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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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Little TacTGN38 is seen in late  endosomes. Cells were coincubated with  fluorescein dextran, and either with Cy3-IgG (a) or rhodamine dextran (b) for 10  min and then chased for 20 min. Images  were obtained by confocal microscopy,  and single slices are shown. Fluorescein  dextran is shown in green, Cy3-IgG and  rhodamine dextran are shown in red, and  colocalized areas range from orange to  yellow. After a 20-min chase, the fluorescein dextran is much less colocalized with  the Cy3-IgG (a) than with the rhodamine  dextran (b). Bar, 10 μm.
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Figure 8: Little TacTGN38 is seen in late endosomes. Cells were coincubated with fluorescein dextran, and either with Cy3-IgG (a) or rhodamine dextran (b) for 10 min and then chased for 20 min. Images were obtained by confocal microscopy, and single slices are shown. Fluorescein dextran is shown in green, Cy3-IgG and rhodamine dextran are shown in red, and colocalized areas range from orange to yellow. After a 20-min chase, the fluorescein dextran is much less colocalized with the Cy3-IgG (a) than with the rhodamine dextran (b). Bar, 10 μm.

Mentions: If the late endosome is a major pathway for delivery of internalized TacTGN38 to the TGN, there should be significant colocalization between fluorescein dextran and Cy3-IgG after 20 min. Fig. 8 shows single confocal slices at a focal plane that contains several late endosomes. Relatively little colocalization is seen at this time (Fig. 8 a), and structures labeled with only one probe were readily detected (red and green labeling in Fig. 8 a). Some fluorescein–dextran-containing spots also contained Cy3-IgG, but we are unable to determine if these are late endosomes or sorting endosomes that have not as yet matured. As a positive control we cointernalized R-dex and F-dex for 10 min, followed by a 20-min chase. We see good colocalization of R-dex and F-dex because the majority of spots range from orange to yellow in Fig. 8 b. Although R-dex and F-dex traffic similarly and are colocalized in the same compartments, the amount of both dextrans in an individual endosome may not be equal. These data suggest that late endosomes are not major intermediates in the delivery of TacTGN38 from the recycling pathway to the TGN, or that TacTGN38 passes through late endosomes too rapidly to be detected by our methods. Further experiments are necessary to distinguish between these possibilities.


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)

Little TacTGN38 is seen in late  endosomes. Cells were coincubated with  fluorescein dextran, and either with Cy3-IgG (a) or rhodamine dextran (b) for 10  min and then chased for 20 min. Images  were obtained by confocal microscopy,  and single slices are shown. Fluorescein  dextran is shown in green, Cy3-IgG and  rhodamine dextran are shown in red, and  colocalized areas range from orange to  yellow. After a 20-min chase, the fluorescein dextran is much less colocalized with  the Cy3-IgG (a) than with the rhodamine  dextran (b). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 8: Little TacTGN38 is seen in late endosomes. Cells were coincubated with fluorescein dextran, and either with Cy3-IgG (a) or rhodamine dextran (b) for 10 min and then chased for 20 min. Images were obtained by confocal microscopy, and single slices are shown. Fluorescein dextran is shown in green, Cy3-IgG and rhodamine dextran are shown in red, and colocalized areas range from orange to yellow. After a 20-min chase, the fluorescein dextran is much less colocalized with the Cy3-IgG (a) than with the rhodamine dextran (b). Bar, 10 μm.
Mentions: If the late endosome is a major pathway for delivery of internalized TacTGN38 to the TGN, there should be significant colocalization between fluorescein dextran and Cy3-IgG after 20 min. Fig. 8 shows single confocal slices at a focal plane that contains several late endosomes. Relatively little colocalization is seen at this time (Fig. 8 a), and structures labeled with only one probe were readily detected (red and green labeling in Fig. 8 a). Some fluorescein–dextran-containing spots also contained Cy3-IgG, but we are unable to determine if these are late endosomes or sorting endosomes that have not as yet matured. As a positive control we cointernalized R-dex and F-dex for 10 min, followed by a 20-min chase. We see good colocalization of R-dex and F-dex because the majority of spots range from orange to yellow in Fig. 8 b. Although R-dex and F-dex traffic similarly and are colocalized in the same compartments, the amount of both dextrans in an individual endosome may not be equal. These data suggest that late endosomes are not major intermediates in the delivery of TacTGN38 from the recycling pathway to the TGN, or that TacTGN38 passes through late endosomes too rapidly to be detected by our methods. Further experiments are necessary to distinguish between these possibilities.

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