Figure 1.

EEA1 is associated with the sorting endosomes of A431 and CHO cells. (A–D) Control or BFA-treated A431 cells were labeled for the transferrin receptor (B and D) and for EEA1 (A and C). EEA1 and transferrin receptor colocalize partially in the untreated cells (A and B, arrows), although some transferrin-positive structures are negative for EEA1 (B, arrowhead). After BFA treatment, the transferrin receptor–containing elements are converted into an extensive tubular system that runs throughout the cell (D). The EEA1 labeling remains as discrete puncta (C). The only colocalization with transferrin receptor–positive elements is at foci from which tubules appear to emanate (C and D, arrows). (E–H) CHO cells were incubated with FITC–transferrin for 30 min at 37°C and then labeled for EEA1. (E and G) FITC–transferrin; (F and H) EEA1 labeling. EEA1 is distributed throughout the CHO cells (F), and some colocalization with peripheral transferrin (G) is evident (F, small arrows). However, there is no colocalization with internalized transferrin labeling the pericentriolar recycling endosome (E and F, large arrows). In G and H, cells were incubated with transferrin as in E and then further incubated for 20 min at 37°C in the presence or absence of 5 μg/ml BFA. In the absence of BFA, the bulk of the FITC–transferrin labeling was lost, but in the presence of BFA, the transferrin remains within the pericentriolar region (G, arrowheads). EEA1 shows a redistribution toward the pericentriolar region but negligible overlap with pericentriolar transferrin (H). Bars, 10 μm.