Figure 2.

Immunoelectron microscopic localization of EEA1 in BHK and A431 cells. BHK (A and B) or A431 cells (C and D) were incubated with endocytic markers (BHK cells with 5 nm BSA–gold for 10 min at 37°C; A431 cells with 14 nm CT-B–gold) and then processed for frozen sectioning. Sections were labeled with antibodies to EEA1 followed by 10 nm protein A–gold. EEA1 labeling is predominantly associated with endosomal vacuoles (asterisks). (A) Labeling of two early endosomal vacuoles containing internalized 5 nm BSA–gold. Note that the labeling is clearly located at some distance from the membrane of the endosome in the cytoplasm (arrows). A putative late endosomal structure filled with internal membranes at the lower edge of the figure is unlabeled by EEA1. (B) Labeling for EEA1, which is in the area of the BSA–gold-labeled endosomal vacuole, but labeling is also associated with the tubular elements surrounding (and presumably emanating from) the endosomal vacuole (arrows). (C and D) CT-B–gold-labeled endosomes in A431 cells. (C) An extracted cell in which EEA1 labeling of the vacuole and associated membranes is particularly high. Arrowheads indicate internalized CT-B–gold. (D) An endosome with particularly high CT-B–gold labeling. EEA1 (arrows) is associated with the CT-B–gold-labeled structure. Also note the labeling of surface caveolae by the CT-B–gold (arrowheads). P, plasma membrane. Bars, 100 nm.