Limits...
GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.

Wei ML, Bonzelius F, Scully RM, Kelly RB, Herman GA - J. Cell Biol. (1998)

Bottom Line: Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation.Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes.These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, The Hormone Research Institute, University of California, San Francisco, California 94143, USA.

ABSTRACT
The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

Show MeSH

Related in: MedlinePlus

Immunofluorescence microscopy of CHO/G4myc cells  after internalization of anti-myc mAb or transferrin. Cells were  incubated with either anti-myc mAb or Texas red–coupled human transferrin as indicated below, and fixed with paraformaldehyde. Cells with internalized antibody were permeabilized with  saponin and incubated with fluorescently labeled anti–mouse antibodies before processing. Cells with internalized transferrin  were processed immediately. Incubation with Texas red–coupled  human transferrin was for 30 min at 37°C (A and B), or for 2.5 h  at 15°C (E and F), or for 2.5 h at 15°C followed by a chase in  transferrin-free media for 10 min at 37°C (I and J). GLUT4-containing compartments were labeled by internalizing anti-myc antibodies for 30 min at 37°C (C and D) or for 2.5 h at 15°C (G and  H) or for 2.5 h at 15°C, followed by a chase in antibody-free media for 10 min at 37°C (K and L). Images were obtained by laser  scanning confocal microscopy. Low (A, C, E, G, I, and K) and  high power (B, D, F, H, J, and L) views are shown. Note that at  37°C, surface-labeled GLUT4 and transferrin both reach peripheral and juxtanuclear structures (A–D). However, at 15°C,  GLUT4 is observed primarily in peripheral compartments, with a  concomitant loss of labeling of the juxtanuclear structures (G and  H), whereas the transferrin pattern is unchanged from that seen  at 37°C (E and F). With the 37°C chase, labeling of GLUT4 in  juxtanuclear structures is restored (K and L). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140164&req=5

Figure 4: Immunofluorescence microscopy of CHO/G4myc cells after internalization of anti-myc mAb or transferrin. Cells were incubated with either anti-myc mAb or Texas red–coupled human transferrin as indicated below, and fixed with paraformaldehyde. Cells with internalized antibody were permeabilized with saponin and incubated with fluorescently labeled anti–mouse antibodies before processing. Cells with internalized transferrin were processed immediately. Incubation with Texas red–coupled human transferrin was for 30 min at 37°C (A and B), or for 2.5 h at 15°C (E and F), or for 2.5 h at 15°C followed by a chase in transferrin-free media for 10 min at 37°C (I and J). GLUT4-containing compartments were labeled by internalizing anti-myc antibodies for 30 min at 37°C (C and D) or for 2.5 h at 15°C (G and H) or for 2.5 h at 15°C, followed by a chase in antibody-free media for 10 min at 37°C (K and L). Images were obtained by laser scanning confocal microscopy. Low (A, C, E, G, I, and K) and high power (B, D, F, H, J, and L) views are shown. Note that at 37°C, surface-labeled GLUT4 and transferrin both reach peripheral and juxtanuclear structures (A–D). However, at 15°C, GLUT4 is observed primarily in peripheral compartments, with a concomitant loss of labeling of the juxtanuclear structures (G and H), whereas the transferrin pattern is unchanged from that seen at 37°C (E and F). With the 37°C chase, labeling of GLUT4 in juxtanuclear structures is restored (K and L). Bars, 10 μm.

Mentions: Pulse-chase experiments were performed by incubating cells with radioiodinated mAb 9E10 for 80 min at 15°C, washing extensively on ice, and then rewarming the cells in media in the absence of labeled antibody for various periods of time before processing as above. Kinetic data were obtained by determining the GSV-associated radioactivity for each time point. The GSV-associated radioactivity was calculated by plotting the gradient profile of 125I-9E10 for each time point and integrating the GSV peaks using NIH Image 1.6 software (National Institutes of Health, Bethesda, MD). Relative units were then calculated to facilitate comparison between independent experiments. An arbitrary value of 1 was assigned to the area of the peak at 30 min (see Fig. 4), 10 min (see Fig. 7), and at time zero (see Fig. 12, A and B).


GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.

Wei ML, Bonzelius F, Scully RM, Kelly RB, Herman GA - J. Cell Biol. (1998)

Immunofluorescence microscopy of CHO/G4myc cells  after internalization of anti-myc mAb or transferrin. Cells were  incubated with either anti-myc mAb or Texas red–coupled human transferrin as indicated below, and fixed with paraformaldehyde. Cells with internalized antibody were permeabilized with  saponin and incubated with fluorescently labeled anti–mouse antibodies before processing. Cells with internalized transferrin  were processed immediately. Incubation with Texas red–coupled  human transferrin was for 30 min at 37°C (A and B), or for 2.5 h  at 15°C (E and F), or for 2.5 h at 15°C followed by a chase in  transferrin-free media for 10 min at 37°C (I and J). GLUT4-containing compartments were labeled by internalizing anti-myc antibodies for 30 min at 37°C (C and D) or for 2.5 h at 15°C (G and  H) or for 2.5 h at 15°C, followed by a chase in antibody-free media for 10 min at 37°C (K and L). Images were obtained by laser  scanning confocal microscopy. Low (A, C, E, G, I, and K) and  high power (B, D, F, H, J, and L) views are shown. Note that at  37°C, surface-labeled GLUT4 and transferrin both reach peripheral and juxtanuclear structures (A–D). However, at 15°C,  GLUT4 is observed primarily in peripheral compartments, with a  concomitant loss of labeling of the juxtanuclear structures (G and  H), whereas the transferrin pattern is unchanged from that seen  at 37°C (E and F). With the 37°C chase, labeling of GLUT4 in  juxtanuclear structures is restored (K and L). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140164&req=5

Figure 4: Immunofluorescence microscopy of CHO/G4myc cells after internalization of anti-myc mAb or transferrin. Cells were incubated with either anti-myc mAb or Texas red–coupled human transferrin as indicated below, and fixed with paraformaldehyde. Cells with internalized antibody were permeabilized with saponin and incubated with fluorescently labeled anti–mouse antibodies before processing. Cells with internalized transferrin were processed immediately. Incubation with Texas red–coupled human transferrin was for 30 min at 37°C (A and B), or for 2.5 h at 15°C (E and F), or for 2.5 h at 15°C followed by a chase in transferrin-free media for 10 min at 37°C (I and J). GLUT4-containing compartments were labeled by internalizing anti-myc antibodies for 30 min at 37°C (C and D) or for 2.5 h at 15°C (G and H) or for 2.5 h at 15°C, followed by a chase in antibody-free media for 10 min at 37°C (K and L). Images were obtained by laser scanning confocal microscopy. Low (A, C, E, G, I, and K) and high power (B, D, F, H, J, and L) views are shown. Note that at 37°C, surface-labeled GLUT4 and transferrin both reach peripheral and juxtanuclear structures (A–D). However, at 15°C, GLUT4 is observed primarily in peripheral compartments, with a concomitant loss of labeling of the juxtanuclear structures (G and H), whereas the transferrin pattern is unchanged from that seen at 37°C (E and F). With the 37°C chase, labeling of GLUT4 in juxtanuclear structures is restored (K and L). Bars, 10 μm.
Mentions: Pulse-chase experiments were performed by incubating cells with radioiodinated mAb 9E10 for 80 min at 15°C, washing extensively on ice, and then rewarming the cells in media in the absence of labeled antibody for various periods of time before processing as above. Kinetic data were obtained by determining the GSV-associated radioactivity for each time point. The GSV-associated radioactivity was calculated by plotting the gradient profile of 125I-9E10 for each time point and integrating the GSV peaks using NIH Image 1.6 software (National Institutes of Health, Bethesda, MD). Relative units were then calculated to facilitate comparison between independent experiments. An arbitrary value of 1 was assigned to the area of the peak at 30 min (see Fig. 4), 10 min (see Fig. 7), and at time zero (see Fig. 12, A and B).

Bottom Line: Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation.Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes.These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, The Hormone Research Institute, University of California, San Francisco, California 94143, USA.

ABSTRACT
The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15 degrees and 37 degrees C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.

Show MeSH
Related in: MedlinePlus