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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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ARF6 recruits clathrin to the AP PM. A, Cells were either uninfected or infected with adenovirus for ARF6–WT or mutants, then stained for clathrin. Confocal sections are at the level of the arrowhead in Fig. 1 A. B, Examples of shallow and invaginated clathrin-coated pits, as seen by EM. C, Total coated pits/μm AP PM. D, Shallow coated pits. E, Invaginated coated pits.
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Figure 4: ARF6 recruits clathrin to the AP PM. A, Cells were either uninfected or infected with adenovirus for ARF6–WT or mutants, then stained for clathrin. Confocal sections are at the level of the arrowhead in Fig. 1 A. B, Examples of shallow and invaginated clathrin-coated pits, as seen by EM. C, Total coated pits/μm AP PM. D, Shallow coated pits. E, Invaginated coated pits.

Mentions: As ARF6-stimulated endocytosis is clathrin-mediated, we asked if ARF6 overexpression recruited clathrin to the AP PM. Confocal sections were taken at the level indicated by the solid arrowhead in Fig. 1 A. Cells expressing ARF6–WT have a small increase in AP PM clathrin (Fig. 4 A). Overexpression of either ARF6–Q67L or ARF6–T27N gave a greater increase in clathrin.


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)

ARF6 recruits clathrin to the AP PM. A, Cells were either uninfected or infected with adenovirus for ARF6–WT or mutants, then stained for clathrin. Confocal sections are at the level of the arrowhead in Fig. 1 A. B, Examples of shallow and invaginated clathrin-coated pits, as seen by EM. C, Total coated pits/μm AP PM. D, Shallow coated pits. E, Invaginated coated pits.
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Related In: Results  -  Collection

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Figure 4: ARF6 recruits clathrin to the AP PM. A, Cells were either uninfected or infected with adenovirus for ARF6–WT or mutants, then stained for clathrin. Confocal sections are at the level of the arrowhead in Fig. 1 A. B, Examples of shallow and invaginated clathrin-coated pits, as seen by EM. C, Total coated pits/μm AP PM. D, Shallow coated pits. E, Invaginated coated pits.
Mentions: As ARF6-stimulated endocytosis is clathrin-mediated, we asked if ARF6 overexpression recruited clathrin to the AP PM. Confocal sections were taken at the level indicated by the solid arrowhead in Fig. 1 A. Cells expressing ARF6–WT have a small increase in AP PM clathrin (Fig. 4 A). Overexpression of either ARF6–Q67L or ARF6–T27N gave a greater increase in clathrin.

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