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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 endocytic recycling  compartment. Cells were coincubated for 10 min with Cy3-Tf (a)  and Alexa488-anti-Tac IgG (b). Images were obtained by confocal microscopy, and maximum x-y projections are shown in a and  b. The long slender arrows show colocalization in punctate endosomes, and the short thick arrows show colocalization in the perinuclear ERC. An x-z maximum projection of the same cells is  shown in c. Cy3-Tf is in red, Alexa488-IgG is in green, and their  colocalization in the ERC is seen ranging from orange to yellow  (thick arrows). Bar, 10 μm.
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Figure 3: TacTGN38 overlaps with Tf in the endocytic recycling compartment. Cells were coincubated for 10 min with Cy3-Tf (a) and Alexa488-anti-Tac IgG (b). Images were obtained by confocal microscopy, and maximum x-y projections are shown in a and b. The long slender arrows show colocalization in punctate endosomes, and the short thick arrows show colocalization in the perinuclear ERC. An x-z maximum projection of the same cells is shown in c. Cy3-Tf is in red, Alexa488-IgG is in green, and their colocalization in the ERC is seen ranging from orange to yellow (thick arrows). Bar, 10 μm.

Mentions: To follow TacTGN38's initial internalization steps, we compared its trafficking with Tf, which trafficks through sorting endosomes and the ERC before returning to the plasma membrane. Cells were coincubated with 1 μg/ml Alexa488 anti-Tac IgG and 3 μg/ml Cy3-Tf for 10 min. The cells were fixed, and 3-D images through the entire thickness of the cells were obtained by confocal microscopy. Tf (Fig. 3 a) and anti-Tac IgG (Fig. 3 b) are colocalized in small punctate structures that are presumably sorting endosomes (thin arrows) as well as in the juxtanuclear ERC (thick arrows). To ensure that the large structures (thick arrows) in Fig. 3, a and b were not at different elevations in the cell and were truly colocalized, we also viewed the cells as a maximum vertical (x-z) projection (Fig. 3 c). The Cy3-Tf (red) and the Alexa488 anti-Tac IgG (green) are colocalized in the vertical dimension, as seen by the orange-yellow staining (thick arrows).


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 endocytic recycling  compartment. Cells were coincubated for 10 min with Cy3-Tf (a)  and Alexa488-anti-Tac IgG (b). Images were obtained by confocal microscopy, and maximum x-y projections are shown in a and  b. The long slender arrows show colocalization in punctate endosomes, and the short thick arrows show colocalization in the perinuclear ERC. An x-z maximum projection of the same cells is  shown in c. Cy3-Tf is in red, Alexa488-IgG is in green, and their  colocalization in the ERC is seen ranging from orange to yellow  (thick arrows). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 3: TacTGN38 overlaps with Tf in the endocytic recycling compartment. Cells were coincubated for 10 min with Cy3-Tf (a) and Alexa488-anti-Tac IgG (b). Images were obtained by confocal microscopy, and maximum x-y projections are shown in a and b. The long slender arrows show colocalization in punctate endosomes, and the short thick arrows show colocalization in the perinuclear ERC. An x-z maximum projection of the same cells is shown in c. Cy3-Tf is in red, Alexa488-IgG is in green, and their colocalization in the ERC is seen ranging from orange to yellow (thick arrows). Bar, 10 μm.
Mentions: To follow TacTGN38's initial internalization steps, we compared its trafficking with Tf, which trafficks through sorting endosomes and the ERC before returning to the plasma membrane. Cells were coincubated with 1 μg/ml Alexa488 anti-Tac IgG and 3 μg/ml Cy3-Tf for 10 min. The cells were fixed, and 3-D images through the entire thickness of the cells were obtained by confocal microscopy. Tf (Fig. 3 a) and anti-Tac IgG (Fig. 3 b) are colocalized in small punctate structures that are presumably sorting endosomes (thin arrows) as well as in the juxtanuclear ERC (thick arrows). To ensure that the large structures (thick arrows) in Fig. 3, a and b were not at different elevations in the cell and were truly colocalized, we also viewed the cells as a maximum vertical (x-z) projection (Fig. 3 c). The Cy3-Tf (red) and the Alexa488 anti-Tac IgG (green) are colocalized in the vertical dimension, as seen by the orange-yellow staining (thick arrows).

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