Abstract

The general aim of this proposal is to elucidate the role of glycosylphosphatidylinositol (GPI), glycosphingolipids (GSLs) and cholesterol in the processes that lead to the polarized structure of epithelial cells and neurons. Our laboratory has shown that proteins anchored via GPI are apically targeted in various kidney and intestinal epithelial cells (MDCK, LLC-PK, Caco-2), and that this is due to specific sorting mechanisms operating in the TGN. This proposal aims to investigate the nature of the mechanisms that regulate the incorporation of GPI- proteins and GSLs into apical carrier vesicles and to identify sorter molecules in the TGN that participate in this process. For this purpose, we will utilize cell lines characterized by our laboratory that sort differentially GPI-proteins and GSLs. VIP21/caveolin, a protein present in TGN-derived vesicles and in plasma membrane caveolae, appears as a major player in the clustering of GPI-anchored proteins with GSLs and cholesterol, and in the poorly understood interactions of these clusters with the apical sorting machinery. The role of VIP21 /caveolin in TGN sorting will be studied by transfection of the protein into a rat thyroid cell line (FRT) which does not express it, fails to sort GPI-anchored proteins apically and fails to cluster GPI-proteins with GSLs and cholesterol. Deep-etch electron microscopy will be used to look for the characteristic striated coat of caveolae in the plasma membrane and Golgi complex in wild type FRT and in FRT cells expressing caveolin. A video microscopy assay will be used to search for endogenous cross-linking factors that may be necessary for the recruitment of GPI-proteins into caveolae or their equivalent post-TGN vesicles. Cross-linking experiments with bifunctional reagents and mechanical isolation of plasma membrane and Golgi caveolae will be attempted to identify components of the apical sorting machinery that interact with GSL patches and GPI-anchored proteins. We will also compare the polarity of GPI-proteins in epithelial cells and neurons and develop procedures to characterize the surface distribution of free GPI and CSLs in epithelial cells. It is expected that these studies will clarify important aspects of the biology of epithelial cells and neurons.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041771-09
Application #
2608892
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1989-12-01
Project End
1999-05-31
Budget Start
1997-12-01
Budget End
1999-05-31
Support Year
9
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
Weill Medical College of Cornell University
Department
Anatomy/Cell Biology
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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