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- W4319915608 abstract "Regulated vesicular exocytosis is governed by the SNARE complex, consisting of synaptobrevin2, SNAP25 and syntaxin. However, the number of copies of SNARE complexes that are cooperating in membrane fusion is highly debated. To estimate the number of SNARE complexes changing conformation preceding a single fusion event we used a SNAP25-based SNARE Complex Reporter (SCORE2) incorporating mCeruelan-3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. We demonstrated that all parameters of exocytosis in SNAP25 knockout cells are completely rescued by SCORE2 (SNARE Complex REporter2) and unchanged compared to 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 of SCORE2 conformational changes we detected a rapid FRET increase specifically associated with individual fusion events. This conformational change precedes fusion by ∼60 ms. Calibrated by the SCORE2 overexpression factor, we estimate that in wt cells ∼7 copies of SNAP25 change conformation preceding a fusion event. This number increased by ∼20% in L-Dopa-loaded vesicles which exhibited 1.67 fold larger quantal size and presumably 21% larger diameter. The number of SNAP25 copies undergoing a conformational change increases with vesicle size in parallel to the estimated area of the vesicle-plasma membrane contact zone. The results suggest that the FRET change of SCORE2 reports the docking of the vesicle, which is associated with a conformational of all SNAP25 copies in the contact zone." @default.
- W4319915608 created "2023-02-11" @default.
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- W4319915608 date "2023-02-01" @default.
- W4319915608 modified "2023-09-25" @default.
- W4319915608 title "The number of SNAP25 molecules changing conformation preceding a fusion event increases with vesicle size" @default.
- W4319915608 doi "https://doi.org/10.1016/j.bpj.2022.11.2478" @default.
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