Spatiotemporal coordination of GEF-H1 and RhoA at the edge of migrating cells. (A and B) Individual time points from the migration studies used to generate panels C–E, showing MDA-MD-231 cells expressing the GEF-H1 (A) or RhoA (B) biosensors with superimposed sampling windows (right panels). Scale bar = 10 µm. (C) Time courses of biosensor activity (red) recorded in one sampling window and velocity (black) of the edge adjacent to the window. (D and E) Biosensor activity (left panel) and edge velocity (middle panel) maps of representative cells expressing the GEF-H1 (D) and RhoA (E) biosensors. Sections marked “A” and “B” in panels D and E and delimited with black dotted lines indicate ranges of windows corresponding to “A” and “B” in panels A and B. Right panels show in each sampling window the cross-correlation between the edge velocity and biosensor activity, as a function of the time lag between them. (F and G) Cross-correlation of edge velocity and GEF-H1 (F) or RhoA (G) as a function of their time lag. Black curves display the per-cell average cross-correlation (n = 9 for GEF-H1; n = 7 for RhoA). Red curves display average values. The total number of windows (m) from cells (n) imaged in multiple independent experiments (five for GEF-H1 and three for RhoA) is indicated. (H and I) Fluctuation of GEF-H1 (H) and RhoA (I) activity during major cell edge motion events. Fluctuation curves from different cells (n = 9 for GEF-H1; n = 7 for RhoA) are averaged (solid lines), and shaded confidence bands indicate ±2 × SEM. The total number of edge motion events sampled (l) is indicated. (J) Summary of GEF-H1 and RhoA activation during a protrusion and retraction cycle.