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ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

D'Souza-Schorey C, van Donselaar E, Hsu VW, Yang C, Stahl PD, Peters PJ - J. Cell Biol. (1998)

Bottom Line: We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments.Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system.Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

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(A and B) Biotinylation of the surface of cells expressing ARF6(Q67L). Cells were either infected with vector virus  and incubated with HRP for 10 min (A) or infected with recombinant virus encoding ARF6(Q67L) (B), and then incubated with  sulfo-NHS-biotin and processed for immunolabeling. Sections  were labeled first with an antibiotin antibody followed by protein  A–gold (15 nm), and then with an antibody against HRP followed by protein A–gold (5 nm, A), or with antibody against  ARF6 followed by protein A–gold (10 nm, B). (A) Only the  plasma membrane and cross-sections across membrane invaginations were labeled with biotin. Early endocytic structures labeled  with HRP were inaccessible to biotin. (B) In ARF6(Q67L)- expressing cells, plasma membrane invaginations were accessible  to biotin (arrowheads) and thus are in continuity with the extracellular milieu. i, invaginations; p, plasma membrane; e, endosomes. Bar, 200 nm.
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Figure 2: (A and B) Biotinylation of the surface of cells expressing ARF6(Q67L). Cells were either infected with vector virus and incubated with HRP for 10 min (A) or infected with recombinant virus encoding ARF6(Q67L) (B), and then incubated with sulfo-NHS-biotin and processed for immunolabeling. Sections were labeled first with an antibiotin antibody followed by protein A–gold (15 nm), and then with an antibody against HRP followed by protein A–gold (5 nm, A), or with antibody against ARF6 followed by protein A–gold (10 nm, B). (A) Only the plasma membrane and cross-sections across membrane invaginations were labeled with biotin. Early endocytic structures labeled with HRP were inaccessible to biotin. (B) In ARF6(Q67L)- expressing cells, plasma membrane invaginations were accessible to biotin (arrowheads) and thus are in continuity with the extracellular milieu. i, invaginations; p, plasma membrane; e, endosomes. Bar, 200 nm.

Mentions: On analysis of the distribution of the ARF6(Q67L), the GTP-bound mutant, consistent with previous findings in HEK 293 cells, we observed that the mutant protein localized almost exclusively to discrete sites on the plasma membrane of CHO cells, in regions that exhibited numerous membrane folds that lacked clathrin-coated pits and to sac-like structures (see Table I). To determine whether these sac-like structures were cross-sections through plasma membrane folds or were internalized vesicular compartments, we tested for their accessibility to cell surface biotin. For these experiments, cells expressing ARF6(Q67L) were treated with sulpho-NHS-biotin, a biotinylating agent, at 4°C, fixed, and then double labeled with antibodies against ARF6 and biotin. Biotin couples to the entire accessible surface of the fibroblasts. In control cells, labeling for biotin was observed on the plasma membrane and in some invaginations along the cell periphery (Fig. 2 A). In cells expressing ARF6(Q67L), the membrane folds and sac-like structures that labeled positively for ARF6, also labeled with antibiotin antibodies (Fig. 2 B). This accessibility to surface biotinylation suggests that these structures were indeed sections through plasma membrane folds/invaginations and hence continuous with the extracellular environment.


ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

D'Souza-Schorey C, van Donselaar E, Hsu VW, Yang C, Stahl PD, Peters PJ - J. Cell Biol. (1998)

(A and B) Biotinylation of the surface of cells expressing ARF6(Q67L). Cells were either infected with vector virus  and incubated with HRP for 10 min (A) or infected with recombinant virus encoding ARF6(Q67L) (B), and then incubated with  sulfo-NHS-biotin and processed for immunolabeling. Sections  were labeled first with an antibiotin antibody followed by protein  A–gold (15 nm), and then with an antibody against HRP followed by protein A–gold (5 nm, A), or with antibody against  ARF6 followed by protein A–gold (10 nm, B). (A) Only the  plasma membrane and cross-sections across membrane invaginations were labeled with biotin. Early endocytic structures labeled  with HRP were inaccessible to biotin. (B) In ARF6(Q67L)- expressing cells, plasma membrane invaginations were accessible  to biotin (arrowheads) and thus are in continuity with the extracellular milieu. i, invaginations; p, plasma membrane; e, endosomes. Bar, 200 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140168&req=5

Figure 2: (A and B) Biotinylation of the surface of cells expressing ARF6(Q67L). Cells were either infected with vector virus and incubated with HRP for 10 min (A) or infected with recombinant virus encoding ARF6(Q67L) (B), and then incubated with sulfo-NHS-biotin and processed for immunolabeling. Sections were labeled first with an antibiotin antibody followed by protein A–gold (15 nm), and then with an antibody against HRP followed by protein A–gold (5 nm, A), or with antibody against ARF6 followed by protein A–gold (10 nm, B). (A) Only the plasma membrane and cross-sections across membrane invaginations were labeled with biotin. Early endocytic structures labeled with HRP were inaccessible to biotin. (B) In ARF6(Q67L)- expressing cells, plasma membrane invaginations were accessible to biotin (arrowheads) and thus are in continuity with the extracellular milieu. i, invaginations; p, plasma membrane; e, endosomes. Bar, 200 nm.
Mentions: On analysis of the distribution of the ARF6(Q67L), the GTP-bound mutant, consistent with previous findings in HEK 293 cells, we observed that the mutant protein localized almost exclusively to discrete sites on the plasma membrane of CHO cells, in regions that exhibited numerous membrane folds that lacked clathrin-coated pits and to sac-like structures (see Table I). To determine whether these sac-like structures were cross-sections through plasma membrane folds or were internalized vesicular compartments, we tested for their accessibility to cell surface biotin. For these experiments, cells expressing ARF6(Q67L) were treated with sulpho-NHS-biotin, a biotinylating agent, at 4°C, fixed, and then double labeled with antibodies against ARF6 and biotin. Biotin couples to the entire accessible surface of the fibroblasts. In control cells, labeling for biotin was observed on the plasma membrane and in some invaginations along the cell periphery (Fig. 2 A). In cells expressing ARF6(Q67L), the membrane folds and sac-like structures that labeled positively for ARF6, also labeled with antibiotin antibodies (Fig. 2 B). This accessibility to surface biotinylation suggests that these structures were indeed sections through plasma membrane folds/invaginations and hence continuous with the extracellular environment.

Bottom Line: We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments.Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system.Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

Show MeSH
Related in: MedlinePlus