F-actin assembly onto the secretory granules precedes the recruitment of NMIIA and NMIIB. (A–D) ISMic of salivary acini in mice expressing RFP-LF and GFP-NMIIA (A, C, and D), GFP-NMIIB (B, C, and D), or mTomato (mT; D). (A and B, left) Low magnification of individual acini shows the localization of LF and the NMII isoforms at the APM (arrows). Bars, 10 µm. (Right) Time-lapse imaging of the recruitment of LF and the NMII isoforms onto the secretory granules after the injection of 0.03 mg/kg ISOP. Bars 1 µm. (C) Kinetics of the recruitment of RFP-LF and the NMII isoforms onto the secretory granules. Curves are from one representative granule (5–10 granules per animal, total of three animals). Norm. Fluor. Int., normalized fluorescence intensity. (D) The mean lag time ± SD between the appearance onto the fused secretory granules of LF and mT, GFP-NMIIA, or GFP-NMIIB was measured in mice expressing GFP-LF/mT (red dots), RFP-LF/GFP-NMIIA (green dots), and RFP-LF/GFP-NMIIB (cyan dots). **, P < 0.01; ****, P < 0.0001; ANOVA.