Molecular Mechanism of T Cell Signaling

Abstract

T cell activation begins with the formation of signaling complexes at the cell surface involving among others, the T cell antigen receptor (TCR), the Src family kinase Lck and the adaptor protein, linker for activation of T cells (LAT). We have established photo-activatable localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), dual channel PALM/direct STORM (dSTORM), live cell PALM and quantitative statistical cluster analysis to determine how TCR engagement reorganizes the T cell membrane on the molecular scale.In quiescent T cells, LAT is already segregated into clusters on the cell surface raising the question how TCR triggering initiates signaling through these pre-existing domains. We demonstrated that pre-existing LAT domains are neither phosphorylated nor laterally transported to the TCR activation site suggesting that these clusters do not participate in TCR signaling. Instead, TCR activation results in the recruitment, and phosphorylation of new LAT clusters from sub-synaptic vesicles. Our data suggests that TCR ligation preconditions the membrane for vesicle recruitment and bulk activation of the LAT signaling network.We next demonstrated that TCR triggering re-organizes Lck into distinct signaling clusters. Lck clustering, driven by self-association was independent of the kinase activation state but TCR activation segregated Lck clusters from clusters of the phosphatase CD45. In addition, de-phosphorylation of Lck, either at Tyr 394 or Tyr 505, resulted in larger but fewer clusters. Phosphorylated TCR and Lck co-clustered at the centre of the activation area suggesting that molecular re-organization may result in unique TCR signaling domains in the plasma membrane.In conclusion, single-molecule imaging provides insights into T cell signaling that could not have been obtained by any other means.