BAR domain proteins regulate the fusion pore. (A) Qualitative phenotypic RNAi screen for BAR domain-containing proteins. MIM^KD, Amph^KD, SNX1^KD, and SNX6^KD showed frequent occurrences of full collapse (FC) events; SNX1^KD, SNX6^KD, and CIP4^KD displayed frequent occurrences of kiss-and-run (KAR) events; and CenB1A^KD, SNX1^KD, SNX6^KD, and Synd^KD showed events of compound exocytosis. (B) Mean frequency (%) of stalling, FC, KAR, and membrane crumpling (MC) in CIP4^KD, SNX1^KD, and MIM^KD. CIP4^KD resulted in an increase of kiss-and-run event frequency (53 ± 4% (****)), while SNX1^KD and MIM^KD displayed an increase of full collapse event frequency (51 ± 4% (****) and 46 ± 19% (***), respectively). Error bars = SEM. The statistical significance presented is compared with WT SGs. N (SGs) ≥ 3, n (events) ≥ 150. P values for CIP4^KD: ****(KAR and MC) <0.00001, for SNX1^KD: ****(FC and MC) <0.000001, for MIM^KD: ***(FC) = 0.000111, **(MC) = 0.001267. Two-tailed, unpaired multiple t tests corrected using the Holm-Sidak method. (C) Time-lapse sequence of representative CK666^{500 µM} treated WT (left) and MIM^KD (right) SGs (confocal intensity-projection). Glue-GFP (green) and LifeAct-Ruby (magenta). Inhibiting the Arp2/3 complex in MIM^KD resulted in LSV stalling. Asterisks denote stalled LSVs. Time mm:ss; relative to CK666 treatment. Scale bars = 20 µm. (D) Mean frequency (%) of stalling, FC, KAR, and MC modes in Y-27632 treated WT, MIM^KD, and SNX1^KD SGs. Compared with stalling in Y-27632-treated WT SGs (70 ± 9% of fusion events), significantly less stalling was observed, and full collapse persists at a high frequency in Y-27632 treated MIM^KD (6 ± 6% (****) and 32 ± 8% (*), respectively) and SNX1^KD (20 ± 4% (****) and 35 ± 6% (***), respectively) SGs. Error bars = SEM. The statistical significance presented is compared with WT-treated SGs. N (SGs) ≥ 3, n (events) ≥ 150. P values for MIM^KD: ****(Stalling) = 0.000022, *(FC) = 0.011445, for SNX1^KD: ****(Stalling) = 0.000006, ***(FC) = 0.000441. Two-tailed, unpaired multiple t tests corrected using the Holm-Sidak method. WT and WT Y-27632 treated (from Fig. 2 C and Fig. 3 A; semitransparent) are shown again for comparison convenience in B and D. SGs in A–D express Glue-GFP and LifeAct-Ruby. RNAi expression under UAS control. UAS-based expression in A driven by fkh-GAL4, in B and C by c135-GAL4.