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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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Time course for TacTGN38 delivery to the cell surface  and internalization from the cell surface. (A) Cells were incubated either with [125I]anti-Tac IgG (○) or Cy3 anti-Tac Fab (•)  for the times shown. The cells were fixed, and the amount of endocytosed anti-Tac antibody in the cell was assessed either by radioactivity counting or quantifying the fluorescence intensity in 3-D  stacks of images taken of the whole cell by confocal microscopy.  Background values were subtracted (0-min value), and the data  were normalized so that the fluorescence and 125I curves could be  superimposed. It takes over 1 h for endocytosed anti-Tac to reach  steady state, and fluorescence quantification gives the same kinetic data as 125I measurements. (B) Cells were incubated with  [125I]anti-Tac IgG for brief periods of time, and the surface-bound and internalized counts were determined. The ratio of internalized-to-surface counts is plotted, and it increases linearly  over time with a slope of 0.154 min−1. Error bars represent SEM.
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Figure 2: Time course for TacTGN38 delivery to the cell surface and internalization from the cell surface. (A) Cells were incubated either with [125I]anti-Tac IgG (○) or Cy3 anti-Tac Fab (•) for the times shown. The cells were fixed, and the amount of endocytosed anti-Tac antibody in the cell was assessed either by radioactivity counting or quantifying the fluorescence intensity in 3-D stacks of images taken of the whole cell by confocal microscopy. Background values were subtracted (0-min value), and the data were normalized so that the fluorescence and 125I curves could be superimposed. It takes over 1 h for endocytosed anti-Tac to reach steady state, and fluorescence quantification gives the same kinetic data as 125I measurements. (B) Cells were incubated with [125I]anti-Tac IgG for brief periods of time, and the surface-bound and internalized counts were determined. The ratio of internalized-to-surface counts is plotted, and it increases linearly over time with a slope of 0.154 min−1. Error bars represent SEM.

Mentions: The TacTGN38 internalization rate constant (ki) was derived from plots of the internal/surface ratio at different times, as described previously (31, 48). Cells were cultured to confluence in 24-well plates. Triplicate wells of TacTGN38-transfected cells were incubated with 0.5 ml 125I-labeled anti-Tac IgG (4 μg/ml, 107 cpm/well) from 1 to 5 min at 37°C. To determine nonspecific binding, single wells of TRVb1 cells not transfected with TacTGN38 were incubated with IgG over the same time course. After incubations, cells were placed into a 4°C ice bath and washed rapidly with prechilled 4× 1 ml Medium 1 + 1% (wt/vol) BSA, and then with 1 ml Medium 1. Surface-bound IgG was eluted with 2× 1-ml washes of 0.1 M acetic acid, 0.5 M NaCl; 10 min each wash at 0°C. This procedure eluted ∼98% of all surface-bound counts, as determined from control samples that were incubated with IgG at 4°C to prevent internalization. After the elutions, cells were washed with 1 ml Medium 1 and solubilized by 2× 1 ml incubations with 0.1 M NaOH, 0.1% (wt/vol) SDS at 37°C, 15 min each incubation. Surface and internal counts were obtained using a gamma counter. The counts were corrected for nonspecific binding and the efficiency of the low pH elution, and then the internal-to-surface ratio was calculated for each sample. The ratios were plotted as a function of time, and the slope of the line (ki) was calculated by linear regression (see Fig. 2 b).


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)

Time course for TacTGN38 delivery to the cell surface  and internalization from the cell surface. (A) Cells were incubated either with [125I]anti-Tac IgG (○) or Cy3 anti-Tac Fab (•)  for the times shown. The cells were fixed, and the amount of endocytosed anti-Tac antibody in the cell was assessed either by radioactivity counting or quantifying the fluorescence intensity in 3-D  stacks of images taken of the whole cell by confocal microscopy.  Background values were subtracted (0-min value), and the data  were normalized so that the fluorescence and 125I curves could be  superimposed. It takes over 1 h for endocytosed anti-Tac to reach  steady state, and fluorescence quantification gives the same kinetic data as 125I measurements. (B) Cells were incubated with  [125I]anti-Tac IgG for brief periods of time, and the surface-bound and internalized counts were determined. The ratio of internalized-to-surface counts is plotted, and it increases linearly  over time with a slope of 0.154 min−1. Error bars represent SEM.
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Related In: Results  -  Collection

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Figure 2: Time course for TacTGN38 delivery to the cell surface and internalization from the cell surface. (A) Cells were incubated either with [125I]anti-Tac IgG (○) or Cy3 anti-Tac Fab (•) for the times shown. The cells were fixed, and the amount of endocytosed anti-Tac antibody in the cell was assessed either by radioactivity counting or quantifying the fluorescence intensity in 3-D stacks of images taken of the whole cell by confocal microscopy. Background values were subtracted (0-min value), and the data were normalized so that the fluorescence and 125I curves could be superimposed. It takes over 1 h for endocytosed anti-Tac to reach steady state, and fluorescence quantification gives the same kinetic data as 125I measurements. (B) Cells were incubated with [125I]anti-Tac IgG for brief periods of time, and the surface-bound and internalized counts were determined. The ratio of internalized-to-surface counts is plotted, and it increases linearly over time with a slope of 0.154 min−1. Error bars represent SEM.
Mentions: The TacTGN38 internalization rate constant (ki) was derived from plots of the internal/surface ratio at different times, as described previously (31, 48). Cells were cultured to confluence in 24-well plates. Triplicate wells of TacTGN38-transfected cells were incubated with 0.5 ml 125I-labeled anti-Tac IgG (4 μg/ml, 107 cpm/well) from 1 to 5 min at 37°C. To determine nonspecific binding, single wells of TRVb1 cells not transfected with TacTGN38 were incubated with IgG over the same time course. After incubations, cells were placed into a 4°C ice bath and washed rapidly with prechilled 4× 1 ml Medium 1 + 1% (wt/vol) BSA, and then with 1 ml Medium 1. Surface-bound IgG was eluted with 2× 1-ml washes of 0.1 M acetic acid, 0.5 M NaCl; 10 min each wash at 0°C. This procedure eluted ∼98% of all surface-bound counts, as determined from control samples that were incubated with IgG at 4°C to prevent internalization. After the elutions, cells were washed with 1 ml Medium 1 and solubilized by 2× 1 ml incubations with 0.1 M NaOH, 0.1% (wt/vol) SDS at 37°C, 15 min each incubation. Surface and internal counts were obtained using a gamma counter. The counts were corrected for nonspecific binding and the efficiency of the low pH elution, and then the internal-to-surface ratio was calculated for each sample. The ratios were plotted as a function of time, and the slope of the line (ki) was calculated by linear regression (see Fig. 2 b).

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