The EEA1-cycle in living HeLa cells expressing EEA1-GFP.

EGFR endocytosis was stimulated by EGF-Cy3 (red channel) in HeLa cells transiently expressing EEA1-GFP (green channel) according to the pre-binding protocol (described in the Material and methods section) to synchronize endocytic events. Time-lapse imaging at +37°C was performed. The following stages of the cycle are presented: (A) formation of hybrid EGF/EEA1-endosome (5–10 min), (B) fusion of hybrid endosomes (5–15 min), (C) aggregated hybrid endosomes (15–40 min), (D) EGF-vesicle segregation from hybrid endosome (25–60 min). Scale bars– 10 μm (whole cell) or 3 μm (ROI time-lapse). Arrowheads indicate vesicles of interest. Full movies are presented as S3, S4, S6 and S8 Movies, respectively.