Cortical F-actin accumulates in segregating EphB2-expressing cells in a ROCK-dependent manner. (A) Individual frames from Videos 1 and 2 showing HEK293 cells overexpressing ephrin-B1 and LifeAct-mCherry (magenta; cells marked with yellow arrowheads) mixed with HEK293 cells overexpressing EphB2, membrane-GFP (green), and LifeAct-mCherry (magenta; cells marked with white arrowheads) in the absence (top) or presence (bottom) of Y-27632. Bars, 20 µm. Note the strong increase in cortical F-actin in the EphB2 population (white arrowheads) in the control, but not the Y-27632-treated, condition. (B) 2.5D plots derived from the images in A depicting LifeAct fluorescence intensity on the z axis. (C) Cell tracking analysis of overall EphB2 cell movement over 16 h. (D–E and G–H) LifeAct fluorescence intensities of the cell interiors (D and G) and cell membranes (E and H) of the EphB2-expressing (D–E) and ephrin-B1–expressing (G and H) populations. (F) Ratio of the LifeAct fluorescence intensity at the outside of an EphB2-positive cluster of cells to other cell membranes located inside the cluster. *, P < 0.05; **, P < 0.01; ***, P < 0.0001 compared with EphB2+ephrin-B1 DMSO; ^#, P < 0.01; ^##, P < 0.001 compared with ephrin-B1+EphB2 DMSO. Graphs represent means ± SEM. In A and B, the GFP signal was adjusted in a nonlinear fashion to assist discrimination of the EphB2-expressing population. See also Figs. S2–S5 and Videos 1–6.