Local Rho activity peaks correlate with actomyosin localization and cell contraction. (a–l) Temporal cross-correlation analysis of the Rho activity sensor mCherry-Rhotekin-GBD and plasma membrane localization of actin (a–d; EGFP-actin; see also Video 5, top), Myosin IIa (e–l; EGFP-NMHCIIa; see also Video 5, middle and bottom). (m–r) Temporal cross-correlation analysis of Myosin IIa signal and contractile flow. Nocodazole-treated (a–h and m–r) or (i–l) untreated, resting U2OS cells. For additional cross-correlation analyses between Rho activity and Rac1, Cdc42, and FHOD1 localization, see Fig. S3. (a, e, i, and m) TIRF images. (n) Color-coded vector fields depicting Myosin IIa flux in m measured via STICS. (o) Contractile and expansive flow of Myosin IIa structures derived from the divergence of the STICS vector map field in panel n. (b, f, j, and p) Intensity or contractile flow measurements within the regions indicated by white boxes in panels a, e, i, m, and o. Temporal cross-correlation functions (c, g, k, and q) and frequency distribution of cross-correlation maxima (d, h, l, and r) derived from panels c, g, k, and q (n ≥ 15 cells from at least three experiments; error bars represent 95% confidence interval). Frame rate is 20/min. Bars, 10 µm. ***, P < 0.001 (d, h, l, and r, one-sample t test).