunc13-2, but not Munc13-1, traffics to the plasma membrane with a time-course matching Ca2+-dependent secretion, and trafficking is independent of synaptotagmin-7 (syt7). However, in the absence of syt7, phorbolester became inhibitory for both Munc13-1 and ubMunc13-2 driven secretion, indicating that stimulatory phorbolester x Munc13-2 interaction depends obligatorily on functional pairing with syt7. Overall, DAG/phorbolester, ubMunc13-2 and syt7 form a stimulatory triad for dense-core vesicle priming.

Rabphilin 3A binds the N-peptide of SNAP-25 to promote SNARE complex assembly in exocytosis

“…For example, Rab-binding protein RIMs and other vesicle associated proteins, such as Munc13 and CAPS, have been found to support vesicle tethering/docking via mediating the interaction between synaptic vesicles and the plasma membrane and to support vesicle priming via promoting SNARE complex assembly ( Dulubova et al, 2005 ; Imig et al, 2014 ; Quade et al, 2019 ; Wang et al, 2017 ; Wang et al, 2019 ; James et al, 2009 ). However, neuroendocrine cells lack compartmentalized active zones on the plasma membrane, in which RIMs, Munc13, CAPS, and so forth, are dominantly distributed in the cytosol instead of the plasma membrane ( Fukuda, 2004 ; Kabachinski et al, 2014 ; Kabachinski et al, 2016 ; Houy et al, 2022 ), rendering them unlikely to compensate the loss of Rph3A in DCV exocytosis. A second possibility is that other SN25 binding partners in synapses alternatively promote SN25 assembly into the SNARE complex.…”