Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

39-POS Board 13 SYMMETRICAL ARRANGEMENT OF PROTEINS UNDER RELEASE-READY VESICLES VISUALIZED BY CRYO-ELECTRON TOMOGRAPHY Abhijith Radhakrishnan 1 ; Xia Li 2 ; Kirill Grushin 1 ; Shyam S Krishnakumar 1 ; Jun Liu 2 ; James E Rothman 1 ; 1 Yale University, Cell Biology, New Haven, CT, USA 2 Yale University, Microbial Pathogenisis, New Haven, CT, USA Information processing in the brain relies on the precise release of neurotransmitters from synaptic vesicles (SV) at nerve terminals, a process crucial for cognition and development. The SV exocytosis is a tightly regulated event facilitated by several proteins that constitute the fusion machinery most importantly, SNARE proteins, VAMP2 on the SV and Syntaxin1/SNAP25 on the plasma membrane (PM). The SVs undergo a series of sequential steps as it approaches the active zone, wherein vesicles progress from an initial loosely ‘tethered’ state to a final release ready ‘primed’ state awaiting the influx of Ca 2+ ions. Despite the vast knowledge about the structure and function of the individual protein components choreographing these molecular transitions, how they are organized under a release-ready vesicle facilitating ultrafast fusion in a submillisecond remains a mystery. In this study, we employed cryogenic electron tomography analysis of cultured hippocampal neurons in near native conditions to unravel the arrangement of the exocytosis machinery beneath docked SVs. We observed that, as the vesicles approach the PM, they are initially ‘tethered’ to the plasma membrane by a variable number of protein densities ( ∼ 10 to 20 nm long) with no discernible organization. In contrast, when the SVs are about 4 nm away from the PM, we observe exactly six protein densities arranged symmetrically connecting the ‘primed’ vesicles to the PM. Our data indicate that the fusion machinery is likely organized into a highly cooperative framework during the priming process which enables rapid SV fusion and neurotransmitter release following Ca 2+ influx. It is tempting to speculate that exactly six SNAREpins (or a multiple of six) are organized under the primed vesicles and they act cooperatively to drive ultra-fast SV fusion and rapid release of neurotransmitters.

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