Lipid raft proteins have a random distribution during localized activation of the T-cell receptor
Lipid raft proteins have a random distribution during localized activation of the T-cell receptor
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DOI：
10.1038/ncb1103
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发表时间：
2004-03-01
影响因子：
21.3
通讯作者：
Nichols, BJ
中科院分区：
文献类型：
--
作者：
Glebov, OO;Nichols, BJ
脂筏蛋白在多大程度上被组织在质膜内的功能簇中，这是关于脂筏结构和功能的争论的中心(1-3)。糖基磷脂酰肌醇(GPI)连接的蛋白质是生物化学定义的RAFT(1，4，5)的特征成分。几项研究报道了RAFT在T细胞刺激中的作用(6-8)，但尚不清楚参与T细胞受体(TCR)信号传递的分子是通过RAFT微域的不对称分布还是通过特定的蛋白质-蛋白质相互作用被招募到T细胞突触(或排除在突触外)(9，10)。在这里，我们在活细胞中使用FRET分析(11)来确定GPI连接的蛋白是否聚集在未受刺激的细胞的质膜中，以及使用抗体包裹的珠子激活TCR信号的区域。多项标准表明，COS-7或未受刺激的Jurkat T细胞中不同GPI连接的荧光蛋白之间的FRET是由随机的、非聚集的分布产生的。Jurkat细胞中TCR信号的刺激导致GPI连接的荧光蛋白和霍乱毒素B亚单位(CTB)的局部荧光增加(12)。然而，FRET和比率成像的测量表明，在这些区域没有可检测到的聚集，也没有GPI连接蛋白或CTB的整体浓缩。
The extent to which lipid raft proteins are organized in functional clusters within the plasma membrane is central to the debate on structure and function of rafts(1-3). Glycosylphosphatidylinositol (GPI)-linked proteins are characteristic components of biochemically defined rafts(1,4,5). Several studies report a function for rafts in T-cell stimulation(6-8), but it is unclear whether molecules involved in T-cell receptor (TCR) signalling are recruited to (or excluded from) T-cell synapses through asymmetric distribution of raft microdomains or through specific protein-protein interactions(9,10). Here we used FRET analysis(11) in live cells to determine whether GPI-linked proteins are clustered in the plasma membrane of unstimulated cells, and at regions where TCR signalling has been activated using antibody-coated beads. Multiple criteria suggested that FRET between different GPI-linked fluorescent proteins in COS-7 or unstimulated Jurkat T-cells is generated by a random, un-clustered distribution. Stimulation of TCR signalling in Jurkat cells resulted in localized increases in fluorescence of GPI-linked fluorescent proteins and cholera toxin B-subunit (CTB)(12). However, measurements of FRET and ratio imaging showed that there was no detectable clustering and no overall enrichment of GPI-linked proteins or CTB in these regions.