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. Accord-ingly, 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 traf-ficking 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/phorbo-lester, ubMunc13-2 and Syt7 form a stimulatory triad for dense-core vesicle priming.
Originalsprog | Engelsk |
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Artikelnummer | e79433 |
Tidsskrift | eLife |
Vol/bind | 11 |
Antal sider | 26 |
ISSN | 2050-084X |
DOI | |
Status | Udgivet - 2022 |
Bibliografisk note
Funding Information:We thank Dorte Lauritsen for excellent technical assistance, including genotyping of animals. This investigation was supported by the Novo Nordic Foundation (NNF19OC0058298, JBS), the Independent Research Fund Denmark (0134–00141 A, JBS), the Lundbeck Foundation (R277-2018-802, JBS) and the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG; EXC-2049–390688087 and SFB1286/A11, NL). We are thankful to Nils Brose and Sonja Wojcik, Max-Planck-Institute for Multidisciplinary Sciences, Göttingen, for providing knockout mice, EGFP-fused Munc13-1, Munc13-1 H567K and ubMunc13-2 SFV plasmids, and brain tissue from the EYFP-Munc13-2 mouse.
Publisher Copyright:
© Houy, Martins et al.