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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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Effects of F505 Lck and cellular stimulation on the kinetics of plasma membrane acquisition of ZAP-70 GFP and KD ZAP-70 GFP. Individual live Cos 7 cells were examined by confocal microscopy and plasma membrane acquisition of ZAP-70 GFP assessed as described in Materials and  Methods. A single image of each cell is shown before activation (unstim) with subsequent images shown at 1-min intervals after the addition of PV. Arrows highlight specific areas of plasma membrane accumulation of the ZAP-70 GFP fusion proteins.
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Figure 3: Effects of F505 Lck and cellular stimulation on the kinetics of plasma membrane acquisition of ZAP-70 GFP and KD ZAP-70 GFP. Individual live Cos 7 cells were examined by confocal microscopy and plasma membrane acquisition of ZAP-70 GFP assessed as described in Materials and Methods. A single image of each cell is shown before activation (unstim) with subsequent images shown at 1-min intervals after the addition of PV. Arrows highlight specific areas of plasma membrane accumulation of the ZAP-70 GFP fusion proteins.

Mentions: A representative cell, expressing ZAP-70 GFP alone, was observed before and after stimulation with PV (Fig. 3, top). Consistent with the data acquired using fixed cells, the unstimulated cell displayed a diffuse pattern of ZAP-70 expression, with only a small amount of fluorescence at the plasma membrane (unstim). Upon pharmacologic stimulation, little change in ZAP-70 distribution was apparent during the first minute or so after stimulation (1 min PV  ). However, after 2 min, plasma membrane accumulation of ZAP-70 was evident (2 min PV, arrow highlights area of accumulation). This redistribution to the cell surface steadily continued throughout the 5-min stimulation period (5 min PV  ).


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)

Effects of F505 Lck and cellular stimulation on the kinetics of plasma membrane acquisition of ZAP-70 GFP and KD ZAP-70 GFP. Individual live Cos 7 cells were examined by confocal microscopy and plasma membrane acquisition of ZAP-70 GFP assessed as described in Materials and  Methods. A single image of each cell is shown before activation (unstim) with subsequent images shown at 1-min intervals after the addition of PV. Arrows highlight specific areas of plasma membrane accumulation of the ZAP-70 GFP fusion proteins.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Effects of F505 Lck and cellular stimulation on the kinetics of plasma membrane acquisition of ZAP-70 GFP and KD ZAP-70 GFP. Individual live Cos 7 cells were examined by confocal microscopy and plasma membrane acquisition of ZAP-70 GFP assessed as described in Materials and Methods. A single image of each cell is shown before activation (unstim) with subsequent images shown at 1-min intervals after the addition of PV. Arrows highlight specific areas of plasma membrane accumulation of the ZAP-70 GFP fusion proteins.
Mentions: A representative cell, expressing ZAP-70 GFP alone, was observed before and after stimulation with PV (Fig. 3, top). Consistent with the data acquired using fixed cells, the unstimulated cell displayed a diffuse pattern of ZAP-70 expression, with only a small amount of fluorescence at the plasma membrane (unstim). Upon pharmacologic stimulation, little change in ZAP-70 distribution was apparent during the first minute or so after stimulation (1 min PV  ). However, after 2 min, plasma membrane accumulation of ZAP-70 was evident (2 min PV, arrow highlights area of accumulation). This redistribution to the cell surface steadily continued throughout the 5-min stimulation period (5 min PV  ).

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