| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
We prepared a non-cytolytic derivative of perfringolysin O (θ-toxin), a cholesterol-binding cytolysin, as a probe for cholesterol, and demonstrated that the probe (BCθ) binds selectively to cholesterol in cholesterol-rich membrane domains of intact cells. Based on the finding that BCθ binds to a subpopulation of membranes among those recovered in detergent-insoluble, low-density fractions (raft fractions),' we isolated BCθ-bound membranes from the raft fractions of Jurkat T cells. The BCθ-bound membrane subpopulation has a much higher cholesterol/phospholipid (C/P) molar ratio (〜1.0) than the BCθ-unbound population in raft fractions (〜0.3). It contains not only the raft markers GM1 and flotillin, but also some T-cell receptor (TCR) signaling molecules, including Lck, Fyn and LAT. In addition, Csk and PAG, inhibitory molecules of the TCR signaling cascade, are also contained in the BCθ-bound membranes. On the other hand, CD3 and Zap70 are localized in the BCθ-unbound membranes, segregated from other TCR signaling molecules under non-stimulated conditions. However, upon stimulation of TCR, portions of CD3 and Zap70 are recruited to the BCθ-bound membranes. The Triton X-100 concentration used for lipid raft preparation affects neither the C/P ratio nor protein composition of the BCθ-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 signaling cascade.
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