Characterization of Coated Vesicles that Participate in Endocytic Recycling

Peters, Peter J.; Gao, Minggeng; Gaschet, Joëlle; Ambach, Andreas; Donselaar, Elly van; Traverse, John F.; Bos, Erik; Wolffe, Elizabeth J.; Hsu, Victor

doi:10.1034/j.1600-0854.2001.21204.x

Cited by 24 publications

(13 citation statements)

References 50 publications

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“…2A) or with makers of the Golgi, the endoplamic reticulum, or lysosomes (data not shown). Thus ARF6 does not localize to classical early endosomes in LOX cells as described in Chinese hamster ovary (CHO), human embryonic kidney 293 (HEK293), PC12, and Madin-Darby canine kidney (MDCK) cells (9,(15)(16)(17) but to the tubular endosomal compartment that has previously been described in HeLa cells (18). In addition, endogenous ARF6 also localized to membrane protrusions that extended into areas of gelatin degradation (Fig.…”

Section: An Experimental System To Visualize Melanoma Cell Invasionmentioning

confidence: 96%

ADP-ribosylation factor 6 regulates tumor cell invasion through the activation of the MEK/ERK signaling pathway

Tague

Muralidharan

D’Souza‐Schorey

2004

Proc. Natl. Acad. Sci. U.S.A.

156

170

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Tumor cell invasion through the extracellular matrix is accompanied by the formation of invadopodia, which are actin-rich protrusions at the adherent surface of cells at sites of extracellular matrix degradation. Using the invasive human melanoma cell line LOX as a model system, we demonstrate that the ADP-ribosylation factor 6 (ARF6) GTPase is an important regulator of invadopodia formation and cell invasion. We show that ARF6 localizes to invadopodia of LOX cells. Sustained activation of ARF6 significantly enhances the invasive capacity of melanoma as well as breast tumor cell lines, whereas dominant negative ARF6 abolishes basal cell invasive capacity as well as invasion induced by growth factors. Furthermore, using biochemical assays, we show that enhanced invasive capacity is accompanied by the activation of endogenous ARF6. Finally, we provide evidence that ARF6-enhanced melanoma cell invasion depends on the activation of the extracellular signalregulated kinase (ERK), and that the ARF6 GTPase cycle regulates ERK activation. This study describes a vital role for ARF6 in melanoma cell invasion and documents a link between ARF6-mediated signaling and ERK activation.A n important characteristic of metastasizing cells is their ability to degrade and invade the extracellular matrix. Matrix degradation and cell invasion also occur during normal physiological processes, such as development and differentiation (1). The process of cell invasion is tightly regulated by a number of cell-signaling proteins, such as tyrosine kinases, Ras-related GTPases, and mitogen-activated protein kinase (MAPK) family proteins (2, 3). As an invading cell moves through the extracellular matrix, it extends actin-rich membrane protrusions into the matrix. These protrusions, called invadopodia, contain a number of actin-binding proteins and recruit various proteinases, including matrix metalloproteinases and serine proteases, which degrade matrix proteins at sites of cell invasion (4, 5). Studies on breast cancer and melanoma progression have shown that there appears to be a direct correlation between the ability of cells to form invadopodia and degrade matrix and the cells' invasive potential as measured by in vitro and in vivo assays for motility and invasion (4, 6, 7).ADP-ribosylation factor 6 (ARF6) is a member of the Ras superfamily of small GTPases, and like most GTPases, ARF6 alternates between its active GTP-bound and inactive GDP-bound conformations. The ARF6 GTPase cycle has been shown to regulate endosome membrane trafficking, regulated exocytosis, and actin remodeling at the cell surface (8). These processes are important for controlling cell shape changes and can impinge on the acquisition of an invasive phenotype. In fact, previous work in our laboratory has shown that ARF6 promotes cell migration in epithelial cells by facilitating adherens junction disassembly through its effect on endocytosis (of adhesion molecules) and by inducing peripheral actin rearrangements (9). In addition, Santy and Casanova (10) have shown that...

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Section: An Experimental System To Visualize Melanoma Cell Invasionmentioning

confidence: 96%

ADP-ribosylation factor 6 regulates tumor cell invasion through the activation of the MEK/ERK signaling pathway

Tague

Muralidharan

D’Souza‐Schorey

2004

Proc. Natl. Acad. Sci. U.S.A.

156

170

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“…Thus it is possible that Arf1 and Arf6 have a more generic and widespread function in the regulation of apical endocytosis in the proximal tubule. Finally, another possibility is that Arf1 may have a role in early endosome trafficking (25), while Arf6 could be involved in the function of recycling endosomes (45). Both populations of early and recycling endosomes are present in apical pole of proximal tubules and cannot be distinguished by immunofluorescence analysis (36).…”

Section: Discussionmentioning

confidence: 99%

“…It is generally accepted that Arf1 is involved in the formation of Golgi-derived COP-coated vesicles, is an important regulator of the intra-Golgi or Golgiendoplasmic reticulum vesicular trafficking and thus is involved in regulation of the exocytotic pathway (48,51). In contrast, Arf6 has been implicated in coat formation on endosome-derived carrier vesicles (45,54) and in actin cytoskeleton rearrangement (15). It appears to be a critical regulator of endocytotic pathways (16,42).…”

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confidence: 99%

Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ

Annan

Brown

Breton

et al. 2004

American Journal of Physiology-Cell Physiology

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ADP-ribosylation factors (Arfs) are small GTPases that regulate vesicular trafficking in exo- and endocytotic pathways. As a first step in understanding the role of Arfs in renal physiology, immunocytochemistry and Western blotting were performed to characterize the expression and targeting of Arf1 and Arf6 in epithelial cells in situ. Arf1 and Arf6 were associated with apical membranes and subapical vesicles in proximal tubules, where they colocalized with megalin. Arf1 was also apically expressed in the distal tubule, connecting segment, and collecting duct (CD). Arf1 was abundant in intercalated cells (IC) and colocalized with V-ATPase in A-IC (apical) and B-IC (apical and/or basolateral). In contrast, Arf6 was associated exclusively with basolateral membranes and vesicles in the CD. In the medulla, basolateral Arf6 was detectable mainly in A-IC. Expression in principal cells became weaker throughout the outer medulla, and Arf6 was not detectable in principal cells in the inner medulla. In some kidney epithelial cells Arf1 but not Arf6 was also targeted to a perinuclear patch, where it colocalized with TGN38, a marker of the trans-Golgi network. Quantitative Western blotting showed that expression of endogenous Arf1 was 26–180 times higher than Arf6. These data indicate that Arf GTPases are expressed and targeted in a cell- and membrane-specific pattern in kidney epithelial cells in situ. The results provide a framework on which to base and interpret future studies on the role of Arf GTPases in the multitude of cellular trafficking events that occur in renal tubular epithelial cells.

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“…For example, Arf1 is involved in regulating ER (endoplasmic reticulum)-Golgi trafficking [24,25], whereas Arf6 is involved in the regulation of endocytosis [26]. In particular, Arf6 has been implicated in endocytosis by regulating: (i) actin cytoskeleton remodelling [27]; (ii) lipid modifications and rearrangements [28]; and (iii) carrier vesicle coat formation [29,30]. While the role of Arf1 in COPI (coatamer protein I) coat recruitment has been well documented, the role of Arf6 in this process is just emerging.…”

Section: Arf-family Small Gtpases and Their Gefsmentioning

confidence: 99%

The V-ATPase a2-subunit as a putative endosomal pH-sensor

Marshansky

2007

Biochemical Society Transactions

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V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

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Characterization of Coated Vesicles that Participate in Endocytic Recycling

Cited by 24 publications

References 50 publications

ADP-ribosylation factor 6 regulates tumor cell invasion through the activation of the MEK/ERK signaling pathway

ADP-ribosylation factor 6 regulates tumor cell invasion through the activation of the MEK/ERK signaling pathway

Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ

The V-ATPase a2-subunit as a putative endosomal pH-sensor

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