Fig. 5: Flip-out relieves Stx2 inhibition of the fusogenic SNARE complex.

a Representative images of human β-cell infected with FAP-Stx2 and NPY-tdOrange2. White arrowheads indicate sites where exocytosis and flipping occurs (n = 3 independent experiments). b Representative fluorescence time course of single FAP-Stx2 flipping events followed by exocytosis, the latter characterized by a transient peak increase and rapid decline. c Relationship between delay time and exocytosis probability at fusion sites. Time delay defined by rising phase of Stx2 flipping and onset of NPY-tdOrange2 as shown in b. Symbols denote average fusion probability measured by the proportion of predocked and newcomer granules undergoing fusion at sites where flipping occurring; solid line shows the fitting. The shaded areas indicate ± s.e.m. The bottom axis “…” denotes delay time longer than 5 s. d Average granule fusion probability at sites of Stx2 flipping. Exocytosis occurred immediately after flipping within a delay time of 2.5 s vs. that of longer delay time. Each dot denotes an average probability from three independent experiments, data are presented as mean ± s.d., two-tailed unpaired Student’s t-test. e Representative live-cell single-molecule imaging of Stx2-mScarlet showing that Stx2 molecules reversibly assemble and disassemble at nanodomain, an example depicted by the circled Stx2 molecule in e that is displayed in f as intensity trace and corresponding montage images. Scale bar in e is 2 µm. g Histogram of time τ that Stx2 spent to re-assemble. Source data are provided as a Source Data file.