Figure 2. Inactivation of Ena/VASP proteins in various fibroblasts impairs 2D cell migration.

(A) MV^D7 cells lack Mena and VASP, but still express Evl. Elimination of Evl by CRISPR/Cas9 in MV^D7 fibroblasts was confirmed by immunoblotting in independent clonal cell lines (MVE-KO). GAPDH was used as loading control. Asterisk indicates nonspecific band in Mena blot. (B) Elimination of Evl in MV^D7 cells decreased cell speed on fibronectin-coated glass and could be rescued by expression of Evl. (C) Analyses of mean square displacement of MV^D7, MVE-KO and reconstituted cells as indicated. Data points represent arithmetic means ± SEM. At least three time-lapse movies from three independent experiments were analyzed for each cell line. (D) Representative frames from wound healing movies of MV^D7, MVE-KO and reconstituted cells as indicated. MVE-KO cells were not able to close the wound after 17 hr. Bar, 200 µm. (E) Reduction of wound area over time. n, number of movies analyzed. Data are means ± SD. (F) Average wound closure rate. n, number of movies analyzed. (G) Immunoblot of independent single and double-KO mutants derived from NIH 3T3 fibroblasts lacking Mena (M–KO) or Mena and VASP (MV-KO) by CRISPR/Cas9 technology. GAPDH was used as loading control. (H) Consistent with findings in B16-F1 cells, consecutive gene disruption of these two Ena/VASP paralogues in NIH 3T3 fibroblasts again increasingly diminished cell migration on fibronectin. (I) Analyses of mean square displacement of NIH 3T3, M-KO and MV-KO cells as indicated. Data points represent arithmetic means ± SEM. n, number of cells tracked. At least three time-lapse movies from three independent experiments were analyzed for each cell line. (J) Total cytoplasmic concentrations of Ena/VASP proteins in investigated cell lines. (B, F and H) Boxes in box plots indicate 50% (25–75%) and whiskers (5–95%) of all measurements, with dashed black lines depicting the medians, arithmetic means are highlighted in red. Non-parametric, Kruskal-Wallis test and Dunn’s Multiple Comparison test were used to reveal statistically significant differences between datasets. *p≤0.05, **p≤0.01, ***p≤0.001; n.s.; not significant. n, number of cells analyzed from at least three independent experiments.

Figure 2—figure supplement 1. Elimination of Evl in MV^D7 cells affects directionality.

(A) Quantification of directionality. Bars represent arithmetic means ± SD. ***p≤0.001 and n.s., not significant by Kruskal-Wallis test and Dunn’s Multiple Comparison test and n, number of cells analyzed. (B) Radar plots showing trajectories of 15 randomly migrating MV^D7, MVE-KO and reconstituted cells as indicated. Note higher directional persistence of MVE-KO cells compared to MV^D7 cells, which was diminished again by expression of Evl. Scale bar, 100 µm.