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Sci. Signal., 11 May 2010
Vol. 3, Issue 121, p. ra36
[DOI: 10.1126/scisignal.2000645]

RESEARCH ARTICLES

Dynamics of Subsynaptic Vesicles and Surface Microclusters at the Immunological Synapse

Marco A. Purbhoo1, Hebin Liu2, Stephane Oddos3,4, Dylan M. Owen4, Mark A. A. Neil4, Sophie V. Pageon3, Paul M. W. French4, Christopher E. Rudd2,5, and Daniel M. Davis3*

1 Section of Hepatology and Gastroenterology, Department of Medicine, Medical School Building, Imperial College London, London W2 1PG, UK.
2 Cell Signaling Section, Division of Immunology, Department of Pathology, Tennis Court Road, University of Cambridge, Cambridge CB2 1QP, UK.
3 Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK.
4 Department of Physics, Imperial College London, London SW7 2AZ, UK.
5 Cambridge Institute for Medical Research, Addenbrookes Hospital, Hills Road, Cambridge CB2 0XY, UK.

Abstract: Imaging studies have identified clusters of kinases and adaptor proteins that serve as centers of signaling at the contact points between T cells and antigen-presenting cells (APCs). Here, we report that the kinase ZAP-70 and the adaptor proteins LAT and SLP-76 accumulated in separate clusters at the interface between T cells and coverslips coated with a stimulatory antibody against CD3, a component of the T cell antigen receptor complex. A fraction of LAT was detected in motile vesicles that repeatedly moved to surface microclusters of SLP-76 and the adaptor protein GADS (growth factor receptor–bound protein–related adaptor downstream of Shc), where they exhibited decreased motility. LAT molecules in which the residues tyrosine 171 and tyrosine 191 (which are required for the binding of LAT to GADS) were mutated to phenylalanine did not dwell at clusters of SLP-76. At immunological synapses, LAT-containing vesicles also colocalized with microclusters of SLP-76, as detected in experiments in which laser tweezers were used to position T cell–APC conjugates vertically for high-resolution imaging. Phosphorylation of LAT was most prominent when vesicular LAT colocalized with SLP-76. Indeed, the abundance of phosphorylated LAT within a microcluster of SLP-76 was greatest in those clusters that had more recent interactions with LAT-containing vesicles. Finally, negative signals by the inhibitory receptor ILT2 disrupted the assembly of SLP-76–containing microclusters. Together, these data show that the movement of LAT-containing vesicles is linked to the organization of protein microclusters and suggest an important role for vesicular LAT in the SLP-76 signalosome.

* To whom correspondence should be addressed. E-mail: d.davis{at}imperial.ac.uk

Citation: M. A. Purbhoo, H. Liu, S. Oddos, D. M. Owen, M. A. A. Neil, S. V. Pageon, P. M. W. French, C. E. Rudd, D. M. Davis, Dynamics of Subsynaptic Vesicles and Surface Microclusters at the Immunological Synapse. Sci. Signal. 3, ra36 (2010).

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