The RUSH system applied to integrin α5. (A) Principles of the RUSH-α5 integrin. In all experiments, SBP-EGFP-ITGA5 (RUSH-α5) is co-expressed with streptavidin-KDEL (ER-hook). In the absence of biotin, this combined complex is retained within the ER. Biotin addition displaces the ER-hook and releases RUSH-α5 into the cytoplasm. (B) The number of contacts between EGFP and FN during simulations of the coarse-grained model. Left: simulation of EGFP being pulled towards the FN-binding site, starting when the C-terminus of the EGFP and the N-terminus of the integrin α5 are <1 nm apart, the linker included, leading to the formation of contacts (Video 2 A). Right: simulation of a fully stretched EGFP, initially in close proximity to the FN-binding site, that is allowed to relax without a biasing force resulting in a spontaneous and rapid loss of contacts (<100 ns; Video 2 B). The pulling process spanned 8 nm and 80 ns. The relaxation spanned 3200 ns. Contacts were calculated between EGFP and FN with a cutoff of 0.6 nm. (C and D) RUSH-α5 is expressed on the cell surface and forms a functional heterodimer with integrin β1. (C) Representative flow cytometry analysis of cell surface RUSH-α5 levels (detected with the anti-GFP-AF647 antibody) in RUSH-α5–expressing U2OS cells ± biotin. (D) Representative immunoblots of GFP pulldowns performed in RUSH-α5 or control transfected cells ± biotin treatment for the indicated times and probed for endogenous integrin β1. The faster migrating band of immature integrin β1 is indicated by a green arrow and box and the slower migrating band of mature integrin β1 with a magenta arrow and box. Source data are available for this figure: SourceData FS1.