Association of early endosomal autoantigen 1 with macropinocytosis in EGF-stimulated A431 cells.
Association of early endosomal autoantigen 1 (EEA1) with macropinosomes was examined in EGF-stimulated A431 cells by dual labeling with immunofluorescence of anti-EEA1 and FITC-dextran (FDx), a fluid-phase endocytic marker. Addition of EGF to A431 cells drastically enhanced macropinosome formation. Newly formed macropinosomes labeled with 5-min pulse of FDx were located at the cell periphery and labeled weakly for EEA1. After a 5-min chase, these macropinosomes aggregated and frequently fused with each other. Immunofluorescence showed that EEA1 appeared on the membrane of FDx-labeled macropinosomes at that time, suggesting that EEA1 functioned in homotypic macropinosome fusion. With longer chase (30-60 min), macropinosomes decreased in number and size, indicating that FDx was largely exocytosed via recycling compartments. A small amount of FDx-labeled macropinosomes remained in the perinuclear region even at 60 min after pulse labeling. They were EEA1-positive but negative for cathepsin D, a lysosomal enzyme. This indicates that macropinosomes do not mature to late endosomes or fuse with lysosomes. Instead, EEA1 continuously mediates homotypic fusion as long as the macropinosomes persist.
Hamasaki
Masao
M
Department of Histology and Cell Biology, School of Medicine, Kagawa University, Kagawa, Japan.
Araki
Nobukazu
N
Hatae
Tanenori
T
D002460:Cell Line; D004705:Endocytosis / Q000502:physiology; D000078404:Epidermal Cells; D004815:Epidermal Growth Factor / Q000502:physiology*; D004817:Epidermis / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D010873:Pinocytosis / Q000502:physiology*; D013997:Time Factors; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D004815:Epidermal Growth Factor