Abstract

The polArity of epithelial cells is key to the function of adult epithelial organs, such as kidney, liver, intestine and exocrine glands. The hallmark of epithelial cell polarity is the asymmetric distribution of structural features and functional markers between the apical and the basolateral domains of the plasma membrane. The molecular basis of this asymmetric distribution is currently under intensive investigation. This laboratory has recently shown that a novel group of integral plasma membrane proteins anchored to the bilayer via glycosylphosphatidylinositol (GPI) is targeted preferentially to the apical surface of the polarized dog kidney epithelial cell line MDCK. This proposal aims to study the possible role of GPI as an apical targeting signal in epithelial cells. The surface localization of GPI-anchored proteins in epithelial cells of diverse tissue origins will be analyzed by a biotin polarity assay and immunocytochemistry. Antibodies against GPI anchors will be raised and used to localize them in frozen sections of epithelial monolayers. The intracellular sorting and surface expression of exogenous GPI-anchored proteins, (e.g., Thy-1, Decay Accelerating Factor -DAF-, etc.) will be studied after transfection of their c-DNAs into MDCK cells). GPI-deficient mutants of these proteins either naturally occurring or constructed in vitro by deletion of a C- terminal signal for GPI-addition will be transferred and tested for polarity of secretion. GPI will be added, by recombinant DNA procedures, to the C-terminal end of a variety of secretory and truncated plasma membrane proteins and the polarized expression of these fusion proteins studied after transfection into MDCK cells. The hypothesis that lateral clustering of glycolipids, including GPI, contribute to the sorting of apical glycoproteins will be analysed by biophysical techniques (fluorescence recovery after photobleaching -FRAP-, and fluorescence energy transfer -FET) and video enhanced microscopy in the apical surface of intact monolayers and after incorporation of glycolipids and GPI-anchored proteins into large unilamellar liposomes. It is expected that these studies will contribute fundamental information to understand the process of epithelial cell differentiation.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041771-03
Application #
3300164
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1989-12-01
Project End
1994-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
3
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
Weill Medical College of Cornell University
Department
Type
Schools of Medicine
DUNS #
201373169
City
New York
State
NY
Country
United States
Zip Code
10065

  Publications

Mora, R; Bonilha, V L; Marmorstein, A et al. (1999) Caveolin-2 localizes to the golgi complex but redistributes to plasma membrane, caveolae, and rafts when co-expressed with caveolin-1. J Biol Chem 274:25708-17
Lipardi, C; Mora, R; Colomer, V et al. (1998) Caveolin transfection results in caveolae formation but not apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins in epithelial cells. J Cell Biol 140:617-26
Yeaman, C; Le Gall, A H; Baldwin, A N et al. (1997) The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells. J Cell Biol 139:929-40
Le Gall, A H; Powell, S K; Yeaman, C A et al. (1997) The neural cell adhesion molecule expresses a tyrosine-independent basolateral sorting signal. J Biol Chem 272:4559-67
van't Hof, W; Malik, A; Vijayakumar, S et al. (1997) The effect of apical and basolateral lipids on the function of the band 3 anion exchange protein. J Cell Biol 139:941-9
Burdick, D; Le Gall, A H; Rodriguez-Boulan, E (1996) Vesicular transport: implications for cell polarity. Biocell 20:343-53
Le Gall, A H; Yeaman, C; Muesch, A et al. (1995) Epithelial cell polarity: new perspectives. Semin Nephrol 15:272-84
van't Hof, W; Rodriguez-Boulan, E; Menon, A K (1995) Nonpolarized distribution of glycosylphosphatidylinositols in the plasma membrane of polarized Madin-Darby canine kidney cells. J Biol Chem 270:24150-5
Zurzolo, C; van't Hof, W; van Meer, G et al. (1994) VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells. EMBO J 13:42-53
Hannan, L A; Lisanti, M P; Rodriguez-Boulan, E et al. (1993) Correctly sorted molecules of a GPI-anchored protein are clustered and immobile when they arrive at the apical surface of MDCK cells. J Cell Biol 120:353-8

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