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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

Quantitation of nuclear ZAP-70 GFP, and its relationship to  expression levels, in individual cells of stably transfected P116 subclones.  The middle section from a complete Z series of 0.5 μM optical sections  through a field of F4 (a) and H9 (b) subclones is shown. Numbered cells  correlate with the corresponding quantitation analyses reported in (c) for  F4 and (d) for H9. Mean pixel intensity throughout individual cells is reported as a method to accurately compare GFP expression levels between  cells. Percent nuclear ZAP-70 GFP was determined as described in Materials and Methods.
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Figure 6: Quantitation of nuclear ZAP-70 GFP, and its relationship to expression levels, in individual cells of stably transfected P116 subclones. The middle section from a complete Z series of 0.5 μM optical sections through a field of F4 (a) and H9 (b) subclones is shown. Numbered cells correlate with the corresponding quantitation analyses reported in (c) for F4 and (d) for H9. Mean pixel intensity throughout individual cells is reported as a method to accurately compare GFP expression levels between cells. Percent nuclear ZAP-70 GFP was determined as described in Materials and Methods.

Mentions: Transfected Cos 7 cells were grown on sterile glass coverslips (10 or 25 mM diameter, No. 1 thickness). ZAP-70 GFP subclones were adhered to coverslips precoated with poly L-lysine (100 μg/ml; Sigma Chemical Co., St. Louis, MO; see Figs. 5 and 6) for 2 h at 37°C. Cells were stimulated directly on the coverslips (PV × 10 min for Cos 7 or F(ab′)2 of OKT3 × 2 min for 2G1), fixed in 2% formaldehyde in PBS for 15 min at RT, and examined directly or permeabilized and stained with the appropriate antibody. Antibody staining was performed with T cells in suspension in all other experiments, and cells were mounted onto coverslips immediately before microscopy analysis. The cells were fixed using 3.7% paraformaldehyde in PBS for 30 min at RT, washed (three times) in PBS containing 10% fetal bovine serum (PBS/FBS), permeabilized using 0.1% Triton X-100 in PBS for 4 min at RT, washed (three times), and incubated for 45 min in PBS/FBS for preblocking. Cells were then incubated with first stage antibody (anti– ZAP-70, anti-GFP, or anti-Lck) in PBS/FCS for 45 min at RT, washed and incubated with second stage antibody (rhodamine-coupled goat anti–mouse or anti–rabbit IgG or (see Fig. 7) CyTM 3 donkey anti–rabbit IgG) for 45 min, followed by washing with PBS (three times). Cells were resuspended in Fluoromount G added (Southern Biotechnology) and pipetted onto microscope slides with a coverslip mounted on top. Hoechst stain (20 μg/ml) was included during the second antibody incubation (See Fig. 7, c and d). These cells were viewed using a Zeiss Axioskop microscope equipped with both UV and rhodamine optics and cells photographed directly. All other fixed cells were viewed as 0.5 μM dual color optical sections, or composites of a complete Z section analysis in (see Fig. 2) using a Zeiss lasor scanning microscope 410 confocal microscope having a 100 × Zeiss planapo objective (numerical aperature 1.4) and optics for both fluorescein (GFP) and rhodamine (antibody stains). For experiments using live cells, the coverslips were affixed to a Leiden coverslip dish and mounted on a custom-made 37°C stage of the confocal microscope using the 100× objective. The GFP molecule was excited with the 488 line of a krypton-argon laser and imaged using a 515–540-nm bandpass filter. Images were averaged 16 times to improve image quality. Two images of each cell were taken before addition of stimulant, and subsequent images were taken at 20-s intervals thereafter until 5 min after stimulation, with the same cell slice being viewed in each image.


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)

Quantitation of nuclear ZAP-70 GFP, and its relationship to  expression levels, in individual cells of stably transfected P116 subclones.  The middle section from a complete Z series of 0.5 μM optical sections  through a field of F4 (a) and H9 (b) subclones is shown. Numbered cells  correlate with the corresponding quantitation analyses reported in (c) for  F4 and (d) for H9. Mean pixel intensity throughout individual cells is reported as a method to accurately compare GFP expression levels between  cells. Percent nuclear ZAP-70 GFP was determined as described in Materials and Methods.
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Related In: Results  -  Collection

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Figure 6: Quantitation of nuclear ZAP-70 GFP, and its relationship to expression levels, in individual cells of stably transfected P116 subclones. The middle section from a complete Z series of 0.5 μM optical sections through a field of F4 (a) and H9 (b) subclones is shown. Numbered cells correlate with the corresponding quantitation analyses reported in (c) for F4 and (d) for H9. Mean pixel intensity throughout individual cells is reported as a method to accurately compare GFP expression levels between cells. Percent nuclear ZAP-70 GFP was determined as described in Materials and Methods.
Mentions: Transfected Cos 7 cells were grown on sterile glass coverslips (10 or 25 mM diameter, No. 1 thickness). ZAP-70 GFP subclones were adhered to coverslips precoated with poly L-lysine (100 μg/ml; Sigma Chemical Co., St. Louis, MO; see Figs. 5 and 6) for 2 h at 37°C. Cells were stimulated directly on the coverslips (PV × 10 min for Cos 7 or F(ab′)2 of OKT3 × 2 min for 2G1), fixed in 2% formaldehyde in PBS for 15 min at RT, and examined directly or permeabilized and stained with the appropriate antibody. Antibody staining was performed with T cells in suspension in all other experiments, and cells were mounted onto coverslips immediately before microscopy analysis. The cells were fixed using 3.7% paraformaldehyde in PBS for 30 min at RT, washed (three times) in PBS containing 10% fetal bovine serum (PBS/FBS), permeabilized using 0.1% Triton X-100 in PBS for 4 min at RT, washed (three times), and incubated for 45 min in PBS/FBS for preblocking. Cells were then incubated with first stage antibody (anti– ZAP-70, anti-GFP, or anti-Lck) in PBS/FCS for 45 min at RT, washed and incubated with second stage antibody (rhodamine-coupled goat anti–mouse or anti–rabbit IgG or (see Fig. 7) CyTM 3 donkey anti–rabbit IgG) for 45 min, followed by washing with PBS (three times). Cells were resuspended in Fluoromount G added (Southern Biotechnology) and pipetted onto microscope slides with a coverslip mounted on top. Hoechst stain (20 μg/ml) was included during the second antibody incubation (See Fig. 7, c and d). These cells were viewed using a Zeiss Axioskop microscope equipped with both UV and rhodamine optics and cells photographed directly. All other fixed cells were viewed as 0.5 μM dual color optical sections, or composites of a complete Z section analysis in (see Fig. 2) using a Zeiss lasor scanning microscope 410 confocal microscope having a 100 × Zeiss planapo objective (numerical aperature 1.4) and optics for both fluorescein (GFP) and rhodamine (antibody stains). For experiments using live cells, the coverslips were affixed to a Leiden coverslip dish and mounted on a custom-made 37°C stage of the confocal microscope using the 100× objective. The GFP molecule was excited with the 488 line of a krypton-argon laser and imaged using a 515–540-nm bandpass filter. Images were averaged 16 times to improve image quality. Two images of each cell were taken before addition of stimulant, and subsequent images were taken at 20-s intervals thereafter until 5 min after stimulation, with the same cell slice being viewed in each image.

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