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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) Localization of wild-type ARF6.  CHO cells expressing wild-type ARF6 were fixed and  processed for ultrathin cryosections. Sections were labeled with anti-ARF6 polyclonal antibody followed by  protein A–gold (10 nm). (A)  ARF6 localizes to the plasma  membrane and intracellular  vesicles. (B) At relatively  lower levels of expression,  ARF6 localizes predominantly to intracellular vesicles that are situated near the  TGN. i, invaginations; p,  plasma membrane; er, endoplasmic reticulum; m, mitochondria; G, Golgi complex;  TGN, trans-Golgi network; e,  endosome; c, centriole; n, nucleus. Bar, 200 nm.
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Figure 1: (A and B) Localization of wild-type ARF6. CHO cells expressing wild-type ARF6 were fixed and processed for ultrathin cryosections. Sections were labeled with anti-ARF6 polyclonal antibody followed by protein A–gold (10 nm). (A) ARF6 localizes to the plasma membrane and intracellular vesicles. (B) At relatively lower levels of expression, ARF6 localizes predominantly to intracellular vesicles that are situated near the TGN. i, invaginations; p, plasma membrane; er, endoplasmic reticulum; m, mitochondria; G, Golgi complex; TGN, trans-Golgi network; e, endosome; c, centriole; n, nucleus. Bar, 200 nm.

Mentions: In a previous study on the subcellular distribution of epitope-tagged ARF6 at the ultrastructural level in HEK 293 fibroblasts, ARF6 was shown to be present on the plasma membrane and on intracellular compartments (Peters et al., 1995). However, high levels of protein were expressed using the calcium phosphate method of transfection, followed by analysis of expressed proteins 48 h after transfection. In this study, we have investigated the distribution of wild-type ARF6 in CHO cells using the Sindbis virus expression system, which allows for protein detection at relatively shorter intervals after virus infection. Therefore, we could analyze the subcellular distribution of untagged ARF6 at varying levels of protein expression, by the technique of cryoimmunogold electron microscopy using antibodies directed against ARF6. At relatively high levels of ARF6 expression, implicit by higher amounts of gold labeling, the plasma membrane exhibited tortuous invaginations resembling membrane alterations seen in cells expressing the GTPase-defective mutant, ARF6(Q67L). 50–60% of gold label was seen along these invaginations (Fig. 1 A, Table I). Gold label was also seen on intracellular vesicles, some of which were peripherally distributed while most were localized around the pericentriolar region of the cell (see below). About 20% of the ARF6 label was found scattered throughout the cytosol. Interestingly, in cells expressing ARF6 at the lowest detectable level, gold label was seen predominantly on vesicles around the centriole, juxtaposed to the TGN (Fig. 1 B, Table I), whereas <15% of the label was detected on the plasma membrane. Also, the plasma membrane of these cells was relatively unperturbed compared with the surface of cells expressing relatively higher levels of ARF6. Thus, ARF6 localization in CHO cells is predominantly intracellular; with increased expression, the cell phenotype resembles that observed in cells expressing the GTPase-defective mutant, most likely due to sequestration of factors required for GTP hydrolysis by overexpressed protein. The ARF6-labeled intracellular vesicles were small and ranged in diameter from 60 to 200 nm and resembled the recycling endosomal compartment previously described in CHO cells (Yashimoro et al., 1984; Johnson et al., 1996). This compartment is distinct from the TGN, late endosomes, or lysosomes. These findings were unexpected since a previous study that used subcellular fractionation procedures, had found endogenous ARF6 to be localized exclusively to the plasma membrane of CHO cells (Cavenagh et al., 1996). This point is addressed further below.


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) Localization of wild-type ARF6.  CHO cells expressing wild-type ARF6 were fixed and  processed for ultrathin cryosections. Sections were labeled with anti-ARF6 polyclonal antibody followed by  protein A–gold (10 nm). (A)  ARF6 localizes to the plasma  membrane and intracellular  vesicles. (B) At relatively  lower levels of expression,  ARF6 localizes predominantly to intracellular vesicles that are situated near the  TGN. i, invaginations; p,  plasma membrane; er, endoplasmic reticulum; m, mitochondria; G, Golgi complex;  TGN, trans-Golgi network; e,  endosome; c, centriole; n, nucleus. Bar, 200 nm.
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Related In: Results  -  Collection

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Figure 1: (A and B) Localization of wild-type ARF6. CHO cells expressing wild-type ARF6 were fixed and processed for ultrathin cryosections. Sections were labeled with anti-ARF6 polyclonal antibody followed by protein A–gold (10 nm). (A) ARF6 localizes to the plasma membrane and intracellular vesicles. (B) At relatively lower levels of expression, ARF6 localizes predominantly to intracellular vesicles that are situated near the TGN. i, invaginations; p, plasma membrane; er, endoplasmic reticulum; m, mitochondria; G, Golgi complex; TGN, trans-Golgi network; e, endosome; c, centriole; n, nucleus. Bar, 200 nm.
Mentions: In a previous study on the subcellular distribution of epitope-tagged ARF6 at the ultrastructural level in HEK 293 fibroblasts, ARF6 was shown to be present on the plasma membrane and on intracellular compartments (Peters et al., 1995). However, high levels of protein were expressed using the calcium phosphate method of transfection, followed by analysis of expressed proteins 48 h after transfection. In this study, we have investigated the distribution of wild-type ARF6 in CHO cells using the Sindbis virus expression system, which allows for protein detection at relatively shorter intervals after virus infection. Therefore, we could analyze the subcellular distribution of untagged ARF6 at varying levels of protein expression, by the technique of cryoimmunogold electron microscopy using antibodies directed against ARF6. At relatively high levels of ARF6 expression, implicit by higher amounts of gold labeling, the plasma membrane exhibited tortuous invaginations resembling membrane alterations seen in cells expressing the GTPase-defective mutant, ARF6(Q67L). 50–60% of gold label was seen along these invaginations (Fig. 1 A, Table I). Gold label was also seen on intracellular vesicles, some of which were peripherally distributed while most were localized around the pericentriolar region of the cell (see below). About 20% of the ARF6 label was found scattered throughout the cytosol. Interestingly, in cells expressing ARF6 at the lowest detectable level, gold label was seen predominantly on vesicles around the centriole, juxtaposed to the TGN (Fig. 1 B, Table I), whereas <15% of the label was detected on the plasma membrane. Also, the plasma membrane of these cells was relatively unperturbed compared with the surface of cells expressing relatively higher levels of ARF6. Thus, ARF6 localization in CHO cells is predominantly intracellular; with increased expression, the cell phenotype resembles that observed in cells expressing the GTPase-defective mutant, most likely due to sequestration of factors required for GTP hydrolysis by overexpressed protein. The ARF6-labeled intracellular vesicles were small and ranged in diameter from 60 to 200 nm and resembled the recycling endosomal compartment previously described in CHO cells (Yashimoro et al., 1984; Johnson et al., 1996). This compartment is distinct from the TGN, late endosomes, or lysosomes. These findings were unexpected since a previous study that used subcellular fractionation procedures, had found endogenous ARF6 to be localized exclusively to the plasma membrane of CHO cells (Cavenagh et al., 1996). This point is addressed further below.

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