Inhibition of Ezrin delays the integration of the secretory granules. (A–H) WT (G and H) or GFP-LF/mTom (E and F) mice were treated with 250 µM of NSC668394 or left untreated (A–D). Animals were stimulated with 0.01 mg/kg ISOP and then processed for immunofluorescence (A–D, G, and H) or ISMic (E and F). (A–D) Immunofluorescence staining of phalloidin (green), Ezrin (magenta, A and B), and p-Ezrin (magenta, C and D). (E) ISMic time-lapse of granule integration (left panels) and quantification of % of granules exhibiting the F-actin inner ring (right panels). Bar, 1 µm. (F) Time course of granule integration. Diameters, S.A. and rate of S.A. changes were measured. Data are averages ± SD (NSC: N = 36 granules, 15 cells, 3 animals; CK666: N = 44 granules, 19 cells, 5 animals in Fig. 5); DMSO: N = 48 granules, 16 cells, 4 animals). (G and H) Immunofluorescence staining of phalloidin (green) and Ezrin (magenta, G), NMIIA (magenta, H left panel), or ARPC2 (magenta, H right panel). (I) Model for Ezrin’s role during granule integration.