Figure 5

Exocyst/GEF-H1 interaction is required for dissemination, RhoA activity and generation of traction forces of TGFβ-treated cells.

(A) Exocyst/GEF-H1 uncoupling impairs TGFβ-induced dissemination in CIA. TGFβ-treated cells stably expressing Cherry (control) or Cherry-GEF-H1^aa119–236 (the peptide aa119–236 competes for the binding of endogenous GEF-H1 to Sec5) were submitted to CIA. Dissemination speeds were measured by manual tracking: Cherry n = 60, Cherry-GEF-H1^aa119–236 n = 45, from two experiments. (B) Exocyst/GEF-H1 uncoupling perturbs RhoA activity. TGFβ-treated cells stably expressing Cherry or Cherry-GEF-H1^aa119–236 were transfected with a FRET biosensor to visualize RhoA activity. Graphic shows mean whole-cell FRET values: Cherry n = 54, Cherry-GEF-H1^aa119–236 n = 39, from two experiments. (C) Exocyst/GEF-H1 uncoupling impairs generation of traction forces. The ability of Cherry or Cherry-GEF-H1^aa119–236 stably expressing cells to generate forces was investigated by Traction Force Microscopy (TFM). Phase contrast images and corresponding traction force maps are shown. Color scale bar denotes traction stress (Pa). Scale bar, 50 μm. (D) Quantification of the strain energy. Cherry-GEF-H1^aa119–236 cells generate significantly less strain energies compared to Cherry. Number of Cherry cells n = 72, Cherry-GEF-H1^aa119–236 cells n = 51, from two experiments. Error bars represent SEM. p values come from two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.