Cortactin controls MVE docking at the PM. (A–E) TIRFM of fixed and live cells. For analyses of TIRF images in B–E, the center of the cell (demarcated by the yellow circle in A) was excluded. (A and B) Cells were immunostained for CD63 (green) and cortactin (red). (A) Representative images. Bars: (main images) 10 µm; (zoom) 5 µm. Note increased vesicle number in Sc and OE cells compared with KD in zooms. (B) Number of CD63-positive vesicles per square micrometer cell area. n ≥ 20 cells per condition from three or more independent experiments. (C–E) Cells were transiently transfected with GFP-CD63 and imaged live. Individual vesicles were tracked to yield quantitation of total number of MVEs (C), percentage of immobile MVEs (D), and number of immobile MVEs (E) present at the PM. See Fig. S4 B for representative images. n ≥ 8 cells for each condition and n ≥ 3 independent experiments. Bar graphs represent mean ± SE. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Student’s t test determined statistical significance.