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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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CD treatment blocks the recycling of Tac from the tubular compartment back out to the PM. Anti-Tac antibodies  bound to the surface of cells expressing ARF6 and Tac were  loaded into the tubular compartment by incubation with 1 μM  CD at 37°C for 30 min. The cells were chilled to 4°C, rinsed with  low pH buffer to remove the remaining surface anti-Tac antibodies, and then either fixed immediately (Load) and assessed for  the Tac antibody loaded, or warmed to 37°C for 30 min in the absence or presence of CD before fixation. Tac antibody that reappeared on the cell surface was detected by incubation with fluorescently labeled secondary antibodies in the absence of detergent  permeabilization (Surface Reappearance); ARF6 was subsequently  localized in these cells after permeabilization.
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Figure 7: CD treatment blocks the recycling of Tac from the tubular compartment back out to the PM. Anti-Tac antibodies bound to the surface of cells expressing ARF6 and Tac were loaded into the tubular compartment by incubation with 1 μM CD at 37°C for 30 min. The cells were chilled to 4°C, rinsed with low pH buffer to remove the remaining surface anti-Tac antibodies, and then either fixed immediately (Load) and assessed for the Tac antibody loaded, or warmed to 37°C for 30 min in the absence or presence of CD before fixation. Tac antibody that reappeared on the cell surface was detected by incubation with fluorescently labeled secondary antibodies in the absence of detergent permeabilization (Surface Reappearance); ARF6 was subsequently localized in these cells after permeabilization.

Mentions: Having demonstrated that the low pH wash could efficiently remove surface-bound Tac antibody, we examined whether internalized Tac recycled from the tubular compartment back to the PM. Cells were labeled with Tac antibody at 4°C and then warmed to 37°C in the presence of CD to accumulate Tac antibody in the tubular compartment. Antibodies remaining at the cell surface were then removed with a low pH wash at 4°C (as in Fig. 6), leaving only the internalized Tac antibody (Fig. 7, Load).


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

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

CD treatment blocks the recycling of Tac from the tubular compartment back out to the PM. Anti-Tac antibodies  bound to the surface of cells expressing ARF6 and Tac were  loaded into the tubular compartment by incubation with 1 μM  CD at 37°C for 30 min. The cells were chilled to 4°C, rinsed with  low pH buffer to remove the remaining surface anti-Tac antibodies, and then either fixed immediately (Load) and assessed for  the Tac antibody loaded, or warmed to 37°C for 30 min in the absence or presence of CD before fixation. Tac antibody that reappeared on the cell surface was detected by incubation with fluorescently labeled secondary antibodies in the absence of detergent  permeabilization (Surface Reappearance); ARF6 was subsequently  localized in these cells after permeabilization.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: CD treatment blocks the recycling of Tac from the tubular compartment back out to the PM. Anti-Tac antibodies bound to the surface of cells expressing ARF6 and Tac were loaded into the tubular compartment by incubation with 1 μM CD at 37°C for 30 min. The cells were chilled to 4°C, rinsed with low pH buffer to remove the remaining surface anti-Tac antibodies, and then either fixed immediately (Load) and assessed for the Tac antibody loaded, or warmed to 37°C for 30 min in the absence or presence of CD before fixation. Tac antibody that reappeared on the cell surface was detected by incubation with fluorescently labeled secondary antibodies in the absence of detergent permeabilization (Surface Reappearance); ARF6 was subsequently localized in these cells after permeabilization.
Mentions: Having demonstrated that the low pH wash could efficiently remove surface-bound Tac antibody, we examined whether internalized Tac recycled from the tubular compartment back to the PM. Cells were labeled with Tac antibody at 4°C and then warmed to 37°C in the presence of CD to accumulate Tac antibody in the tubular compartment. Antibodies remaining at the cell surface were then removed with a low pH wash at 4°C (as in Fig. 6), leaving only the internalized Tac antibody (Fig. 7, Load).

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