ZAP-70 ASSOCIATION WITH T-CELL RECEPTOR ZETA (TCR-ZETA) - FLUORESCENCE IMAGING OF DYNAMIC CHANGES UPON CELLULAR-STIMULATION

The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme re quired for successful T lymphocyte activation. After antigenic stimula tion, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. T he kinetics of its translocation to the cell surface, the properties o f its specific interaction with the TCR zeta chain expressed as a chim eric protein (TT zeta and T zeta zeta), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and T zeta zeta fused to green fluorescent protein (ZAP- 70 GFP and T zeta zeta-GFP, respectively). Time-lapse imaging using co nfocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long d uration. The presence of the TCR zeta chain is critical for the redist ribution, which is enhanced when an active form of the protein tyrosin e kinase Lck is coexpressed. Binding specificity to TT zeta was indica ted using mutant ZAP-70 GFPs and a truncated zeta chimera. Photobleach ing 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 zeta zeta-GFP is relatively immobile, while peripherally lo cated ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respec tive diffusion constants. Examination of the specific molecular associ ation of signaling proteins using these approaches has provided new in sights into the TCR zeta-ZAP-70 interaction and will be a powerful too l for continuing studies of lymphocyte activation.