RA disassembly is coupled to cell migration. (A) U2Os-AP2-GFP cells were grown on FN-precoated or non-coated micropatterns (1,100 mm2) and stained for integrin αvβ5 and p-Pax. Representative TIRF images. See Fig. S5 K for the same staining of H-shaped patterns. (B) Analysis of RA coverage from samples in A. N (images): Arrow non-coated = 28, Arrow FN = 27, H non-coated = 25, H FN = 20 from one representative image. One-way ANOVA with Tukey’s multiple comparison, F(2, 79) = 38.09, P < 0.0001. (C) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on FN-coated or non-coated micropatterns (1,100 mm2) and stained for active integrin β1 12G10. Representative TIRF images. See Fig. S5 L for the same staining with H-shaped patterns. (D) Top: U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on non-coated dishes for 2 d, wounded, and let to migrate for 80 min or 4 h in fresh complete medium, and stained for p-Pax. Representative TIRF images (stitched tile of five side-by-side fields of view). The wound is exactly at the right edge of the images. Bottom: Analysis of normalized RA coverage from tiles in D. RA coverage was calculated in 11-µm-wide sliding windows from the wound edge. N (tiles): 0 min = 48, 80 min = 36, 4 h = 15. One-way ANOVA with Tukey’s multiple comparison, F(2, 79) = 38.09, P < 0.0001. (E) U2Os-AP2-GFP-ITGB5-mScarlet cells were grown on non-coated dishes for 2 d, wounded, and let to migrate for 30 min or 80 min in fresh complete medium, and stained for FN. Representative TIRF images from the migrating front (indicated as yellow lines). (F) Analysis of FN fluorescent intensity from samples in E. N (images): 0 min = 10, 30 min = 25, 80 min = 22. One-way ANOVA with Tukey’s multiple comparison, F(2, 56) = 13.77, P < 0.0001. (G) A schematic illustration of results shown in this figure. Data are the mean ± SD, ** P value < 0.01, *** P value < 0.001. Scale bars, 10 µm, except in D (50 µm) and inset (10 µm).
