Figure 3.
Myosin II is essential for efficient pore expansion and constriction. (A) Mean frequency (%) of stalling, full collapse (FC), kiss-and-run (KAR), and membrane crumpling (MC) in WT untreated, ZipperKD (myosin II heavy chain), and SGs treated with the ROCK inhibitor Y-27632100 µM. Exocytosis by membrane crumpling is the dominant mode of exocytosis in WT SGs (from Fig. 2 C, semi-transparent). ZipperKD and Y-27632100 µM treatment resulted in significant LSV stalling (36 ± 4% (****) and 70 ± 9% (***) of fusion events, respectively). Error bars show SEM. Statistical significance with respect to WT, untreated frequencies. N (SGs) ≥ 3, n (events) ≥ 140. P values: **** (stalling and MC) < 0.0001. Two-tailed unpaired multiple t-tests corrected using the Holm-Sidak method. (B) Change in mean pore diameter over time in WT untreated (control, diamonds; n [pores] = 12; from Fig. 2 F; green; mean maximal pore diameter = 1.6 ± 0.1 µm) compared to ZipperKD (stars; n [pores] = 6; mean maximal pore diameter = 1.4 ± 0.2 µm), Y-27632100 µM (circles; n [pores] = 10; mean arrested pore diameter = 1.4 ± 0.1 µm) or Y-27632100 µM + CK666500 µM (triangles; n [pores] = 9; mean arrested pore diameter = 0.6 ± 0.05 µm) SGs. In the presence of myosin II perturbation, the fusion pore exhibited slower expansion (WT - 68 ± 6 s, and Y-27632100 µM - 266 ± 38 s(***), ZipperKD - 111 ± 32 s(ns), P values = 0.0002 and 0.323, respectively) and constricted partially (ZipperKD) or remained expanded (Y-27632100 µM). Perturbation of both Arp2/3 and myosin resulted in a failure of pore expansion, leading to an arrested narrow pore (Y-27632100 µM + CK666500 µM). Fusion = time 0. Significance calculated using two-tailed, non-parametric, unpaired t tests using the Kolmogorov–Smirnov method. Green or shades of gray = SEM. (C) Representative images of stalled LSVs with wide pores after Y-27632100 µM treatment. Glue-GFP (green) and LifeAct-Ruby (magenta; also in Video 5). (D) Time-lapse sequence of representative LSVs in WT SG expressing Sqh-mCherry (myosin II light chain; green) undergoing membrane crumpling (MC, top), full collapse (FC, second row), kiss-and-run (KAR w or w/o actomyosin, third and fourth rows; also in Video 6). LSVs are visible in the background of the cytoplasmic Sqh-mCherry signal (dashed line in FC) and vesicle swelling severs as a proxy for fusion as in Glue-GFP (asterisk). In FC, Sqh-mCherry is recruited after vesicle integration (arrowhead; 02:00). In KAR w actomyosin, vesicles undergo deformation and displacement from the apical membrane upon Sqh-mCherry recruitment (dashed line; 02:00–04:15). In KAR w/o actomyosin, vesicles fuse multiple times before recruiting Sqh-mCherry (00:00 and 02:30) before proceeding to membrane crumpling. SGs express Glue-GFP and LifeAct-Ruby in A–C. UAS expression driven by c135-GAL4. Time mm:ss in C and D relative to fusion. Scale bars in C and D, 1 µm.

Myosin II is essential for efficient pore expansion and constriction. (A) Mean frequency (%) of stalling, full collapse (FC), kiss-and-run (KAR), and membrane crumpling (MC) in WT untreated, ZipperKD (myosin II heavy chain), and SGs treated with the ROCK inhibitor Y-27632100 µM. Exocytosis by membrane crumpling is the dominant mode of exocytosis in WT SGs (from Fig. 2 C, semi-transparent). ZipperKD and Y-27632100 µM treatment resulted in significant LSV stalling (36 ± 4% (****) and 70 ± 9% (***) of fusion events, respectively). Error bars show SEM. Statistical significance with respect to WT, untreated frequencies. N (SGs) ≥ 3, n (events) ≥ 140. P values: **** (stalling and MC) < 0.0001. Two-tailed unpaired multiple t-tests corrected using the Holm-Sidak method. (B) Change in mean pore diameter over time in WT untreated (control, diamonds; n [pores] = 12; from Fig. 2 F; green; mean maximal pore diameter = 1.6 ± 0.1 µm) compared to ZipperKD (stars; n [pores] = 6; mean maximal pore diameter = 1.4 ± 0.2 µm), Y-27632100 µM (circles; n [pores] = 10; mean arrested pore diameter = 1.4 ± 0.1 µm) or Y-27632100 µM + CK666500 µM (triangles; n [pores] = 9; mean arrested pore diameter = 0.6 ± 0.05 µm) SGs. In the presence of myosin II perturbation, the fusion pore exhibited slower expansion (WT - 68 ± 6 s, and Y-27632100 µM - 266 ± 38 s(***), ZipperKD - 111 ± 32 s(ns), P values = 0.0002 and 0.323, respectively) and constricted partially (ZipperKD) or remained expanded (Y-27632100 µM). Perturbation of both Arp2/3 and myosin resulted in a failure of pore expansion, leading to an arrested narrow pore (Y-27632100 µM + CK666500 µM). Fusion = time 0. Significance calculated using two-tailed, non-parametric, unpaired t tests using the Kolmogorov–Smirnov method. Green or shades of gray = SEM. (C) Representative images of stalled LSVs with wide pores after Y-27632100 µM treatment. Glue-GFP (green) and LifeAct-Ruby (magenta; also in Video 5). (D) Time-lapse sequence of representative LSVs in WT SG expressing Sqh-mCherry (myosin II light chain; green) undergoing membrane crumpling (MC, top), full collapse (FC, second row), kiss-and-run (KAR w or w/o actomyosin, third and fourth rows; also in Video 6). LSVs are visible in the background of the cytoplasmic Sqh-mCherry signal (dashed line in FC) and vesicle swelling severs as a proxy for fusion as in Glue-GFP (asterisk). In FC, Sqh-mCherry is recruited after vesicle integration (arrowhead; 02:00). In KAR w actomyosin, vesicles undergo deformation and displacement from the apical membrane upon Sqh-mCherry recruitment (dashed line; 02:00–04:15). In KAR w/o actomyosin, vesicles fuse multiple times before recruiting Sqh-mCherry (00:00 and 02:30) before proceeding to membrane crumpling. SGs express Glue-GFP and LifeAct-Ruby in A–C. UAS expression driven by c135-GAL4. Time mm:ss in C and D relative to fusion. Scale bars in C and D, 1 µm.

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