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. Anat Rec Part A 277A: 298 -306, 2004.
ABSTRACT. In the process of receptor-mediated endocytosis, the fusion of endosomes in vitro is known to be inhibited by wortmannin or LY294002; inhibitors of phosphoinositide 3-kinase (PI3K), suggesting that the activity of PI3K is required for the fusion of early endosomes. In macropinocytosis, a process of bulk fluid-phase endocytosis, however, it remains unclear whether PI3K is required for the fusion of macropinosomes, since the macropinosome formation is inhibited by the PI3K inhibitors. In this study, we examined the effect of 3-methlyadenine (3-MA), which shows a distinct specificity to the PI3K classes from wortmannin and LY294002, on the macropinosome formation and fusion in EGF-stimulated A431 cells. Unlike wortmannin or LY294002, 3-MA did not inhibit the uptake of fluorescent dextran by macropinocytosis. However, the fusion of macropinosomes was inhibited by 3-MA. By imaging of live-cells expressing fluorescent protein-fused tandem FYVE domains, we found that PtdIns(3)P appeared on the macropinosomal membrane shortly after the closure of macropinocytic cups and remained on macropinosomes even at 60-min age. The production of PtdIns(3)P and the recruitment of EEA1 to macropinosomes were abolished by the 3-MA treatment. Therefore, it is likely that 3-MA impairs recruitment of EEA1 by inhibiting PtdIns(3)P production and resultantly blocks the fusion of macropinosomes. These results suggest that the local production of PtdIns(3)P implicates the fusion of macropinosomes via EEA1 as well as conventional early endosomes. However, the long association of PtdIns(3)P with macropinosomes may well be a cell-type specific feature of A431 cells.
Cubilin was recently shown to function as an endocytic receptor for high density lipoprotein (HDL) holoparticles and apolipoprotein A-I (apo A-I), the main protein constituent of HDL. In the present study, we analyzed the distribution and intracellular trafficking of cubilin and HDL in rat visceral yolk sac epithelial cells. After epithelial cells were loaded with apolipoprotein E-free HDL for 30 min in vitro, double immunofluorescence showed that the apical cytoplasm of the cells was strongly stained with anti-cubilin antibodies and anti-apo A-I/HDL. Furthermore, double immunogold electron-microscopic observations revealed the distinct localization of cubilin and HDL in endocytic vacuoles. In early endosomes, both were colocalized on the membrane. Although, in late endosomes, cubilin was also localized on the membrane, HDL was mainly located in the matrix. Both were found in the matrix in lysosomes. In addition, cubilin was markedly localized in apical tubules (ATs), which are generally accepted as being receptor recycling compartments. Thus, HDL is internalized through cubilin-mediated endocytosis and is finally transported to lysosomes. By contrast, cubilin is mainly translocated to ATs for recycling, although some of the cubilin is degraded in lysosomes. Quantitative analysis further revealed that cubilin was not concentrated on the membranes of ATs, although it accumulated in the AT area. Some HDL were also observed in the AT area. These findings suggest that the translocation of cubilin and HDL to ATs from early endosomes occurs through a simple sorting mechanism based on the geometry of these compartments and the bulk membrane and volume flow.
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