UNC-45a localizes to contractile stress fibers with rapid association–dissociation dynamics. (A) IF of endogenous actin, NM-IIA, and UNC-45a in U2OS cells detected by phalloidin, polyclonal NM-IIA heavy chain and polyclonal UNC-45a antibodies, respectively. Magnified regions (indicated by numbers) illustrate the colocalization of NM-IIA and UNC-45a. Bars: (main) 20 µm; (insets) 10 µm. (B) Time-lapse images of photoactivation experiments revealing the dynamics of photoactivatable–GFP fusion constructs (paGFP) of NM-IIA and UNC-45a in stress fibers. U2OS cells were cotransfected with mApple–NM-IIA together with either NM-IIA–paGFP or UNC-45a–paGFP. The fluorescence of paGFP fusion proteins at discrete spots (indicated by orange circles) of mApple–NM-IIA–positive stress fibers were activated with a 405-nm laser pulse, and the dispersion of the photoactivated fluorophore was followed. Bar, 15 µm. (C) Quantification of the dynamics of stress fiber associated NM-IIA (green line), UNC-45a (orange line), and cytosolic UNC-45a (gray line). The rates of the photoactivated fusion protein dissociation from stress fibers, and UNC-45a dissipation from cytosolic regions devoid of stress fibers were measured by following the paGFP signal dispersion on activated foci (exemplified with orange squares in B) and normalized to the background (cytoplasmic) signal at each time point. Data acquisition was continued until fluorescence reached the preactivation level. The data are presented as means ± SEM. For cytosolic and stress fiber–bound UNC-45a, n = 5 photoactivated foci from two UNC-45a–paGFP transfected cells and n = 7 photoactivated foci from two NM-IIA–paGFP transfected U2OS cells.