Localization and activation of the ARF6 GTPase during cleavage furrow ingression and cytokinesis

The ARF6 GTPase mediates cell shape changes in interphase cells through its effects on membrane cycling and actin remodeling. In this study, we focus our attention on the dynamics of cell division and present evidence supporting a novel role for ARF6 during cleavage furrow ingression and cytokinesis. We demonstrate that endogenous ARF6 redistributes during mitosis and concentrates near the cleavage furrow during telophase. Constitutively activated ARF6 localizes to the plasma membrane at the site of cleavage furrow ingression and midbody formation, and dominant negative ARF6 remains cytoplasmic. By using a novel pull-down assay for ARF6-GTP, we find an abrupt, but transient, increase in ARF6-GTP levels as cells progress through cytokinesis. Whereas high levels of expression of a GTPase-defective ARF6 mutant induce aberrant phenotypes in cells at cytokinesis, cells expressing low levels of ARF6 mutants do not display a significant mitotic delay or cytokinesis defect, presumably due to compensatory or redundant mechanisms that allow cytokinesis to proceed when the ARF6 GTPase cycle is disrupted. Finally, actin accumulation and phospholipid metabolism at the cleavage furrow are unchanged in cells expressing ARF6 mutants, suggesting that ARF6 may be involved in membrane remodeling during cytokinesis via effector pathways that are distinct from those operative in interphase cells.     

Arfaptin 2 regulates the aggregation of mutant huntingtin protein

Huntington's disease (HD) is an inherited neurodegenerative disorder. Here we demonstrate that expression of arfaptin 2/POR1 (partner of Rac1) in cultured cells induces the formation of pericentriolar and nuclear aggregates, which morphologically resemble mutant huntingtin aggregates characteristic of HD. Endogenous arfaptin 2 localizes to aggregates induced by expression of an abnormal amino-terminal fragment of huntingtin that contains polyglutamine (polyQ) expansions. A dominant inhibitory mutant of arfaptin 2 inhibits aggregation of mutant huntingtin, but not in the presence of proteasome inhibitors. Using cell-free biochemical assays, we show that arfaptin 2 inhibits proteasome activity. Finally, we show that expression of arfaptin 2 is increased at sites of neurodegeneration and the protein localizes to huntingtin aggregates in HD transgenic mouse brains. Our data suggest that arfaptin 2 is involved in regulating huntingtin protein aggregation, possibly by impairing proteasome function.     

Elevated phospholipase D activity in H-Ras- but not K-Ras-transformed cells by the synergistic action of RalA and ARF6

Phospholipase D (PLD) activity is elevated in response to the oncogenic stimulus of H-Ras but not K-Ras. H-Ras and K-Ras have been reported to localize to different membrane microdomains, with H-Ras localizing to caveolin-enriched light membrane fractions. We reported previously that PLD activity elevated in response to mitogenic stimulation is restricted to the caveolin-enriched light membrane fractions. PLD activity in H-Ras-transformed cells is dependent upon RalA, and consistent with a lack of elevated PLD activity in K-Ras-transformed cells, RalA was not activated in K-Ras-transformed cells. Although H-Ras-induced PLD activity is dependent upon RalA, an activated mutant of RalA is not sufficient to elevate PLD activity. We reported previously that RalA interacts with PLD activating ADP ribosylation factor (ARF) proteins. In cells transformed by H-Ras, we found increased coprecipitation of ARF6 with RalA. Moreover, ARF6 colocalized with RalA in light membrane fractions. Interestingly, ARF6 protein levels were elevated in H-Ras- but not K-Ras-transformed cells. A dominant-negative mutant of ARF6 inhibited PLD activity in H-Ras-transformed NIH 3T3 cells. Activated mutants of either ARF6 or RalA were not sufficient to elevate PLD activity in NIH 3T3 cells; however, expression of both activated RalA and activated ARF6 in NIH 3T3 cells led to increased PLD activity. These data suggest a model whereby H-Ras stimulates the activation of both RalA and ARF6, which together lead to the elevation of PLD activity.     

Establishing epithelial glandular polarity: interlinked roles for ARF6, Rac1, and the matrix microenvironment

Epithelial cysts comprise the structural units of the glandular epithelium. Although glandular inversion in epithelial tumors is thought to be a potential mechanism for the establishment of metastatic disease, little is known about the morphogenic cues and signaling pathways that govern glandular polarity and organization. Using organotypic cultures of Madin-Darby canine kidney cells in reconstituted basement membrane, we show that cellular depletion of the small GTP-binding protein ARF6 promotes the formation of inverted cysts, wherein the apical cell membrane faces the cyst exterior, and the basal domain faces the central lumen, while individual cell polarity is maintained. These cysts are also defective in interactions with laminin at the cyst–matrix interface. This inversion of glandular orientation is accompanied by Rac1 inactivation during early cystogenesis, and temporal activation of Rac1 is sufficient to recover the normal cyst phenotype. In an unnatural collagen I microenvironment, ARF6-depleted, inverted epithelial cysts exhibit some loss of cell polarity, a marked increase in Rho activation and Rac1 inactivation, and striking rearrangement of the surrounding collagen I matrix. These studies demonstrate the importance of ARF6 as a critical determinant of glandular orientation and the matrix environment in dictating structural organization of epithelial cysts.     

A Highlights from MBoC Selection: Unregulated ARF6 Activation in Epithelial Cysts Generates Hyperactive Signaling Endosomes and Disrupts Morphogenesis

Tumor development in glandular tissues is associated with structural alterations in the hollow ducts and spherical structures that comprise such tissues. We describe a signaling axis involving sustained activation of the GTP-binding protein, ARF6, that provokes dramatic changes in the organization of epithelial cysts, reminiscent of tumorigenic glandular phenotypes. In reconstituted basement membrane cultures of renal epithelial cysts, enhanced ARF6 activation induces the formation of cell-filled glandular structures with multiple lumens and disassembled cadherin-based cell–cell contacts. All of these alterations are accompanied by growth factor receptor internalization into signaling endosomes and reversed by blocking ARF6 activation or receptor endocytosis. Receptor localization in signaling endosomes results in hyperactive extracellular signal-regulated kinase signaling leading to Bcl-2 stabilization and aberrant cysts. Similarly, formation of hyperproliferative and disorganized mammary acini induced by chronic stimulation of colony-stimulating factor 1 receptor is coupled to endogenous ARF6 activation and constitutive receptor internalization and is reversed by ARF6 inhibition. These findings identify a previously unrecognized link between ARF6-regulated receptor internalization and events that drive dramatic alterations in cyst morphogenesis providing new mechanistic insight into the molecular processes that can promote epithelial glandular disruption.     

Annexin 2 binding to phosphatidylinositol 4,5-bisphosphate on endocytic vesicles is regulated by the stress response pathway

Annexin 2 is a Ca(2+)-binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitogen-activated protein (MAP) kinase, and calcium, suggesting the involvement of phosphoinositide signaling. Since various phosphoinositides are enriched on inwardly mobile vesicles, we examined whether or not annexin 2 binds to any of this class of phospholipid. In liposome sedimentation assays, we show that recombinant annexin 2 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5P(2)) but not to other poly- and mono-phosphoinositides. The affinity of annexin 2 for PtdIns-4,5P(2) (K(D) approximately 5 microm) is comparable with those reported for a variety of PtdIns-4,5P(2)-binding proteins and is enhanced in the presence of Ca(2+). Although annexin 1 also bound to PtdIns-4,5P(2), annexin 5 did not, indicating that this is not a generic annexin property. To test whether annexin 2 binds to PtdIns-4,5P(2) in vivo, we microinjected rat basophilic leukemia cells stably expressing annexin 2-green fluorescent protein (GFP) with fluorescently tagged antibodies to PtdIns-4,5P(2). Annexin 2-GFP and anti-PtdIns-4,5P(2) IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P(2) synthesis on pinosomes through activation of phosphatidylinositol 5 kinase. These results establish a novel phospholipid-binding specificity for annexin 2 consistent with a role in mediating the interaction between the macropinosome surface and the polymerized actin tail.     

ARF6-GTP recruits Nm23-H1 to facilitate dynamin-mediated endocytosis during adherens junctions disassembly

ARF6-regulated endocytosis of E-cadherin is essential during the disassembly of adherens junctions in epithelial cells. Here, we show that activation of ARF6 promotes clathrin-dependent internalization of E-cadherin and caveolae at the basolateral cell surface. Furthermore, we demonstrate that ARF6-GTP, a constitutively activate form of ARF6, interacts with and recruits Nm23-H1, a nucleoside diphosphate (NDP) kinase that provides a source of GTP for dynamin-dependent fission of coated vesicles during endocytosis. Finally, we show that ARF6-mediated recruitment of Nm-23-H1 to cell junctions is accompanied by a decrease in the cellular levels of Rac1-GTP, consistent with previous findings that Nm23-H1 down-regulates activation of Rac1. These studies provide a molecular basis for ARF6 function in polarized epithelia during adherens junction disassembly.     

An essential role for ARF6-regulated membrane traffic in adherens junction turnover and epithelial cell migration

We describe a novel role for the ARF6 GTPase in the regulation of adherens junction (AJ) turnover in MDCK epithelial cells. Expression of a GTPase-defective ARF6 mutant, ARF6(Q67L), led to a loss of AJs and ruffling of the lateral plasma membrane via mechanisms that were mutually exclusive. ARF6-GTP-induced AJ disassembly did not require actin remodeling, but was dependent on the internalization of E-cadherin into the cytoplasm via vesicle transport. ARF6 activation was accompanied by increased migratory potential, and treatment of cells with hepatocyte growth factor (HGF) induced the activation of endogenous ARF6. The effect of ARF6(Q67L) on AJs was specific since ARF6 activation did not perturb tight junction assembly or cell polarity. In contrast, dominant-negative ARF6, ARF6(T27N), localized to AJs and its expression blocked cell migration and HGF-induced internalization of cadherin-based junctional components into the cytoplasm. Finally, we show that ARF6 exerts its role downstream of v-Src activation during the disassembly of AJs. These findings document an essential role for ARF6- regulated membrane traffic in AJ disassembly and epithelial cell migration.     

Role for a Cindr-Arf6 axis in patterning emerging epithelia

Patterning of the Drosophila pupal eye is characterized by precise cell movements. In this paper, we demonstrate that these movements require an Arf regulatory cycle that connects surface receptors to actin-based movement. dArf6 activity-regulated by the Arf GTPase-activating proteins (ArfGAPs) dAsap and dArfGAP3 and the Arf GTP exchange factors Schizo and dPsd-promoted large cellular extensions; time-lapse microscopy indicated that these extensions presage cell rearrangements into correct epithelial niches. During this process, the Drosophila eye also requires interactions between surface Neph1/nephrin adhesion receptors Roughest and Hibris, which bind the adaptor protein Cindr (CD2AP). We provide evidence that Cindr forms a physical complex with dArfGAP3 and dAsap. Our data suggest this interaction sequesters ArfGAP function to liberate active dArf6 elsewhere in the cell. We propose that a Neph1/nephrin-Cindr/ArfGAP complex accumulates to limit local Arf6 activity and stabilize adherens junctions. Our model therefore links surface adhesion via an Arf6 regulatory cascade to dynamic modeling of the cytoskeleton, accounting for precise cell movements that organize the functional retinal field. Further, we demonstrate a similar relationship between the mammalian Cindr orthologue CD2AP and Arf6 activity in cell motility assays. We propose that this Cindr/CD2AP-mediated regulation of Arf6 is a widely used mechanism in emerging epithelia.     

The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.