Tài liệu Báo cáo khoa học: Separation of a cholesterol-enriched microdomain involved in T-cell

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 Medical Science, Bunkyo-ku, Tokyo, Japan

Cholesterol is one of the major constituents of the

plasma membrane, and is involved in the formation

of the membrane bilayer. The distribution of choles￾terol in the plasma membrane is not uniform, sug￾gesting that cholesterol is also involved in the

construction of functional membrane domains. One

such functional membrane domain is called lipid

rafts [1,2]. Lipid rafts are lateral lipid clusters formed

of sphingolipids and cholesterol, in which particular

molecules are concentrated to form platforms for

intracellular transport and signal transduction. Cho￾lesterol depletion reduces the association of these

molecules with lipid rafts [3,4], indicating that choles￾terol is necessary for the partitioning of these partic￾ular molecules into functional domains in the plasma

membrane.

Several reports have suggested that lipid rafts are

platforms for signal transduction in T-cells [5]. Lipid

rafts obtained from resting T-cells are enriched in Src￾family kinases, Lck and Fyn [6,7], and the linker for

the activation of T-cells (LAT) [8]. Minor amounts of

CD3f are associated with rafts, but the data concern￾ing other T-cell receptor (TCR)⁄ CD3 constituents

remains contradictory [6,7]. In addition, the partition￾ing or recruitment of CD3e to lipid rafts after TCR

stimulation with antibodies remains uncertain [9,10].

These inconsistent results might be due mainly to the

different methods for isolating lipid rafts. Currently,

Keywords

raft; cholesterol; T-cell signalling;

perfringolysin O

Correspondence

Y. Shimada, Biomembrane Research Group,

Tokyo Metropolitan Institute of Gerontology,

35-2 Sakae-cho, Itabashi-ku, Tokyo 173-

0015, Japan

Fax: +81 3 3579 4776

Tel: +81 3 3964 3241 extn 3063, 3068

E-mail: [email protected]

(Received 29 June 2005, revised 15 August

2005, accepted 24 August 2005)

doi:10.1111/j.1742-4658.2005.04938.x

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 (
1.0) than the BCh-unbound population in raft fractions

(
0.3). 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 BCh-bound membranes. On the other

hand, CD3e, CD3f and Zap70 are localized in the BCh-unbound mem￾branes, segregated from other TCR signalling molecules under nonstimulat￾ed conditions. However, upon stimulation of TCR, portions of CD3e, CD3f

and Zap70 are recruited to the BCh-bound membranes. The Triton X-100

concentration used for lipid raft preparation affects neither the C ⁄ P ratio

nor protein composition of the BCh-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 signalling cascade.

Abbreviations

C ⁄ P, cholesterol ⁄ phospholipid molar ratio; DRM, detergent-resistant membrane; LAT, linker for activation of T-cells; PAG, phosphoprotein

associated with glycosphingolipid-enriched membrane microdomains; PC, phosphatidylcholine; PE, phosphatidylethanolamine;

PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; TCR, T-cell antigen receptor.

5454 FEBS Journal 272 (2005) 5454–5463 ª 2005 FEBS