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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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18% of TacTGN38 is delivered to TGN with each  round of endocytosis. Cells were labeled with 1 μg/ml F-anti-Tac  IgG on ice, and were then warmed for 5 min, washed, and chased  with 10 μg/ml anti-fluorescein antibodies. Fluorescence images  were obtained by wide-field microscopy, quantified, and fit to a  monoexponential decay. Data from a representative experiment  are shown. The asymptote gave the residual fluorescence F∞, and  a cell that was fixed and imaged without warming gave the initial  cell-associated fluorescence, Fi*. Fq is the residual fluorescence  after quenching surface bound anti-Tac. Error bars represent SEM.
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Figure 7: 18% of TacTGN38 is delivered to TGN with each round of endocytosis. Cells were labeled with 1 μg/ml F-anti-Tac IgG on ice, and were then warmed for 5 min, washed, and chased with 10 μg/ml anti-fluorescein antibodies. Fluorescence images were obtained by wide-field microscopy, quantified, and fit to a monoexponential decay. Data from a representative experiment are shown. The asymptote gave the residual fluorescence F∞, and a cell that was fixed and imaged without warming gave the initial cell-associated fluorescence, Fi*. Fq is the residual fluorescence after quenching surface bound anti-Tac. Error bars represent SEM.

Mentions: To quantify the fluorescence from images in the fluorescein quenching experiments (see Figs. 6, 7, and 11), the background fluorescence was first subtracted from each image. This background was determined as the mean intensity from a cell-free region. The remaining cell-associated fluorescence was summed and divided by the number of cells in the field to derive the fluorescence power per cell. Ten fields of cells were imaged for each experimental condition or time point, typically with about twenty cells per field.


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)

18% of TacTGN38 is delivered to TGN with each  round of endocytosis. Cells were labeled with 1 μg/ml F-anti-Tac  IgG on ice, and were then warmed for 5 min, washed, and chased  with 10 μg/ml anti-fluorescein antibodies. Fluorescence images  were obtained by wide-field microscopy, quantified, and fit to a  monoexponential decay. Data from a representative experiment  are shown. The asymptote gave the residual fluorescence F∞, and  a cell that was fixed and imaged without warming gave the initial  cell-associated fluorescence, Fi*. Fq is the residual fluorescence  after quenching surface bound anti-Tac. Error bars represent SEM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132871&req=5

Figure 7: 18% of TacTGN38 is delivered to TGN with each round of endocytosis. Cells were labeled with 1 μg/ml F-anti-Tac IgG on ice, and were then warmed for 5 min, washed, and chased with 10 μg/ml anti-fluorescein antibodies. Fluorescence images were obtained by wide-field microscopy, quantified, and fit to a monoexponential decay. Data from a representative experiment are shown. The asymptote gave the residual fluorescence F∞, and a cell that was fixed and imaged without warming gave the initial cell-associated fluorescence, Fi*. Fq is the residual fluorescence after quenching surface bound anti-Tac. Error bars represent SEM.
Mentions: To quantify the fluorescence from images in the fluorescein quenching experiments (see Figs. 6, 7, and 11), the background fluorescence was first subtracted from each image. This background was determined as the mean intensity from a cell-free region. The remaining cell-associated fluorescence was summed and divided by the number of cells in the field to derive the fluorescence power per cell. Ten fields of cells were imaged for each experimental condition or time point, typically with about twenty cells per field.

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
Related in: MedlinePlus