Figure 10. Pak1TE expression recapitulates protrusiveness and rapid migration at nonoptimal ECM density. (A) Activity maps of cell edge movement over time in migrating PtK1 cells plated at high FN concentration (30 µg/ml) and expressing GFP alone (noted FN30 control) or GFP–Pak1TE (noted FN30 Pak1). Edge displacements are encoded with warm color (red) for protrusion and cold color (blue) for retraction. (B) Average protrusion efficiency ± SEM. n ≥ 5 cells for each condition. *, P < 0.05 compared with a protrusion efficiency of 1. A protrusion efficiency value >1, represented by the red line, indicates a net advancement of the entire leading edge. (C) Individual tracks transposed to a common origin of 15 cells plated on FN30 and expressing GFP–Pak1TE (noted FN30 Pak1) or not (noted FN30 control). Pak1TE expression led to longer 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 (G) cell velocity. The experiment was repeated at least four times, and n ≥ 17 cells analyzed for each condition. **, P < 0.05 and ***, P = 0.001 compared with control (Ct) cells. a.u., arbitrary units. (H) A model of Pak regulation of FA and actomyosin organization. In the presence of active Pak (left), lamellipodium (LP) and lamella (LA) networks present little overlap within the tip of the lamella, adjacent to the cell edge. Myosin IIA is distributed throughout the lamella. FAs appear at the cell edge, mature in the lamella, and disassemble. Coordination between actin dynamics, myosin IIA contractility, and FA turnover leads to efficient protrusion and cell migration. Inhibition of Pak activity (right) widens the lamellipodium and accelerates its F-actin treadmilling rate. In contrast, F-actin retrograde flow in the lamella is inhibited. Myosin IIA is displaced from the leading edge, and FA maturation is decreased. Thus, Pak inhibition reduces the dynamics of edge movements and cell motility.
Figure 10.

Pak1TE expression recapitulates protrusiveness and rapid migration at nonoptimal ECM density. (A) Activity maps of cell edge movement over time in migrating PtK1 cells plated at high FN concentration (30 µg/ml) and expressing GFP alone (noted FN30 control) or GFP–Pak1TE (noted FN30 Pak1). Edge displacements are encoded with warm color (red) for protrusion and cold color (blue) for retraction. (B) Average protrusion efficiency ± SEM. n ≥ 5 cells for each condition. *, P < 0.05 compared with a protrusion efficiency of 1. A protrusion efficiency value >1, represented by the red line, indicates a net advancement of the entire leading edge. (C) Individual tracks transposed to a common origin of 15 cells plated on FN30 and expressing GFP–Pak1TE (noted FN30 Pak1) or not (noted FN30 control). Pak1TE expression led to longer 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 (G) cell velocity. The experiment was repeated at least four times, and n ≥ 17 cells analyzed for each condition. **, P < 0.05 and ***, P = 0.001 compared with control (Ct) cells. a.u., arbitrary units. (H) A model of Pak regulation of FA and actomyosin organization. In the presence of active Pak (left), lamellipodium (LP) and lamella (LA) networks present little overlap within the tip of the lamella, adjacent to the cell edge. Myosin IIA is distributed throughout the lamella. FAs appear at the cell edge, mature in the lamella, and disassemble. Coordination between actin dynamics, myosin IIA contractility, and FA turnover leads to efficient protrusion and cell migration. Inhibition of Pak activity (right) widens the lamellipodium and accelerates its F-actin treadmilling rate. In contrast, F-actin retrograde flow in the lamella is inhibited. Myosin IIA is displaced from the leading edge, and FA maturation is decreased. Thus, Pak inhibition reduces the dynamics of edge movements and cell motility.

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