Dynamics of actin assembly on secretory granules during regulated exocytosis indicates multiple populations of F-actin. (A–F and H) Diagram of the GFP-LF/mTom mouse and ISMic set up for the imaging of the submandibular salivary glands in live mice. Upon ISOP stimulation secretory granules fuse with the APM allowing the mTom (magenta) to diffuse into the granule and the GFP-LF (green) to be recruited onto the membranes. GFP-LF/mTom (B–F) or mTom (E, F, and H) mice were injected with 0.01 mg/kg ISOP. Acinar cells were either exposed and imaged by time-lapse spinning disk ISMic (B–D) or excised, fixed, and processed for imaging using spinning disk microscopy (E–H). (B) Acinar cells before (CTRL) and after stimulation (ISOP). Arrows in the right panel point to fused granules. mTom (magenta) and GFP-LF (green). Insets show a close-up of the APM and a fused granule. Bar, 10 µm. (C) Still images from a time-lapse of a secretory granule integration event (Video 1). The focal plane was chosen to show the mid-cross section of the granule. Asterisk marks the first time point in which mTom is detected, arrows point to F-actin lattice, arrowheads to the F-actin thickening, and double arrowheads to the inner actin structure. Bar, 1 µm. (D) Lattice (green symbols) and granule (magenta symbols) diameters were measured. Data are averages ± SD of N = 55 granules, 24 cells, 5 animals. (E and F) High-resolution spinning disk images of fused secretory granules before (E) and during (F) integration. Upper panels show maximal projections (MP) of the granules. Lower panels show cross-sections (CS) of the granules. The lattice and the thicker layer of actin are highlighted by arrows and arrowheads, respectively. Bar, 1 µm. (G and H) Correlation between granule diameter and either LF (G: R2 = 0.7565 P < 0.0001; N = 25 granules in 3 animals) or phalloidin (H: R2 = 0.7407 P < 0.0001; N = 28 granules in 3 animals) thickness was calculated as described in Fig. S1 F.
