tailieunhanh - Báo cáo khoa học: Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction

We isolated a cholesterol-enriched membrane subpopulation from the so-called lipid raft fractions of Jurkat T-cells by taking advantage of its selective binding to a cholesterol-binding probe, BCh. The BCh-bound mem-brane subpopulation has a much higher cholesterol⁄phospholipid (C⁄P) molar ratio ( ) than the BCh-unbound population in raft fractions ( ). It contains not only the raft markers GM1 and flotillin, but also some T-cell receptor (TCR) signalling molecules, including Lck, Fyn and LAT. . | iFEBS Journal Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction Yukiko Shimada1 Mitsushi Inomata1 Hidenori Suzuki2 Masami Hayashi1 A. Abdul Waheed1 and Yoshiko Ohno-Iwashita1 1 Biomembrane Research Group Tokyo Metropolitan Institute of Gerontology Itabashi-ku Tokyo Japan 2 Center for Electron Microscopy Tokyo Metropolitan Institute of MedicalScience Bunkyo-ku Tokyo Japan Keywords raft cholesterol T-cellsignalling perfringolysin O Correspondence Y. Shimada Biomembrane Research Group Tokyo Metropolitan Institute of Gerontology 35-2 Sakae-cho Itabashi-ku Tokyo 1730015 Japan Fax 81 3 3579 4776 Tel 81 3 3964 3241 extn 3063 3068 E-mail yshimada@ Received 29 June 2005 revised 15 August 2005 accepted 24 August 2005 doi We isolated a cholesterol-enriched membrane subpopulation from the so-called lipid raft fractions of Jurkat T-cells by taking advantage of its selective binding to a cholesterol-binding probe BC0. The BC0-bound membrane subpopulation has a much higher cholesterol phospholipid C P molar ratio w than the BC0-unbound population in raft fractions w . It contains not only the raft markers GM1 and flotillin but also some T-cell receptor TCR signalling molecules including Lck Fyn and LAT. In addition Csk and PAG inhibitory molecules of the TCR signalling cascade are also contained in the BC0-bound membranes. On the other hand CD3e CD3f and Zap70 are localized in the BC0-unbound membranes segregated from other TCR signalling molecules under nonstimulated conditions. However upon stimulation of TCR portions of CD3e CD3f and Zap70 are recruited to the BC0-bound membranes. The Triton X-100 concentration used for lipid raft preparation affects neither the C P ratio nor protein composition of the BC0-bound membranes. These results show that our method is useful for isolating a particular cholesterol-rich membrane domain of T-cells which could be a core domain controlling the TCR .