Immunocytochemical analysis of cubilin-mediated endocytosis of high density lipoproteins (HDL) in epithelial cells of the rat visceral yolk sac.
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.
Ishida
Tetsuya
T
Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, 761-0793, Kagawa, Japan. ishidat@kms.ac.jp
Hatae
Tanenori
T
Nishi
Nozomu
N
Araki
Nobukazu
N
Hamasaki
Masao
M
D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism / Q000648:ultrastructure; D007150:Immunohistochemistry / Q000379:methods*; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism / Q000648:ultrastructure; D015017:Yolk Sac / Q000378:metabolism* / Q000648:ultrastructure
D008075:Lipoproteins, HDL; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C029486:intrinsic factor-cobalamin receptor