Pak inhibition decreases cell migration and protrusiveness. (A) Activity maps of cell edge movement over time in migrating PtK1 cells expressing PID or an inactive mutant of the PID (PID L107F, noted as control). Edge displacements are encoded with warm color (red) for protrusion and cold color (blue) for retraction. (B) Average protrusion efficiency ± SEM. A protrusion efficiency value >1, represented by the red line, indicates a net advancement of the entire leading edge. Pak-inhibited cells did not protrude efficiently (P = 0.95 for PID-expressing cells). n ≥ 7 cells for each condition. *, P < 0.05 compared with a protrusion efficiency of 1. (C) Individual tracks of 15 cells expressing PID L107F as a control or PID WT transposed to a common origin. Pak inhibition led to shorter migration paths. (D–G) Quantification of motility parameters in C, including net path length (D), the net distance that the cells traversed from the first to the last frame; total path length (E), the total distance traversed by cells over time; directionality (F), the ratio of net to total path length; and cell velocity (G). The experiment was repeated at least four times, and n ≥ 30 cells analyzed for each condition. **, P < 0.01; ***, P = 0.0001 compared with control (Ct) cells. a.u., arbitrary units.