Pak1TE expression rescues F-actin phenotypes at a high adhesion strength. (A and B) Phase-contrast (A) and FSM images of X-rhodamine actin (B) in migrating PtK1 cells plated at high FN concentration (30 µg/ml) and expressing GFP alone (noted as FN30 control) or GFP–Pak1TE (noted as FN30 Pak1). Insets show GFP expression of the GFP or GFP-Pak1TE constructs. (C) Kymographs taken from lines oriented along the axis of F-actin flow (indicated in B). The 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 increased flow in the lamella of the cell expressing Pak1TE (black asterisk) compared with the control cell (white 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 control and Pak1TE-expressing cells ± SEM. The expression of Pak1TE significantly decreased F-actin flow in the lamellipodium, whereas it significantly increased the flow in the lamella (*, P = 0.002 and *, P < 1.10−5, respectively, compared with control cells; Student’s t test). (G) Average width of cellular regions measured from kymographs ± SEM. Pak1TE expression decreased lamellipodium width. *, P < 1.10−5 compared with control; Student’s t test. (F and G) n ≥ 9 cells were analyzed for each condition, with a minimum of 125 measurements per condition.
