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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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Effect of ARF6 on AP endocytosis. A, Effect on rate of endocytosis. B, Effects of varying expression level of ARF6 mutants on AP endocytosis. Cells were infected with the indicated quantity of virus. Endocytosis was assayed for 5 min at 37°C, 1 μl of virus ∼25 pfu/cell. C, Effect of coexpression of ARF6 mutants. Cells were either infected with virus for ARF6–Q67L plus virus for β-gal (to keep the total virus constant), ARF6–T27N plus β-gal virus, or doubly infected with viruses encoding both ARF6 mutants. To improve temporal resolution, this experiment was conducted at 32°C. D, Effect of ARF6 on endocytosis of endocytosis-deficient mutant pIgR. Endocytosis of pIgR with both cytoplasmic Tyr mutated to Ala was assayed in control cells or cells expressing ARF6–WT or mutants. In this experiment only, normal MDCK cells stably expressing the pIgR-664A, 734A, or wild-type pIgR were coinfected with the virus for β-gal or ARF6 mutant, plus 70–80 pfu/cell of adenovirus encoding the transactivator. The level of expression of the ARF6 mutant proteins was comparable to other experiments. E, Cells were doubly infected with ARF6–Q67L and the indicated quantity of virus for dynamin-I K44A. Endocytosis over 5 min was measured. Expression of dynamin did not alter expression of the ARF6, as assayed by immunoblot (not shown).
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Figure 2: Effect of ARF6 on AP endocytosis. A, Effect on rate of endocytosis. B, Effects of varying expression level of ARF6 mutants on AP endocytosis. Cells were infected with the indicated quantity of virus. Endocytosis was assayed for 5 min at 37°C, 1 μl of virus ∼25 pfu/cell. C, Effect of coexpression of ARF6 mutants. Cells were either infected with virus for ARF6–Q67L plus virus for β-gal (to keep the total virus constant), ARF6–T27N plus β-gal virus, or doubly infected with viruses encoding both ARF6 mutants. To improve temporal resolution, this experiment was conducted at 32°C. D, Effect of ARF6 on endocytosis of endocytosis-deficient mutant pIgR. Endocytosis of pIgR with both cytoplasmic Tyr mutated to Ala was assayed in control cells or cells expressing ARF6–WT or mutants. In this experiment only, normal MDCK cells stably expressing the pIgR-664A, 734A, or wild-type pIgR were coinfected with the virus for β-gal or ARF6 mutant, plus 70–80 pfu/cell of adenovirus encoding the transactivator. The level of expression of the ARF6 mutant proteins was comparable to other experiments. E, Cells were doubly infected with ARF6–Q67L and the indicated quantity of virus for dynamin-I K44A. Endocytosis over 5 min was measured. Expression of dynamin did not alter expression of the ARF6, as assayed by immunoblot (not shown).

Mentions: To examine the effects of ARF6 on endocytosis, we used 125I-IgA as an endocytic marker, which can be endocytosed via the polymeric immunoglobulin receptor (pIgR) at either PM. Strikingly, overexpression of either ARF6–Q67L or ARF6–T27N stimulated endocytosis of 125I-IgA from the AP PM (Fig. 2 A). ARF6–Q67L and ARF6–T27N increased endocytosis during the first minute by ∼3-fold or 1.5-fold, respectively. In contrast, AP endocytosis of IgA in cells overexpressing ARF6–WT did not markedly differ from control cells infected with a virus encoding β-gal or uninfected cells. Overexpression of ARF6–WT or either mutant did not significantly alter the rate or extent of BL endocytosis, recycling back to the AP PM, BL to AP transcytosis, or BL to BL recycling (our unpublished data). Therefore, only AP endocytosis was affected by ARF6.


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)

Effect of ARF6 on AP endocytosis. A, Effect on rate of endocytosis. B, Effects of varying expression level of ARF6 mutants on AP endocytosis. Cells were infected with the indicated quantity of virus. Endocytosis was assayed for 5 min at 37°C, 1 μl of virus ∼25 pfu/cell. C, Effect of coexpression of ARF6 mutants. Cells were either infected with virus for ARF6–Q67L plus virus for β-gal (to keep the total virus constant), ARF6–T27N plus β-gal virus, or doubly infected with viruses encoding both ARF6 mutants. To improve temporal resolution, this experiment was conducted at 32°C. D, Effect of ARF6 on endocytosis of endocytosis-deficient mutant pIgR. Endocytosis of pIgR with both cytoplasmic Tyr mutated to Ala was assayed in control cells or cells expressing ARF6–WT or mutants. In this experiment only, normal MDCK cells stably expressing the pIgR-664A, 734A, or wild-type pIgR were coinfected with the virus for β-gal or ARF6 mutant, plus 70–80 pfu/cell of adenovirus encoding the transactivator. The level of expression of the ARF6 mutant proteins was comparable to other experiments. E, Cells were doubly infected with ARF6–Q67L and the indicated quantity of virus for dynamin-I K44A. Endocytosis over 5 min was measured. Expression of dynamin did not alter expression of the ARF6, as assayed by immunoblot (not shown).
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Related In: Results  -  Collection

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Figure 2: Effect of ARF6 on AP endocytosis. A, Effect on rate of endocytosis. B, Effects of varying expression level of ARF6 mutants on AP endocytosis. Cells were infected with the indicated quantity of virus. Endocytosis was assayed for 5 min at 37°C, 1 μl of virus ∼25 pfu/cell. C, Effect of coexpression of ARF6 mutants. Cells were either infected with virus for ARF6–Q67L plus virus for β-gal (to keep the total virus constant), ARF6–T27N plus β-gal virus, or doubly infected with viruses encoding both ARF6 mutants. To improve temporal resolution, this experiment was conducted at 32°C. D, Effect of ARF6 on endocytosis of endocytosis-deficient mutant pIgR. Endocytosis of pIgR with both cytoplasmic Tyr mutated to Ala was assayed in control cells or cells expressing ARF6–WT or mutants. In this experiment only, normal MDCK cells stably expressing the pIgR-664A, 734A, or wild-type pIgR were coinfected with the virus for β-gal or ARF6 mutant, plus 70–80 pfu/cell of adenovirus encoding the transactivator. The level of expression of the ARF6 mutant proteins was comparable to other experiments. E, Cells were doubly infected with ARF6–Q67L and the indicated quantity of virus for dynamin-I K44A. Endocytosis over 5 min was measured. Expression of dynamin did not alter expression of the ARF6, as assayed by immunoblot (not shown).
Mentions: To examine the effects of ARF6 on endocytosis, we used 125I-IgA as an endocytic marker, which can be endocytosed via the polymeric immunoglobulin receptor (pIgR) at either PM. Strikingly, overexpression of either ARF6–Q67L or ARF6–T27N stimulated endocytosis of 125I-IgA from the AP PM (Fig. 2 A). ARF6–Q67L and ARF6–T27N increased endocytosis during the first minute by ∼3-fold or 1.5-fold, respectively. In contrast, AP endocytosis of IgA in cells overexpressing ARF6–WT did not markedly differ from control cells infected with a virus encoding β-gal or uninfected cells. Overexpression of ARF6–WT or either mutant did not significantly alter the rate or extent of BL endocytosis, recycling back to the AP PM, BL to AP transcytosis, or BL to BL recycling (our unpublished data). Therefore, only AP endocytosis was affected by ARF6.

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