Sensitive detection of GM1 lipid rafts and TCR partitioning in the T cell membrane.
The cholesterol-rich lipid rafts on T cell membrane play important role in the formation of T cell receptor (TCR) signalosome upon receptor ligation. Analytical studies on the kinetics of lipid rafts formation and recruitment of protein receptors to lipid rafts are still limited by the use of a large number of cells. Herein, we describe a strategy for detecting fine alterations in the amount and distribution of glycosphingolipid (GM1) lipid rafts, and in the formation of GM1-TCR complexes in detergent-insoluble and -soluble compartments of the T cell membrane from a relative low number of cells. Using this strategy, we found that the GM1 moiety was physically associated with TCR in both detergent-insoluble and -soluble fractions. Shortly after ligation of CD3/TCR complex with a soluble CD3- epsilon mAb, the TCR was found mainly in the detergent-soluble fraction of the T cell membrane.
Thomas
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Department of Microbiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA.
Kumar
R S
RS
Casares
S
S
Brumeanu
T-D
TD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017252:CD3 Complex / Q000378:metabolism; D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D003902:Detergents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry; D005677:G(M1) Ganglioside / Q000378:metabolism*; D015151:Immunoblotting; D066298:In Vitro Techniques; D021962:Membrane Microdomains / Q000276:immunology* / Q000378:metabolism*; D018613:Microscopy, Confocal; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D003432:Cross-Linking Reagents; D003902:Detergents; D011948:Receptors, Antigen, T-Cell; D005677:G(M1) Ganglioside