Distinct functional clusters along the RHOA-GEFs/RHOA/actomyosin pathway. (A) Summary of all phenotypic alterations by validated hits of our GEF-screen and for experiments using inhibition of contractility. Number of arrows corresponds to most significant p-value for a given attribute (P ≤ 0.01, P ≤ 0.001, or P ≤ 0.0001). Empty table bins indicate no phenotype. (B) Pairwise distance matrix for knockdown of RhoA, RhoA-GEFs, and contractility experiments. Y, Y27632; B, blebbistatin; number indicates concentration applied over 48 h before wounding; a.u., arbitrary units. Dashed white boxes show functional clusters as defined by close phenotypic similarity. (C) Treatment with Rhosin (5, 25, 50, 100, or 200 µM) induced motility in a dose-dependent fashion. Each point represents the difference in PC of the indicated variable between drug treatment in one well location and the mean of the daily control; N = 2 and n = 12 for 5 µM and N = 3 and n = 18 for the other concentrations, one control well per daily experiment. Statistics via Wilcoxon signed rank test: *, P ≤ 0.01; **, P ≤ 0.001. (D) Western blots to identify expression crosstalk between RhoC and RhoA depletion. (E) Effect on PCs by depletion of RhoC. N = 9 and n = 52. Statistics via Wilcoxon signed rank test. Coord, coordination; Direct, directionality. (F) Pairwise distance matrix for knockdown of RhoA, RhoC, and RhoA-GEFs. (G) Pairwise distance matrix for the mean clusters phenotype: contractility (Contract.) applied for 48 h, RhoA and Arhgef18, RhoC, Arhgef3, Arhgef28, and Arhgef11. (H) Working model emerging from the phenotypic similarity established in this study and existing literature. Dotted black arrows are biochemical associations described in the literature (see citations in text), continuous black arrows indicate functional similarities, and cyan arrows link protein knockdown to a specific phenotype.