Figure 5.
Figure 5. EVL-mediated actin polymerization is required for mechanically directed motility. (a–d) Control (LKO vector) and EVL KD MCF7 cells, plated on 35-kPa hydrogels, were mechanically stimulated. (a) Still images from representative time-lapse videos of control and EVL KD cells (Video 2). Scale bars are 10 µm. (b) Corresponding cell traces at 0, 5, 10, 15, 20, 25, and 30 min, with starting positions in tan and final positions in blue. Crosshairs denote micropipette positions. (c) Rose plots show cumulative turning angles for control and EVL KD cells. Black sectors denote turns in the direction of the force gradient, and gray sectors denote turns away from the force gradient. Data are collected from six independent experiments (n = 15 per condition). (d) Sensing indices of control and EVL KD cells over time. Two-way ANOVA shows a significant difference in sensing index between control and EVL KD cells (P < 0.0001). Data are collected from six independent experiments; all data points are shown (n = 15 per condition; violin plot shows median and quartiles of sensing indices). (e) Illustration depicting PDAI analysis. (f) Dot plot showing quantification of average PDAI per cell over time. Data are collected from six independent experiments; all data points are shown (n = 15 per condition; P values were determined using two-sample t test; ***, P ≤ 0.001; n.s., not significant; exact P values are found in Table S2; mean ± SEM). (g) Dot plot showing quantification of the number of de-adhesion events occurring within the proximal half of the cell. Data are collected from six independent experiments; all data points are shown (n = 15 per condition; P values were determined using Mann-Whitney test; ****, P ≤ 0.0001; n.s., not significant; exact P values are found in Table S2; mean ± SD). (h–k) Control (LKO vector) + GFP, EVL KD + GFP and EVL KD + GFP-ΔGF-PFN EVL MCF7 cells, plated on 35-kPa hydrogels, were mechanically stimulated. (h) Still images from representative time-lapse videos of control + GFP, EVL KD + GFP and EVL KD + GFP-ΔGF-PFN EVL cells (Video 5). Scale bars are 10 µm. (i) Corresponding cell traces at 0, 5, 10, 15, 20, 25 and 30 min, with starting positions in tan and final positions in blue. Crosshairs denote micropipette positions. (j) Rose plots show cumulative turning angles for control + GFP, EVL KD + GFP and EVL KD + GFP-ΔGF-PFN EVL cells. Black sectors denote turns in the direction of the force gradient and gray sectors denote turns away from the force gradient. Data are collected from three independent experiments (n = 15 per condition). (k) Sensing indices of control + GFP, EVL KD + GFP and EVL KD + GFP-ΔGF-PFN EVL cells over time. Two-way ANOVA shows a significant difference in sensing index between control, EVL KD, and EVL KD + GFP-ΔGF-PFN EVL cells (P < 0.0001). Data are collected from three independent experiments; all data points are shown (n = 15 per condition; violin plot shows median and quartiles of sensing indices).

EVL-mediated actin polymerization is required for mechanically directed motility. (a–d) Control (LKO vector) and EVL KD MCF7 cells, plated on 35-kPa hydrogels, were mechanically stimulated. (a) Still images from representative time-lapse videos of control and EVL KD cells (Video 2). Scale bars are 10 µm. (b) Corresponding cell traces at 0, 5, 10, 15, 20, 25, and 30 min, with starting positions in tan and final positions in blue. Crosshairs denote micropipette positions. (c) Rose plots show cumulative turning angles for control and EVL KD cells. Black sectors denote turns in the direction of the force gradient, and gray sectors denote turns away from the force gradient. Data are collected from six independent experiments (n = 15 per condition). (d) Sensing indices of control and EVL KD cells over time. Two-way ANOVA shows a significant difference in sensing index between control and EVL KD cells (P < 0.0001). Data are collected from six independent experiments; all data points are shown (n = 15 per condition; violin plot shows median and quartiles of sensing indices). (e) Illustration depicting PDAI analysis. (f) Dot plot showing quantification of average PDAI per cell over time. Data are collected from six independent experiments; all data points are shown (n = 15 per condition; P values were determined using two-sample t test; ***, P ≤ 0.001; n.s., not significant; exact P values are found in Table S2; mean ± SEM). (g) Dot plot showing quantification of the number of de-adhesion events occurring within the proximal half of the cell. Data are collected from six independent experiments; all data points are shown (n = 15 per condition; P values were determined using Mann-Whitney test; ****, P ≤ 0.0001; n.s., not significant; exact P values are found in Table S2; mean ± SD). (h–k) Control (LKO vector) + GFP, EVL KD + GFP and EVL KD + GFP-ΔGF-PFN EVL MCF7 cells, plated on 35-kPa hydrogels, were mechanically stimulated. (h) Still images from representative time-lapse videos of control + GFP, EVL KD + GFP and EVL KD + GFP-ΔGF-PFN EVL cells (Video 5). Scale bars are 10 µm. (i) Corresponding cell traces at 0, 5, 10, 15, 20, 25 and 30 min, with starting positions in tan and final positions in blue. Crosshairs denote micropipette positions. (j) Rose plots show cumulative turning angles for control + GFP, EVL KD + GFP and EVL KD + GFP-ΔGF-PFN EVL cells. Black sectors denote turns in the direction of the force gradient and gray sectors denote turns away from the force gradient. Data are collected from three independent experiments (n = 15 per condition). (k) Sensing indices of control + GFP, EVL KD + GFP and EVL KD + GFP-ΔGF-PFN EVL cells over time. Two-way ANOVA shows a significant difference in sensing index between control, EVL KD, and EVL KD + GFP-ΔGF-PFN EVL cells (P < 0.0001). Data are collected from three independent experiments; all data points are shown (n = 15 per condition; violin plot shows median and quartiles of sensing indices).

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