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ADP-ribosylation factor 6 and endocytosis at the apical surface of Madin-Darby canine kidney cells.

Altschuler Y, Liu S, Katz L, Tang K, Hardy S, Brodsky F, Apodaca G, Mostov K - J. Cell Biol. (1999)

Bottom Line: Surprisingly, overexpression of the mutant ARF6-T27N, which is predicted to be in the GDP-bound form, also stimulated apical endocytosis, though to a lesser extent.ARF6-stimulated endocytosis is inhibited by a dominant-negative form of dynamin, or a dominant-negative hub fragment of clathrin heavy chain, indicating that it is mediated by clathrin.When ARF6-Q67L is overexpressed in the presence of the dominant-negative dynamin, the ARF6-Q67L colocalizes with clathrin and with IgA bound to its receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, University of California, San Francisco, California 94143-0452, USA. yoram@itsa.ucsf.edu

ABSTRACT
We report that the small GTPase, ADP-ribosylation factor 6 (ARF6), is present only on the apical surface of polarized MDCK epithelial cells. Overexpression of a mutant of ARF6, ARF6-Q67L, which is predicted to be in the GTP-bound form, stimulates endocytosis exclusively at this surface. Surprisingly, overexpression of the mutant ARF6-T27N, which is predicted to be in the GDP-bound form, also stimulated apical endocytosis, though to a lesser extent. ARF6-stimulated endocytosis is inhibited by a dominant-negative form of dynamin, or a dominant-negative hub fragment of clathrin heavy chain, indicating that it is mediated by clathrin. Correspondingly, overexpression of either mutant of ARF6 leads to an increase in the number of clathrin-coated pits at the apical plasma membrane. When ARF6-Q67L is overexpressed in the presence of the dominant-negative dynamin, the ARF6-Q67L colocalizes with clathrin and with IgA bound to its receptor. We conclude that ARF6 is an important modulator of clathrin-mediated endocytosis at the apical surface of epithelial cells.

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Colocalization of ARF6, clathrin, and IgA in cells expressing dynamin-I K44A. Left, Cells were infected with viruses for dynamin-I K44A (100 pfu/cell), and either ARF6–WT or ARF6–Q67L. Cells were treated for 5 min at 4°C with 120 μg/ml digitonin 10 to extract cytoplasmic clathrin, washed three times with cold PBS, fixed, and processed for immunofluorescence to detect ARF6 and endogenous clathrin. Right, Cells were infected with viruses for pIgR, dynamin-I K44A, and either β-gal, ARF6–WT, or ARF6–Q67L. IgA was bound to the AP PM for 1 h at 4°C. Cells were warmed to 15°C for 10 min. Unpermeabilized cells were stained for IgA, then cells were extracted with digitonin, fixed, permeabilized, and ARF6 stained.
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Figure 5: Colocalization of ARF6, clathrin, and IgA in cells expressing dynamin-I K44A. Left, Cells were infected with viruses for dynamin-I K44A (100 pfu/cell), and either ARF6–WT or ARF6–Q67L. Cells were treated for 5 min at 4°C with 120 μg/ml digitonin 10 to extract cytoplasmic clathrin, washed three times with cold PBS, fixed, and processed for immunofluorescence to detect ARF6 and endogenous clathrin. Right, Cells were infected with viruses for pIgR, dynamin-I K44A, and either β-gal, ARF6–WT, or ARF6–Q67L. IgA was bound to the AP PM for 1 h at 4°C. Cells were warmed to 15°C for 10 min. Unpermeabilized cells were stained for IgA, then cells were extracted with digitonin, fixed, permeabilized, and ARF6 stained.

Mentions: We next investigated if ARF6 is present, even transiently, in coated pits by trapping ARF6 in the deeply invaginated coated pits induced by dynamin-I K44A 9. As shown in Fig. 5, there was significant colocalization of ARF6–WT and ARF6–Q67L with endogenous clathrin at the AP PM of cells expressing dynamin-I K44A. This indicates that ARF6 may normally cycle through coated pits, and when coated pit pinching-off is prevented, a significant fraction of the ARF6–WT or ARF6–Q67L can be trapped.


ADP-ribosylation factor 6 and endocytosis at the apical surface of Madin-Darby canine kidney cells.

Altschuler Y, Liu S, Katz L, Tang K, Hardy S, Brodsky F, Apodaca G, Mostov K - J. Cell Biol. (1999)

Colocalization of ARF6, clathrin, and IgA in cells expressing dynamin-I K44A. Left, Cells were infected with viruses for dynamin-I K44A (100 pfu/cell), and either ARF6–WT or ARF6–Q67L. Cells were treated for 5 min at 4°C with 120 μg/ml digitonin 10 to extract cytoplasmic clathrin, washed three times with cold PBS, fixed, and processed for immunofluorescence to detect ARF6 and endogenous clathrin. Right, Cells were infected with viruses for pIgR, dynamin-I K44A, and either β-gal, ARF6–WT, or ARF6–Q67L. IgA was bound to the AP PM for 1 h at 4°C. Cells were warmed to 15°C for 10 min. Unpermeabilized cells were stained for IgA, then cells were extracted with digitonin, fixed, permeabilized, and ARF6 stained.
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Figure 5: Colocalization of ARF6, clathrin, and IgA in cells expressing dynamin-I K44A. Left, Cells were infected with viruses for dynamin-I K44A (100 pfu/cell), and either ARF6–WT or ARF6–Q67L. Cells were treated for 5 min at 4°C with 120 μg/ml digitonin 10 to extract cytoplasmic clathrin, washed three times with cold PBS, fixed, and processed for immunofluorescence to detect ARF6 and endogenous clathrin. Right, Cells were infected with viruses for pIgR, dynamin-I K44A, and either β-gal, ARF6–WT, or ARF6–Q67L. IgA was bound to the AP PM for 1 h at 4°C. Cells were warmed to 15°C for 10 min. Unpermeabilized cells were stained for IgA, then cells were extracted with digitonin, fixed, permeabilized, and ARF6 stained.
Mentions: We next investigated if ARF6 is present, even transiently, in coated pits by trapping ARF6 in the deeply invaginated coated pits induced by dynamin-I K44A 9. As shown in Fig. 5, there was significant colocalization of ARF6–WT and ARF6–Q67L with endogenous clathrin at the AP PM of cells expressing dynamin-I K44A. This indicates that ARF6 may normally cycle through coated pits, and when coated pit pinching-off is prevented, a significant fraction of the ARF6–WT or ARF6–Q67L can be trapped.

Bottom Line: Surprisingly, overexpression of the mutant ARF6-T27N, which is predicted to be in the GDP-bound form, also stimulated apical endocytosis, though to a lesser extent.ARF6-stimulated endocytosis is inhibited by a dominant-negative form of dynamin, or a dominant-negative hub fragment of clathrin heavy chain, indicating that it is mediated by clathrin.When ARF6-Q67L is overexpressed in the presence of the dominant-negative dynamin, the ARF6-Q67L colocalizes with clathrin and with IgA bound to its receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, University of California, San Francisco, California 94143-0452, USA. yoram@itsa.ucsf.edu

ABSTRACT
We report that the small GTPase, ADP-ribosylation factor 6 (ARF6), is present only on the apical surface of polarized MDCK epithelial cells. Overexpression of a mutant of ARF6, ARF6-Q67L, which is predicted to be in the GTP-bound form, stimulates endocytosis exclusively at this surface. Surprisingly, overexpression of the mutant ARF6-T27N, which is predicted to be in the GDP-bound form, also stimulated apical endocytosis, though to a lesser extent. ARF6-stimulated endocytosis is inhibited by a dominant-negative form of dynamin, or a dominant-negative hub fragment of clathrin heavy chain, indicating that it is mediated by clathrin. Correspondingly, overexpression of either mutant of ARF6 leads to an increase in the number of clathrin-coated pits at the apical plasma membrane. When ARF6-Q67L is overexpressed in the presence of the dominant-negative dynamin, the ARF6-Q67L colocalizes with clathrin and with IgA bound to its receptor. We conclude that ARF6 is an important modulator of clathrin-mediated endocytosis at the apical surface of epithelial cells.

Show MeSH
Related in: MedlinePlus