Tpm3.1 and NMIIA colocalize with the F-actin lattice, but not with the inner branched actin pool. (A–F) WT (A and D), mTom (B and E), Tpm3.1- NG/RFP-LF (C), or RFP-LF/NMIIA (F) mice were injected with 0.01 mg/kg ISOP and salivary glands either exposed and imaged by ISMic (C and F) or after 10 min they were excised, processed for immunofluorescence, and stained with Alexa488 phalloidin and antibodies against Tpm3.1 (A and B) or NMIIA (D and E). (A, B, D, and E) Distribution of Tpm3.1 (A and B arrowheads), NMIIA (D and E arrowheads) on the secretory granules at early or late stage of integration. Dashed lines show the granules’ membranes. Bar, 1 µm. (C and F) Time-lapse of recruitment of Tpm3.1 and LifeAct (C; Video 4) or NMIIA and LifeAct (F; Video 5). Bar, 1 µm. (G) Model for assembly of Tpm3.1 and NMIIA on the external F-actin lattice.