Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events

Abstract

Engaged T cell antigen receptors (TCRs) initiate signaling through the adaptor protein Lat. In quiescent T cells, Lat is segregated into clusters on the cell surface, which raises the question of how TCR triggering initiates signaling. Using super-resolution fluorescence microscopy, we found that pre-existing Lat domains were neither phosphorylated nor laterally transported to TCR activation sites, which suggested that these clusters do not participate in TCR signaling. Instead, TCR activation resulted in the recruitment and phosphorylation of Lat from subsynaptic vesicles. Studies of Lat mutants confirmed that recruitment preceded and was essential for phosphorylation and that both processes were independent of surface clustering of Lat. Our data suggest that TCR ligation preconditions the membrane for vesicle recruitment and bulk activation of the Lat signaling network.

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Figure 1: Mapping of single Lat molecules in resting and activated T cells.
Figure 2: Quantitative statistical analysis of Lat clustering in resting and activated T cells.
Figure 3: Clustering of endogenous Lat and phosphorylated Lat in activated wild-type Jurkat cells and primary mouse T cells.
Figure 4: Localized activation of T cells on micro-patterned surfaces.
Figure 5: Live-cell PALM imaging of Lat in activated T cells.
Figure 6: Crosslinking of Lat at the cell surface.
Figure 7: Cluster analysis of Lat mutants in resting and activated T cells.

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Acknowledgements

Supported by the National Health and Medical Research Council of Australia (D.J.W., J.J.G. and K.G.), the Australian Research Council (D.M.O., J.J.G. and K.G.) and Human Frontier Science Program (K.G.).

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Contributions

D.J.W., molecular biology, PALM, crosslinking experiments and analysis, and manuscript preparation; D.M.O., conceptualization and PALM-STORM analysis; J.R., STORM experiments and analysis; A.M., PALM experiments and analysis of surface-patterning experiments; M.W., surface-patterning experiments; J.J.G., conceptualization of surface patterning; and K.G., conceptualization and manuscript preparation.

Corresponding author

Correspondence to Katharina Gaus.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Methods (PDF 1612 kb)

41590_2011_BFni2049_MOESM20_ESM.avi

Lat cluster map movie. (AVI 7297 kb)

Supplementary Video 1

Lat cluster map movie. (AVI 7297 kb)

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Williamson, D., Owen, D., Rossy, J. et al. Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat Immunol 12, 655–662 (2011). https://doi.org/10.1038/ni.2049

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