FAK and Src control the asymmetric distribution of adhesive signaling and actomyosin bundles to the front and rear, respectively. (A) TIRF microscopy time-lapse movie of a CHO.K1 cell expressing RLC-D,D–mCherry and mGFP-dSH2 migrating on fibronectin before and after treatment with the Src inhibitor PP2. Note the rapid disappearance of the GFP signal from the front. (B) Localization of p130(Cas) and phospho-Tyr(165) p130(Cas) in RLC-D,D–expressing cells treated with 10 µM PP2 (Src inhibitor) or 0.1 µM PF-562,271 (FAK inhibitor) imaged using a confocal microscope (FV1000; Olympus). Note the almost complete disappearance of phospho-Tyr(165) p130(Cas) in inhibited cells. Representative cells are shown. Bars, 10 µm. (C) Quantification of the axis ratio (top) and accumulation of RLC-D,D–containing bundles to the rear (bottom) in cells treated with 10 µM PP2 or 0.1 µM PF-562,271. n > 200 cells scored from three independent experiments. P represents significance using the nonparametric Mann-Whitney U test.