Myosin IIa acts both downstream and upstream of Rho activity patterns. (a–d) Temporal cross-correlation analysis of GEF-H1 (EGFP-GEF-H1 WT) and Myosin IIa (mCherry-NMHCIIa) in nocodazole-treated U2OS cells. (a) Individual images of TIRF measurements. (b) Intensity measurements within white boxes in panel a. (c and d) Temporal cross-correlation functions (c) and frequency distribution (d) of anticorrelation maxima derived from panel c (n = 37 cells from three experiments; error bars represent 95% confidence interval). (e) Schematic of the proposed Rho activity based signaling network and inhibition by blebbistatin. The inhibition of ROCK with Y27632 is shown in Fig. S5. (f) TIRF images of the Rho activity sensor mCherry-Rhotekin-GBD and Myosin IIa (EGFP-NMHCIIa) before and after 50 µM blebbistatin treatment (1 h; see also Video 7). Kymographs correspond to the white lines in the images above. (g) Intensity measurements within white regions indicated in f. (h and i) Standard deviation of local Rho activity and myosin signal (n ≥ 10 cells from three experiments; see Fig. S5 a for vehicle controls). Frame rate is 20/min (a–d) or 3/min (f–j). Bars, 10 µm. *, P < 0.05; ***, P < 0.001 (d, one-sample t test; h and I, paired t test).