Fig. 6: Spatiotemporal regulation of ARHGEF25 by caveolae.

a–e Representative time-lapse images of Hs578t cells expressing optogenetic constructs (Lck-mTurquoise2-iLID, SspB-HaloTag-ARHGEF25-DH (ARHGEF25), and Cav1-RFP), in the following conditions: (a) WT, (b) cavin1-KO, (c) cavin1-KO+cavin1, (d) Cav1-KO, (e) Cav1-KO+wtCav1. Insets show zoomed ROI corresponding to optogenetic activation. Timepoints span from -2 min (pre-activation) to +16 min (opto-activation). Normalized intensities of ARHGEF25 (orange), Cav1 (gray), and cell area (blue) in the opto-activated ROI are plotted over time. Data are presented as mean values ± SEM., n = 21 (WT), 16 (cavin1-KO), 22 (cavin1-KO+cavin1), 19 (Cav1-KO), and 23 (Cav1-KO+wtCav1) cells from 3 independent experiments. f Representative time-lapse images of WT- and cavin1-KO Hs578t cells expressing Halo-ARHGEF25 and Cav1-RFP. White arrow indicate direction of migration. g Quantification of ARHGEF25 and Cav1 fluorescence intensity at the front and rear. h Quantification at the cell periphery. Data are presented as mean values ± SEM., n = 32 (WT) and 43 (cavin1-KO) cells from 3 independent experiments. Statistical tests: Two-tailed, Wilcoxon matched-pairs signed-rank test (g); Two-tailed, Mann-Whitney t-test (h); ****P  <  0.0001; ns = not significant. i Representative time-lapse images of random contracting events in WT- and cavin1-KO Hs578t cells expressing Halo-ARHGEF25 and Cav1-RFP. Plots show normalized intensity and cell area: ARHGEF25 (black), Cav1 (purple), and cell area (blue). Data are presented as mean values ± SEM., n = 90 (WT) and 42 (cavin1-KO) contraction events pooled from 3 independent experiments. Scale bars, 10 µm. F, front; R, rear. Fluo., fluorescence. Source numerical data are provided.