Size and dynamic of β3 integrin FAs are dependent on β1 integrins and ICAP-1. (A) Staining area of β3 integrins was carried out on osteoblast cells spread on fibronectin-coated coverglass for 4 h using LucA5 antibody. Scale bar, 20 µm. (B) Quantification of the adhesive area of β3 integrins FA, normalized to the cellular surface area. Error bars represent SD. N ≥ 208 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (C) Quantification of the mean of β3 integrin FAs area. The deletion of ICAP-1 in β1 deficient cell line (grey box) drives massive leap of β3 integrin stained FAs size. Error bars represent SD. N ≥ 208 cells. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (D) Representative time series of the lifetime of eGFP-β3 integrin FAs of the four osteoblastic cell lines. Asterix points out typical eGFP-β3 integrin FAs in the cells with typical disassembly lifetime. Note the translocation of FA to the cell center in β1 integrin^−/−/icap-1^−/− cells. Scale bar, 2 µm. (E) TIRF lifetime analysis on the eGFP-β3 integrin FAs. The lifetime of the eGFP-β3 integrin containing FA is increased in β1 integrin^−/−/icap-1^−/− cells. Spinning disk videos of eGFP-β3 integrin were taken for the duration of 2 h. The adhesion lifetime was analyzed by Focal Adhesion Analysis Server (FAAS; Berginski and Gomez, 2013) and verified visually. Error bars represent SD. N ≥ 50 focal adhesions. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001. (F) FRAP analysis on the eGFP-β3 integrin FAs reveal that the β3 integrin mobility at the plasma membrane is halted in the absence of ICAP-1 and β1 integrin. At least six FAs per cell lines were bleached and their recovery was monitored for 5 min. Error bars represent SD. ns, adjusted P value > 0.05; *, P value ≤ 0.05; **, P value ≤ 0.01; ***, P value ≤ 0.001; ****, P value ≤ 0.0001.