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Regulation of ZAP-70 intracellular localization: visualization with the green fluorescent protein.

Sloan-Lancaster J, Zhang W, Presley J, Williams BL, Abraham RT, Lippincott-Schwartz J, Samelson LE - J. Exp. Med. (1997)

Bottom Line: Subsequent studies in T cells confirmed this phenotype.Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells.Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.

ABSTRACT
To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

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Related in: MedlinePlus

Demonstration of inaccessibility of Abs to nuclei. Cos 7 cells,  expressing ZAP-70 GFP, were immunostained with anti-GFP (a–c) or  anti–ZAP-70 (d–f ). ZAP-70 GFP (a and d), Ab signals (b and e) and the  dual-color overlays (c and f ) are shown for a 0.5 μM slice through an individual cell stained with either anti-GFP (a–c) or anti–ZAP-70 (d–f ).
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Figure 8: Demonstration of inaccessibility of Abs to nuclei. Cos 7 cells, expressing ZAP-70 GFP, were immunostained with anti-GFP (a–c) or anti–ZAP-70 (d–f ). ZAP-70 GFP (a and d), Ab signals (b and e) and the dual-color overlays (c and f ) are shown for a 0.5 μM slice through an individual cell stained with either anti-GFP (a–c) or anti–ZAP-70 (d–f ).

Mentions: To formally test this hypothesis, Cos 7 cells expressing ZAP-70 GFP were immunostained with either anti-GFP or anti–ZAP-70 and a rhodamine-coupled secondary Ab. Individual cells were then viewed, using dual-color optics for fluorescein and rhodamine, to compare the location of ZAP-70 using the two detection methods. A complete Z series analysis was performed, and the center slice of a typical cell is shown for cells immunostained for GFP (Fig. 8, a–c) or ZAP-70 (d–f  ). Although GFP indicated a high concentration of the chimera in the nucleus (Fig. 8 a), this was completely unrecognized by the anti-GFP (Fig. 8 b). In contrast, expression of the protein elsewhere in the cell was detected comparably by either method (c, yellow indicates overlay of both signals). Although the anti–ZAP-70 appeared to be somewhat more accessible to the nucleus (e), some exclusion was apparent since the GFP signal was much stronger than the Ab signal (compare d, e, and overlay in f  ). The partial exclusion of anti–ZAP-70 is consistent with the detection of only ∼50% of examined T cells displaying nuclear ZAP-70 using this Ab. Our studies demonstrate two strengths of the ZAP-70 GFP chimeric system. Intracellular locations of proteins, which are overlooked by conventional IF techniques, are efficiently detected. Also, accurate quantitative intracellular distribution measurements can be made.


Regulation of ZAP-70 intracellular localization: visualization with the green fluorescent protein.

Sloan-Lancaster J, Zhang W, Presley J, Williams BL, Abraham RT, Lippincott-Schwartz J, Samelson LE - J. Exp. Med. (1997)

Demonstration of inaccessibility of Abs to nuclei. Cos 7 cells,  expressing ZAP-70 GFP, were immunostained with anti-GFP (a–c) or  anti–ZAP-70 (d–f ). ZAP-70 GFP (a and d), Ab signals (b and e) and the  dual-color overlays (c and f ) are shown for a 0.5 μM slice through an individual cell stained with either anti-GFP (a–c) or anti–ZAP-70 (d–f ).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199132&req=5

Figure 8: Demonstration of inaccessibility of Abs to nuclei. Cos 7 cells, expressing ZAP-70 GFP, were immunostained with anti-GFP (a–c) or anti–ZAP-70 (d–f ). ZAP-70 GFP (a and d), Ab signals (b and e) and the dual-color overlays (c and f ) are shown for a 0.5 μM slice through an individual cell stained with either anti-GFP (a–c) or anti–ZAP-70 (d–f ).
Mentions: To formally test this hypothesis, Cos 7 cells expressing ZAP-70 GFP were immunostained with either anti-GFP or anti–ZAP-70 and a rhodamine-coupled secondary Ab. Individual cells were then viewed, using dual-color optics for fluorescein and rhodamine, to compare the location of ZAP-70 using the two detection methods. A complete Z series analysis was performed, and the center slice of a typical cell is shown for cells immunostained for GFP (Fig. 8, a–c) or ZAP-70 (d–f  ). Although GFP indicated a high concentration of the chimera in the nucleus (Fig. 8 a), this was completely unrecognized by the anti-GFP (Fig. 8 b). In contrast, expression of the protein elsewhere in the cell was detected comparably by either method (c, yellow indicates overlay of both signals). Although the anti–ZAP-70 appeared to be somewhat more accessible to the nucleus (e), some exclusion was apparent since the GFP signal was much stronger than the Ab signal (compare d, e, and overlay in f  ). The partial exclusion of anti–ZAP-70 is consistent with the detection of only ∼50% of examined T cells displaying nuclear ZAP-70 using this Ab. Our studies demonstrate two strengths of the ZAP-70 GFP chimeric system. Intracellular locations of proteins, which are overlooked by conventional IF techniques, are efficiently detected. Also, accurate quantitative intracellular distribution measurements can be made.

Bottom Line: Subsequent studies in T cells confirmed this phenotype.Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells.Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.

ABSTRACT
To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

Show MeSH
Related in: MedlinePlus