Sensitive detection of GM1 lipid rafts and TCR partitioning in the T cell membrane S. Thomas, R.S. Kumar, S. Casares, T.-D. Brumeanu * Department of Microbiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA Received 24 July 2002; received in revised form 9 January 2003; accepted 20 January 2003 Abstract 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-q mAb, the TCR was found mainly in the detergent- soluble fraction of the T cell membrane. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Glycosphingolipid GM1; CD3-mediated TCR cross-linking; GM1 and TCR distribution; ELISA 1. Introduction The plasma membrane contains a lipid bilayer made of diffuse patches of cholesterol and sphingoli- pid microdomains, which refers to the detergent- insoluble glycolipid-enriched complexes (DIGs), gly- cosphingolipid-enriched membranes (GEM), deter- gent-resistant membranes (DRMs), or lipid rafts (Simons and Ikonen, 1997). The lipid rafts play an important role in membrane trafficking and signal transduction, by means of reorganizing their compo- sition in protein receptors with signaling ability (Harder and Simons, 1997). During the formation of immunological synapse following TCR engagement by MHC–peptide complexes on the surface of APCs, the lipid rafts in T cells recruit various protein recep- tors like CD2, CD3, CD4, CD28, and TCR (Ohtani et al., 2000) together with protein tyrosine kinases, i.e., p56lck, p59fyn, PI-3K (Hope and Pike, 1996; Simons and Ikonen, 1997; Zhang et al., 1998), and adaptor molecules like LAT and Grb2 (Montixi et al., 1998, 0022-1759/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-1759(03)00014-0 Abbreviations: BSA, bovine serum albumin; CTB – HRP, cholera toxin subunit B conjugated with horse radish peroxidase; CD, cluster of differentiation; CLSM, confocal laser scanning microscopy; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; IP, immunoprecipitation; IDB, immunodot blot; mAb, monoclonal antibody; MHC, major histocompatibility com- plex; MWCO, molecular weight cutoff; PBS, phosphate-buffered saline; PVDF, polyvinylidene fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TcH, T cell hybridoma; TCR, T cell receptor; WB, Western blot. * Corresponding author. Tel.: +1-212-241-7551; fax: +1-212- 828-4151. E-mail address: teodor.brumeanu@mssm.edu (T.-D. Brumeanu). www.elsevier.com/locate/jim Journal of Immunological Methods 275 (2003) 161 – 168