Requirement of ICAP-1 for NME function in CCP upstream of dynamin and auxillin steps to allow optimal clathrin-coated vesicle budding in the vicinity of focal adhesion. (A) Highly inclined illumination fluorescence microscopy analysis was performed at the basal face of adherent cell on FN coated coverslide. Left panel shows cells immunostained for vinculin (green) and β1 integrin/NME duolink signal (PLA event, red), and the right panel shows cells stained for vinculin (green) and β3 integrin/NME duolink signal (PLA event, red). Scale bar, 5 µm. (B) Boxplot representation of the fraction of the hits estimated for β1 integrin/NME-vinculin and for β3 integrin/NME-vinculin and for randomized images (rand.). (C and D) Representative images of PLA with quantification performed with antibodies against NME and β3 integrin. Red dots denote regions of signal amplification revealing ICAP-1/β3 integrin proximity in β1 integrin deficient cells. PLA performed on ICAP-1 deficient cell line is used as control. Nuclei are stained in blue with DAPI. N ≥ 25 cells/condition from three independent experiments. Scale bar, 20 µm. (E and F) Spinning disk highly magnified video micrographs (every 4 s) of an FA, (vinculin - green) and auxillin bursts in red (indicated by yellow arrow) in β1 integrin^+/+-ICAP-1^+/+ cells. The quantification in F shows that ICAP-1 deletion slows down the auxillin bursts/FA by 50%. At least 35 FAs and 3 squares in at least 4 cells per condition were imaged in 3 independent experiments. 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.