O'Donoghue Geoff P, Pielak Rafal M, Smoligovets Alexander A, Lin Jenny J, Groves Jay T
Department of Chemistry , Howard Hughes Medical Institute, University of California, Berkeley , Berkeley , United States ; Physical Biosciences Division , Lawrence Berkeley National Laboratory, University of California, Berkeley , Berkeley , United States.
Elife. 2013 Jul 3;2:e00778. doi: 10.7554/eLife.00778.
T cells discriminate between self and foreign antigenic peptides, displayed on antigen presenting cell surfaces, via the TCR. While the molecular interactions between TCR and its ligands are well characterized in vitro, quantitative measurements of these interactions in living cells are required to accurately resolve the physical mechanisms of TCR signaling. We report direct single molecule measurements of TCR triggering by agonist pMHC in hybrid junctions between live primary T cells and supported lipid membranes. Every pMHC:TCR complex over the entire cell is tracked while simultaneously monitoring the local membrane recruitment of ZAP70, as a readout of TCR triggering. Mean dwell times for pMHC:TCR molecular binding of 5 and 54 s were measured for two different pMHC:TCR systems. Single molecule measurements of the pMHC:TCR:ZAP70 complex indicate that TCR triggering is stoichiometric with agonist pMHC in a 1:1 ratio. Thus any signal amplification must occur downstream of TCR triggering. DOI:http://dx.doi.org/10.7554/eLife.00778.001.
T细胞通过TCR区分呈递在抗原呈递细胞表面的自身和外来抗原肽。虽然TCR与其配体之间的分子相互作用在体外已得到充分表征，但需要对活细胞中的这些相互作用进行定量测量，以准确解析TCR信号传导的物理机制。我们报告了在活的原代T细胞与支持脂质膜之间的杂交连接处，通过激动剂pMHC对TCR触发进行的直接单分子测量。在整个细胞上追踪每个pMHC:TCR复合物，同时监测ZAP70的局部膜募集情况，作为TCR触发的读数。对于两种不同的pMHC:TCR系统，测得pMHC:TCR分子结合的平均停留时间分别为5秒和54秒。pMHC:TCR:ZAP70复合物的单分子测量表明，TCR触发与激动剂pMHC呈化学计量比的1:1关系。因此，任何信号放大都必须发生在TCR触发的下游。DOI:http://dx.doi.org/10.7554/eLife.00778.001
