1. Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery.
- Author
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Kalyana Sundaram, R, Chatterjee, Atrouli, Bera, Manindra, Grushin, Kirill, Panda, Aniruddha, Li, Feng, Coleman, Jeff, Lee, Seong, Ramakrishnan, Sathish, Gupta, Kallol, Rothman, James, Krishnakumar, Shyam, and Ernst, Andreas
- Subjects
Munc13 ,Munc18 ,SNARE protein ,neurotransmitter release ,vesicle priming ,Humans ,Diglycerides ,Synaptic Vesicles ,Exocytosis ,Synaptic Transmission ,Synaptotagmins ,Blister - Abstract
Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 template complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.
- Published
- 2023