Pak inhibition displaces myosin IIA from the leading edge. (A) MHC immunofluorescence (green) and F-actin phalloidin staining (red) in migrating PtK1 expressing PID or an inactive mutant of the PID (PID L107F, noted control). Red lines in the MHC column indicate the leading edge of the cells as determined by the F-actin staining. White boxes in the merge column indicate the positions of insets for higher magnifications shown in the rightmost column. (B) Quantification of fluorescence intensity of MHC and F-actin for each indicated condition, measured from the cell edge (0 µm) into the cell center (10 µm). The data shown represent one experiment and are averaged from n ≥ 14 cells for each condition. The experiment was repeated at least three times with similar results. The arrows indicate the MHC-depleted zone from the cell edge. MHC was depleted in the first 2 µm in control cells, a distance corresponding to the size of the lamellipodium, whereas it was absent from at least 5 µm in Pak-inhibited cells.