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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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Internalized anti-Tac colocalizes with furin after a 60-min chase. Cells were incubated with 4 mg/ml anti-Tac monoclonal antibodies conjugated to Alexa 488 fluorescent dye (a and  c) for 10 min. Cells were fixed immediately (a and b) or chased in  medium for 60 min (c and d). After fixation, cells were permeabilized and stained with polyclonal antibodies against furin (b and  d) followed by rhodamine-conjugated goat anti–rabbit secondary  antibodies. In the absence of a chase, anti-Tac was detected in a  distribution that resembles the endocytic recycling compartment,  and is surrounded by the TGN (arrows) as labeled by anti-furin  antibodies. With a 60-min chase, anti-Tac predominantly colocalized with furin (arrows). Bar, 5 μm.
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Figure 4: Internalized anti-Tac colocalizes with furin after a 60-min chase. Cells were incubated with 4 mg/ml anti-Tac monoclonal antibodies conjugated to Alexa 488 fluorescent dye (a and c) for 10 min. Cells were fixed immediately (a and b) or chased in medium for 60 min (c and d). After fixation, cells were permeabilized and stained with polyclonal antibodies against furin (b and d) followed by rhodamine-conjugated goat anti–rabbit secondary antibodies. In the absence of a chase, anti-Tac was detected in a distribution that resembles the endocytic recycling compartment, and is surrounded by the TGN (arrows) as labeled by anti-furin antibodies. With a 60-min chase, anti-Tac predominantly colocalized with furin (arrows). Bar, 5 μm.

Mentions: TRVb1 cells were transfected with TacTGN38, and clonal cell lines were selected by FACS sorting using Cy3-labeled monoclonal anti-Tac IgG. We first examined the distribution of internalized Cy3-labeled anti-Tac IgG and monovalent Fab. Fig. 1 shows confocal microscopy images of Cy3-Fab (Fig. 1 b) and Cy3-IgG (Fig. 1 d) after a 5–10 min incubation with the antibodies and a 30–40-min chase. The cells were also stained with C6-NBD-ceramide which labels the TGN (36), and single optical sections were obtained in regions that showed significant NBD fluorescence (Fig. 1, a and b). Both the IgG and the Fab showed extensive colocalization with the C6-NBD-ceramide, demonstrating that the endocytosed Cy3 labeled anti-Tac Fab and IgG reached the TGN. After an extensive chase, internalized anti-Tac also codistributed with furin, an endoprotease that is localized to the TGN (see Fig. 4, c and d). Endocytosed TacTGN38 trafficked to the TGN in all the clonal lines we examined.


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)

Internalized anti-Tac colocalizes with furin after a 60-min chase. Cells were incubated with 4 mg/ml anti-Tac monoclonal antibodies conjugated to Alexa 488 fluorescent dye (a and  c) for 10 min. Cells were fixed immediately (a and b) or chased in  medium for 60 min (c and d). After fixation, cells were permeabilized and stained with polyclonal antibodies against furin (b and  d) followed by rhodamine-conjugated goat anti–rabbit secondary  antibodies. In the absence of a chase, anti-Tac was detected in a  distribution that resembles the endocytic recycling compartment,  and is surrounded by the TGN (arrows) as labeled by anti-furin  antibodies. With a 60-min chase, anti-Tac predominantly colocalized with furin (arrows). Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132871&req=5

Figure 4: Internalized anti-Tac colocalizes with furin after a 60-min chase. Cells were incubated with 4 mg/ml anti-Tac monoclonal antibodies conjugated to Alexa 488 fluorescent dye (a and c) for 10 min. Cells were fixed immediately (a and b) or chased in medium for 60 min (c and d). After fixation, cells were permeabilized and stained with polyclonal antibodies against furin (b and d) followed by rhodamine-conjugated goat anti–rabbit secondary antibodies. In the absence of a chase, anti-Tac was detected in a distribution that resembles the endocytic recycling compartment, and is surrounded by the TGN (arrows) as labeled by anti-furin antibodies. With a 60-min chase, anti-Tac predominantly colocalized with furin (arrows). Bar, 5 μm.
Mentions: TRVb1 cells were transfected with TacTGN38, and clonal cell lines were selected by FACS sorting using Cy3-labeled monoclonal anti-Tac IgG. We first examined the distribution of internalized Cy3-labeled anti-Tac IgG and monovalent Fab. Fig. 1 shows confocal microscopy images of Cy3-Fab (Fig. 1 b) and Cy3-IgG (Fig. 1 d) after a 5–10 min incubation with the antibodies and a 30–40-min chase. The cells were also stained with C6-NBD-ceramide which labels the TGN (36), and single optical sections were obtained in regions that showed significant NBD fluorescence (Fig. 1, a and b). Both the IgG and the Fab showed extensive colocalization with the C6-NBD-ceramide, demonstrating that the endocytosed Cy3 labeled anti-Tac Fab and IgG reached the TGN. After an extensive chase, internalized anti-Tac also codistributed with furin, an endoprotease that is localized to the TGN (see Fig. 4, c and d). Endocytosed TacTGN38 trafficked to the TGN in all the clonal lines we examined.

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