Src activates GEF-H1. (A) Effect of CA-Src overexpression on the activity of GEF-H1 FLARE212 mutants, measured in populations of suspended HEK293T cells. Results are averages of at least three independent experiments (WT, n = 12; Y198F, n = 7; Y198A, n = 5; Y198E, n = 3; C53A, n = 6). Error bars indicate SD. (B) Example showing the effect of dasatinib (Src inhibitor) on GEF-H1 activity at a cell protrusion, indicated by the white star. Scale bar = 10 µm. (C) Src inhibition with 10 nM dasatinib decreases average GEF-H1 activity in motile cells. Curves show individual cell data where GEF-H1 activity was measured at 10-s intervals before and after treatment. Mean biosensor activity levels are normalized to the first acquisition time point. (D) Statistical analysis of the dasatinib-induced decrease in GEF-H1 activity (paired t test, P = 0.043). (E) Cross-correlation curves of GEF-H1 activity and cell edge motion in the ~1.6–3.2-µm windows before, immediately after, and 30–60 min after dasatinib. (F) Modulation of GEF-H1 activity for the three stages of the treatment at different distances from the edge, quantified by temporal coefficients of variation. Curves of individual cells are averaged (solid lines, n = 4 for each condition), and shaded confidence bands indicate ±2 × SEM in E and F.