RUSH-α5 delivery to the plasma membrane is spatially regulated by the ECM. (A) Model of RUSH-α5 (EGFP-integrin α5)–integrin-β1 heterodimer based on the structure of human α5β1-integrin bound to FN (PDB: 7NWL) (see also Video 1). (B) Representative immunofluorescence timelapse images of a U2OS cell expressing RUSH-α5 (SBP-EGFP-integrin α5) and plated on FN ± biotin treatment (single grayscale images for the indicated time points are shown; see Video 4). (C) Representative immunofluorescence images of RUSH-α5 and active integrin α5β1 (SNAKA51 antibody) in RUSH-α5–expressing U2OS cells plated on FN or collagen + biotin (60 min). Grayscale single-channel images and merged images (white, colocalization; blue, nuclei [DAPI]) are shown. (D) Representative images of RUSH-α5 and RUSH-CD59 release in U2OS cells co-expressing both constructs and plated on dual-coated micropatterns (alternating FN coating (cyan) and collagen-peptide (GFOGER) (non-fluorescent) lines). Nuclei (blue) are co-labeled. Intensity line profiles generated across the yellow line are displayed relative to the position of the FN-coated micropattern lines. White insets represent regions of interest (ROIs) that are magnified for each channel. FN, fibronectin.