Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

31-POS Board 11 COMPLEMENTARY ACTION OF SYNAPTOPHYSIN AND MUNC13 IN LIMITING THE SNARE PIN TO 12 UNDER EACH RELEASE-READY VESICLE.

Manindra Bera 1 ; Ramalingam Venkat Kalyana Sundaram 1 ; Abhijith Radhakrishnan 1 ; James E Rothman 1 ; 1 Yale School of Medicine, Cell Biology, New Haven, CT, USA

Synaptophysin, a synaptic vesicle marker constitutes ~10% of total protein mass. Paradoxically, knocking out of Synaptophysin did not produce any significant perturbation in synaptic vesicle exocytosis. In our well-defined in vitro reconstitution on a suspended bilayer with pre-assembled t-SNAREs, we observed that Synaptophysin can limit the number of SNAREpins (four-helix bundle of Vamp2, Syntaxin and SNAP25) to ~12 underneath each docked vesicle. In the presence of Synaptophysin and Synaptotagmin 1, each docked vesicle produces SNAREpins in two equal waves (6 SNAREpin in each). Furthermore, we have shown that Synaptophysin and Vamp2 can form a complex with 6:12 stoichiometry respectively on a supported bilayer, thereby providing a limit to the number of SNAREpin formations. Munc13 can also (independent of Synaptophysin) constitute ~12 and ~6 SNAREpins in the absence and presence of DHG (a soluble replacement of diacyl glycerol) respectively under each docked vesicle. In addition to limiting the number of SNAREpins, Syp accelerates the vesicle fusion to ~3.5 ms (currently limited by time resolution in the system). In the absence of Syp, the vesicle opens the fusion pore at a slower rate providing an insight into plausible membrane resealing, thereby incomplete fusion. In summary, Munc13 and Synaptophysin orchestrate the SNAREpin formation in a complementary way to achieve complete ultrafast fusion.

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