Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

UNRAVELLING THE STEPS TAKEN BY MUNC13 TO CHAPERONE VESICLE PRIMING FOR NEUROTRANSMITTER RELEASE Kirill S. Grushin 1 ; R. Venkat Kalyana Sundaram 1 ; Jasmine Shahanoor 2 ; Atrouli Chatterjee 1 ; Jeff Coleman 1 ; Jeremy Dittman 2 ; James Rothman 1 ; 1 Yale University, Department of Cell Biology, New Haven, CT, USA 2 Weill Cornell Medicine, Department of Biochemistry, New York, NY, USA Multiple proteins play a critical role in synaptic vesicle docking and priming, the key steps in neurotransmitter release process. Munc13-1, a crucial chaperone, participates in tethering vesicles to PIP2-microdomains on the plasma membrane and templating SNARE proteins (Syntaxin1A, VAMP2 and SNAP25). The precise series of steps taken by Munc13-1 during the priming process remains elusive. Recently we reported that the functional core of Munc13-1, consisting of C1–C2B–MUN (D1408-1452, EF)–C2C (Munc13C) spontaneously crystallizes between phosphatidylserine-rich bilayers in two distinct conformations, each in a radically different oligomeric state: an "open" conformation forming trimers and a "closed" conformation forming hexamers, with trimers connecting hexamers into a 2D lattice. Based on these findings, we proposed a model to elucidate the actions of Munc13-1 during the synaptic vesicle docking/priming process. To validate our model, we identified and mutated residues in the MUN domain responsible for the observed oligomerization interfaces. Our cryo-electron microscopy results demonstrate that mutations in trimer interface effectively disrupted continuous crystal formation, yielding separate hexagons between lipid bilayers. Mutations in the hexamer interface have a more profound impact, completely abolishing hexagonal crystal formation and leading to the formation of new rectangular 2D crystals between the membrane bilayers. Notably, the conformations of Munc13C within the resolved crystals resemble the previously described "open" and "closed" molecules, with an intriguing slanted orientation of the "open" conformation potentially reflecting a staging process during Munc13C-driven priming. Preliminary experiments using single vesicle fusion assays and in vivo rescue experiments in C. elegans reveal that the selected mutations impair evoked neurotransmitter release and block fusion in vitro. These findings underscore the significance of the discovered interfaces, necessitating further investigation to determine their precise roles in synaptic vesicle docking/priming.

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