Glebov Oleg O, Nichols Benjamin J
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK.
Nat Cell Biol. 2004 Mar;6(3):238-43. doi: 10.1038/ncb1103. Epub 2004 Feb 8.
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. Glycosylphosphatidylinositol (GPI)-linked proteins are characteristic components of biochemically defined rafts. Several studies report a function for rafts in T-cell stimulation, 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. Here we used FRET analysis 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). 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.
脂筏蛋白在质膜内功能性簇中的组织程度是关于脂筏结构与功能争论的核心。糖基磷脂酰肌醇（GPI）连接蛋白是生化定义脂筏的特征性成分。多项研究报道了脂筏在T细胞刺激中的作用，但尚不清楚参与T细胞受体（TCR）信号传导的分子是通过脂筏微区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（或被排除在外）。在这里，我们在活细胞中使用荧光共振能量转移（FRET）分析来确定GPI连接蛋白在未刺激细胞的质膜中以及在使用抗体包被珠子激活TCR信号的区域是否聚集。多个标准表明，COS - 7细胞或未刺激的Jurkat T细胞中不同GPI连接荧光蛋白之间的FRET是由随机的、未聚集的分布产生的。Jurkat细胞中TCR信号的刺激导致GPI连接荧光蛋白和霍乱毒素B亚基（CTB）的荧光局部增加。然而，FRET测量和比率成像表明，在这些区域没有可检测到的聚集，并且GPI连接蛋白或CTB没有整体富集。
