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Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery.

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.

SUBMITTER: Kalyana Sundaram RV

PROVIDER: S-EPMC10450444 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery.

Kalyana Sundaram R Venkat RV Chatterjee Atrouli A Bera Manindra M Grushin Kirill K Panda Aniruddha A Li Feng F Coleman Jeff J Lee Seong S Ramakrishnan Sathish S Ernst Andreas M AM Gupta Kallol K Rothman James E JE Krishnakumar Shyam S SS

Proceedings of the National Academy of Sciences of the United States of America 20230817 34

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 Ca<sup>2+</sup>. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca<sup>2+</sup>-triggered release involving the SNARE ...[more]

PMID: 37590407

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Project description:Here we introduce the full functional reconstitution of genetically-validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, 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 Ca 2+ . Using this novel setup, we discover new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca 2+- triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca 2+ -dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of ready-release vesicles. Dynamic single-molecule imaging of Complexin binding to ready-release vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by Munc13 and Munc18 chaperones. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 'template' complex is a functional intermediate in the production of primed, ready-release vesicles, which requires the coordinated action of Munc13 and Munc18.Significance statementMunc13 and Munc18 are SNARE-associated chaperones that act as "priming" factors, facilitating the formation of a pool of docked, release-ready vesicles and regulating Ca 2+ -evoked neurotransmitter release. Although important insights into Munc18/Munc13 function have been gained, how they assemble and operate together remains enigmatic. To address this, we developed a novel biochemically-defined fusion assay which enabled us to investigate the cooperative action of Munc13 and Munc18 in molecular terms. We find that Munc18 nucleates the SNARE complex, while Munc13 promotes and accelerates the SNARE assembly in a DAG-dependent manner. The concerted action of Munc13 and Munc18 stages the SNARE assembly process to ensure efficient 'clamping' and formation of stably docked vesicles, which can be triggered to fuse rapidly (∼10 msec) upon Ca 2+ influx.

Project description:Synaptotagmin-1 (SYT1) is a synaptic vesicle resident protein that interacts via its C2 domain with anionic lipids from the plasma membrane in a calcium-dependent manner to efficiently trigger rapid neurotransmitter (NT) release. In addition, SYT1 acts as a negative regulator of spontaneous NT release and regulates synaptic vesicle (SV) priming. How these functions relate to each other mechanistically and what role other synaptotagmin (SYT) isoforms play in supporting and complementing the role of SYT1 is still under intensive investigation. In this work, we analyzed three putative functions of SYT1 in exocytosis by systematically varying its expression in autaptic hippocampal glutamatergic neurons from mice of either sex. We find that regulation of release probability is most sensitive to variation of expression levels, whereas its impact on vesicle priming is least sensitive. Also, loss of SYT1 phenotypes on spontaneous release and vesicle priming is compensated in less mature synaptic cultures by redundant support from SYT7. Overall, our data help in resolving some controversies in SYT1 functions in exocytosis and in our understanding of how SYT1 contributes to the pathophysiology underlying SYT1-related human neurologic disorders.SIGNIFICANCE STATEMENT Our work clarifies the functions of SYT1 protein in synaptic vesicle priming and spontaneous and calcium-evoked neurotransmitter release and analyzes whether these occur at different stages of synaptic responses by examining their relative sensitivity to protein concentration at the synaptic terminal. We demonstrate that these synaptic functions are unequally sensitive to both protein levels and neuronal stage, indicating that they operate under distinct molecular mechanisms. Furthermore, we analyze how these functions are modulated by another synaptotagmin isoform expression. We show that to understand the phenotype displayed by SYT1 knock-out neurons (Syt1 -/-) is necessary to consider the interplay between SYT1 and SYT7 molecules at the presynaptic terminal.

Dataset Information

Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery.

Publications

Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery.

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