Pak inhibition decreases F-actin flow and turnover in the lamella. (A and B) Phase-contrast (A) and FSM images of X-rhodamine actin (B) in motile PtK1 cells expressing PID WT or an inactive mutant of the PID (PID L107F, used as a control). Insets show GFP expression of the GFP–PID WT or GFP–PID L107F constructs. (C) Kymographs taken from lines oriented along the axis of F-actin flow (indicated in B). Lines in C highlight the F-actin flow rates in the lamellipodium (LP) and the lamella (LA). Time bar (t), 2 min; Bar (d), 2 µm. (D) qFSM kinematic maps of the speed of F-actin flow. Note the slower flow in the lamella of the cell treated with PID (white asterisk) compared with the control cell (black asterisk). (E) qFSM kinetic maps of F-actin polymerization (red) and depolymerization (green) rates. Brightness indicates relative rate magnitude. (F) Average rates of F-actin retrograde flow in the lamellipodium and the lamella of Pak-inhibited cells (expressing PID) and control (Ct) cells (expressing an inactive mutant of PID, PID L107F) ± SEM. Pak inhibition significantly increased F-actin flow in the lamellipodium, whereas it significantly decreased the flow in the lamella. *, P < 0.0001 versus control cells; Student’s t test. (G) Average width of cellular regions measured from kymographs ± SEM. **, P = 0.0008; Student’s t test. (F and G) n ≥ 11 cells for each condition, with a minimum of 125 measurements per condition.