Figure 3.

Immunoelectron microscopic localization of EEA1 in MDCK cells. MDCK cells grown on polycarbonate filters were perforated with the use of nitrocellulose to remove parts of the apical surface. Cells were labeled for EEA1, fixed, and processed for Epon embedding with the use of tannic acid to increase staining of cytoplasmic coat proteins. All images show areas underlying the apical plasma membrane. Labeling for EEA1 (arrows) is associated with both tubular structures (A, B, C, and E) and vesicular elements (asterisks in C, D, and B inset) of the endocytic system. Mitochondria (M), Golgi (G), and other membranes are completely unlabeled. In addition, the plasma membrane (P) and clathrin-coated pits and vesicles (arrowheads in B and C; note clathrin lattice in B) are invariably completely negative for EEA1. EEA1 labeling is apparent on filamentous material surrounding the membrane compartments, particularly in B, C, and E. This filamentous material does not contain actin, as shown by double labeling for actin (A in panel B indicates anti-actin 5 nm gold labeling; arrows indicate 10 nm anti-EEA1 labeling). T, tight junction. Bars, 100 nm.