Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Tuesday Speaker Abstracts

ALL SNAP25 COPIES IN THE VESICLE-PLASMA MEMBRANE CONTACT ZONE CHANGE CONFORMATION DURING VESICLE PRIMING Ying Zhao 2 ; Qinghua Fang 2 ; Satyan Sharma 2,4 ; Shrutee Jakhanwal 3 ; Reinhard Jahn 3 ; Manfred Lindau 1,2 ; 1 University of Miami Miller School of Medicine, Department of Physiology & Biophysics, Miami, FL, USA 2 Max-Planck-Institute for Biophysical Chemistry, Laboratory for Nanoscale Cell Biology, Göttingen, Germany 3 Max-Planck-Institute for Biophysical Chemistry, Department of Neurobiology, Göttingen, Germany 4 Uppsala University, Department of Cell and Mol. Biology, Uppsala, Sweden In neuronal cell types vesicular exocytosis is governed by the SNARE complex consisting of synaptobrevin2, SNAP25 and syntaxin1. However, the number of copies of SNARE complexes that are cooperating in membrane fusion is controversial. To estimate the number of SNARE complexes changing conformation preceding a fusion event we generated an improved SNAP25 based SNARE Complex Reporter (SCORE2) incorporating mCeruelan-3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming and fusion, and the delay suggests that the FRET change likely reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. The number of SNAP25 molecules changing conformation in this step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells 6-7 copies of SNAP25 change conformation during the priming step.

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