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ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation

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 inter...

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Autores principales: Sloan-Lancaster, Joanne, Presley, John, Ellenberg, Jan, Yamazaki, Tetsuo, Lippincott-Schwartz, Jennifer, Samelson, Lawrence E.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1998
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2148150/
https://www.ncbi.nlm.nih.gov/pubmed/9813084
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author Sloan-Lancaster, Joanne
Presley, John
Ellenberg, Jan
Yamazaki, Tetsuo
Lippincott-Schwartz, Jennifer
Samelson, Lawrence E.
author_facet Sloan-Lancaster, Joanne
Presley, John
Ellenberg, Jan
Yamazaki, Tetsuo
Lippincott-Schwartz, Jennifer
Samelson, Lawrence E.
author_sort Sloan-Lancaster, Joanne
collection PubMed
description 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 TCRζ chain expressed as a chimeric protein (TTζ and Tζζ), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tζζ fused to green fluorescent protein (ZAP-70 GFP and Tζζ–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 TCRζ chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTζ was indicated using mutant ZAP-70 GFPs and a truncated ζ 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. Tζζ– 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 TCRζ–ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.
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spelling pubmed-21481502008-05-01 ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation Sloan-Lancaster, Joanne Presley, John Ellenberg, Jan Yamazaki, Tetsuo Lippincott-Schwartz, Jennifer Samelson, Lawrence E. J Cell Biol Regular Articles 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 TCRζ chain expressed as a chimeric protein (TTζ and Tζζ), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tζζ fused to green fluorescent protein (ZAP-70 GFP and Tζζ–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 TCRζ chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTζ was indicated using mutant ZAP-70 GFPs and a truncated ζ 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. Tζζ– 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 TCRζ–ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation. The Rockefeller University Press 1998-11-02 /pmc/articles/PMC2148150/ /pubmed/9813084 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Regular Articles
Sloan-Lancaster, Joanne
Presley, John
Ellenberg, Jan
Yamazaki, Tetsuo
Lippincott-Schwartz, Jennifer
Samelson, Lawrence E.
ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation
title ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation
title_full ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation
title_fullStr ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation
title_full_unstemmed ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation
title_short ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation
title_sort zap-70 association with t cell receptor ζ (tcrζ): fluorescence imaging of dynamic changes upon cellular stimulation
topic Regular Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2148150/
https://www.ncbi.nlm.nih.gov/pubmed/9813084
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