Figure 4.
Depletion of mDia1 or pharmacological inhibition of formins affects the assembly of the F-actin lattice and the stabilization of the fused granules. (A–D and H) mDia1fl/fl Mist1-Cre, mT/mG (A and B left panels, C, D, and H) or CtrlMist1-Cre, mT/mG (A and B right panels, C and H) mice were treated with TMX. After 4 wk, salivary glands were exposed, mice were injected with 0.01 mg/kg ISOP, and imaged by ISMic (A–D and H). (A) Expanded granules in mDia1−/− (arrows) and normal granules in mDia1+/+ (arrowheads) acinar cells. Bar, 5 µm. (B) ISMic time-lapse of granule integration showing expansion (left panels). Bar, 1 µm. (C) Time course (left panels) of granule diameter measured in mDia1−/− (black symbols; N = 100 granules, 35 cells, 8 animals) and mDia1+/+ (green symbols; N = 61 granules, 24 cells, 5 animals) cells. Diameters of secretory granules right after fusion (center panels). One-way ANOVA (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.1). Insets show the diameter of the granules in the first 10 s after fusion (right panels). Data are averages ± SD. (D) Time-lapse of compound exocytosis. Bar, 1 µm. (E–H) mTom (E and H), GFP-LF/mTom (F), or WT (G) mice were treated with 200 µm SMIFH2 (E–G) or DMSO (vehicle, E), injected with 0.01 mg/kg ISOP, and salivary glands were imaged by ISMic or fixed, processed for immunofluorescence, and imaged by spinning disk microscopy. (E) Time course (left panels) of granule integration was measured in SMIFH2- (black symbols; N = 102 granules, 29 cells, 8 animals) and DMSO-treated (vehicle, green symbols; N = 48 granules, 16 cells, 5 animals) mice. Diameters of the secretory granules after fusion (center panels). One-way ANOVA (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.1). Insets show the diameter of the granules in the first 10 s after fusion (right panels). Data are averages ± SD. (F) Time-lapse of expanded granules in acinar cells. Upper panels—Expanded granule (magenta) that fails to recruit GFP-LF and does not integrate. Bar, 3 µm. Center panels—Expanded granule that recruits GFP-LF (green) and undergoes delayed integration. Bar, 1 µm. Lower panels—Granules that undergo normal integration. Bar, 1 µm. (G) Expanded granules undergoing delayed integration labeled with phalloidin (green) and an antibody against NMIIA (magenta). Left panel shows the F-actin lattice. Bar, 1 µm. (H) Surface area (S.A.) and rate of changes in S.A. were calculated for the mice described in C and E. (I) Model for formin depletion/impairment.

Depletion of mDia1 or pharmacological inhibition of formins affects the assembly of the F-actin lattice and the stabilization of the fused granules. (A–D and H) mDia1fl/fl Mist1-Cre, mT/mG (A and B left panels, C, D, and H) or CtrlMist1-Cre, mT/mG (A and B right panels, C and H) mice were treated with TMX. After 4 wk, salivary glands were exposed, mice were injected with 0.01 mg/kg ISOP, and imaged by ISMic (A–D and H). (A) Expanded granules in mDia1−/− (arrows) and normal granules in mDia1+/+ (arrowheads) acinar cells. Bar, 5 µm. (B) ISMic time-lapse of granule integration showing expansion (left panels). Bar, 1 µm. (C) Time course (left panels) of granule diameter measured in mDia1−/− (black symbols; N = 100 granules, 35 cells, 8 animals) and mDia1+/+ (green symbols; N = 61 granules, 24 cells, 5 animals) cells. Diameters of secretory granules right after fusion (center panels). One-way ANOVA (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.1). Insets show the diameter of the granules in the first 10 s after fusion (right panels). Data are averages ± SD. (D) Time-lapse of compound exocytosis. Bar, 1 µm. (E–H) mTom (E and H), GFP-LF/mTom (F), or WT (G) mice were treated with 200 µm SMIFH2 (E–G) or DMSO (vehicle, E), injected with 0.01 mg/kg ISOP, and salivary glands were imaged by ISMic or fixed, processed for immunofluorescence, and imaged by spinning disk microscopy. (E) Time course (left panels) of granule integration was measured in SMIFH2- (black symbols; N = 102 granules, 29 cells, 8 animals) and DMSO-treated (vehicle, green symbols; N = 48 granules, 16 cells, 5 animals) mice. Diameters of the secretory granules after fusion (center panels). One-way ANOVA (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.1). Insets show the diameter of the granules in the first 10 s after fusion (right panels). Data are averages ± SD. (F) Time-lapse of expanded granules in acinar cells. Upper panels—Expanded granule (magenta) that fails to recruit GFP-LF and does not integrate. Bar, 3 µm. Center panels—Expanded granule that recruits GFP-LF (green) and undergoes delayed integration. Bar, 1 µm. Lower panels—Granules that undergo normal integration. Bar, 1 µm. (G) Expanded granules undergoing delayed integration labeled with phalloidin (green) and an antibody against NMIIA (magenta). Left panel shows the F-actin lattice. Bar, 1 µm. (H) Surface area (S.A.) and rate of changes in S.A. were calculated for the mice described in C and E. (I) Model for formin depletion/impairment.

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