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Published online 23 September 2002. doi:10.1083/jcb.200203043
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© The Rockefeller University Press, 0021-9525/2002/9/1263 $5.00
The Journal of Cell Biology, Volume 158, Number 7, September 30, 2002 1263-1275

Article

 T cell receptor ligation induces the formation of dynamically regulated signaling assemblies

Stephen C. Bunnell1, David I. Hong1, Julia R. Kardon1, Tetsuo Yamazaki1, C. Jane McGlade2, Valarie A. Barr1 and Lawrence E. Samelson1

1 Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
2 Department of Medical Biophysics and Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada

Address correspondence to Lawrence Samelson, Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg. 37, Rm. 1E24, Bethesda, MD 20892. Tel.: (301) 496-9683. Fax: (301) 496-8479. E-mail: samelson{at}helix.nih.gov

Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein–GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70–containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.

Key Words: T cell receptor; adapters; signaling complexes; tyrosine phosphorylation; confocal microscopy


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