Limits...
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.

Show MeSH
Accumulation of TacTGN38 in the TGN. Cells were  incubated with F-anti-Tac IgG for the times shown, fixed, and labeled with Cy3-anti-Tac IgG. Confocal microscopy was used to  obtain the entire 3-D distribution through the cell, and these images were quantified to determine the amount of F-IgG per TGN  volume element (○). The data were fit to a kinetic approach to  steady state with a half-time of 46 min. Cells were also incubated  with Cy3-anti-Tac Fab for different times, and then the TGN was  stained with C6NBD-ceramide. Single slice images were obtained  by confocal microscopy. Quantification of the amount of Cy3-Fab fluorescence per TGN area (see Fig. 9, c–f) also gave a half-time of 46 min (▵). Error bars represent SEM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132871&req=5

Figure 10: Accumulation of TacTGN38 in the TGN. Cells were incubated with F-anti-Tac IgG for the times shown, fixed, and labeled with Cy3-anti-Tac IgG. Confocal microscopy was used to obtain the entire 3-D distribution through the cell, and these images were quantified to determine the amount of F-IgG per TGN volume element (○). The data were fit to a kinetic approach to steady state with a half-time of 46 min. Cells were also incubated with Cy3-anti-Tac Fab for different times, and then the TGN was stained with C6NBD-ceramide. Single slice images were obtained by confocal microscopy. Quantification of the amount of Cy3-Fab fluorescence per TGN area (see Fig. 9, c–f) also gave a half-time of 46 min (▵). Error bars represent SEM.

Mentions: Image processing was done using the MetaMorph image processing package (Universal Imaging Corp., West Chester, PA) on a Pentium PC. To analyze the amount of Cy3 fluorescence per labeled TGN (see Figs. 9 and 10), first a local median background intensity obtained from a 32 × 32 pixel area (0.15 μm/pixel) was subtracted from every pixel of the Cy3 and NBD images, as previously described (6). A mask to identify the TGN was then obtained by retaining those pixels whose intensities in the NBD image were above a specific threshold value. The threshold value was the mean plus 3 standard deviations of all the positive pixels intensities in the NBD image. To complete the mask, these retained pixels were all given the same constant intensity, and the rest of the image was made black. This mask delineates the areas of bright NBD labeling from the dimmer, background, and cellular haze (see Fig. 9, c and e). The selected region was robust to the intensity threshold value chosen, as a lower threshold of the mean intensity plus two standard deviations gave similar results. This mask was applied to the Cy3 image (see Fig. 9 d), and the intensities in only those Cy3 pixels that were in the area delineated by the mask (see Fig. 9 f) were summed. This summed intensity was normalized by the number of pixels in the mask, giving the Cy3 intensity per TGN element (pixel).


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)

Accumulation of TacTGN38 in the TGN. Cells were  incubated with F-anti-Tac IgG for the times shown, fixed, and labeled with Cy3-anti-Tac IgG. Confocal microscopy was used to  obtain the entire 3-D distribution through the cell, and these images were quantified to determine the amount of F-IgG per TGN  volume element (○). The data were fit to a kinetic approach to  steady state with a half-time of 46 min. Cells were also incubated  with Cy3-anti-Tac Fab for different times, and then the TGN was  stained with C6NBD-ceramide. Single slice images were obtained  by confocal microscopy. Quantification of the amount of Cy3-Fab fluorescence per TGN area (see Fig. 9, c–f) also gave a half-time of 46 min (▵). Error bars represent SEM.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Accumulation of TacTGN38 in the TGN. Cells were incubated with F-anti-Tac IgG for the times shown, fixed, and labeled with Cy3-anti-Tac IgG. Confocal microscopy was used to obtain the entire 3-D distribution through the cell, and these images were quantified to determine the amount of F-IgG per TGN volume element (○). The data were fit to a kinetic approach to steady state with a half-time of 46 min. Cells were also incubated with Cy3-anti-Tac Fab for different times, and then the TGN was stained with C6NBD-ceramide. Single slice images were obtained by confocal microscopy. Quantification of the amount of Cy3-Fab fluorescence per TGN area (see Fig. 9, c–f) also gave a half-time of 46 min (▵). Error bars represent SEM.
Mentions: Image processing was done using the MetaMorph image processing package (Universal Imaging Corp., West Chester, PA) on a Pentium PC. To analyze the amount of Cy3 fluorescence per labeled TGN (see Figs. 9 and 10), first a local median background intensity obtained from a 32 × 32 pixel area (0.15 μm/pixel) was subtracted from every pixel of the Cy3 and NBD images, as previously described (6). A mask to identify the TGN was then obtained by retaining those pixels whose intensities in the NBD image were above a specific threshold value. The threshold value was the mean plus 3 standard deviations of all the positive pixels intensities in the NBD image. To complete the mask, these retained pixels were all given the same constant intensity, and the rest of the image was made black. This mask delineates the areas of bright NBD labeling from the dimmer, background, and cellular haze (see Fig. 9, c and e). The selected region was robust to the intensity threshold value chosen, as a lower threshold of the mean intensity plus two standard deviations gave similar results. This mask was applied to the Cy3 image (see Fig. 9 d), and the intensities in only those Cy3 pixels that were in the area delineated by the mask (see Fig. 9 f) were summed. This summed intensity was normalized by the number of pixels in the mask, giving the Cy3 intensity per TGN element (pixel).

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