Figure 5.
Figure 5. F-actin recruits MLCK onto fused secretory granules to promote NMII RLC phosphorylation. (A–E) Wild-type (A and B) or mTomato (mTom; C, D, and E) mice were left untreated (A) or treated with either 10 µM ML-7 (B and C) or 10 µM CytoD (D and E). Mice were injected with 0.03 mg/kg ISOP, and the SGs were either processed for indirect immunofluorescence (A, B, D, and E) or imaged in time-lapse modality (C). (A and B) Localization of the phosphorylated forms of NMII RLC, MLCK, and NMIIA (red; arrowheads and insets) onto the fused secretory granules revealed by Alexa Fluor 488–phalloidin (Phall) staining (green; arrowheads and insets). Bars: (top) 2 µm; (insets) 1 µm. (C) Quantitative analysis of the integration of the secretory granules in mice treated with ML-7 (red symbols) or DMSO (green symbols). (Left) The diameter (Diam) of the granules (Gran.) was measured during membrane integration and reported as a function of time. N = number of granules in three animals. (Middle left) Fitted curves were also generated. Maximum (Max) diameter (middle right) and the integration time (right) were estimated as described in the Materials and methods. Error bars represent SD. *, P < 0.05; ANOVA; ****, P < 0.0001; Student’s t test. (D) Phosphorylated RLC is recruited on F-actin–coated secretory granules (arrows) but not on expanded secretory granules lacking F-actin (arrowheads and dotted lines). Bars, 2 µm. (E) MLCK recruitment on the expanded granules varies with the levels of F-actin. (Left) Expanded granules lacking both phalloidin and MLCK staining (arrowheads). (Right) Expanded granules with low levels of MLCK and phalloidin (arrowheads). Bars, 3 µm. (F) Proposed model of assembly of the actomyosin complex and role of the two NMII isoforms, based on data from this study (solid blue lines) and speculation (dashed blue lines). F-actin is assembled onto the membrane of the granules, and NMII isoforms are recruited via a not yet defined mechanism. F-actin recruits MLCK, which phosphorylates the RLC initiating the formation of NMII filaments, which in turn bind to F-actin. NMIIB generates sustained contraction on the F-actin scaffold (light blue double arrows), and the resulting forces drive the membranes of the granules into the APM (light blue arrows). This process may be also driven by F-actin cross-linking and depolymerization. NMIIA may generate contractions (light blue double arrows) that facilitate the expansion of the fusion pore (light blue arrows). Alternatively, NMIIA may directly or indirectly control plasma membrane tension and regulate the integration. ELC, essential light chain.

F-actin recruits MLCK onto fused secretory granules to promote NMII RLC phosphorylation. (A–E) Wild-type (A and B) or mTomato (mTom; C, D, and E) mice were left untreated (A) or treated with either 10 µM ML-7 (B and C) or 10 µM CytoD (D and E). Mice were injected with 0.03 mg/kg ISOP, and the SGs were either processed for indirect immunofluorescence (A, B, D, and E) or imaged in time-lapse modality (C). (A and B) Localization of the phosphorylated forms of NMII RLC, MLCK, and NMIIA (red; arrowheads and insets) onto the fused secretory granules revealed by Alexa Fluor 488–phalloidin (Phall) staining (green; arrowheads and insets). Bars: (top) 2 µm; (insets) 1 µm. (C) Quantitative analysis of the integration of the secretory granules in mice treated with ML-7 (red symbols) or DMSO (green symbols). (Left) The diameter (Diam) of the granules (Gran.) was measured during membrane integration and reported as a function of time. N = number of granules in three animals. (Middle left) Fitted curves were also generated. Maximum (Max) diameter (middle right) and the integration time (right) were estimated as described in the Materials and methods. Error bars represent SD. *, P < 0.05; ANOVA; ****, P < 0.0001; Student’s t test. (D) Phosphorylated RLC is recruited on F-actin–coated secretory granules (arrows) but not on expanded secretory granules lacking F-actin (arrowheads and dotted lines). Bars, 2 µm. (E) MLCK recruitment on the expanded granules varies with the levels of F-actin. (Left) Expanded granules lacking both phalloidin and MLCK staining (arrowheads). (Right) Expanded granules with low levels of MLCK and phalloidin (arrowheads). Bars, 3 µm. (F) Proposed model of assembly of the actomyosin complex and role of the two NMII isoforms, based on data from this study (solid blue lines) and speculation (dashed blue lines). F-actin is assembled onto the membrane of the granules, and NMII isoforms are recruited via a not yet defined mechanism. F-actin recruits MLCK, which phosphorylates the RLC initiating the formation of NMII filaments, which in turn bind to F-actin. NMIIB generates sustained contraction on the F-actin scaffold (light blue double arrows), and the resulting forces drive the membranes of the granules into the APM (light blue arrows). This process may be also driven by F-actin cross-linking and depolymerization. NMIIA may generate contractions (light blue double arrows) that facilitate the expansion of the fusion pore (light blue arrows). Alternatively, NMIIA may directly or indirectly control plasma membrane tension and regulate the integration. ELC, essential light chain.

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