(A) Immunolocalization of phosphorylated-cofilin (pcofilin, green) and F-actin phalloidin staining (red) in control cells (empty vector), active Rac1-expressing cells (RacQL) alone or in combination with Pak1 inhibitory domain (RacQL+PID), dominant negative LIMK (RacQL+LIMK DN) or ROCK inhibitor (Y27632). Bar = 10 μm. Boxed regions are magnified to the right of the ‘Merge’ column. Bar = 2 μm. LP: Lp, LM: Lm, TB: transverse bundles.
(B) Fluorescence intensity ratio of pcofilin in injected/control cells at the cell edge (± SEM). The experiment was repeated at least three times; n = 20 to 50 cells for each condition. Red *, P < 0.05 vs. RacQL expressing cells. Black *, P < 0.05 and **, P < 0.01 vs. control cells.

(A) Kymograph analysis of actin retrograde flow in a control cell expressing GFP (empty vector) and cells treated with 100 μM blebbistatin expressing GFP, RacQL alone or RacQL+CFL SA. White lines highlight speckle translocation used to calculate flow velocities. t = 1.5 min; d = 1.5 μm.
(B–C) Average F-actin flow rates measured at the leading edge (B) and 5 μm from the leading edge (C) of injected cells (± SEM). EV = empty vector control. **, P < 0.01 compared to EV + blebbistatin. n ≥ 9 cells for each condition. The experiment was repeated three times.
(D) F-actin flow maps computed from qFSM analysis of time-lapse movies of a control cell expressing GFP and cells treated with 100 μM blebbistatin expressing GFP, RacQL alone or RacQL+CFL SA. Flow rates are color coded, ranging from fast flow in red to slow flow in blue. Flow maps have been averaged over 60 frames, i.e. 5 min, and have been created with the same color-coded speed scale in order to allow comparison of cells under different conditions.

(A) Immunolocalization of Arp3 (green) and F-actin phalloidin staining (red) in control cell, or cell expressing RacQL alone or in combination with PID, LIMK DN or CFL SA. Bar = 10 μm. Boxed regions are magnified to the right of the ‘Merge’ column. Bar = 2 μm.
(B) Arp3/F-actin fluorescence intensity ratio in control (black), RacQL- (pink), RacQL+PID- (green), RacQL+LIMK DN- (red) and RacQL+CFL SA- (blue) expressing cells, measured from the cell edge (0 μm) into the cell center (5 μm).
(C-D) Fluorescence intensity of Arp3 (C) and F-actin (D) in injected cells (control: black, RacQL: pink, RacQL+PID: green, RacQL+LIMK DN: red, RacQL+CFL SA: blue), measured from the cell edge (0 μm) into the cell center (5 μm). In B–D, data shown represent one experiment, and are averaged from n ≥ 9 cells for each condition. The experiment was repeated at least three times, with similar results.

(A) Speckle lifetime and speed distributions. Red lines: speed and velocity thresholds for Lm (blue) and Lp (orange) segmentations. Percentages indicate the relative number of classified speckles compared to the total number of speckles. nc indicates the number of unambiguously classifiable speckles.
(B,C,D,E,F,I,K,N) Lm speckles (blue) and Lp speckles (orange) in control cell (B), or cells expressing RacQL alone (C), or in combination with CIN (D), LIMK DN (E), CFL SA (F and I) or cells expressing CFL SA alone (K and N). Bar = 5 μm.
(G,J,L,O) F-actin flow speed maps in cells expressing RacQL + CFL SA (G and J) or CFL SA alone (L and O). Maps are averaged over 24 frames, i.e. 2 min.
(H,M) F-actin turnover in cell expressing RacQL + CFL SA (H) or CFL SA alone (M). F-actin polymerization: red; depolymerization green. Maps are averaged over 24 frames, i.e. 2 min.
(P) Ratio between the number of fast-moving, short-lived speckles (FM/SL) and slow-moving, long-lived speckles (SM/LL). Measured separately for the leading edge region (circle 1 in B, C, D, E, F, I, K, N; red line, ± SEM) and the region adjacent to the leading edge region (circle 2 in B, C, D, E, F, I, K, N; black line, ± SEM), respectively. n = 4 to 8 cells for each condition.

(A) Single frames of actin fluorescent speckle time-lapse series of PtK1 cell expressing GFP (empty vector), RacQL alone or in combination with PID, LIMK DN, CIN WT, CFL SA, or CFL SA alone. Bar = 5 μm. White arrows indicate the lines used to generate kymographs.
(B) Kymograph analysis of actin retrograde flow in the cells depicted in A. White lines highlight speckle translocation used to calculate flow velocities; steeper streaks = faster flow rates. Time bar (t) = 2 min; scale bar (d) = 2 μm.
(C–D) Average F-actin flow rates measured at the leading edge (C) and 5 μm from the leading edge (D) of injected cells (± SEM). EV = empty vector control. n = 4 to 25 cells for each condition, with a minimum of 100 measurements per condition. *, P < 0.0001 vs. control cells; **, P < 0.0001 vs. RacQL-expressing cells. The experiment was repeated at least three times.

(A) F-actin turnover maps computed from qFSM analysis of time-lapse movies of cells expressing GFP (empty vector), CFL SA, RacQL alone or in combination with PID, LIMK DN, CIN or CFL SA. Turnover maps depict F-actin polymerization (red) and depolymerization (green) rates. Maps have been averaged over 24 frames, i.e. 2 min. Bar = 5 μm. n ≥ 4 cells analyzed for each condition.
(B) Free barbed-end actin incorporation (green) and F-actin phalloidin staining (red) in control cells, cells expressing RacQL alone or in combination with PID, LIMK DN or CFL SA. Bar = 5 μm.
(C) Fluorescence intensity ratio of actin incorporation marking free barbed-ends relative to F-actin in control (black), RacQL (pink), RacQL+PID (green), RacQL+LIMK DN (red) and RacQL+CFL SA (blue), measured from the cell edge (0 μm) into the cell center (5μm).
(D–E) Fluorescence intensity of free barbed-end actin incorporation (D) and F-actin (E) in injected cells (control: black, RacQL: pink, RacQL+PID: green, RacQL+LIMK DN: red, RacQL+CFL SA: blue), measured from the cell edge (0 μm) into the cell center (5 μm). In C–E, data shown represent one experiment, and are averaged from n ≥ 7 cells for each condition. The experiment was repeated at least three times, with similar results.

(A) Kymographs of protrusion and retraction dynamics of the leading edge of a migrating PtK1 control cell, or a cell expressing RacQL alone, RacQL + LIMK DN, RacQL + CFL SA, or CFL SA alone. Edge displacements are encoded with warm color (red) for protrusion and cold color (blue) for retraction. t = 30 sec; d = 5 μm.
(B) Average velocity of a 40 μm leading edge section over 10 min (blue bars, ± SEM). *, P < 0.05 compared to zero average velocity. Average of the absolute velocity of a 40 μm leading edge section over 10 min (purple bars, ± SEM). **, P < 0.05 vs. control cells. Levels of active cofilin as percentage of the level in cells expressing LIMK TE (red line).
(C) Protrusion efficiency. Black *, P < 0.05 compared to a protrusion efficiency of one. n = 4 to 11 cells for each condition.

(A) Electron micrographs of control cells and cells expressing RacQL, RacQL+CFL SA or CFL SA. Bar = 5 μm.
(B and C) Higher magnification images of the inserts in column A. The Lp (yellow), defined by a dense network adjacent to the cell edge, is followed by the Lm (red) containing a large population of actin bundles (highlighted in pink). A region of transverse bundles (green) delineates the cell body from the Lm. Increased levels of active cofilin induce a widening of the Lp (RacQL+CFL SA, CFL SA) and cause Lm actin bundles to visibly extend to the leading edge (RacQL+CFL SA 2^nd set of panels, CFL SA). The overlap between the Lp and the Lm is indicated in orange. White bars = 2.4 μm. Stereo pairs of these images can be seen in Fig. S4. The experiment was repeated at least three times for each condition, with similar results.