We will examine the distribution and transport of lipids in polarized cells (MDCK, Caco-2, WIF12-1, and hepatocytes). The major goals of this project are (i) to study the movement of lipids along the secretory pathway. Fluorescent ceramide analogs will be used to examine the synthesis, sorting, and transport of newly-synthesized fluorescent sphingolipids in these different cell types. Using the perfused liver system, we will determine whether lipids destined for the apical surface of hepatocytes are first transported to the basolateral surface, as is the case for apically- directed proteins; (ii) to study the effects of cellular sphingolipids and cholesterol on the sorting and transport of lipids along the secretory pathway. Cellular cholesterol and endogenous sphingolipids will be modulated, respectively, by altering growth conditions and by use of an inhibitor of glycosphingolipid synthesis. Since the Golgi apparatus is the major site of sphingolipid biosynthesis and cholesterol also accumulates there, these perturbations may have important effects on lipid and protein (Project 3) sorting and transport through this organelle; (iii) to study processing of lipids along the endocytic pathway. Using two different fluorescent analogs of sphingomyelin, we will study the internalization of lipids from the apical and basolateral membrane surfaces of cells, monitor the intermixing of membrane lipids among populations of endosomes derived from the different plasma membrane domains, and study the process of transcytosis; (iv) to determine the lipid composition of several different intracellular membrane systems including the Golgi apparatus. The lipid composition of Golgi membrane subfractions obtained by immunoisolation (Project 4) will be determined by classical methods. We will also attempt to develop an in situ method for determining lipid composition, using fluorescent lipid analogs and the photoactivatable probe, 5- [125I]iodonaphthyl-1-azide. If successful, this methodology will allow us to determine the lipid composition of endosomes and the plasma membrane; and (v) to study the uptake and metabolism of a fluorescent analog of phosphatidylinositol introduced into the apical or basolateral surfaces of polarized cells. The studies proposed in this application are basic to understanding membrane assembly and regulation of membrane lipid composition in polarized cells, and are fundamental to the future development of rational treatments of membrane or cell surface-related disease states.

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Suzuki, Kenichi G N; Fujiwara, Takahiro K; Edidin, Michael et al. (2007) Dynamic recruitment of phospholipase C gamma at transiently immobilized GPI-anchored receptor clusters induces IP3-Ca2+ signaling: single-molecule tracking study 2. J Cell Biol 177:731-42
Suzuki, Kenichi G N; Fujiwara, Takahiro K; Sanematsu, Fumiyuki et al. (2007) GPI-anchored receptor clusters transiently recruit Lyn and G alpha for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1. J Cell Biol 177:717-30
Brown, Christa L; Maier, Kerstin C; Stauber, Tobias et al. (2005) Kinesin-2 is a motor for late endosomes and lysosomes. Traffic 6:1114-24
Berezuk, Matthew A; Schroer, Trina A (2004) Fractionation and characterization of kinesin II species in vertebrate brain. Traffic 5:503-13
Nyasae, Lydia K; Hubbard, Ann L; Tuma, Pamela L (2003) Transcytotic efflux from early endosomes is dependent on cholesterol and glycosphingolipids in polarized hepatic cells. Mol Biol Cell 14:2689-705
Tuma, Pamela L; Hubbard, Ann L (2003) Transcytosis: crossing cellular barriers. Physiol Rev 83:871-932
King, Stephen J; Brown, Christa L; Maier, Kerstin C et al. (2003) Analysis of the dynein-dynactin interaction in vitro and in vivo. Mol Biol Cell 14:5089-97
Bastaki, M; Braiterman, L T; Johns, D C et al. (2002) Absence of direct delivery for single transmembrane apical proteins or their ""Secretory"" forms in polarized hepatic cells. Mol Biol Cell 13:225-37
Tuma, Pamela L; Nyasae, Lydia K; Hubbard, Ann L (2002) Nonpolarized cells selectively sort apical proteins from cell surface to a novel compartment, but lack apical retention mechanisms. Mol Biol Cell 13:3400-15
Quintyne, Nicholas J; Schroer, Trina A (2002) Distinct cell cycle-dependent roles for dynactin and dynein at centrosomes. J Cell Biol 159:245-54

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