Figure 9.

GFP-M6tail is recruited to transferrin-containing uncoated endocytic vesicles, slowing traffic of transferrin to the early endosome. A portion of a transfected cell is shown in each panel, oriented with the nucleus positioned to the left and the cell periphery to the right. (A) Pulse-chase experiments after R-Tsfn in GFP-M6tail–transfected ARPE-19 cells. Transfected cells were incubated with R-Tsfn at 4°C for 30 min (a) and then warmed to 37°C for 2 min (b), 10 min (c), or 30 min (d). Open arrows point to overlap between the R-Tsfn (red) and the GFP-M6tail (green) staining at the periphery of each cell. The boxed region is presented at the same scale as separate M6tail and R-Tsfn images below each panel. The position of GFP-M6tail–associated vesicles is denoted with arrows in both lower panels. PN in d demarcates the position of the perinuclear early endosome. (B) Localization of rab5 in GFP-M6tail–transfected ARPE-19 cells. Open arrows mark the position of GFP-M6tail–associated endocytic vesicles that also contain rab5. (C) Localization of AP-2 (red) in GFP-M6tail (green)–transfected ARPE-19 cells reveals no overlap in CCPs. (D) Localization of EEA1 (red) in GFP-M6tail (green)–transfected ARPE-19 cells reveals that a subset of the GFP-M6tail is associated with peripherally located endosomes. Overlap in location is seen as yellow and is demarcated with an arrow. Bars, 10 μm.