Recognition and binding of peptide-loaded major histocompatibility complex protein (pMHC) by the α and β TCR chains initiates the signal transduction cascade by recruiting Src-family protein tyrosine kinases (PTKs), predominantly Lck, or Fyn, to phosphorylate the immunoreceptor-based tyrosine activation motifs (ITAMs) on the intracellular CD3 and TCRζ subunits of the TCR. T cell activation is mediated by engagement of the TCR, which consists of the α and β chains, the CD3δ, ε, γ, and TCRζ subunits. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation. These delays are regulated by intracellular calcium flux downstream of T cell activation. Kinetic analysis of microcluster assembly reveal surprising delays between the stepwise recruitment of ZAP70 and signaling proteins to the TCR, as well as distinct patterns in their disassociation. Here we show, using TIRF-SIM to examine the organization of microclusters at sub-diffraction resolution, the presence of two spatially distinct domains composed of ZAP70-bound TCR and LAT-associated signaling complex. Whereas microclusters have been studied extensively using confocal microscopy, the spatial and kinetic relationships of their signaling components have not been well characterized due to limits in image resolution and acquisition speed. Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation.

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