J. Cell Biol., Volume 143, Number 3, November 2, 1998 613-624

ZAP-70 Association with T Cell Receptor  (TCR): 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 critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCR chain expressed as a chimeric protein (TT and T), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and T fused to green fluorescent protein (ZAP-70 GFP and T-GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCR chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TT was indicated using mutant ZAP-70 GFPs and a truncated  chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. T- GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCR-ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.

ZAP-70;  TCR;  protein tyrosine kinase;  intracellular signaling;  GFP

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