RUSH-α5 delivery to FAs. (A) Representative immunofluorescence images of U2OS cells co-expressing RUSH-α5 and pmKate2-Paxillin plated on FN or collagen ± biotin treatment for the indicated times. Insets represent ROIs that are magnified and show paxillin-segmented adhesions (red outlines). (B) Quantification of the relative mean intensity of RUSH-α5 in segmented adhesions/cell ± biotin treatment for the indicated times. Data are mean ± SD; n = 64 cells on collagen, 50 cells on FN, pooled from three independent experiments; One-way ANOVA, Holm-Šídák’s multiple comparison test; data distribution was assumed to be normal but this was not formally tested. (C) TIRF imaging of U2OS cells expressing RUSH-α5-pHluorin on a FN-coated surface after biotin release at T = 0. The arrows indicate exocytosis events. Exocytosis events were detected by performing a ratiometric analysis, which consisted of dividing each frame by the previous. All detected events before 19 min of release are indicated in red. The graph indicates the distance between the exocytosis events and the nearest FA segmented on the last frame of Video 6 (37 min after release), compared to the distance of random dots to FAs, showing that the localization to FAs is not random. Individual measurements and the mean ± SD are represented. Unpaired T test. RUSH-α5-pHluorin spots n = 336 spots, random spots n = 116 spots from one experiment.