Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells
Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells
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DOI：
10.1073/pnas.0700411104
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发表时间：
2007-11-06
影响因子：
11.1
通讯作者：
Klenerman, David
中科院分区：
文献类型：
--
作者：
James, John R.;White, Samuel S.;Klenerman, David
在大多数T细胞上表达的T细胞受体（TCR）是由结合抗原的TCR α β异二聚体和启动信号传导的分化簇（CD）3 β δ、β γ和ζ ζ二聚体组成的蛋白质复合物。一个长期存在的争议涉及是否有一个，或一个以上，α β异二聚体每个复合物。我们使用了一种形式的单分子光谱学来研究这个问题的活T细胞杂交瘤。该方法依赖于检测来自用两种不同荧光团标记的单个分子的一致荧光，因为分子扩散通过共聚焦体积。在统计背景之上重合的事件的分数被定义为“关联商”Q。在对照实验中，与用Alexa488和Alexa647双标记的麦胚凝集素孵育的细胞相比，与用单标记的麦胚凝集素孵育的细胞相比，Q显著更高。类似地，当与Alexa488和Alexa647标记的抗体Fab片段的混合物一起孵育时，表达同二聚体CD28的细胞比表达单体CD86的细胞给出更大的Q值。与用每种荧光团标记的抗TCR β Fab片段的混合物孵育的T细胞杂交瘤给出与CID86的Q值不可区分的Q值，表明TCR的主要形式包含单个α β异二聚体。CD86和TCR的Q值较低，但不为零，这表明T细胞表面存在瞬时或非随机限制，或分子扩散聚集。这种分析蛋白质复合物亚基组成的通用方法可以扩展到其他细胞表面或细胞内复合物以及其他活细胞。
The T cell receptor (TCR) expressed on most T cells is a protein complex consisting of TCR alpha beta heterodimers that bind antigen and cluster of differentiation (CD) 3 epsilon delta, epsilon gamma, and zeta zeta dimers that initiate signaling. A long-standing controversy concerns whether there is one, or more than one, alpha beta heterodimer per complex. We used a form of single-molecule spectroscopy to investigate this question on live T cell hybridomas. The method relies on detecting coincident fluorescence from single molecules labeled with two different fluorophores, as the molecules diffuse through a confocal volume. The fraction of events that are coincident above the statistical background is defined as the "association quotient," Q. In control experiments, Q was significantly higher for cells incubated with wheat germ agglutinin dual-labeled with Alexa488 and Alexa647 than for cells incubated with singly labeled wheat germ agglutinin. Similarly, cells expressing the homodimer, CD28, gave larger values of Q than cells expressing the monomer, CD86, when incubated with mixtures of Alexa488- and Alexa647-labeled antibody Fab fragments. T cell hybridomas incubated with mixtures of anti-TCR beta Fab fragments labeled with each fluorophore gave a Q value indistinguishable from the Q value for CID86, indicating that the dominant form of the TCR comprises single alpha beta heterodimers. The values of Q obtained for CD86 and the TCR were low but nonzero, suggesting that there is transient or nonrandom confinement, or diffuse clustering of molecules at the T cell surface. This general method for analyzing the subunit composition of protein complexes could be extended to other cell surface or intracellular complexes, and other living cells.