Dynamic properties of αABD clusters. (a) Dendra photoconversion assay of A431 cells expressing Dn-αABD (αABD) or its K842A or K866A point mutants. The images of green (−2) and red (−1) fluorescence were taken 2 and 1 s before photoconversion, respectively. The encircled areas (d = 2.5 µm) were converted from green to red fluorescence at time 0. After photoconversion, the red fluorescence was imaged in a stream mode with an image acquisition time of 1 s. Selected frames taken 1, 6, or 11 s after photoconversion (+1, +6, and +11) are shown. (b) Red fluorescence intensity over time in the photoconverted spots of A431 cells expressing Dn-αABD or its K842A point mutant. The curves were plotted based on experiments shown in Fig. 8 a (repeated 15 times) in unaffected A431 cells (control), in the cells with cytoskeleton stabilized by ATP depletion (ATP depletion), and in the cells with actin dynamics arrested by a triple-drug cocktail (JYL [Jasplakinolide, Latrunculin B, and Y27632]). Error bars indicate SEs. (c) FSM of A431 cells expressing Dn-αABD or its I792A point mutant. The encircled areas of the cells (d = ∼4 µm) were photoconverted to image the adjacent area (yellow boxes) for 40 s in a stream mode with 200-ms acquisition time (Videos 1 and 2, respectively). Frames taken 20 s after photoconversion (+20) are shown in the images on the right. (d) Spatial localization of αABD speckles plotted based on Video 1. The color of a given speckle corresponds to the moment of its appearance (the time bar is given at the bottom). (e) Lifetime distribution of αABD speckles. (f) Displacement of speckles (in nanometers) during their entire lifetime. Error bars in f and e indicate SDs. (g) The photobleaching curves of the individual speckles. A.U., arbitrary unit. Bars, 10 µm.