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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 reaches the TGN soon after internalization.  Cells were pulse-labeled with Cy3-Fab for 10 min before being  chased for either 0 or 10 min. The cells were then fixed, and the  TGN was stained with C6-NBD-ceramide. Single-slice images  were obtained by confocal microscopy. (a and b, 0-min chase)  Some Cy3-Fab (b, arrows) is already seen in the NBD-stained  TGN (a, arrows). (c and d, 10-min chase) A larger amount of  Cy3-Fab (d, arrows) is in the TGN (c, arrows). (e and f) Image-processing steps used to determine the amount of Cy3-Fab in  C6NBD-ceramide-labeled TGN. Background subtraction and  thresholding of c is used to generate the mask of the C6-NBD-ceramide–labeled TGN as shown in e. After background-subtracting d, the mask in e is applied to d to select those Cy3-Fab  pixels that overlap with the C6-NBD-ceramide labeling, and the  result is shown in f. Arrows in c–f indicate the selected overlapping areas. The intensity of the selected Cy3 pixels are summed  and then normalized by the number of pixels in the mask to give  the Cy3 fluorescence per NBD-labeled pixel. Bar, 10 μm.
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Figure 9: TacTGN38 reaches the TGN soon after internalization. Cells were pulse-labeled with Cy3-Fab for 10 min before being chased for either 0 or 10 min. The cells were then fixed, and the TGN was stained with C6-NBD-ceramide. Single-slice images were obtained by confocal microscopy. (a and b, 0-min chase) Some Cy3-Fab (b, arrows) is already seen in the NBD-stained TGN (a, arrows). (c and d, 10-min chase) A larger amount of Cy3-Fab (d, arrows) is in the TGN (c, arrows). (e and f) Image-processing steps used to determine the amount of Cy3-Fab in C6NBD-ceramide-labeled TGN. Background subtraction and thresholding of c is used to generate the mask of the C6-NBD-ceramide–labeled TGN as shown in e. After background-subtracting d, the mask in e is applied to d to select those Cy3-Fab pixels that overlap with the C6-NBD-ceramide labeling, and the result is shown in f. Arrows in c–f indicate the selected overlapping areas. The intensity of the selected Cy3 pixels are summed and then normalized by the number of pixels in the mask to give the Cy3 fluorescence per NBD-labeled pixel. Bar, 10 μm.

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)

TacTGN38 reaches the TGN soon after internalization.  Cells were pulse-labeled with Cy3-Fab for 10 min before being  chased for either 0 or 10 min. The cells were then fixed, and the  TGN was stained with C6-NBD-ceramide. Single-slice images  were obtained by confocal microscopy. (a and b, 0-min chase)  Some Cy3-Fab (b, arrows) is already seen in the NBD-stained  TGN (a, arrows). (c and d, 10-min chase) A larger amount of  Cy3-Fab (d, arrows) is in the TGN (c, arrows). (e and f) Image-processing steps used to determine the amount of Cy3-Fab in  C6NBD-ceramide-labeled TGN. Background subtraction and  thresholding of c is used to generate the mask of the C6-NBD-ceramide–labeled TGN as shown in e. After background-subtracting d, the mask in e is applied to d to select those Cy3-Fab  pixels that overlap with the C6-NBD-ceramide labeling, and the  result is shown in f. Arrows in c–f indicate the selected overlapping areas. The intensity of the selected Cy3 pixels are summed  and then normalized by the number of pixels in the mask to give  the Cy3 fluorescence per NBD-labeled pixel. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 9: TacTGN38 reaches the TGN soon after internalization. Cells were pulse-labeled with Cy3-Fab for 10 min before being chased for either 0 or 10 min. The cells were then fixed, and the TGN was stained with C6-NBD-ceramide. Single-slice images were obtained by confocal microscopy. (a and b, 0-min chase) Some Cy3-Fab (b, arrows) is already seen in the NBD-stained TGN (a, arrows). (c and d, 10-min chase) A larger amount of Cy3-Fab (d, arrows) is in the TGN (c, arrows). (e and f) Image-processing steps used to determine the amount of Cy3-Fab in C6NBD-ceramide-labeled TGN. Background subtraction and thresholding of c is used to generate the mask of the C6-NBD-ceramide–labeled TGN as shown in e. After background-subtracting d, the mask in e is applied to d to select those Cy3-Fab pixels that overlap with the C6-NBD-ceramide labeling, and the result is shown in f. Arrows in c–f indicate the selected overlapping areas. The intensity of the selected Cy3 pixels are summed and then normalized by the number of pixels in the mask to give the Cy3 fluorescence per NBD-labeled pixel. Bar, 10 μm.
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.

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