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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 goes from the TGN to the plasma membrane with a half-time of 46 min. Cells were pulsed with 1 μg/ml  F-anti-Tac IgG for 10 min, and were then chased for 50 min. The  cells were then incubated with 10 μg/ml anti-fluorescein antibodies that quench the fluorescence of F-IgG that returns to the cell  surface. Images obtained by wide-field microscopy show that  from 10 (a) to 180 min (b) in the presence of anti-fluorescein antibody, fluorescence quenching has occurred. The fluorescence intensity was quantified to determine the loss of fluorescence in the  cell over time (c). When fit to a monoexponential decay, the efflux kinetics show a half-time of 46 min for TacTGN38 to go from  the TGN to the plasma membrane. Bar, 10μm; error bars represent SD.
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Figure 11: TacTGN38 goes from the TGN to the plasma membrane with a half-time of 46 min. Cells were pulsed with 1 μg/ml F-anti-Tac IgG for 10 min, and were then chased for 50 min. The cells were then incubated with 10 μg/ml anti-fluorescein antibodies that quench the fluorescence of F-IgG that returns to the cell surface. Images obtained by wide-field microscopy show that from 10 (a) to 180 min (b) in the presence of anti-fluorescein antibody, fluorescence quenching has occurred. The fluorescence intensity was quantified to determine the loss of fluorescence in the cell over time (c). When fit to a monoexponential decay, the efflux kinetics show a half-time of 46 min for TacTGN38 to go from the TGN to the plasma membrane. Bar, 10μm; error bars represent SD.

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)

TacTGN38 goes from the TGN to the plasma membrane with a half-time of 46 min. Cells were pulsed with 1 μg/ml  F-anti-Tac IgG for 10 min, and were then chased for 50 min. The  cells were then incubated with 10 μg/ml anti-fluorescein antibodies that quench the fluorescence of F-IgG that returns to the cell  surface. Images obtained by wide-field microscopy show that  from 10 (a) to 180 min (b) in the presence of anti-fluorescein antibody, fluorescence quenching has occurred. The fluorescence intensity was quantified to determine the loss of fluorescence in the  cell over time (c). When fit to a monoexponential decay, the efflux kinetics show a half-time of 46 min for TacTGN38 to go from  the TGN to the plasma membrane. Bar, 10μm; error bars represent SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132871&req=5

Figure 11: TacTGN38 goes from the TGN to the plasma membrane with a half-time of 46 min. Cells were pulsed with 1 μg/ml F-anti-Tac IgG for 10 min, and were then chased for 50 min. The cells were then incubated with 10 μg/ml anti-fluorescein antibodies that quench the fluorescence of F-IgG that returns to the cell surface. Images obtained by wide-field microscopy show that from 10 (a) to 180 min (b) in the presence of anti-fluorescein antibody, fluorescence quenching has occurred. The fluorescence intensity was quantified to determine the loss of fluorescence in the cell over time (c). When fit to a monoexponential decay, the efflux kinetics show a half-time of 46 min for TacTGN38 to go from the TGN to the plasma membrane. Bar, 10μm; error bars represent SD.
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