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ADP-ribosylation factor 6 regulates a novel plasma membrane recycling pathway.

Radhakrishna H, Donaldson JG - J. Cell Biol. (1997)

Bottom Line: Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively.Thus, the ARF6 GTP cycle regulates this membrane traffic pathway.The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.

ABSTRACT
ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and to a tubulovesicular compartment in its GDP state in HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions. Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and shifted the distribution of ARF6 to a tubular membrane compartment emanating from the juxtanuclear region of cells, which resembled the compartment where the GTP-binding defective mutant of ARF6 localized. This membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2 cytoplasmic targeting sequences, such as the IL-2 receptor alpha subunit Tac. ARF6 and surface Tac moved into this compartment and back out to the PM in the absence of pharmacologic treatment. Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively. Thus, the ARF6 GTP cycle regulates this membrane traffic pathway. The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.

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Endogenous MHC-I localizes to the tubular compartment with or without ARF6 overexpression. HeLa cells were  transfected with ARF6 plasmid and then incubated in the absence (Unt) or presence (CD) of 1 μM CD for 30 min at 37°C,  and they were fixed and processed for immunofluorescence localization of MHC-I using mouse anti–MHC-I antibodies and for  overexpression of ARF6 using antibody to ARF6. Anti–MHC-I  was detected with sequential FITC-labeled goat anti–mouse and  FITC-labeled donkey anti–goat antibodies, while anti-ARF6 was  detected with rhodamine-conjugated donkey anti–rabbit antibody. The tubular structures (arrowheads) radiating from the  perinuclear region, labeled with anti–MHC-I antibodies, become  more apparent after CD treatment, and are observed in both  ARF6-transfected and untransfected cells.
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Figure 4: Endogenous MHC-I localizes to the tubular compartment with or without ARF6 overexpression. HeLa cells were transfected with ARF6 plasmid and then incubated in the absence (Unt) or presence (CD) of 1 μM CD for 30 min at 37°C, and they were fixed and processed for immunofluorescence localization of MHC-I using mouse anti–MHC-I antibodies and for overexpression of ARF6 using antibody to ARF6. Anti–MHC-I was detected with sequential FITC-labeled goat anti–mouse and FITC-labeled donkey anti–goat antibodies, while anti-ARF6 was detected with rhodamine-conjugated donkey anti–rabbit antibody. The tubular structures (arrowheads) radiating from the perinuclear region, labeled with anti–MHC-I antibodies, become more apparent after CD treatment, and are observed in both ARF6-transfected and untransfected cells.

Mentions: We previously observed that inhibition of actin polymerization, by treatment of cells with CD, inhibited the AlF-induced protrusions in HeLa cells overexpressing ARF6, and additionally, we noticed that CD treatment shifted the distribution of ARF6 to an internal membrane compartment (Radhakrishna et al., 1996). To further investigate this response to CD, HeLa cells transfected with plasmid encoding ARF6 were treated with CD for 30 min. The cells were then fixed and immunolabeled with polyclonal anti-ARF6 antibodies, and the actin filaments were labeled with Oregon green–conjugated phalloidin. In untreated cells, ARF6 localized along the PM at the peripheral edge of cells and in internal structures near the nucleus. Fine tubular elements were observed emanating out of the juxtanuclear region towards the peripheral edge of some cells (Fig. 1, untreated). The extent to which these tubular elements were observed in untreated cells varied (for example, see tubular elements in Figs. 4 and 5); often they were difficult to discern over the cell surface labeling.


ADP-ribosylation factor 6 regulates a novel plasma membrane recycling pathway.

Radhakrishna H, Donaldson JG - J. Cell Biol. (1997)

Endogenous MHC-I localizes to the tubular compartment with or without ARF6 overexpression. HeLa cells were  transfected with ARF6 plasmid and then incubated in the absence (Unt) or presence (CD) of 1 μM CD for 30 min at 37°C,  and they were fixed and processed for immunofluorescence localization of MHC-I using mouse anti–MHC-I antibodies and for  overexpression of ARF6 using antibody to ARF6. Anti–MHC-I  was detected with sequential FITC-labeled goat anti–mouse and  FITC-labeled donkey anti–goat antibodies, while anti-ARF6 was  detected with rhodamine-conjugated donkey anti–rabbit antibody. The tubular structures (arrowheads) radiating from the  perinuclear region, labeled with anti–MHC-I antibodies, become  more apparent after CD treatment, and are observed in both  ARF6-transfected and untransfected cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139810&req=5

Figure 4: Endogenous MHC-I localizes to the tubular compartment with or without ARF6 overexpression. HeLa cells were transfected with ARF6 plasmid and then incubated in the absence (Unt) or presence (CD) of 1 μM CD for 30 min at 37°C, and they were fixed and processed for immunofluorescence localization of MHC-I using mouse anti–MHC-I antibodies and for overexpression of ARF6 using antibody to ARF6. Anti–MHC-I was detected with sequential FITC-labeled goat anti–mouse and FITC-labeled donkey anti–goat antibodies, while anti-ARF6 was detected with rhodamine-conjugated donkey anti–rabbit antibody. The tubular structures (arrowheads) radiating from the perinuclear region, labeled with anti–MHC-I antibodies, become more apparent after CD treatment, and are observed in both ARF6-transfected and untransfected cells.
Mentions: We previously observed that inhibition of actin polymerization, by treatment of cells with CD, inhibited the AlF-induced protrusions in HeLa cells overexpressing ARF6, and additionally, we noticed that CD treatment shifted the distribution of ARF6 to an internal membrane compartment (Radhakrishna et al., 1996). To further investigate this response to CD, HeLa cells transfected with plasmid encoding ARF6 were treated with CD for 30 min. The cells were then fixed and immunolabeled with polyclonal anti-ARF6 antibodies, and the actin filaments were labeled with Oregon green–conjugated phalloidin. In untreated cells, ARF6 localized along the PM at the peripheral edge of cells and in internal structures near the nucleus. Fine tubular elements were observed emanating out of the juxtanuclear region towards the peripheral edge of some cells (Fig. 1, untreated). The extent to which these tubular elements were observed in untreated cells varied (for example, see tubular elements in Figs. 4 and 5); often they were difficult to discern over the cell surface labeling.

Bottom Line: Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively.Thus, the ARF6 GTP cycle regulates this membrane traffic pathway.The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.

ABSTRACT
ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and to a tubulovesicular compartment in its GDP state in HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions. Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and shifted the distribution of ARF6 to a tubular membrane compartment emanating from the juxtanuclear region of cells, which resembled the compartment where the GTP-binding defective mutant of ARF6 localized. This membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2 cytoplasmic targeting sequences, such as the IL-2 receptor alpha subunit Tac. ARF6 and surface Tac moved into this compartment and back out to the PM in the absence of pharmacologic treatment. Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively. Thus, the ARF6 GTP cycle regulates this membrane traffic pathway. The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.

Show MeSH
Related in: MedlinePlus