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An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells.

Ghosh RN, Mallet WG, Soe TT, McGraw TE, Maxfield FR - J. Cell Biol. (1998)

Bottom Line: When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min.Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes.TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.

ABSTRACT
To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and 125I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38's postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.

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TacTGN38 overlaps with Tf in the ERC, as shown by  HRP ablation. Cells were incubated with 5 μg/ml Cy3-Tf for 30  min, and were then chased for 10 min with 50 μg/ml HRP-Tf with  (a) or without (b) excess (5 mg/ml) unlabeled Tf. The cells were  then treated with hydrogen peroxide and DAB. The reaction  products of the DAB reaction quenched fluorescence from molecules contained in the same compartment as the HRP-Tf (b), and  the unlabeled Tf competed with HRP-Tf for binding to the TR  and prevented quenching of the Cy3-Tf fluorescence (a). The cell  boundaries, determined by DIC microscopy, are shown in b.  Cells were also incubated with 1 μg/ml Cy3 anti-Tac IgG for 10  min, and were then chased for 10 min with 50 μg/ml HRP-Tf with  (c) or without (d) 5 mg/ml unlabeled Tf. The DAB reaction with  the HRP-Tf in the ERC quenched the Cy3-IgG fluorescence in  this compartment (d), leaving a dark area (arrows) surrounded  by the ring staining of the Cy3-IgG characteristic of the TGN.  The fluorescence images show a summation projection of slices in  a 3-D stack obtained by confocal microscopy. Bar, 10 μm.
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Figure 5: TacTGN38 overlaps with Tf in the ERC, as shown by HRP ablation. Cells were incubated with 5 μg/ml Cy3-Tf for 30 min, and were then chased for 10 min with 50 μg/ml HRP-Tf with (a) or without (b) excess (5 mg/ml) unlabeled Tf. The cells were then treated with hydrogen peroxide and DAB. The reaction products of the DAB reaction quenched fluorescence from molecules contained in the same compartment as the HRP-Tf (b), and the unlabeled Tf competed with HRP-Tf for binding to the TR and prevented quenching of the Cy3-Tf fluorescence (a). The cell boundaries, determined by DIC microscopy, are shown in b. Cells were also incubated with 1 μg/ml Cy3 anti-Tac IgG for 10 min, and were then chased for 10 min with 50 μg/ml HRP-Tf with (c) or without (d) 5 mg/ml unlabeled Tf. The DAB reaction with the HRP-Tf in the ERC quenched the Cy3-IgG fluorescence in this compartment (d), leaving a dark area (arrows) surrounded by the ring staining of the Cy3-IgG characteristic of the TGN. The fluorescence images show a summation projection of slices in a 3-D stack obtained by confocal microscopy. Bar, 10 μm.

Mentions: Since the TGN is in close proximity to the ERC, it is important to verify that endocytosed anti-Tac IgG colocalizes with Tf in the ERC after short incubation times. We used an HRP fluorescence ablation technique (29a) that extinguishes the fluorescence from any fluorescent probe that is in the same compartment as HRP. We coincubated cells with fluorescent IgG and HRP-Tf, and then treated the cells with hydrogen peroxide and DAB. HRP-Tf binds to the TR and trafficks to the ERC. The HRP-catalyzed DAB reaction product will ablate the fluorescence from fluorophores in the same compartment as the HRP-Tf (29a). We first did a positive control to check that the fluorescence in the ERC was being quenched. We incubated cells with 1 μg/ml Cy3-Tf for 10 min, chased for 10 min with 50 μg/ml HRP-Tf, and fixed and treated the cells with hydrogen peroxide and DAB. The DAB reaction quenched most of the Cy3-Tf in the ERC (Fig. 5 b). To ensure the DAB reaction was not nonspecifically quenching cell fluorescence, we carried out the same protocol but also included excess unlabeled Tf (5 mg/ml) in the chase media along with the HRP-Tf. The excess unlabeled Tf prevents significant HRP-Tf binding to the TR and uptake into cells, and thus the cells are still fluorescent after the DAB reaction (Fig. 5 a). These controls demonstrate that we can quench Cy3 fluorescence in the same compartment as HRP-Tf, and that this quenching is specific. We next incubated cells with 1 μg/ml Cy3 anti-Tac IgG for 10 min, and then chased for 10 min with 50 μg/ml HRP-Tf with or without excess unlabeled Tf. In cells that had internalized Cy3-IgG and were chased with excess unlabeled Tf, the Cy3-IgG was in a large perinuclear area consistent with labeling of both the ERC and the TGN (Fig. 5 c). When there was no unlabeled Tf present in the chase, the HRP-Tf reached the ERC; the DAB reaction quenched the center of the perinuclear staining (arrows), but a ring of Cy3-IgG staining remained (Fig. 5 d). The fluorescence loss in the center of the structure shows that after 20 min, some of the Cy3-IgG was in the ERC. The fluorescence that remained unquenched was in a ring pattern characteristic of the TGN. In cells that had been incubated with Cy3 anti-Tac IgG for 50 min and then with HRP-Tf for an additional 10 min (total time is 60 min), and then treated with DAB and hydrogen peroxide, a similar pattern as seen in Fig. 5 d was obtained, except that the remaining ring stain was much brighter (data not shown). This result is consistent with increased accumulation of Cy3-IgG in the TGN where it would not be quenched by the HRP reaction.


An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells.

Ghosh RN, Mallet WG, Soe TT, McGraw TE, Maxfield FR - J. Cell Biol. (1998)

TacTGN38 overlaps with Tf in the ERC, as shown by  HRP ablation. Cells were incubated with 5 μg/ml Cy3-Tf for 30  min, and were then chased for 10 min with 50 μg/ml HRP-Tf with  (a) or without (b) excess (5 mg/ml) unlabeled Tf. The cells were  then treated with hydrogen peroxide and DAB. The reaction  products of the DAB reaction quenched fluorescence from molecules contained in the same compartment as the HRP-Tf (b), and  the unlabeled Tf competed with HRP-Tf for binding to the TR  and prevented quenching of the Cy3-Tf fluorescence (a). The cell  boundaries, determined by DIC microscopy, are shown in b.  Cells were also incubated with 1 μg/ml Cy3 anti-Tac IgG for 10  min, and were then chased for 10 min with 50 μg/ml HRP-Tf with  (c) or without (d) 5 mg/ml unlabeled Tf. The DAB reaction with  the HRP-Tf in the ERC quenched the Cy3-IgG fluorescence in  this compartment (d), leaving a dark area (arrows) surrounded  by the ring staining of the Cy3-IgG characteristic of the TGN.  The fluorescence images show a summation projection of slices in  a 3-D stack obtained by confocal microscopy. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 5: TacTGN38 overlaps with Tf in the ERC, as shown by HRP ablation. Cells were incubated with 5 μg/ml Cy3-Tf for 30 min, and were then chased for 10 min with 50 μg/ml HRP-Tf with (a) or without (b) excess (5 mg/ml) unlabeled Tf. The cells were then treated with hydrogen peroxide and DAB. The reaction products of the DAB reaction quenched fluorescence from molecules contained in the same compartment as the HRP-Tf (b), and the unlabeled Tf competed with HRP-Tf for binding to the TR and prevented quenching of the Cy3-Tf fluorescence (a). The cell boundaries, determined by DIC microscopy, are shown in b. Cells were also incubated with 1 μg/ml Cy3 anti-Tac IgG for 10 min, and were then chased for 10 min with 50 μg/ml HRP-Tf with (c) or without (d) 5 mg/ml unlabeled Tf. The DAB reaction with the HRP-Tf in the ERC quenched the Cy3-IgG fluorescence in this compartment (d), leaving a dark area (arrows) surrounded by the ring staining of the Cy3-IgG characteristic of the TGN. The fluorescence images show a summation projection of slices in a 3-D stack obtained by confocal microscopy. Bar, 10 μm.
Mentions: Since the TGN is in close proximity to the ERC, it is important to verify that endocytosed anti-Tac IgG colocalizes with Tf in the ERC after short incubation times. We used an HRP fluorescence ablation technique (29a) that extinguishes the fluorescence from any fluorescent probe that is in the same compartment as HRP. We coincubated cells with fluorescent IgG and HRP-Tf, and then treated the cells with hydrogen peroxide and DAB. HRP-Tf binds to the TR and trafficks to the ERC. The HRP-catalyzed DAB reaction product will ablate the fluorescence from fluorophores in the same compartment as the HRP-Tf (29a). We first did a positive control to check that the fluorescence in the ERC was being quenched. We incubated cells with 1 μg/ml Cy3-Tf for 10 min, chased for 10 min with 50 μg/ml HRP-Tf, and fixed and treated the cells with hydrogen peroxide and DAB. The DAB reaction quenched most of the Cy3-Tf in the ERC (Fig. 5 b). To ensure the DAB reaction was not nonspecifically quenching cell fluorescence, we carried out the same protocol but also included excess unlabeled Tf (5 mg/ml) in the chase media along with the HRP-Tf. The excess unlabeled Tf prevents significant HRP-Tf binding to the TR and uptake into cells, and thus the cells are still fluorescent after the DAB reaction (Fig. 5 a). These controls demonstrate that we can quench Cy3 fluorescence in the same compartment as HRP-Tf, and that this quenching is specific. We next incubated cells with 1 μg/ml Cy3 anti-Tac IgG for 10 min, and then chased for 10 min with 50 μg/ml HRP-Tf with or without excess unlabeled Tf. In cells that had internalized Cy3-IgG and were chased with excess unlabeled Tf, the Cy3-IgG was in a large perinuclear area consistent with labeling of both the ERC and the TGN (Fig. 5 c). When there was no unlabeled Tf present in the chase, the HRP-Tf reached the ERC; the DAB reaction quenched the center of the perinuclear staining (arrows), but a ring of Cy3-IgG staining remained (Fig. 5 d). The fluorescence loss in the center of the structure shows that after 20 min, some of the Cy3-IgG was in the ERC. The fluorescence that remained unquenched was in a ring pattern characteristic of the TGN. In cells that had been incubated with Cy3 anti-Tac IgG for 50 min and then with HRP-Tf for an additional 10 min (total time is 60 min), and then treated with DAB and hydrogen peroxide, a similar pattern as seen in Fig. 5 d was obtained, except that the remaining ring stain was much brighter (data not shown). This result is consistent with increased accumulation of Cy3-IgG in the TGN where it would not be quenched by the HRP reaction.

Bottom Line: When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min.Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes.TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.

ABSTRACT
To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and 125I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38's postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.

Show MeSH