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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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Intracellular location of ZAP-70 GFP in stably  transfected P116 T cells, and its  movement to the plasma membrane upon cellular activation.  (a) Nuclear expression of ZAP-70 GFP in P116/2G1 cells is  highlighted by the nucleolar exclusion pattern. Endogenous Lck,  stained with anti-Lck and a  rhodamine-coupled secondary  mAb, is entirely extranuclear in  all cells, and areas of yellow indicate the cytosolic colocalization  of ZAP-70 GFP and Lck. (b and c)  P116/2G1 cells were left untreated (b) or stimulated with  F(ab′)2 of OKT3 for 2 min at 37°C  (c). Arrows and arrowheads indicate peripheral rims and membrane blebs of ZAP-70 GFP, respectively (c).
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Figure 5: Intracellular location of ZAP-70 GFP in stably transfected P116 T cells, and its movement to the plasma membrane upon cellular activation. (a) Nuclear expression of ZAP-70 GFP in P116/2G1 cells is highlighted by the nucleolar exclusion pattern. Endogenous Lck, stained with anti-Lck and a rhodamine-coupled secondary mAb, is entirely extranuclear in all cells, and areas of yellow indicate the cytosolic colocalization of ZAP-70 GFP and Lck. (b and c) P116/2G1 cells were left untreated (b) or stimulated with F(ab′)2 of OKT3 for 2 min at 37°C (c). Arrows and arrowheads indicate peripheral rims and membrane blebs of ZAP-70 GFP, respectively (c).

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)

Intracellular location of ZAP-70 GFP in stably  transfected P116 T cells, and its  movement to the plasma membrane upon cellular activation.  (a) Nuclear expression of ZAP-70 GFP in P116/2G1 cells is  highlighted by the nucleolar exclusion pattern. Endogenous Lck,  stained with anti-Lck and a  rhodamine-coupled secondary  mAb, is entirely extranuclear in  all cells, and areas of yellow indicate the cytosolic colocalization  of ZAP-70 GFP and Lck. (b and c)  P116/2G1 cells were left untreated (b) or stimulated with  F(ab′)2 of OKT3 for 2 min at 37°C  (c). Arrows and arrowheads indicate peripheral rims and membrane blebs of ZAP-70 GFP, respectively (c).
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Related In: Results  -  Collection

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Figure 5: Intracellular location of ZAP-70 GFP in stably transfected P116 T cells, and its movement to the plasma membrane upon cellular activation. (a) Nuclear expression of ZAP-70 GFP in P116/2G1 cells is highlighted by the nucleolar exclusion pattern. Endogenous Lck, stained with anti-Lck and a rhodamine-coupled secondary mAb, is entirely extranuclear in all cells, and areas of yellow indicate the cytosolic colocalization of ZAP-70 GFP and Lck. (b and c) P116/2G1 cells were left untreated (b) or stimulated with F(ab′)2 of OKT3 for 2 min at 37°C (c). Arrows and arrowheads indicate peripheral rims and membrane blebs of ZAP-70 GFP, respectively (c).
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