Cofilin siRNA knockdown cells are elongated and show changes in F-actin structures. (A) Method of length/width (L/W) measurement. Length was scored as Feret's diameter, which is the longest distance between any two points along the boundary, black line; width is the secondary axis of the best fit ellipse of the selected cell, white line. (B) quantification of the L/W ratio of (a) control cells transfected with either (b) scrambled or (c) non-silencing bacterial siRNAs, (d) CF KD cells, (e) cofilin siRNA and human cofilin plasmid cotransfected cells, and (f) cofilin siRNA and control plasmid cotransfected cells. Number of cells scored: control (136), scrambled (67), non-silencing bacterial (51), CF KD cells (123), CF KD + rescue plasmid (51), CF KD + control plasmid (38) cells. *, P < 0.001; **, P < 0.001. (C) control and CF KD cells were subdivided into three categories depending on their actin structures as stained with rhodamine-phalloidin. Cells in each group, were compared with the examples shown in panel, and were subdivided according to the following criteria: cells exhibiting normal F-actin (asterisk; cells exhibiting prominent F-actin, showing an increase in the thickness and number of stress fibers as compared with cells with normal F-actin (arrows); and cells exhibiting F-actin aggregates (arrows in magnified inset mark F-actin aggregates). Bar, 10 μm. (D) cells were double-blind scored as explained in C, and presented as percentage of total. Number of cells scored: control (87), CF KD (67). (E) Total F-actin levels of control (n = 69) and CF KD (n = 67) cells. Results shown here and throughout the paper are the average of cells collected from at least three experiments.

Cofilin knockdown changes the motility behavior of MTC cells. (A) Western blot of whole cell lysate samples taken from MTC cells treated either with oligofectamine (control), or cofilin siRNA (CF KD). (B) quantification of the levels of cofilin in MTC cells treated with cofilin siRNA. (C and D) phase images of control and CF KD MTC cells. Bar, 10 μm. (E) L/W of control and CF KD MTC cells; number of cells scored: control (197) and CF KD (178). F, scoring of the degree of polarity of MTLn3 and MTC cells. Cells were scored as shown in the cartoon on the right, and presented as percentage of total cells scored (n = 350 cells/group). (G and H) quantification of the motility parameters (using DIAS). G, direction change (deg); H, directionality; 8 control and CF KD cells were scored. A–F were analyzed at 36 h after siRNA transfection.

Cofilin knockdown suppresses EGF-induced F-actin assembly and protrusion at the leading edge and the ability of the cells to chemotaxis. (A) representative control and CF KD GFP-actin expressing MTLn3 cells, over a range of 6 min; 0 min (no stimulation), 120 s and 360 s (after EGF stimulation). Arrows refer to global response along the membrane in the control panel compared with localized response mainly at the leading edge in the CF KD cells. Bar, 10 μm. (B) Quantification of F-actin levels at the leading edge after EGF stimulation. (C) Quantification of membrane protrusion, of control, CF KD cells, and CF KD cells + WT(ΔH) cofilin plasmid, after EGF stimulation (standardized over time 0). Number of cells analyzed: B, 15 cells; C, at least 21 cells/group. (D) Cartoon showing the pipette assay technique, and method of calculation of the chemotactic index. Chemotactic index is calculated as the cosine of the angle that is formed between the vector connecting the first and last centroid of the stimulated cells, and the vector between the first centroid and the tip of the pipette. E, quantification of the chemotactic indices of at least 11 cells/ category (control-front, control-back, CF KD-front, and CF KD-back). Cells were either stimulated at the front leading edge or at the back as determined by their shape and the presence of the pipette. *, P < 0.05; **, P < 0.01.

Arp2/3 complex is required for the shape and direction of migration of metastatic MTLn3 cells. (A) Representative images of control (i), CF KD (ii), Arp KD (iii), and DB KD (iv). Bar, 10 μm. (B) Quantification of the L/W of the groups in A. Number of cells scored: control (151), CF KD (214), Arp KD (62), and DB KD (203). (C) Quantification of membrane protrusion after EGF stimulation. Number of cells analyzed: control (24), CF KD (27), Arp KD (21), DB KD (23). (D) F-actin analysis, done as shown in Fig. 2. Quantification of the motility parameters: (E) centroid plots; (F) cell migration velocity and (G) directionality (using DIAS). For F and G, number of cells scored: control (35), CF KD cells (32), Arp KD (6), and DB KD (14). (H) Quantification of the turning frequency; number of cells scored: control (30), CF KD (30), Arp KD (20) and DB KD (20). (I–K) Quantification of the dynamics of protrusions as measured by kymography; number of cells analyzed in I: Control (19 cells, 106 measurements), CF KD (26 cells, 107 measurements), Arp KD (15 cells, 81 measurements), and DB KD (12 cells, 109 measurements). For J and K, number of cells analyzed: control (21 cells, 105 measurements), CF KD (26 cells, 126 measurements), and (12 cells, 123 measurements) for both Arp and DB KD groups. In I and K, the groups at the underlined asterisk are each significantly different from the control group. In J, Arp and DB KD are both different from both control and CF KD.

Cofilin sets the directionality of cancer cells. In comparing untreated (left) and cofilin suppressed (right) cells, current work and previous reports (Ghosh et al., 2004), have shown that cofilin is important for the motility for mammary cancer cells. This study shows that decreasing cofilin levels using siRNA or by LIM kinase overexpression, changes the cell “random walking” behavior resulting from frequent turning into a directional motility resulting from infrequent turning. The change in phenotype seen is related to the ability of cofilin to determine the location of Arp2/3 complex by supplying new filaments for dendritic nucleation (left). Cofilin suppression causes the localization of Arp2/3 complex to one region of the cell which becomes the front of the cell causing linear walking. This relocalization is determined by the availibilty of tropomyosin-free filaments in cofilin suppressed cells (right). Lower cofilin levels also caused changes in membrane protrusion dynamics; CF KD cells had lower frequency and velocity of protrusion than control cells.

Suppression of cofilin using siRNA. (A) Western blotting of whole cell lysates of cells treated with the different siRNAs, blotted for cofilin. The western was repeated four times, and the corresponding quantification is shown in B. (C) representative figures of (a) control cells (treated with oligofectamine only), (b) cells treated with cofilin siRNA, (c) cells treated with scrambled siRNA, and (d) cells treated with non-silencing bacterial siRNA. Bar, 10 μm.

Cofilin knockdown cells exhibit directional migration. (A) Centroid plots of representative control and CF KD cells show the trajectory of cells over time (4 h), analyzed using DIAS. Black dots represent path of cells over time (centroid). (B) Perimeter plots of cells in A shown as a stack of the shapes of the cells as they move over time. (C–H) Quantification of the motility parameters. Net path length is the net distance that the cells traversed from the first to the last frame; total path length is the total distance traversed by cells over time; directionality is the ratio of net to total path length, and persistence is the instantaneous velocity divided by direction change. Cartoon: quantification method of the turning frequency (degree of turning) of cells. Solid lines represent the path followed by cells between frames (every 10 min), and the broken lines represent the imaginary straight path, the cells would have followed if they did not turn. The angle of turning calculated is the angle between the followed path and the imaginary straight one. For C–H, number of cells scored: control (17), CF KD (16). (I) is the average of (11) control and (12) CF KD cells.

Importance of cofilin to the dynamics of protrusions in MTLn3 cells. (A) shape analysis of representative control, and CF KD cells, taken from a time lapse recorded over 4 h (y-axis), shown as unwrapped stacked shapes of cells. Unwrapping was done by angle conversion (x-axis) (see Materials and methods). (B) Tabular display of representative control and CF KD cells. Centroid of cells (black), protrusions (green), retractions (red), and direction of movement (arrow). Consecutive frames are shown as out takes from videos recorded over 4 h (time between frames shown is 4 min). Frequency (C), persistence (D), and lifetime (E) of protrusions were calculated by analyzing the protrusions (green) of cells like ones in B. C, frequency of protrusions (protrusions/min); D average persistence of these protrusions; E, quantification of the number of protrusions lasting for different time intervals. Number of protrusions analyzed in C: control (258) and CF KD cells, (327). For D and E, (13) cells were analyzed. F, phase images of control and CF KD cells. Bar, 10 μm. Two representative kymographs of control (a and b) and CF KD (c and d) are shown. ΔX = persistence of protrusions, ΔY = Distance of protrusions, and ΔY1/ΔX1 = protrusion velocity (rate). (G–I) Quantification of the dynamics of protrusions as measured by kymographs. Number of cells analyzed in G: Control (19 cells, 106 measurements), and CF KD (26 cells, 107 measurements). For H and I, number of cells analyzed: control (21 cells, 105 measurements), and CF KD (26 cells, 126 measurements).

Cofilin knockdown causes relocalization of Arp2/3 to the front of the elongated MTLn3 cells. (A) Arp2/3 staining of control, and CF KD cells. Insets show the front (f) and the back (b) at higher magnification; asterisks represent the regions that are shown in the magnified images and white arrows refer to the leading edge of the cell. Bar, 10 μm. (B) 1-pixel-line intensity analysis of Arp2/3 complex staining in (30) control and (30) CF KD cells; **, P < 0.001. The front and back, were defined by drawing a line through the geometrical center of the cell. The regions were assigned randomly for control cells due lack of polarized shape, while for CF KD cells, the front was assigned to the leading edge of the cell, and the back was assigned to the tail of the cell (the end being geometrically opposite to the leading edge of the cell).

Activated Rac rescues the elongated phenotype of the cofilin siRNA knockdown cells. (A) Constitutively active GFP-RacQ61L was able to rescue the elongated phenotype of the CF KD cells. (A) L/W ratio of control and CF KD cells transfected with GFP-RacQ61L. CF KD Cells transfected with control GFP plasmid showed elongated phenotype compared with control cells treated with the same plasmid. (B) CF KD cells transfected with GFP-RacQ61L (red arrowheads in right panel), have normal phenotype as opposed to CF KD cells that are not transfected with the plasmid (white arrows in left panel). Bar, 10 μm. For A, at least 70 cells/group were analyzed. (C–E) Quantification of the dynamics of protrusions as measured by kymography (analyzed for cells in serum, as explained in Fig. 5 legend). (C) Distance of protrusion, (D) protrusion rate, and (E) persistence of protrusion.