Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

23-POS Board 8 USING A SINGLE-VESICLE ASSAY TO DISSECT THE ROLE OF SNARE-MUNC13 INTERACTIONS IN SYNAPTIC VESICLE DOCKING AND THE ACTIVATION OF MUNC13 IN RAPID CALCIUM-MEDIATED FUSION

Atrouli Chatterjee 1,2 ; Venkat R Kalyana Sundaram 1,2 ; Shyam S Krishnakumar 1,3 ; James E Rothman 1,2 ; 1 Yale University School of Medicine, Nanobiology Institute, New Haven, CT, USA 2 Yale University School of Medicine, Department of Cell Biology, New Haven, CT, USA 3 Yale University School of Medicine, Department of Neurology, New Haven, CT, USA

Brain and central nervous system functionality are critically dependent on the efficient transfer of information at the synaptic junctions of nerve terminals, which involves the Ca 2+ -mediated release of neurotransmitters stored in the synaptic vesicles (SVs). Although the proteins involved in synaptic transmission have been characterized, the precise biochemical pathway that enables the synchronous stimulated fusion and release of SVs in sub-millisecond timescales in vivo still remain unclear. To obtain a more precise understanding of the molecular interactions that are involved in vesicle docking and subsequently in Ca 2+ -triggered release, we introduce a full reconstitution of the genetically-validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, Complexin) within a geometry the enables the detailed characterization of vesicle fate. Specifically, we leveraged a silicon-chip-based suspended-bilayer system in conjunction with fluorescence microscopy to monitor vesicle fate with millisecond resolution. Using this set up, we performed mutational studies on the chaperones (Munc13 and Munc18) to elucidate their roles in vesicle fate before and after Ca 2+ -mediated release as well as to validate the overall function of our assay with respect to the phenotypic observations from in vivo tests. We found that: (i) SNARE-binding to Munc13 is required to form a “readily-releasable pool”; (ii) using mutations to destabilize the Munc18-Syntaxin-VAMP2 complex leads to fewer “clamped” vesicles; and that (iii) both the activation of Munc13 and the co-operative interaction of Munc13 and Munc18 was required to achieve fast (millisecond) fusion kinetics. Altogether, the new methodology establishes a more complete in vitro analogue of the neuronal SNARE machinery and thereby provides a platform by which to study the molecular interactions and biochemical pathways that enable information transfer in the brain.

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