Figure 1: Integrin-beta3 recruits Dab2 when the cell adheres on mobile RGD membranes. | Nature Communications

Figure 1: Integrin-beta3 recruits Dab2 when the cell adheres on mobile RGD membranes.

From: Integrin-beta3 clusters recruit clathrin-mediated endocytic machinery in the absence of traction force

Figure 1

(a) Dab2-mCherry is absent at integrin-beta3-GFP clusters when the cell is plated on RGD-glass. (b) Dab2-mCherry is recruited to a subpopulation of integrin-beta3-GFP clusters on RGD membranes. (c) RGD membranes activate both integrin-beta1 and beta3. Dab2-mCherry is only localized with integrin-beta3-GFP, not integrin-beta1 clusters (anti-beta1 clone 9EG7 staining). (d) Pearson’s correlation analysis of Dab2 and activated integrin-beta3, RGD clusters and activated integrin-beta1. Dab2 colocalizes with integrin-beta3 and RGD clusters. (e) Pearson’s correlation analysis of RGD and activated integrin-beta3 and integrin-beta3-GFP. RGD clusters colocalize with activated integrin-beta3 and integrin-beta3-GFP. (f) Two-colour structured illumination super-resolution microscopy of Dab2-mCherry and Integrin-beta3-GFP. Dab2 colocalizes with integrin-beta3 when the cell adheres on RGD membranes (red arrows). Bottom panel represents the boxed area in the upper panel. (g) During the initial phase of cell adhesion on RGD membranes (0–90 s), Dab2-mCherry is absent from the integrin-beta3-GFP cluster (empty white arrow). Dab2 recruitment comes after RGD-integrin-beta3 clustering (90 s and after, solid white arrows). (h) The area ratio between Dab2-mCherry and integrin-beta3-GFP during the initial adhesion formation. With an initial delay, Dab2 binding to integrin-beta3 increases as integrin-beta3 clusters develop. More than 1,000 RGD-integrin-beta3 clusters were analysed (n=6). (i) When the cell adheres on micropatterned RGD substrates, Dab2-mCherry is only recruited to mobile RGD-integrin-beta3 clusters. Right panel represents boxed area in the left panel. (j) Pearson’s correlation analysis of Dab2 and integrin-beta3 colocalization on mobile or fixed RGD regions. Dab2 and integrin-beta3 are colocalized on mobile RGD regions. All data are the mean of more than three independent experiments (n>20 in each condition, except panel 1 h). P values by unpaired Kruskal–Wallis test are: ****P<0.0001 and ns P>0.05. Error bars represent s.d. (s.e.m. in h), and all samples represent biological replicates. Detailed statistical test results are available in Supplementary Table 1. Scale bar, 10 μm (except f) and 1 μm (f).

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