Effects of the mutants of Rho-kinase and α-adducin on cell migration in a wound healing assay. (A) The localization of Thr445-phosphorylated α-adducin in the migrating NRK49F cell. NRK49F cells which migrated in response to the wounding were stained with TRITC-phalloidin (panel a) and anti-pT445 (panel b). Arrows indicate the direction of migration. Arrowheads indicate the membrane ruffling in the leading edge. Bar, 20 μm. (B) A confluent monolayer of NRK49F cells was linearly wounded with a white chip. Soon after wounding, MBP (5 mg/ml; panels a and b), C3 (0.1 mg/ml; panels c and d), MBP-RB/PH (TT) (5 mg/ml; panels e and f), HA-α-adducin (WT) (5 mg/ml; panels g and h), HA-α-adducin-AA (5 mg/ml; panels i and j), or HA-α-adducin-DD (5 mg/ml; panels k and l) was microinjected along with a marker protein (rabbit IgG, 1.0 mg/ml) into the cytoplasm of only the cells along the wound edge. The original wound edge was determined by taking photographs soon after the cells were wounded and microinjected, and then the cells that moved (>20 μm) from the original wound edge were counted by taking photographs of the same fields after 6 h. The cells were doubly labeled by staining with TRITC-phalloidin (panels a, c, e, g, i, and k) and FITC anti–rabbit Ig Ab (panels b, d, f, h, j, and l). Arrows indicate the positions of the migrating wound edge cells. Arrowheads indicate the injected cells. Morphologies of the injected cells that showed the typical phenotypes were shown in insets. Bar, 20 μm. These results are representative of three independent experiments. (C) The ratios of migrating cells to the total injected cells are indicated. Data are means ± SEM of triplicate determinations.