Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor
Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor
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DOI：
10.1042/0264-6021:3630645
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发表时间：
2002-05-01
影响因子：
4.1
通讯作者：
Aussel, C
中科院分区：
文献类型：
--
作者：
Rouquette-Jazdanian, AK;Pelassy, C;Aussel, C
细胞膜含有鞘脂和胆固醇，它们聚集在称为筏的不同区域中。这些特殊的膜结构域的外膜小叶含有糖基磷脂酰肌醇锚定蛋白，而内小叶含有与信号传导有关的蛋白质，如酰化蛋白激酶p56（lck）和棕榈酰化衔接子LAT（T细胞活化接头）。我们在这里提出了一种方法来研究筏的脂质组成和T细胞活化后的变化。我们的方法是基于Jurkat T细胞与甘油磷脂合成的不同前体的代谢标记。包括甘油和具有不同长度和饱和度的脂肪酸以及磷脂极性头基。所获得的结果表明，通过使用Triton X-100提取后，在寒冷的蔗糖密度梯度离心分离的脂筏，除了鞘脂和胆固醇，含有明确所有类别的甘油磷脂：磷脂酰丝氨酸，磷脂酰肌醇，磷脂酰乙醇胺和磷脂酰胆碱。脂肪酸标记表明，脂筏优先标记饱和脂肪酸，而其余的质膜主要是长链多不饱和脂肪酸。通过磷脂的代谢标记来观察筏组成是否参与T细胞活化。我们研究了在CD 3活化的细胞中sn-1，2-二酰基甘油（DAG）的产生。DAG的生产发生在筏，证实了以前的证明蛋白激酶C易位到膜微区。我们的数据表明，筏解体的甲基-β-环糊精损害CD 3诱导的DAG生产和细胞内Ca 2+浓度的变化。这些证据支持T细胞活化的主要事件发生在脂筏内或由于脂筏的结论。
Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outermembrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56(lck) and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis. including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation. we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-beta-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca2+ concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts.