TCR microclusters form spatially segregated domains and sequentially assemble in calcium-dependent kinetic steps

Jason Yi,Katherine M Mcintire,Tiffany Nguyen,Lakshmi Balagopalan,Lawrence E Samelson

doi:10.1038/s41467-018-08064-2

Jason Yi, Katherine M Mcintire + Show 3 more

Open Access

https://doi.org/10.1038/s41467-018-08064-2

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Abstract

Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation. 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. 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. 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. These delays are regulated by intracellular calcium flux downstream of T cell activation. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation.

Highlights

Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation
Jurkat T cells expressing TCRζ-Halo conjugated to Janelia Fluor 646 ligand (JF646) and ZAP70-Emerald were activated on anti-CD3ε-coated glass and imaged using TIRF-SIM
TCRζ-Halo and ZAP70-Emerald signals co-localized at each microcluster (Fig. 1a) as expected from the known binding of ZAP70 to TCRζ immunoreceptorbased tyrosine activation motifs (ITAMs) residues

Summary

Introduction

Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites 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 These delays are regulated by intracellular calcium flux downstream of T cell activation. T cells display remarkable sensitivity to antigen despite the relatively weak affinity of TCRs for pMHCs and low numbers of stimulatory ligand on the antigen presenting cell (APC) surface[3,4]. This sensitivity is thought to be, in part, the result of signal amplification from the transiently engaged TCRs through a multi-protein structure at the membrane called the TCR microcluster[5]. The dynamic interaction between TCR microclusters, actin cytoskeleton, and adhesion molecules leads to the formation of an immunological synapse between the T cell and APC to facilitate lysis of target cells, directed cytokine secretion, and other effector functions[3,8,9]

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