Figure 6.

Immunoelectron microscopic analysis of eps15 redistribution during EGFR endocytosis. NIH-EGFR cells were treated with EGF for 1 h at 4°C and warmed to 37°C for 10 min (a) or 30 min (b and c). After 10 min of warming, gold immunolabeling appears associated with endosomal membranes (a, arrows) and vesicles mostly localized in proximity to Golgi cisternae (a). Colocalization of eps15 (large golds) and EGFR (small golds) is observed on clathrin-coated pits at the cell surface (Figure 5a, left inset; eps15: arrow, EGFR: arrowhead), whereas clathrin-coated vesicles juxtaposed to the plasma membrane appear positively labeled only for EGFR (Figure 5a, right inset, arrowhead). At 30 min of warming, gold particles are present in the central perinuclear region of the cell (b and c), close to Golgi complexes (c), and around late endosomes (b and c) positive for cathepsin D (small golds in c). In b, the arrow points to a vesicle positively labeled for eps15 in the process of fusion with a late endosome. Nu, Nucleus; LE, late endosome; cv, clathrin-coated vesicle. Bars, 0.2 μm; insets in a, 0.1 μm.