Figure 3.

Myo6 is recruited to uncoated endocytic vesicles. ARPE-19 cells were incubated with R-Tsfn at 4°C for 30 min. After surface labeling, the cells were either formaldehyde fixed (a and b) or warmed to 37°C for a 1-min chase (c–f) or a 10-min chase (g and h) before fixation. Cells were stained using antibodies to the tail domain of myo6 (a, c, d, and g), the clathrin-adapter AP-2 (b and e), or the early endosome protein EEA1 (f and h). Antibodies were visualized with a fluorescein-conjugated secondary antibody (green) and compared with the endocytosed rhodamine-conjugated transferrin (red) by immunofluorescence microscopy. Overlap between the R-Tsfn and either Myo6, AP-2, or EEA1 is shown in yellow. Cell peripheries of flat cells were chosen for analysis to allow clear visualization of the R-Tsfn as it moved through the endocytic pathway. Boxed areas in each top panel are enlarged in the bottom panel. The perinuclear early endosome is shown with arrows in f and h. Bars and brackets found in enlarged bottom panels are 10 μm.