mDia1 and ARPC2 are recruited sequentially on fused secretory granules. (A and B) WT or mTom (only panels vi) mice were injected with 0.01 mg/kg ISOP, and after 10 min, salivary glands were processed for immunofluorescence and stained with Alexa488 phalloidin and antibodies against mDia1 (A) or ARPC2 (B). (i–vi) Images were acquired by spinning disk and processed. Cross sections of large granules before the wave (i, ii, and vi), intermediate size granules (iii and iv), and small granules (v). Insets show 3D view of the granules. Bar, 1.5 µm (overview), 0.5 µm (high magnification and vi). (C and D) Wistar rats were transfected through the salivary duct with RFP-LF and either mDia1-Emerald (C) or Arp2-Emerald (D). After 24 h, salivary glands were exposed, animals were injected with 0.025 mg/kg ISOP, and imaged using confocal ISMic. Still images from time-lapses (left panels; Video 2), kymographs (center panels; Video 3), and quantitation of recruitment of RFP-LifeAct, mDia1, and Arp2 (right panels). The inset graphs show the expansion of the first 10 s of the integration shown in the adjacent plots. Arrow in C points to the second pool of mDia1 recruited on the granules. Data are averages ± SD: N = 10 granules, 4 cells, 2 animals for mDia1/LifeAct; and N = 12 granules, 4 cells, 2 animals for Arp2/LifeAct. Bar, 1 µm. (E) Model of step-wise recruitment for linear and branched filaments.