D122K cofilin expression in KD cells is incapable of restoring the normal migration mode of MTLn3. (A) Mean directionality of MTLn3 control cell population (n = 19 cells) and of populations of KD (n = 17), W-KD (n = 15), and M-KD (n = 15) cells. Directionality is here the ratio of net to total path length. Unlike WT cofilin, D122K cofilin expression does not rescue the migration defects characteristic for KD cells. *p < 0.05 calculated using t test. Experimental setup and cofilin expression constructs used are as in Figure 5. Supplemental Figure S5 (videos 1–4) shows cell migration of cells from the four populations. (B) Migration parameters (mean net path length, mean cell velocity, and mean persistence) of M-KD cells compared with control, KD, and W-KD cells for same cell populations as in A. Persistence is here cell speed divided by direction change, as in Sidani et al. (2007).

D122K cofilin translocates slower from the plasma membrane than WT cofilin. (A) FLIP was measured in MTLn3 cells overexpressing eGFP-WT cofilin (black) or eGFP-D122K cofilin (gray) after bleaching in the cytosol at time 0 as indicated. The FLIP signal is expressed as Per/Cyt (ratio of peripheral vs. cytosolic fluorescence) in time (the initial ratio is set to 0, the first measuring point after bleaching is set to 1). Data shown represent the average of four independent experiments with a total number of 31 and 33 cells for WT and D122K, respectively. The solid lines represent the two-phase exponential decay fitting based on the first 20s. (B) τ_PM value ± SEM (τ values or mean time constants associated with fast component of 2-phase exponential decay) derived from data for cells with eGFP-WT or eGFP-D122K cofilin shown in A; *p < 0.05 calculated using t test. Based on van Rheenen et al. (2007), τ_PM is representative of the exchange kinetics of plasma membrane-bound cofilin; a higher τ represents slower translocation kinetics.

D122K cofilin has an increased affinity for micellar PI(4,5)P₂ but displays a WT effect on actin. (A) Percentage of GST-WT cofilin (squares) and GST-D122K cofilin (triangles) bound to homogenous PI(4,5)P₂ micelles at pH 7.5 as a function of total PI(4,5)P₂ concentration, based on gel filtration analysis as in Van Troys et al. (2000). C₅₀ values ([PI(4,5)P₂] for 50% cofilin binding) for GST-D122K cofilin are substantially lower than for GST-WT cofilin: 11 versus 84 μM (experiment shown), 10–12 μM versus 100–125 μM in a second independent experiment. (B) SDS-PAGE analysis and Coomassie staining of the flow-through of microfiltration of mixtures of 5 μM cofilin (WT or D122K) and PI(4,5)P₂ (at the indicated concentrations and in homogenous micelles) incubated at pH 7.5. The analyzed flow-through contains unbound, free cofilin. Bottom, quantification for three independent experiments (including the one shown); levels are normalized to the sample without PI(4,5)P₂. (C) pH dependency of PI(4,5)P₂ binding of GST-WT and D122K cofilin (15 μM) by using the same experimental setup as in A except for different buffer conditions (either pH 6.5 or 7.5). The PI(4,5)P₂-concentration used is 25 μM. Average values with standard deviations for two experiments are shown. (D) Depolymerizing effect of adding WT or D122K cofilin (3 μM) to 4.5 μM steady state F-actin at the time indicated by the arrow. Depolymerization was recorded in time by measuring 90° light scattering at 350 nm. (E) Effect of cofilin on elongation kinetics of 1 μM unlabeled F-actin nuclei (seeds) when added to 4 μM G-actin (10% pyrene labeled). The unlabeled seeds were added as such (control) or treated with 0.5 μM of WT or D122K cofilin before addition. (F) Comparison analysis of normalized elongation rates calculated from experiments as described in E performed for a range of WT (diamonds) or D122K (squares) cofilin concentrations. Mean of three data sets with SD is shown. Normalization is done versus the elongation rate of non-cofilin-treated seeds.

Localization of WT and D122K cofilin-eGFP in MTLn3 cells and effect on PI(4,5)P₂ hydrolysis rate. (A) Localization of eGFP-cofilin (WT (left), D122K (middle), and eGFP (right) in MTLn3 cells. The insets show enlarged peripheral regions of the lamellipodia (left, middle) and a phase contrast image (right). The graphs show the eGFP intensity along the indicated lines (yellow) through the flat lamellipodia. Intensities are normalized to the intensity of the total cell area. eGFP-WT and D122K mutant are both targeted to the extreme leading edge; eGFP is not enriched in this region (note different y-axis for eGFP). Bars, 20 μm. (B) Immunofluorescence signal of endogenous cofilin in MTln3 showing peripheral enrichment. In the graph, the cofilin intensity along the indicated line normalized to the intensity of the total cell area is shown. Bar, 20 μm. (C) EGF-induced PI(4,5)P₂ hydrolysis was measured using the PI(4,5)P₂ probe mRFP-PH(PLCδ1) in MTLn3 cells transfected with eGFP-WT or eGFP-D122K cofilin and mRFP-PH(PLCδ1). Insets, confocal mRFP images before and after 5 nM EGF stimulation. The plots represent the relative PI(4,5)P₂ levels over time expressed as the fluorescence ratio of plasma membrane to cytosol. To calibrate, 5 μM ionomycin was added to induce a full translocation. (D) The mean rate of PI(4,5)P₂ hydrolysis (n = 5) as measured by linearly fitting the EGF-induced PI(4,5)P₂ decrease between the time of EGF addition and the lowest level of PI(4,5)P₂; the rate is expressed as the change in fluorescence ratio at plasma membrane to cytosol (expressed in percentage of the ratio at start of measurement) per minute. (E) The expression levels of eGFP-WT or D122K cofilin in the cells analyzed in D and E. Note that there is no significant difference.

D122K cofilin and WT cofilin are phosphorylated to a similar extent. (A) Representative Western blot signals for endogenous and/or exogenous (WT or D122K) cofilin in NIH3T3 and MTLn3 cells overexpressing WT or D122K cofilin(eGFP) after 2D-SDS PAGE analysis on whole cell lysates. For resolving spots of endogenous cofilin and exogenous cofilin, cell lysates were separated using pI ranges of 6–11 and 3–10, respectively. The spots corresponding to unphosphorylated and phosphorylated cofilin are indicated with open and closed arrows, respectively. (B and C) Graphs showing the percentages of unphosphorylated active cofilin for n independent experiments for NIH3T3 (B) and MTLn3 (C). Significant differences are not observed between WT and D122K cofilin(eGFP) nor between exogenous and endogenous cofilin.

D122K cofilin overexpression results in faster and more directional fibroblast migration. (A) Cell trajectories (a selection is shown of the cell populations considered) of NIH3T3, migrating on fibronectin and either overexpressing WT (top) or D122K cofilin-eGFP (bottom). The initial position of each track is superimposed on (0,0). (B) Quantification of expression levels of WT or D122K cofilin-eGFP and ratio of cofilin-eGFP to endogenous cofilin in transfected NIH3T3 cells. Western blot with anti-cofilin signal is shown for WT (lanes 1–3) and D122K (lanes 4–6) cofilin–eGFP-expressing cells (3 independent samples). The GAPDH signal is used to correct for equal loading on the gel. In comparing WT and D122K cofilin expression levels, the cofilin-eGFP/GADPH ratio was set to 1 for WT (C) Cartoon illustrating migration parameters: cell trajectory in red, separate migration steps in black. s(0), origin, s(N), end position of trajectory. MDO and Hull area (gray) are measures of distance and area covered by a cell, respectively (see Supplemental Data). Mean value for Hull area (D), mean MDO (E), and average speed (F) for a population of NIH3T3 cells expressing eGFP (n = 155 cells), overexpressing WT (n = 390) or D122K cofilin-eGFP (n = 140). (G) Mean SLOPE, a parameter for persistence (see Supplemental Data), for NIH3T3 cell populations overexpressing WT (n = 204) or D122K cofilin-eGFP (n = 71). A less negative SLOPE value reflects a higher directionality of cell movement. (D–G) Mean or average values ± SEM; statistical significance of differences between mean values are calculated using Mann–Whitney tests and indicated by *, p < 0.05 and ***, p < 0.001.

D122K cofilin expression does not rescue the cofilin KD morphological phenotype. (A) Morphological phenotype of control MTLn3, KD (cofilin siRNA treated), W-KD (cofilin siRNA + WT cofilin expression), and M-KD (cofilin siRNA + D122K cofilin expression) cells. Bar, 10 μm. The constructs code for V5-tagged cofilin (WT or D122K), as also used in Sidani et al. (2007). (B) Western Blot analysis demonstrating equal expression levels for WT and D122K cofilin-V5 used in A and C. Level of GAPDH is used to correct for equal loading on the gel. Below the blot, the average ratio ± SD of V5 over GAPDH signal is shown for three independent experiments; ratio for WT set to one. (C) Mean length to width ratios for the four cell populations in A; control (n = 70 cells); KD (n = 93 cells); W-KD (n = 125 cells); M-KD (n = 156 cells). Significantly different mean values, calculated between indicated pairs using t test, are shown by ***, p <0.001.

M-KD cells retain polarized protrusive activity upon EGF stimulation. The expression constructs are coding for WT and D122K cofilin-V5 (also used in Figures 5 and 6). (A) Mean-fold increase in cell area (as measure for protrusive activity induced by EGF) for the morphologically similar KD and M-KD cell population in function of time. The fold increase is expressed in relation to the cell area at time 0. (B) Cartoon clarifying the mode of protrusion, based upon measurements along axes. Control MTLn3 and W-KD cells follow scheme 1, whereas KD and M-KD cells follow either schemes 2 or 3. Vertical and horizontal axes pass through the geometrical center of the cells. (C) Measurement method of axial increase, t seconds after EGF stimulation (only illustrated along axis 2). Time 0 is time of EGF (5 nM) addition via bath stimulation. (D) Mean increases in distance along cell axes 1–4 in response to EGF for cells of the four MTLn3 cell populations. In total, 20 cells were used for each group: i) control cells, ii) KD cells, iii) W-KD cells, and iv) M-KD cells.

PI(4,5)P₂–cofilin interaction controls the mode of EGF-induced protrusion and migration in MTLn3 cells: model of underlying mechanisms in cofilin KD cells expressing either WT cofilin or D122K cofilin. The extent of release of active cofilin from PI(4,5)P₂ within the timeframe of PLC activation and resulting PI(4,5)P₂ hydrolysis is the key control point in lamellipodia initiation. Released cofilin is modeled to sever actin filaments in the periphery, an event followed by Arp2/3-mediated actin nucleation and lamellipodia extension. Uncompromised cofilin activation and lamellipodia initiation in WT cells results in protrusion along the entire periphery (apolar protrusion) and random walking. Due to its higher affinity for PI(4,5)P₂, D122K is released slower resulting in insufficient release of cofilin and no new side protrusion formation. In the established lamellipodia in the pole, activated PLC is enriched (Chou et al., 2003; Mouneimne et al., 2006). This will locally result in stronger PI(4,5)P₂ decrease, which we suggest compensates the slower D122K cofilin release and results in normal cofilin-mediated protrusion at the pole. In combination, this results in directional migration of cells expressing the mutant. EGFR-P, phosphorylated EGF receptor; PLC-P, phosphorylated (active) phospholipase C; DAG, diacylglycerol; and IP₃, inositol-1,4,5-trisphosphate.