ZAP-70 Association with T Cell Receptor {zeta} (TCR{zeta}): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation

doi:10.1083/jcb.143.3.613

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© The Rockefeller University Press, 0021-9525/1998//613 $5.00
The Journal of Cell Biology, Volume 143, Number 3, , 1998 613-624

Regular Articles

ZAP-70 Association with T Cell Receptor ${zeta}$ (TCR ${zeta}$ ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation

Joanne Sloan-Lancaster^*, John Presley, Jan Ellenberg, Tetsuo Yamazaki^*, Jennifer Lippincott-Schwartz, and Lawrence E. Samelson^*

^* The Section on Lymphocyte Signaling, The Unit of Organelle Biology, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

The nonreceptor protein tyrosine kinase ZAP-70 is a criticalenzyme required for successful T lymphocyte activation. Afterantigenic stimulation, ZAP-70 rapidly associates with T cellreceptor (TCR) subunits. The kinetics of its translocationto the cell surface, the properties of its specific interactionwith the TCR ${zeta}$ chain expressed as a chimeric protein (TT ${zeta}$ and T ${zeta}$ ${zeta}$ ), and its mobility in different intracellular compartmentswere studied in individual live HeLa cells, using ZAP-70 andT ${zeta}$ ${zeta}$ fused to green fluorescent protein (ZAP-70 GFP and T ${zeta}$ ${zeta}$ –GFP,respectively). Time-lapse imaging using confocal microscopyindicated that the activation-induced redistribution of ZAP-70to the plasma membrane, after a delayed onset, is of long duration.The presence of the TCR ${zeta}$ chain is critical for the redistribution,which is enhanced when an active form of the protein tyrosinekinase Lck is coexpressed. Binding specificity to TT ${zeta}$ was indicatedusing mutant ZAP-70 GFPs and a truncated ${zeta}$ chimera. Photobleachingtechniques revealed that ZAP-70 GFP has decreased mobility atthe plasma membrane, in contrast to its rapid mobility in thecytosol and nucleus. T ${zeta}$ ${zeta}$ – GFP is relatively immobile, whileperipherally located ZAP-70 in stimulated cells is less mobilethan cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examinationof the specific molecular association of signaling proteinsusing these approaches has provided new insights into the TCR ${zeta}$ –ZAP-70interaction and will be a powerful tool for continuing studiesof lymphocyte activation.

Key Words: ZAP-70 • TCR ${zeta}$ • protein tyrosine kinase • intracellular signaling • GFP

Abbreviations used in this paper: FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; ITAM, immunoreceptor tyrosine-based activation motif; MHC, major histocompatibility complex; PV, pervanadate; ROI, region of interest; TCR, T cell antigen receptor; TT ${zeta}$ , Tac Tac zeta; T ${zeta}$ ${zeta}$ , Tac zeta zeta.

J. Sloan-Lancaster is a fellow of the Damon Runyon-Walter Winchell Cancer Research Fund. T. Yamazaki is a fellow of the JapanSociety for the Promotion of Science.

J. Sloan-Lancaster's current address is Division of ResearchTechnologies and Proteins, Eli Lilly and Company, Lilly CorporateCenter, Indianapolis, IN 46285.

Address all correspondence to Lawrence E. Samelson, NICHD, CBMB, Bldg. 18T, Rm 101, Bethesda, MD 20892. Tel.: (301) 496-6368. Fax: (301) 402-0078. E-mail: samelson{at}helix.nih.gov

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