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 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 on functional pairing with Syt7. Overall, DAG/phorbolester, ubMunc13-2 and Syt7 form a stimulatory triad for dense-core vesicle priming.

High-throughput assay for regulated secretion of neuropeptides in mouse and human neurons

“…Next, we tested the ability of the NPY-Nanoluc assay to detect effects of known modulators of DCV exocytosis. First, we tested the diacylglycerol analog PMA, known to increase priming and release of secretory granules in PC12 and chromaffin cells ( 34 , 35 , 36 ). The addition of 1 μM PMA produced a small but significant increase in evoked release from DCVs (released fraction in dimethyl sulfoxide (DMSO) and PMA was 6.3% and 6.9%, respectively, Fig.…”

“…In addition, NPY-Nanoluc reported changes in basal exocytosis of DCVs and the total number of DCVs in cell lysates. The release of NPY-Nanoluc was successfully blocked by the L-type calcium channel blocker nimodipine ( 29 , 30 , 31 ) or the endoplasmic-reticulum (ER)-Golgi protein trafficking inhibitor brefeldin A (BFA) ( 32 , 33 ) and enhanced by the diacylglycerol analog phorbol 12-myristate 13-acetate (PMA) ( 34 , 35 , 36 ). Genetic inactivation of STXBP1 or Rab3, essential genes for DCV exocytosis ( 19 , 37 ) also lead to blockade of DCV exocytosis measured by NPY-Nanoluc.…”

Pre‐fusion motion state determines the heterogeneity of membrane fusion dynamics for large dense‐core vesicles

“…Subpopulations of LDCVs could be cataloged according to some particular molecular markers during docking and priming, such as Syt7 versus Syt1 11 or Munc13-1 versus ubMunc13-2. 12 However, regarding the complexity of the molecular mechanism of exocytosis, a single protein marker is usually insufficient to reflect the overall priming state of a vesicle. Indeed, SVs were cataloged into two subgroups, "superprimed" and normally primed, based on their priority of release upon neuron excitation, which is largely determined by the priming state.…”

“…Emerging evidence has revealed robust heterogeneity for either SVs or LDCVs, but current understanding about the heterogeneity of vesicle population is still obscure, especially for LDCV. Subpopulations of LDCVs could be cataloged according to some particular molecular markers during docking and priming, such as Syt7 versus Syt1 11 or Munc13‐1 versus ubMunc13‐2 12 . However, regarding the complexity of the molecular mechanism of exocytosis, a single protein marker is usually insufficient to reflect the overall priming state of a vesicle.…”