Branched actin polymerization is essential for pore expansion and stabilization. (A) Time-lapse sequence of representative LSVs from CK666^{100 µM} SGs undergoing full collapse (FC) and kiss-and-run (KAR; SRRF intensity-projection; also in Video 2; see Fig. S2 and Video 3 for WT and Arp3^KD). Top: FC appears as a content release that precedes actomyosin assembly. The fusion pore expands and the vesicle appears to integrate into the apical surface. Middle: KAR w actomyosin, appearing as deformation and displacement of the LSV from the apical membrane upon actomyosin contraction (dashed line outlining the LSV during deformation). The same LSV fused again at 11:28, suggesting it detached between fusion events. Bottom: KAR w/o actomyosin, appearing as consecutive fusion events (asterisk) without content release. Actomyosin was recruited after the second fusion event (01:52), leading to membrane crumpling. (B and C) Schematic model of full collapse (FC) and kiss-and-run (KAR w. and w/o actomyosin) exocytosis (C) mean frequency (%) of stalling, FC, KAR, and membrane crumpling (MC) in WT, Arp3^KD, and CK666^{100 µM} SGs. Exocytosis by membrane crumpling is the dominant mode of exocytosis in WT SGs (67 ± 3%) with lower frequencies of FC and KAR and stalling (6 ± 3%, 23 ± 4%, and 4 ± 1% respectively). Arp3^KD and CK666^{100 µM} present a higher frequency of full collapse (27 ± 3% (***) and 19 ± 9% (ns), respectively) and kiss-and-run (50 ± 6% (****) and 42 ± 4% (**), respectively). Error bars show SEM. Statistical significance with respect to WT frequencies. N (SGs) ≥ 3, n (events) ≥ 200. P values for CK666^{100 µM}: **(KAR) = 0.004044, ****(MC) = 0.00002, for Arp3^KD ***(FC) = 0.000199, ****(KAR) = 0.000007, ****(MC) < 0.000001. Two-tailed unpaired multiple t tests corrected using the Holm-Sidak method (also in Video 2). (D) Time-lapse sequence of a representative SG treated with CK666^{500 µM} (confocal intensity-projection). Stalled, actin-coated LSVs (white asterisks) accumulate at the cell apical membrane (also in Video 4, left). (E) Representative image of stalled LSVs with a narrow pore after CK666^{500 µM} treatment (also in Video 4, right). (F) Change in mean pore diameter over time in control and CK666^{500 µM} treated SGs showing that the pore expanded and constricted in control SGs (from Fig. S1 B; control, diamonds; mean maximal diameter = 1.6 ± 0.2 µm; n [pores] = 12; light gray = SEM) but arrest with a narrow diameter upon Arp2/3 perturbation (CK666^{500 µM}; squares; mean arrested pore diameter = 0.5 ± 0.02 µm; n [pores] = 10; gray = SEM). Fusion = time 0. SGs express Glue-GFP (green) and LifeAct-Ruby (magenta) in A and C–E. RNAi expression in A and C under GAL4/UAS control. UAS expression is driven in A and C by fkh-GAL4, and in D and E by c135-GAL4. Time mm:ss in A and E, relative to fusion, in D relative to treatment. Scale bars in A and E, 1 µm; D, 20 µm.