Figure 7: Internalization of RGD and integrin-beta3.

(a) Endocytosis of RGD-neutravidin. Images represent inverted maximum projection of the confocal planes (1–4 μm above the substrate) in CHO-α5β1 and CHO-α5β1 cells with integrin-beta3-GFP expressed on RGD membranes. Addition of integrin-beta3 in CHO-α5β1 cells promotes endocytosis of RGD ligands. (b) Knockdown of Dab2 and Numb, or inhibition of clathrin coat assembly by chlorpromazine suppresses RGD-neutravidin endocytosis in MEFs on RGD membranes. Within the same time course (3 h), RGD endocytosis is not observed in MEFs that are spread on RGD-glass. Also see Supplementary Fig. 13. (c,d) Integrin-beta3-GFP and RGD-neutravidin are internalized after 3 h of cell adhesion on RGD membranes. Internalized RGD-neutravidin is sorted to mRFP-Rab5-containing vesicles. Three-dimensional confocal images are rendered with 500 nm in each z-step and total 6 μm in height. (e) Proposed mechanism of integrin-beta3 turnover in a cell-matrix force-dependent manner. Without cell matrix force development, activated integrin-beta3 will be internalized by clathrin-mediated endocytosis. Also see Supplementary Fig. 15. All data are the mean of three independent experiments (n>20 in each condition). P values by unpaired Kruskal–Wallis test are ****P<0.0001 and **P<0.01. Error bars represent s.d., and all samples represent biological replicates. Detailed statistical results are available in Supplementary Table 1. Scale bar, 5 μm.