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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...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
1998
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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. |
format | Text |
id | pubmed-2148150 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 1998 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
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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