Figure 2.
Figure 2. GEF-H1 mediates Rho activity self-amplification. (a) TIRF images of the Rho activity sensor mCherry-Rhotekin-GBD in U2OS cells coexpressing EGFP (control) or EGFP-GEF-H1 C53R (see also Video 2). Kymographs correspond to arrows. (b) Standard deviation of local Rho signal (n ≥ 55 cells from four experiments). (c) Proposed mechanism of Rho activity amplification via GEF-H1. (d–k) Temporal cross-correlation analysis of the Rho activity sensor mCherry-Rhotekin-GBD and EGFP-GEF-H1 WT (d–g) or EGFP localization (h–k) in nocodazole-treated cells. (d and h) Individual images of TIRF measurements (see also Video 4). (e and i) Intensity measurements from boxes in d and h. (f and j) Temporal cross-correlation functions and frequency distribution (g and k) of cross-correlation maxima derived from f and j (n ≥ 30 cells from three experiments; error bars represent 95% confidence interval). (l) Standard deviation of local Rho activity signal in cells coexpressing GEF-H1 C53R or GEF-H1 C53R F539A I541E (n ≥ 124 cells from three experiments). (m) TIRF images of the Rho activity sensor mCherry-Rhotekin-GBD in U2OS cells treated with nontargeting (nt) or GEF-H1 siRNA. Kymographs correspond to arrows (see also Video 3, bottom). (n) Standard deviation of local Rho activity signal in cells treated with nt or GEF-H1 siRNA and coexpressing control (EGFP), siRNA-resistant GEF-H1 WT, siRNA-resistant GEF-H1 F539A I541E, or siRNA-resistant GEF-H1 Y393A (n ≥ 82 cells from four experiments). (o) Standard deviation of local Rho activity signal in cells coexpressing control (EGFP) or Ect2 constructs (n ≥ 36 cells from three experiments; measurements from a small subpopulation of cells that show wave propagation are marked in magenta). (p) Representative TIRF images from the subpopulation of cells marked magenta in panel o. Kymographs correspond to yellow circular line. (q) Intensity measurements within the regions indicated by white boxes in panel p. (r) Temporal cross-correlation function from subpopulation of cells marked magenta in panel n (n = 6 cells from two experiments; error bars represent 95% confidence interval). (s) Standard deviation of local Rho activity signal in cells treated with control or Ect2 siRNA (n ≥ 75 cells from three experiments). For representative Western blots of GEF-H1 and Ect2 siRNA knockdown, see Fig. S2 (i and k). Frame rate is 3/min (a, b, and l–s) or 20/min (d–k). Bars, 10 µm. **, P < 0.01; ***, P < 0.001 (b, n, and o, analysis of variance, Dunnett’s post-test; g and k, one-sample t test; and l and s, unpaired t test).

GEF-H1 mediates Rho activity self-amplification. (a) TIRF images of the Rho activity sensor mCherry-Rhotekin-GBD in U2OS cells coexpressing EGFP (control) or EGFP-GEF-H1 C53R (see also Video 2). Kymographs correspond to arrows. (b) Standard deviation of local Rho signal (n ≥ 55 cells from four experiments). (c) Proposed mechanism of Rho activity amplification via GEF-H1. (d–k) Temporal cross-correlation analysis of the Rho activity sensor mCherry-Rhotekin-GBD and EGFP-GEF-H1 WT (d–g) or EGFP localization (h–k) in nocodazole-treated cells. (d and h) Individual images of TIRF measurements (see also Video 4). (e and i) Intensity measurements from boxes in d and h. (f and j) Temporal cross-correlation functions and frequency distribution (g and k) of cross-correlation maxima derived from f and j (n ≥ 30 cells from three experiments; error bars represent 95% confidence interval). (l) Standard deviation of local Rho activity signal in cells coexpressing GEF-H1 C53R or GEF-H1 C53R F539A I541E (n ≥ 124 cells from three experiments). (m) TIRF images of the Rho activity sensor mCherry-Rhotekin-GBD in U2OS cells treated with nontargeting (nt) or GEF-H1 siRNA. Kymographs correspond to arrows (see also Video 3, bottom). (n) Standard deviation of local Rho activity signal in cells treated with nt or GEF-H1 siRNA and coexpressing control (EGFP), siRNA-resistant GEF-H1 WT, siRNA-resistant GEF-H1 F539A I541E, or siRNA-resistant GEF-H1 Y393A (n ≥ 82 cells from four experiments). (o) Standard deviation of local Rho activity signal in cells coexpressing control (EGFP) or Ect2 constructs (n ≥ 36 cells from three experiments; measurements from a small subpopulation of cells that show wave propagation are marked in magenta). (p) Representative TIRF images from the subpopulation of cells marked magenta in panel o. Kymographs correspond to yellow circular line. (q) Intensity measurements within the regions indicated by white boxes in panel p. (r) Temporal cross-correlation function from subpopulation of cells marked magenta in panel n (n = 6 cells from two experiments; error bars represent 95% confidence interval). (s) Standard deviation of local Rho activity signal in cells treated with control or Ect2 siRNA (n ≥ 75 cells from three experiments). For representative Western blots of GEF-H1 and Ect2 siRNA knockdown, see Fig. S2 (i and k). Frame rate is 3/min (a, b, and l–s) or 20/min (d–k). Bars, 10 µm. **, P < 0.01; ***, P < 0.001 (b, n, and o, analysis of variance, Dunnett’s post-test; g and k, one-sample t test; and l and s, unpaired t test).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal