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. 2022 Dec 15;122(18):3600–3610. doi: 10.1016/j.bpj.2022.12.018

Figure 2.

Figure 2

FL2 depletion increases GEF-H1 sequestration by MTs at the leading edge. (A) Violin plot of GEF-H1 fluorescence intensity after control or FL2 siRNA treatment (n = 41,39 cells; Welch’s t-test, ∗∗∗p < 0.001). Data points represent individual cells. (B) Violin plot of Spearman r correlation of GEF-H1 and tubulin immunofluorescence (indicating colocalization) after control or FL2 siRNA treatment. (n = 33, 32 cells; Welch’s t-test, ∗p < 0.05) Datapoints represent individual cells. (C) Representative immunofluorescence images of U2OS cells along a scratch stained for GEF-H1 (red) and tubulin (green). Colored lines indicate 5.6-μm-wide region analyzed in (A) and (B). (D and E) Curves showing correlation between GEF-H1 FRET biosensor activation and edge velocity relative to edge protrusion initiation (x = 0) after either (D) control or (E) FL2 siRNA treatment. Curves plotted are spline fits from pooled correlation coefficients of individual cells. Individual curves represent various distances (up to 2.8 μm) away from the cell edge (Fig. S1B). Dashed and dotted lines show 95% confidence intervals, estimated by a nonparametric bootstrap method. Horizontal lines indicate significance of Pearson’s correlation coefficient at p < 0.05. (D: n = 341 windows, 22 cells; E: 457 windows, 25 cells). (D′–E′) Insets show higher magnification of peak correlation coefficients. All experiments performed in triplicate. (F) Bar graphs showing PACC of GEF-H1 FRET biosensor activation irrespective of time to edge protrusion at various distances (up to 5.6 μm) (Fig. S1B) away from the cortex after control or FL2 siRNA treatment. (n = 341 windows/22 cells (siN control), 457 windows/25 cells (siFL2); Student's t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (G) Quantitative schematic representing correlation of local GEF-H1 FRET biosensor activation to leading edge protrusion at various distances away from the leading edge (up to 5.6 μm) (Fig. S1B) (341 windows, 22 cells (siN control); 457 windows, 25 cells (siFL2)). To see this figure in color, go online.