Spontaneous pulses of Rho activity. (a) Representative TIRF image of Rho activity in resting U2OS cells (see also Video 1, top). (b) Local Rho activity dynamics within white boxes in a, normalized to global mean intensity values. No pulses are observed in cells expressing the control sensor (see also Fig. S1, a–d, for direct comparison of raw signal data). (c) Standard deviation of local Rho activity signal dynamics (percentage of mean intensity; n ≥ 37 cells from three experiments). (d–f) Representative spatiotemporal autocorrelation function derived from the cell shown in panel a. (e and f) Values represent half-time (e) and half-distance (f) ± SEM (n = 39 cells from three experiments). (g–k) Rho activity was stimulated by nocodazole-induced microtubule depolymerization. This induced recurrent local Rho activity pulses with increased amplitude. (g) TIRF images of the Rho activity sensor before and after nocodazole treatment (see also Video 1, bottom). (h) Kymographs corresponding to regions in g (yellow rectangles). (i) Rho activity signal within the white boxes in g. (j and k) Baseline Rho activity (j) and standard deviation of local Rho signal (k; percentage of mean intensity) before and after nocodazole addition (n = 43 cells from three experiments). Frame rate is 3/min. Bars, 10 µm. ***, P < 0.001 (c, unpaired t test; j and k, paired t test). For detailed analyses of unperturbed, spreading cells, see Fig. S1 (e–i).