Skip to main content
SpringerLink
Log in

Immunocytochemical analysis of cubilin-mediated endocytosis of high density lipoproteins (HDL) in epithelial cells of the rat visceral yolk sac

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M (1996) Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 271:518–520

    Google Scholar 

  • Barth JL, Argraves WS (2001) Cubilin and megalin: partners in lipoprotein and vitamin metabolism. Trends Cardiovasc Med 11:26–31

    Article  CAS  PubMed  Google Scholar 

  • Berryman M, Rodewald R (1995) Beta 2-microglobulin co-distributes with the heavy chain of the intestinal IgG-Fc receptor throughout the transepithelial transport pathway of the neonatal rat. J Cell Sci 108:2347–2360

    CAS  PubMed  Google Scholar 

  • Brent RL, Fawcett LB (1998) Nutritional studies of the embryo during early organogenesis with normal embryos and embryos exhibiting yolk sac dysfunction. J Pediatr 132:S6–S16

    CAS  PubMed  Google Scholar 

  • Brent RL, Johnson AJ, Jensen M (1971) The production of congenital malformations using tissue antisera. VII. Yolk-sac antiserum. Teratology 4:255–275

    CAS  PubMed  Google Scholar 

  • Chiba H, Mitamura T, Matsuno K, Kobayashi K (1991) A sensitive sandwich enzyme-linked immunosorbent assay of rat apolipoprotein A–I: effect of various sample treatments on apolipoprotein A–I immunoreactivity and an application to young and aged rat sera. Biochem Med Metab Biol 46:380–390

    Article  CAS  PubMed  Google Scholar 

  • Christensen EI (1982) Rapid membrane recycling in renal proximal tubule cells. Eur J Cell Biol 29:43–49

    CAS  PubMed  Google Scholar 

  • Christensen EI, Birn H (2002) Megalin and cubilin: multifunctional endocytic receptors. Nat Rev Mol Cell Biol 3:256–266

    CAS  PubMed  Google Scholar 

  • Cui S, Christensen EI (1993) Three-dimensional organization of the vacuolar apparatus involved in endocytosis and membrane recycling of rat kidney proximal tubule cells. An electron-microscopic study of serial sections. Exp Nephrol 1:175–184

    CAS  PubMed  Google Scholar 

  • Deurs B van, Petersen OW, Olsnes S, Sandvig K (1989) The ways of endocytosis. Int Rev Cytol 117:131–177

    PubMed  Google Scholar 

  • Dyer CA, Curtiss LK (1988) Apoprotein E-rich high density lipoproteins inhibit ovarian androgen synthesis. J Biol Chem 263:10965–10973

    CAS  PubMed  Google Scholar 

  • Farese RV Jr, Cases S, Ruland SL, Kayden HJ, Wong JS, Young SG, Hamilton RL (1996) A novel function for apolipoprotein B: lipoprotein synthesis in the yolk sac is critical for maternal-fetal lipid transport in mice. J Lipid Res 37:347–360

    CAS  PubMed  Google Scholar 

  • Geuze HJ, Slot JW, Strous GJ, Lodish HF, Schwartz AL (1983) Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell 32:277–287

    Article  CAS  PubMed  Google Scholar 

  • Geuze HJ, Slot JW, Schwartz AL (1987) Membranes of sorting organelles display lateral heterogeneity in receptor distribution. J Cell Biol 104:1715–1723

    Article  CAS  PubMed  Google Scholar 

  • Gruenberg J, Maxfield FR (1995) Membrane transport in the endocytic pathway. Curr Opin Cell Biol 7:552–563

    Article  CAS  PubMed  Google Scholar 

  • Hammad SM, Stefansson S, Twal WO, Drake CJ, Fleming P, Remaley A, Brewer HB Jr, Argraves WS (1999) Cubilin, the endocytic receptor for intrinsic factor-vitamin B (12) complex, mediates high-density lipoprotein holoparticle endocytosis. Proc Natl Acad Sci USA 96:10158–10163

    Article  CAS  PubMed  Google Scholar 

  • Hammad SM, Barth JL, Knaak C, Argraves WS (2000) Megalin acts in concert with cubilin to mediate endocytosis of high density lipoproteins. J Biol Chem 275:12003–12008

    Article  CAS  PubMed  Google Scholar 

  • Hatae T, Fujita M, Sagara H, Okuyama K (1986) Formation of apical tubules from large endocytic vacuoles in kidney proximal tubule cells during absorption of horseradish peroxidase. Cell Tissue Res 246:271–278

    CAS  PubMed  Google Scholar 

  • Hatae T, Fujita M, Okuyama K (1988) Study on the origin of apical tubules in ileal absorptive cells of suckling rats using concanavalin-A as a membrane-bound tracer. Cell Tissue Res 251:511–521

    Article  CAS  PubMed  Google Scholar 

  • Hatae T, Fujita M, Okuyama K (1989) Electron-microscopic demonstration of carbohydrates and proteins in the apical tubules of kidney proximal tubule cells. Acta Histochem Cytochem 22:35–46

    Google Scholar 

  • Hatae T, Ichimura T, Ishida T, Sakurai T (1997) Apical tubular network in the rat kidney proximal tubule cells studied by thick-section and scanning electron microscopy. Cell Tissue Res 288:317–325

    Article  CAS  PubMed  Google Scholar 

  • Hatzopoulos AK, Rigotti A, Rosenberg RD, Krieger M (1998) Temporal and spatial pattern of expression of the HDL receptor SR-BI during murine embryogenesis. J Lipid Res 39:495–508

    CAS  PubMed  Google Scholar 

  • Havel RJ, Eder HA, Bragdon JH (1955) The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34:1345–1353

    CAS  PubMed  Google Scholar 

  • Jollie WP (1990) Development, morphology, and function of the yolk-sac placenta of laboratory rodents. Teratology 41:361–381

    CAS  PubMed  Google Scholar 

  • Kawai Y, Hatae T (1991) Three-dimensional representation and quantification of endosomes in the rat kidney proximal tubule cell. J Electron Microsc (Tokyo) 40:411–415

    CAS  Google Scholar 

  • Koval M, Pagano RE (1989) Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts. J Cell Biol 108:2169–2181

    Article  CAS  PubMed  Google Scholar 

  • Koval M, Pagano RE (1991) Intracellular transport and metabolism of sphingomyelin. Biochim Biophys Acta 1082:113–125

    Article  CAS  PubMed  Google Scholar 

  • Kozyraki R (2001) Cubilin, a multifunctional epithelial receptor: an overview. J Mol Med 79:161–167

    Article  CAS  PubMed  Google Scholar 

  • Kozyraki R, Fyfe J, Kristiansen M, Gerdes C, Jacobsen C, Cui S, Christensen EI, Aminoff M, Chapelle A de la, Krahe R, Verroust PJ, Moestrup SK (1999) The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A–I receptor facilitating endocytosis of high-density lipoprotein. Nat Med 5:656–661

    Article  CAS  PubMed  Google Scholar 

  • Kristiansen M, Kozyraki R, Jacobsen C, Nexo E, Verroust PJ, Moestrup SK (1999) Molecular dissection of the intrinsic factor-vitamin B12 receptor, cubilin, discloses regions important for membrane association and ligand binding. J Biol Chem 274:20540–20544

    Article  CAS  PubMed  Google Scholar 

  • Kugler P, Miki A (1985) Study on membrane recycling in the rat visceral yolk-sac endoderm using concanavalin-A conjugates. Histochemistry 83:359–367

    CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Le Panse S, Ayani E, Nielsen S, Ronco P, Verroust P, Christensen EI (1997) Internalization and recycling of glycoprotein 280 in epithelial cells of yolk sac. Eur J Cell Biol 72:257–267

    PubMed  Google Scholar 

  • Lloyd JB (1990) Cell physiology of the rat visceral yolk sac: a study of pinocytosis and lysosome function. Teratology 41:383–393

    CAS  PubMed  Google Scholar 

  • Marsh M, Griffiths G, Dean GE, Mellman I, Helenius A (1986) Three-dimensional structure of endosomes in BHK-21 cells. Proc Natl Acad Sci USA 83:2899–2903

    CAS  PubMed  Google Scholar 

  • Mayor S, Presley JF, Maxfield FR (1993) Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process. J Cell Biol 121:1257–1269

    Article  CAS  PubMed  Google Scholar 

  • Mellman I (1996) Endocytosis and molecular sorting. Annu Rev Cell Dev Biol 12:575–625

    Article  CAS  PubMed  Google Scholar 

  • Mellman I, Howe CL, Helenius A (1987) The control of membrane traffic on the endocytic pathway. Curr Top Membr Transpl 29:255–288

    CAS  Google Scholar 

  • Moestrup SK, Verroust PJ (2001) Megalin- and cubilin-mediated endocytosis of protein-bound vitamins, lipids, and hormones in polarized epithelia. Annu Rev Nutr 21:407–428

    Article  CAS  PubMed  Google Scholar 

  • Moestrup SK, Kozyraki R, Kristiansen M, Kaysen JH, Rasmussen HH, Brault D, Pontillon F, Goda FO, Christensen EI, Hammond TG, Verroust PJ (1998) The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins. J Biol Chem 273:5235–5242

    Article  CAS  PubMed  Google Scholar 

  • Plonne D, Winkler L, Franke H, Dargel R (1992) The visceral yolk sac—an important site of synthesis and secretion of apolipoprotein B containing lipoproteins in the feto-placental unit of the rat. Biochim Biophys Acta 1127:174–185

    Article  CAS  PubMed  Google Scholar 

  • Sahali D, Mulliez N, Chatelet F, Dupuis R, Ronco P, Verroust P (1988) Characterization of a 280-kD protein restricted to the coated pits of the renal brush border and the epithelial cells of the yolk sac. Teratogenic effect of the specific monoclonal antibodies. J Exp Med 167:213–218

    Article  CAS  PubMed  Google Scholar 

  • Terasawa Y, Cases SJ, Wong JS, Jamil H, Jothi S, Traber MG, Packer L, Gordon DA, Hamilton RL, Farese RV Jr (1999) Apolipoprotein B-related gene expression and ultrastructural characteristics of lipoprotein secretion in mouse yolk sac during embryonic development. J Lipid Res 40:1967–1977

    CAS  PubMed  Google Scholar 

  • Woollett LA (1996) Origin of cholesterol in the fetal golden Syrian hamster: contribution of de novo sterol synthesis and maternal-derived lipoprotein cholesterol. J Lipid Res 37:1246–1257

    CAS  PubMed  Google Scholar 

  • Wyne KL, Woollett LA (1998) Transport of maternal LDL and HDL to the fetal membranes and placenta of the golden Syrian hamster is mediated by receptor-dependent and receptor-independent processes. J Lipid Res 39:518–530

    CAS  PubMed  Google Scholar 

  • Xu S, Laccotripe M, Huang X, Rigotti A, Zannis VI, Krieger M (1997) Apolipoproteins of HDL can directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediates selective lipid uptake. J Lipid Res 38:1289–1298

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are grateful to Mr. K. Yokoi and Mr. M. Urushihara for expert technical support.

Author information

Authors and Affiliations

  1. Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa, 761-0793, Japan

    Tetsuya Ishida, Tanenori Hatae, Nobukazu Araki & Masao Hamasaki

  2. Department of Endocrinology, School of Medicine, Kagawa University, Miki, Kagawa, 761-0793, Japan

    Nozomu Nishi

Authors
  1. Tetsuya Ishida
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tanenori Hatae
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nozomu Nishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nobukazu Araki
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Masao Hamasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tetsuya Ishida.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ishida, T., Hatae, T., Nishi, N. et al. Immunocytochemical analysis of cubilin-mediated endocytosis of high density lipoproteins (HDL) in epithelial cells of the rat visceral yolk sac. Cell Tissue Res 318, 533–543 (2004). https://doi.org/10.1007/s00441-004-0962-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-004-0962-y

Keywords

Search

Navigation