Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

2-POS Board 1 UNRAVELING THE MOLECULAR STEPS OF SNAREPIN FORMATION R Venkat Kalyana Sundaram 1,2 ; Manindra Bera 1,2 ; Kirill S Grushin 1,2 ; Abhijith Radhakrishnan 1,2 ; Andrew R Huehn 1,2 ; Atrouli Chatterjee 1,2 ; Jeff Coleman 1,2 ; Sathish Ramakrishnan 2,3 ; James E Rothman 1,2 ; 1 Yale University, Cell Biology, New Haven, CT, USA 2 Yale University, Nanobiology Institute, West Haven, CT, USA 3 Yale University, Pathology, New Haven, CT, USA SNAREpin formation is a crucial process for neurotransmitter release. It involves the assembly of Syntaxin1, SNAP25, and VAMP2 into a protein complex. The chaperones Munc13 and Munc18 play vital roles in recruiting and activating the SNARE proteins during vesicle docking and priming. However, the molecular details underlying this process, which prepares vesicles for neurotransmitter release, remain poorly understood. In our study, we employed an innovative reconstitution approach using co-expressed Munc18 and Syntaxin on planar bilayers suspended on our patented silicon platform. Using TIRF microscopy, we visualized the formation of template complexes by combining vesicles containing VAMP2 with bilayers containing Munc18/STX. Our results revealed that Complexin (CPX), binds to this complex through its central helix, independently of SNAP25. Adding Synaptotagmin 1 and/or Synaptophysin to the vesicle reconstitution enhanced Complexin binding, while mutations hindering template complex formation (Munc13 N1128A/F1131A and VAMP2 F77E) significantly reduced it. Moreover, SNAP25 exhibited a Munc13-dependent binding to this complex. Based on our observations, we propose a model for SNAREpin formation that begins with the assembly of the template complex, stabilized by Complexin. Subsequently, SNAP25 binds to this complex, ultimately leading to the formation of the functional SNAREpin. Furthermore, we suggest a dual role for Complexin, acting as both a stabilizer for template complex formation and a clamp for the SNAREpin. This study provides valuable insights into the molecular interactions and sequential steps involved in SNAREpin assembly, advancing our understanding of neurotransmitter release mechanisms and shedding light on the intricate processes underlying synaptic function.

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