Polarized delivery of newly synthesized integrin to the cell protruding edge. (A–C) RUSH-α5 intensity in U2OS cells plated on 9 µm-wide micropatterns coated with FN and anti-GFP or collagen and anti-GFP ± biotin treatment for the indicated times was analyzed at both cell edges (the predominantly protruding edge was denoted ROI1 and the other edge ROI2; Videos 7 and 8). Representative intensity coded images (A) and quantification of RUSH-α5 release on FN (B; Video 7) and collagen (C; Video 8) (normalized first to the total intensity of the cell and then to 0 min biotin) are shown. Data are mean ± SEM. (D) Representative images and spatiotemporal track maps of cell edge contours over time in U2OS cells expressing RUSH-α5 ± biotin treatment for the indicated times. Red insets represent protruding ROIs that are magnified. Blue insets represent retracting ROIs that are magnified. Spatiotemporal track maps: blue colors represent early time points and magenta colors represent late time points in the time-lapse series. (E) Quantifications of RUSH-α5 intensity in ROIs (retracting or protruding areas determined from spatiotemporal track maps). Data are mean ± SD. (B and C)N = 33 cells on FN and 38 cells on collagen, pooled from three independent experiments, two-way ANOVA, Holm-Šídák’s multiple comparison test. (E)N = 53 cells on collagen, 49 cells on FN, pooled from three independent experiments; one-way ANOVA, Holm-Šídák’s multiple comparisons test; data distribution was assumed to be normal but this was not formally tested.