Sphingolipids are found in all animal cell membranes and recently have been suggested to play important roles in a wide variety of cellular functions, including cell-cell interactions, cell growth and differentiation, transformation, and signal transduction. In order to better understand the roles of sphingolipids in these complex processes, it is necessary to elucidate mechanisms responsible for generating and maintaining the intracellular distribution of these molecules in cell membranes. In this application, five broad projects pertaining to the molecular mechanisms underlying sphingolipid synthesis, sorting, and transport in animal cells will be pursued: (i) We will study the mechanism of Golgi targeting of ceramide (a key intermediate in sphingolipid biosynthesis) using a novel collection of ceramide analogs in which the chain length of the sphingosine backbone is varied from 6 to 20 carbon atoms and will test several hypotheses for retention of ceramide at this organelle; (ii) We will study plasma membrane lipid organization using fluorescent sphingolipid analogs whose spectral properties allow both qualitative and quantitative assessments of their distribution and concentration within various compartments of the living cell. We will evaluate the ability of these molecules to form """"""""microdomains"""""""" at the plasma membrane and study their potential to cluster in caveolae. We will also examine the internalization and """"""""sorting"""""""" of these sphingolipid analogs into subpopulations of vesicles during the first seconds of endocytosis; (iii) We will carry out detailed molecular studies on glucosylceramide synthase, a key enzyme in glycosphingolipid biosynthesis, which we recently cloned from rat. Specifically, we will study oligomerization of the enzyme in the Golgi, perform site-directed mutagenesis and use deletion mutants to map the catalytic site and possible Golgi retention motifs. We will also study the localization of the enzyme at the EM level; (iv) Using a novel fluorescence screening assay which we recently developed, we will attempt to identify and sequence sphingomyelin synthase using a yeast complementation or rescue approach; and (v) We will study """"""""lipid sorting"""""""" at the Golgi apparatus and further examine the mechanisms of sphingolipid transport from this organelle to the cell surface. The studies proposed in this application are basic to understanding membrane assembly and regulation of membrane lipid composition in cells and are fundamental to the development of rational treatments of membrane or cell surface related disease states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022942-28
Application #
6518932
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Chin, Jean
Project Start
1979-03-01
Project End
2003-02-28
Budget Start
2002-03-01
Budget End
2003-02-28
Support Year
28
Fiscal Year
2002
Total Cost
$398,882
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
Ohmine, Seiga; Singh, Raman Deep; Marks, David L et al. (2013) Viral attachment induces rapid recruitment of an innate immune sensor (TRIM5?) to the plasma membrane. J Innate Immun 5:414-24
Singh, Raman Deep; Schroeder, Andreas S; Scheffer, Luana et al. (2013) Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis. Biochem Biophys Res Commun 434:466-72
Marks, David L; Holicky, Eileen L; Wheatley, Christine L et al. (2012) Role of protein kinase d in Golgi exit and lysosomal targeting of the transmembrane protein, Mcoln1. Traffic 13:565-75
Wang, Shaohua; Singh, Raman Deep; Godin, Lindsay et al. (2011) Endocytic response of type I alveolar epithelial cells to hypertonic stress. Am J Physiol Lung Cell Mol Physiol 300:L560-8
Cheng, Zhi-Jie; Singh, Raman Deep; Wang, Teng-Ke et al. (2010) Stimulation of GLUT4 (glucose transporter isoform 4) storage vesicle formation by sphingolipid depletion. Biochem J 427:143-50
Penheiter, Sumedha G; Singh, Raman Deep; Repellin, Claire E et al. (2010) Type II transforming growth factor-beta receptor recycling is dependent upon the clathrin adaptor protein Dab2. Mol Biol Cell 21:4009-19
Singh, Raman Deep; Marks, David L; Holicky, Eileen L et al. (2010) Gangliosides and beta1-integrin are required for caveolae and membrane domains. Traffic 11:348-60
Watzlawik, J; Holicky, E; Edberg, D D et al. (2010) Human remyelination promoting antibody inhibits apoptotic signaling and differentiation through Lyn kinase in primary rat oligodendrocytes. Glia 58:1782-93
Bachar, Adi R; Scheffer, Lea; Schroeder, Andreas S et al. (2010) Humanin is expressed in human vascular walls and has a cytoprotective effect against oxidized LDL-induced oxidative stress. Cardiovasc Res 88:360-6
Cheng, Zhi-Jie; Singh, Raman Deep; Holicky, Eileen L et al. (2010) Co-regulation of caveolar and Cdc42-dependent fluid phase endocytosis by phosphocaveolin-1. J Biol Chem 285:15119-25

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