Nuffield Department of Clinical Medicine and Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom.
J Biol Chem. 2011 Sep 16;286(37):31993-2001. doi: 10.1074/jbc.M111.219212. Epub 2011 Jul 13.
Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.
了解 T 细胞抗原受体 (TCR) 触发装置的组成化学计量对于构建信号起始的现实模型至关重要。最近的研究表明，TCR 和其他信号相关蛋白在静止 T 细胞上预先聚集，这依赖于测量与功能化玻璃表面相互作用的膜蛋白的行为。我们使用光漂白后荧光恢复法表明，与顶端表面相比，Jurkat T 细胞/玻璃界面处 TCR 的流动性以信号敏感的方式显著降低。使用两种减轻这些影响的生物物理方法，生物发光共振能量转移和双色符合检测显微镜，我们表明，在方法的不确定性范围内，TCR 触发装置的膜成分，即 TCR 复合物、MHC 分子、CD4/Lck 和 CD45，在人 T 细胞系中均为单价或单体，这意味着 TCR 触发仅取决于 TCR/pMHC 相互作用的动力学。这些分析还表明，将蛋白质约束在细胞表面的二维空间中会极大地增强随机相互作用，而不是膜与细胞质之间的相互作用。基于这些发现的 TCR-pMHC 复合物形成模拟表明，未聚集的 TCR 触发相关蛋白如何能够通过将细胞质效应物差异募集到细胞膜上来产生复杂的信号输出。
