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Loss of apical-basal polarity and downregulation of cell-cell contacts is a critical step during the pathogenesis of cancer. Both processes are regulated by the scaffolding protein Pals1, however, it is unclear whether the expression of Pals1 is affected in cancer cells and whether Pals1 is implicated in the pathogenesis of the disease. Using mRNA expression data and immunostainings of cancer specimen, we show that Pals1 is frequently downregulated in colorectal cancer, correlating with poorer survival of patients. We further found that Pals1 prevents cancer cell metastasis by controlling Rac1-dependent cell migration through inhibition of Arf6, which is independent of the canonical binding partners of Pals1. Loss of Pals1 in colorectal cancer cells results in increased Arf6 and Rac1 activity, enhanced cell migration and invasion in vitro and increased metastasis of transplanted tumor cells in mice. Thus, our data reveal a new function of Pals1 as a key inhibitor of cell migration and metastasis of colorectal cancer cells. Notably, this new function is independent of the known role of Pals1 in tight junction formation and apical-basal polarity. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-021-01354-2.

Sphingolipids are found in all kingdoms of life. In mammals, sphingolipids regulate multiple cellular processes. Ceramide is a sphingolipid of interest to cancer biologists due to its regulation of cell cycle, membrane morphology, cell migration, differentiation, and cell death. Disruptions in ceramide metabolism are associated with disease. Reduced ceramide in cancer cells leads to increased malignancy and metastasis. Conversely, increasing cellular ceramide leads to differentiation, proliferative arrest, and cell death. Our lab has developed synthetic sphingolipids that effectively and selectively induce cancer cell death in vivo. We have recently described one such synthetic sphingolipid, SH-BC-893 (893), that is selective and effective in murine cancer models without notable toxicity to the animal. Our publications show that the selectivity of sphingolipids for cancer cells is due to oncogene-driven constitutive anabolism of cancer cells. Highly anabolic TSC2 knockout cells are resistant to sphingolipid-induced death but are sensitized by expression of oncogenic KRAS. 893 inhibits multiple, parallel cellular nutrient acquisition mechanisms. One mechanism is by decreasing surface nutrient transporter expression, an activity shared with the endogenous sphingolipid ceramide. However, 893 further limits nutrient acquisition by reducing lysosomal fusion to prevent access to intracellular metabolites from LDL, autophagosomes, and macropinosomes. These phenotypes were not observed with ceramide.The molecular mechanism through which ceramide and synthetic sphingolipids induce loss of surface nutrient transporters is not yet known. The work presented here suggests that recycling of nutrient transporter proteins is inhibited. We show that actin remodeling, ARF6 inactivation, PKC and Rac1 activation all appear to contribute to sphingolipid-induced nutrient transporter loss. ARF6 inactivation may be downstream of sphingolipid-induced PP2A activation similar to other effects. Preliminary data suggests ARF6 inactivation may result from PP2A-mediated de-phosphorylation of the ARF6 guanine nucleotide exchange factor Grp1, which activates ARF6. Both endogenous sphingolipids and 893 inactivate ARF6. Interestingly, ARF6 activation is associated with tumor progression, grade, and metastasis in multiple cancer classes. We found that combining sphingolipid with ARF6 inhibitors synergistically and selectively kill cancer cells. These findings clarify the mechanism of sphingolipid-induced cell death and identify inactivation of ARF6 playing a role in nutrient transporter loss.

Background Not merely the onset of immune evasion, but other factors, such as acidosis and fibrosis, are also major barriers in cancer therapeutics. Dense fibrosis is a hallmark of pancreatic ductal carcinoma (PDAC), in which hyperactivation of focal adhesion kinase (FAK) in tumor cells was shown to be crucial. Double mutations of KRAS/ TP53 are characteristic to PDAC. We previously showed that high protein expression of ARF6 and its downstream effector AMAP1, as well as processes involved in the ARF6 activation by cell surface tyrosine kinase receptors, are major targets of the KRAS/TP53 mutations to promote PDAC invasion, metastasis, and immune evasion. This notion was recaptured by KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre). Mechanistically, the ARF6-AMAP1 pathway is primarily involved in cellular dynamics of PD-L1, β1-integrins, and E-cadherin; and hence modulates cell-adhesion properties when ARF6 is activated. Here, with an aim to understand whether the ARF6-AMAP1 pathway is critically involved in the elevated levels of PD-L1 and fibrosis of PDAC, we analyzed relationship between AMAP1 and these malignant phenotypes. Moreover, because the ARF6 pathway may closely be related to focal adhesion dynamics and hence to FAK, we also investigated whether AMAP1 employs FAK in fibrosis. Methods Clinical specimens, as well as KPC cells/tumors and their shAMAP1 or shFAK derivatives were analyzed. Results Elevated levels of PD-L1 and fibrosis correlated with poor outcome of our patient cohort, to be consistent with previous reports; in which high AMAP1 expression statistically correlated with the elevated PD-L1 and fibrosis. To be consistent, silencing of AMAP1 (shAMAP1) in KPC cells resulted in reduced PD-L1 expression and fibrosis in their tumors. On the other hand, shAMAP1 only slightly affected FAK activation in KPC cells, and phosphorylated FAK did not correlate with enhanced fibrosis or with poor outcome of our patients. Conclusions Together with our previous data, our results collectively indicated that the ARF6-AMAP1 pathway, empowered by the KRAS/TP53 mutations, is closely associated with elevated PD-L1 expression and fibrosis of human PDACs, to be recaptured in the KPC mouse model. The ARF6 pathway may promote fibrosis independent of FAK.

BackgroundChordoma, an extremely rare malignant tumor, remains difficult to be cured because of its strong local invasiveness and high recurrence rate. Long non-coding RNAs (lncRNAs) have been demonstrated to play multiple roles in various cancers. The purpose of this study was to investigate the modulatory function of lncRNA MDFIC-7 in chordoma and to elucidate its underlying mechanisms.MethodsQuantitative real-time polymerase chain reaction was performed to detect the expression of lncRNA MDFIC-7 in tumor tissues and adjacent nontumorous tissues collected from 15 chordoma patients, as well as in chordoma cell lines. Gene silencing and overexpression experiments were carried out by RNA interference and lentiviral transduction. The effect of lncRNA MDFIC-7 on the proliferation of chordoma cells was evaluated by cell counting kit-8 assay, colony formation assay and xenograft tumor experiments. RNA immunoprecipitation and dual luciferase reporter assays were conducted to evaluate the binding between lncRNA MDFIC-7 and miRNA-525-5p and the interaction between miR-525-5p and the 3′ untranslated region of ADP-ribosylation factor 6 (ARF6) mRNA. The glycolytic capacity and mitochondrial function of chordoma cells were measured by the Seahorse Bioscience XF96 Extracellular Flux Analyzer.ResultsThe expression of lncRNA MDFIC-7 was higher in chordoma tumor tissues than in adjacent non-tumor tissues. Downregulation of lncRNA MDFIC-7 reduced colony formation and cell proliferation in chordoma cells and decreased xenograft tumor growth in a nude mouse model. Moreover, lncRNA MDFIC-7 knockdown attenuated the Warburg effect in chordoma cells and xenograft tumors. LncRNA MDFIC-7 knockdown elevated miR-525-5p levels and decreased ARF6 expressions. Overexpression of ARF6 reversed the inhibitory effect of lncRNA MDFIC-7 knockdown on cell proliferation and the Warburg effect in chordoma cells and xenograft tumors. Mechanistically, lncRNA MDFIC-7, as a molecular sponge of miR-525-5p, negatively regulated miR-525-5p expression and promoted the gene expression of ARF6, a miR-525-5p target.ConclusionOur findings demonstrate that lncRNA MDFIC-7 acts as a molecular sponge to competitively bind to miR-525-5p and promote expression of ARF6. The lncRNA MDFIC-7/miR-525-5p/ARF6 axis regulates chordoma progression and the Warburg effect in chordoma, suggesting that lncRNA MDFIC-7 and miR-525-5p could be promising therapeutic targets for the treatment of chordoma.

Wnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa. In vitro studies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findings in vivo, we analysed the consequence of the absence of Arf6 activity on Drosophila wing patterning, a developmental model of Wnt/Wingless signalling. We observed a dominant loss of wing margin bristles and Senseless expression in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, we show that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. Our data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. Our findings represent a novel regulatory role for Arf6 in Wingless signalling., Summary: Arf6 is necessary for the patterning of the wing margin by Wingless in Drosophila melanogaster, acting downstream of stabilized Armadillo but upstream or at the level of Pangolin activity.

Although KRAS and TP53 mutations are major drivers of pancreatic ductal adenocarcinoma (PDAC), the incurable nature of this cancer still remains largely elusive. ARF6 and its effector AMAP1 are often overexpressed in different cancers and regulate the intracellular dynamics of integrins and E-cadherin, thus promoting tumor invasion and metastasis when ARF6 is activated. Here we show that the ARF6-AMAP1 pathway is a major target by which KRAS and TP53 cooperatively promote malignancy. KRAS was identified to promote eIF4A-dependent ARF6 mRNA translation, which contains a quadruplex structure at its 5'-untranslated region, by inducing TEAD3 and ETV4 to suppress PDCD4; and also eIF4E-dependent AMAP1 mRNA translation, which contains a 5'-terminal oligopyrimidine-like sequence, via up-regulating mTORC1. TP53 facilitated ARF6 activation by platelet-derived growth factor (PDGF), via its known function to promote the expression of PDGF receptor beta (PDGFR beta) and enzymes of the mevalonate pathway (MVP). The ARF6-AMAP1 pathway was moreover essential for PDGF-driven recycling of PD-L1, in which KRAS, TP53, elF4A/4E-dependent translation, mTOR, and MVP were all integral. We moreover demonstrated that the mouse PDAC model KPC cells, bearing KRAS/TP53 mutations, express ARF6 and AMAP1 at high levels and that the ARF6-based pathway is closely associated with immune evasion of KPC cells. Expression of ARF6 pathway components statistically correlated with poor patient outcomes. Thus, the cooperation among elF4A/4E-dependent mRNA translation and MVP has emerged as a link by which pancreatic driver mutations may promote tumor cell motility, PD-L1 dynamics, and immune evasion, via empowering the ARF6-based pathway and its activation by external ligands.

Axon regeneration after injury is a conserved biological process that involves a large number of molecular pathways, including rapid calcium influx at injury sites, retrograde injury signaling, epigenetic transition, transcriptional reprogramming, polarized transport, and cytoskeleton reorganization. Despite the numerous efforts devoted to understanding the underlying cellular and molecular mechanisms of axon regeneration, the search continues for effective target molecules for improving axon regeneration. Although there have been significant historical efforts towards characterizing pro-regenerative factors involved in axon regeneration, the pursuit of intrinsic inhibitors is relatively recent. EFA6 (exchange factor for ARF6) has been demonstrated to inhibit axon regeneration in different organisms. EFA6 inhibition could be a promising therapeutic strategy to promote axon regeneration and functional recovery after axon injury. This review summarizes the inhibitory role on axon regeneration through regulating microtubule dynamics and through affecting ARF6 (ADP-ribosylation factor 6) GTPase-mediated integrin transport.

Ciliogenesis is a coordinated process initiated by the recruitment and fusion of pre-ciliary vesicles at the distal appendages of the mother centriole through mechanisms that remain unclear. Here, we report that EFA6A (also known as PSD), an exchange factor for the small G protein Arf6, is involved in early stage of ciliogenesis by promoting the fusion of distal appendage vesicles forming the ciliary vesicle. EFA6A is present in the vicinity of the mother centriole before primary cilium assembly and prior to the arrival of Arl13B-containing vesicles. During ciliogenesis, EFA6A initially accumulates at the mother centriole and later colocalizes with Arl13B along the ciliary membrane. EFA6A depletion leads to the inhibition of ciliogenesis, the absence of centrosomal Rab8-positive structures and the accumulation of Arl13B-positive vesicles around the distal appendages. Our results uncover a novel fusion machinery, comprising EFA6A, Arf6 and Arl13B, that controls the coordinated fusion of ciliary vesicles docked at the distal appendages of the mother centriole.

The scaffolding protein family Fe65, composed of Fe65, Fe65L1, and Fe65L2, was identified as an interaction partner of the amyloid precursor protein (APP), which plays a key function in Alzheimer’s disease. All three Fe65 family members possess three highly conserved interaction domains, forming complexes with diverse binding partners that can be assigned to different cellular functions, such as transactivation of genes in the nucleus, modulation of calcium homeostasis and lipid metabolism, and regulation of the actin cytoskeleton. In this article, we rule out putative new intracellular signaling mechanisms of the APP-interacting protein Fe65 in the regulation of actin cytoskeleton dynamics in the context of various neuronal functions, such as cell migration, neurite outgrowth, and synaptic plasticity.

ADAP1/Centaurin-α1 (CentA1) functions as an Arf6 GTPase-activating protein highly enriched in the brain. Previous studies demonstrated the involvement of CentA1 in brain function as a regulator of dendritic differentiation and a potential mediator of Alzheimer’s disease pathogenesis. To better understand the neurobiological functions of CentA1 signaling in the brain, we developed Centa1 knockout mice. The knockout animals showed neither brain development nor synaptic ultrastructure deficits in the hippocampus. However, they exhibited significantly higher density and enhanced structural plasticity of dendritic spines in the CA1 region of the hippocampus compared to non-transgenic littermates. Moreover, the deletion of Centa1 improved performance in the object-in-place spatial memory task. These results suggest that CentA1 functions as a negative regulator of spine density and plasticity, and of hippocampus-dependent memory formation. Thus, CentA1 and its downstream signaling may serve as a potential therapeutic target to prevent memory decline associated with aging and brain disorders. SIGNIFICANCE STATEMENT ADAP1/Centaurin-α1 (CentA1) is highly enriched in the brain and has been shown to be involved in the developmental regulation of dendritic differentiation. Although increased CentA1 level has been linked to Alzheimer’s disease, the underlying neurological mechanisms are unknown. We found that the genetic deletion of Centa1 leads to increased dendritic spine plasticity and enhanced spine structural plasticity in the hippocampus, accompanied by behavioral improvements in a location recognition task. This is the first study of CentA1 role in the brain, and our findings will facilitate the understanding of neurobiological mechanisms underlying the regulation of dendritic spine morphology and plasticity in healthy brains and in neurological disorders.

The auxin response factor (ARF) transcription factors are a key component in auxin signaling and play diverse functions in plant growth, development, and stress response. ARFs are regulated at the transcript level and posttranslationally by protein modifications. However, relatively little is known regarding the control of ARF protein levels. We expressed five different ARFs with an HA (hemagglutinin) tag and observed that their protein levels under the same promoter varied considerably. Interestingly, their protein levels were affected by several hormonal and environmental conditions, but not by the auxin treatment. ABA (abscisic acid) as well as 4 &deg, C and salt treatments decreased the levels of HA-ARF5, HA-ARF6, and HA-ARF10, but not that of HA-ARF19, while 37 &deg, C treatment increased the levels of the four HA-ARFs, suggesting that the ARF protein levels are regulated by multiple factors. Furthermore, MG132 inhibited the reduction of HA-ARF6 level by ABA and 4 &deg, C treatments, suggesting that these treatments decrease HA-ARF6 level through 26S proteasome-mediated protein degradation. It was also found that ABA treatment drastically increased HA-ARF6 ubiquitination, without strongly affecting the ubiquitination profile of the total proteins. Together, these results reveal another layer of control on ARFs, which could serve to integrate multiple hormonal and environmental signals into the ARF-regulated gene expression.

The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.

Hypocotyl cell elongation related to photomorphogenesis in Arabidopsis seedlings is regulated by a network involving ethylene, auxin, and brassinosteroid signalling that is mediated by interactions among ERF72, ARF6, and BZR1, forming a revised BZR-ARF-PIF/DELLA-ERF (BAP/DE) module., The phytohormones brassinosteroid (BR), auxin, and gibberellin (GA) regulate photomorphogenesis-related hypocotyl elongation in Arabidopsis via the co-operative interaction of BZR-ARF-PIF/DELLA (BAP/D) transcription factors/regulators. In addition, ethylene activates the PIF3 or ERF1 pathway through EIN3/EIL1 to balance hypocotyl elongation in Arabidopsis seedlings. However, the mechanism by which ethylene is co-ordinated with other phytohormones to produce light-regulated hypocotyl growth remains elusive. In this study, we found that hypocotyl cell elongation is regulated by a network involving ethylene, auxin, and BR signalling, which is mediated by interactions among ERF72, ARF6, and BZR1. ERF72 interacted directly with ARF6 and BZR1 in vitro and in vivo, and it antagonised regulation by ARF6 and BZR1 of the transcription of BEE3 and XTH7. In addition, light modulated the subcellular localisation of ERF72 and transcription of ERF72 through the EIN2-EIN3/EIL1 pathway, facilitating the function of ERF72 in photomorphogenesis. The expression of BEE3 and XTH7 was also regulated by the EIN2-EIN3/EIL1 pathway. Our findings indicate that a revised BZR-ARF-PIF/DELLA-ERF (BAP/DE) module integrates light and hormone signals to regulate hypocotyl elongation in Arabidopsis.

Rab35 and the Rab35 network of GAPs, GEFs, and effectors are important regulators of membrane trafficking for a variety of cellular processes, from cytokinesis and phagocytosis to neurite outgrowth. In the past five years, components of this signaling network have also been implicated as critical mediators of synaptic vesicle (SV) recycling and protein homeostasis. Recent studies by several groups, including our own, have demonstrated that Rab35-mediated endosomal sorting is required for the degradation of SV proteins via the ESCRT pathway, thereby eliminating old or damaged proteins from the SV pool. This sorting process is regulated by Rab35 activation in response to neuronal activity, and potentially by an antagonistic signaling relationship between Rab35 and the small GTPase Arf6 that directs SVs into distinct recycling pathways depending on neuronal activity levels. Furthermore, mutations in genes encoding Rab35 regulatory proteins are emerging as causative factors in human neurologic and neurodegenerative diseases, consistent with their important roles in synaptic and neuronal health. Here, we review these recent findings and offer our perspective on how the Rab35 signaling network functions to maintain neurotransmission and synaptic fitness.

Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with b-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis.

We have recently described an A350V mutation in IQSEC2 associated with intellectual disability, autism and epilepsy. We sought to understand the molecular pathophysiology of this mutation with the goal of developing targets for drug intervention. We demonstrate here that the A350V mutation results in interference with the binding of apocalmodulin to the IQ domain of IQSEC2. We further demonstrate that this mutation results in constitutive activation of the guanine nucleotide exchange factor (GEF) activity of IQSEC2 resulting in increased production of the active form of Arf6. In a CRISPR generated mouse model of the A350V IQSEC2 mutation, we demonstrate that the surface expression of GluA2 AMPA receptors in mouse hippocampal tissue was significantly reduced in A350V IQSEC2 mutant mice compared to wild type IQSEC2 mice and that there is a significant reduction in basal synaptic transmission in the hippocampus of A350V IQSEC2 mice compared to wild type IQSEC2 mice. Finally, the A350V IQSEC2 mice demonstrated increased activity, abnormal social behavior and learning as compared to wild type IQSEC2 mice. These findings suggest a model of how the A350V mutation in IQSEC2 may mediate disease with implications for targets for drug therapy. These studies provide a paradigm for a personalized approach to precision therapy for a disease that heretofore has no therapy.

Major histocompatibility complex class I (MHC I) molecules are glycoproteins that display peptide epitopes at the cell surface of nucleated cells for recognition by CD8+ T cells. Like other cell surface receptors, MHC class I molecules are continuously removed from the surface followed by intracellular degradation or recycling to the cell surface, in a process likely involving active quality control the mechanism of which remains unknown. The molecular players and pathways involved in internalization and recycling have previously been studied in model cell lines such as HeLa. However, dendritic cells (DCs), which rely on a specialized endocytic machinery that confers them the unique ability to “cross”-present antigens acquired by internalization, may use distinct MHC I recycling pathways and quality control mechanisms. By providing MHC I molecules cross-presenting antigens, these pathways may play an important role in one of the key functions of DCs, priming of T cell responses against pathogens and tumors. In this review, we will focus on endocytic recycling of MHC I molecules in various experimental conditions and cell types. We discuss the organization of the recycling pathway in model cell lines compared to DCs, highlighting the differences in the recycling rates and pathways of MHC I molecules between various cell types, and their putative functional consequences. Reviewing the literature, we find that conclusive evidence for significant recycling of MHC I molecules in primary DCs has yet to be demonstrated. We conclude that endocytic trafficking of MHC class I in DCs remains poorly understood and should be further studied because of its likely role in antigen cross-presentation.

Although several lines of evidence have shown that the small GTPase ADP-ribosylation factor 6 (Arf6) plays pivotal roles in cancer progression of several types of cancers, little is known about the functions of Arf6 in tumor microenvironment. We demonstrated that Arf6 in vascular endothelial cells (VECs) plays a crucial role in tumor angiogenesis and growth using endothelial cell-specific Arf6 conditional knockout mice into which B16 melanoma and Lewis lung carcinoma cells were implanted. It was also found that Arf6 in VECs positively regulates hepatocyte growth factor (HGF)-induced β1 integrin recycling, which is a critical event for tumor angiogenesis by promoting cell migration. Importantly, pharmacological inhibition of HGF-induced Arf6 activation significantly suppresses tumor angiogenesis and growth in mice, suggesting that Arf6 signaling would be a potential target for anti-angiogenic therapy. In this manuscript, we summarize the multiple roles of Arf6 in cancer progression, particularly in cancer cell invasion/metastasis and our recent findings on tumor angiogenesis, and discuss a possible approach to develop innovative anti-cancer drugs.

The maintenance of cell surface proteins is critical to the ability of a cell to sense and respond to information in its environment. As such, modulation of cell surface composition and receptor trafficking is a potentially important target of control in virus infection. Sorting endosomes (SEs) are control stations regulating the recycling or degradation of internalized plasma membrane proteins. Here we report that human cytomegalovirus (HCMV), a ubiquitous betaherpesvirus, alters the fate of internalized clathrin-independent endocytosis (CIE) cargo proteins, retaining them in virally reprogrammed SEs. We show that the small G protein ARF6 (ADP ribosylation factor 6), a regulator of CIE trafficking, is highly associated with SE membranes relative to uninfected cells. Combined with the observation of accumulated CIE cargo at the SE, these results suggest that infection diminishes the egress of ARF6 and its cargo from the SE. Expression of ubiquitin-specific protease 6 (USP6), also known as TRE17, was sufficient to restore ARF6 and some ARF6 cargo trafficking to the cell surface in infected cells. The USP activity of TRE17 was required to rescue both ARF6 and associated cargo from SE retention in infection. The finding that TRE17 expression does not rescue the trafficking of all CIE cargos retained at SEs in infection suggests that HCMV hijacks the normal sorting machinery and selectively sorts specific cargos into endocytic microdomains that are subject to alternative sorting fates., IMPORTANCE Cells maintain their surface composition, take up nutrients, and respond to their environment through the internalization and recycling of cargo at the cell surface through endocytic trafficking pathways. During infection with human cytomegalovirus (HCMV), host endocytic membranes are reorganized into a juxtanuclear structure associated with viral assembly and egress. Less appreciated is the effect of this reorganization on the trafficking of host proteins through the endocytic pathway. We show that HCMV retains internalized cargo and the effector of clathrin-independent endocytosis at sorting endosomes. The retention of some cargo, but not all, was reversed by overexpression of a ubiquitin-specific protease, TRE17. Our results demonstrate that HCMV induces profound reprogramming of endocytic trafficking and influences cargo sorting decisions. Further, our work suggests the presence of a novel ubiquitin-regulated checkpoint for the recycling of cargo from sorting endosome. These findings have important implications for host signaling and immune pathways in the context of HCMV infection.

The study of cancer has allowed researchers to describe some biological characteristics that tumor cells acquire during their development, known as the “hallmarks of cancer” but more research is needed to expand our knowledge about cancer biology and to generate new strategies of treatment. The role that RabGTPases might play in some hallmarks of cancer represents interesting areas of study since these proteins are frequently altered in cancer. However, their participation is not well known. Recently, Rab35was recognized as an oncogenic RabGTPase and and because of its association with different cellular functions, distinctly important in immune cells, a possible role of Rab35 in leukemia can be suggested. Nevertheless, the involvement of Rab35 in cancer remains poorly understood and its possible specific role in leukemia remains unknown. In this review, we analyze general aspects of the participation of RabGTPases in cancer, and especially, the plausible role of Rab35 in leukemia.

The data is related to the research article entitled “Arf6 mediates Schwann cell differentiation and myelination” [1]. To further investigate the role of Arf6 in promoting myelination by Schwann cells in vivo, we have characterized an another line (#2) of small-hairpin (sh)RNA transgenic mice targeting Arf6. The number of transgenes per one allele in this line was very low (2 transgenes), comparing with high copies in the previous line (#1, 20 transgenes) [1]. In 4 days of neonatal age, transgenic mice exhibited decreased myelin thickness; however, decreased levels were not as much as those in the line #1, likely depending on transgene copy number. In 60-day-old mice, the difference became smaller. On the other hand, transgene׳s effect was not related to cell proliferation and apoptosis. These data support the key role of Arf6 in Schwann cell myelination, especially in the initiation.

Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells.

Significance Viral-like nanovesicles of endosomal origin, or “exosomes,” are newly recognized vehicles of signals that cells use to communicate, in various systemic diseases, including cancer. Yet the molecular mechanisms that regulate the biogenesis and activity of exosomes remain obscure. Here, we establish that the oncogenic protein SRC stimulates the secretion of exosomes loaded with syntenin and syndecans, known co-receptors for a plethora of signaling and adhesion molecules. SRC phosphorylates conserved tyrosine residues in the syndecans and syntenin and stimulates their endosomal budding. Moreover, SRC-dependent exosomes have a promigratory activity that strictly depends on syntenin expression. This work sheds light on a function of SRC in cell-to-cell communication and mechanisms of exosome biogenesis and activity, with potential broad impact for physiopathology., The cytoplasmic tyrosine kinase SRC controls cell growth, proliferation, adhesion, and motility. The current view is that SRC acts primarily downstream of cell-surface receptors to control intracellular signaling cascades. Here we reveal that SRC functions in cell-to-cell communication by controlling the biogenesis and the activity of exosomes. Exosomes are viral-like particles from endosomal origin that can reprogram recipient cells. By gain- and loss-of-function studies, we establish that SRC stimulates the secretion of exosomes having promigratory activity on endothelial cells and that syntenin is mandatory for SRC exosomal function. Mechanistically, SRC impacts on syndecan endocytosis and on syntenin–syndecan endosomal budding, upstream of ARF6 small GTPase and its effector phospholipase D2, directly phosphorylating the conserved juxtamembrane DEGSY motif of the syndecan cytosolic domain and syntenin tyrosine 46. Our study uncovers a function of SRC in cell–cell communication, supported by syntenin exosomes, which is likely to contribute to tumor–host interactions.

The small GTase Arf6 has several important functions in intracellular vesicular trafficking and regulates the recycling of different types of cargo internalized via clathrin-dependent or -independent endocytosis. It activates the lipid modifying enzymes PIP 5-kinase and phospholipase D, promotes actin polymerization, and affects several functionally distinct processes in the cell. Arf6 is used for the phagocytosis of pathogens and can be directly or indirectly targeted by various pathogens to block phagocytosis or induce the uptake of intracellular pathogens. Arf6 is also used in the signaling of Toll-like receptors and in the activation of NADPH oxidases. In this review, we first give an overview of the different roles and mechanisms of action of Arf6 and then focus on its role in innate immunity and host–pathogen interactions.

Recycling of synaptic vesicles (SVs) is a fundamental step in the process of neurotransmission. Endocytosed SV can travel directly into the recycling pool or recycle through endosomes but little is known about the molecular actors regulating the switch between these SV recycling routes. ADP ribosylation factor 6 (Arf6) is a small GTPase known to participate in constitutive trafficking between plasma membrane and early endosomes. Here, we have morphologically and functionally investigated Arf6-silenced hippocampal synapses and found an activity dependent accumulation of synaptic endosome-like organelles and increased release-competent docked SVs. These features were phenocopied by pharmacological blockage of Arf6 activation. The data reveal an unexpected role for this small GTPase in reducing the size of the readily releasable pool of SVs and in channeling retrieved SVs toward direct recycling rather than endosomal sorting. We propose that Arf6 acts at the presynapse to define the fate of an endocytosed SV. DOI: http://dx.doi.org/10.7554/eLife.10116.001, eLife digest Communication between neurons takes place at cell-to-cell contacts called synapses. Each synapse is formed between one neuron that sends the message, and another neuron that receives it. The neuron before the synapse – called the presynaptic neuron – contains packets called synaptic vesicles, which are full of chemical messengers ready to be released upon activity. Accurate communication between neurons relies on the exact composition, and organized trafficking, of the synaptic vesicles when the neuron is active. Synapses also contain bigger structures, called endosomal structures, which may represent an intermediate station in which synaptic vesicle composition is controlled. However, the trafficking of synaptic vesicles through the endosomal structures is poorly understood. Now, Tagliatti, Fadda et al. have revealed that a protein called Arf6 plays an important role in presynaptic neurons. The experiments involved rat neurons grown in the laboratory, and showed that Arf6 controls both the number of synaptic vesicles ready to be released and the trafficking of synaptic vesicles via endosomal structures in active neurons. The next step following on from these findings is to understand how Arf6 exerts its effects and how this protein is regulated in the presynaptic neuron. DOI: http://dx.doi.org/10.7554/eLife.10116.002

Upon wounding, syndecan-4 detects the appearance of fibronectin in the wound bed and mediates regulation of the small GTPases, Rac1, RhoA and RhoG. Cohesive regulation of these molecules results in cycles of membrane protrusion and cytoskeletal contraction, and triggers the endocytosis of α 5β 1-integrin, which collectively lead to immigration of fibroblasts into the wound bed. In this manuscript we identify the regulation of a fourth GTPase, Arf6 that is responsible for α 5β 1-integrin recycling and thereby completes the cycle of syndecan-4-regulated integrin trafficking. We demonstrate that each of the GTPase signals can be regulated by syndecan-4, but that they are independent of one another. By doing so we identify syndecan-4 as the coordinating center of pro-migratory signals.

Various receptors navigate through the endocytic recycling compartment (ERC) on route to the plasma membrane. They are transported through recycling endosomes that emanate from the ERC that display distinct tubular morphology. A key question in the field is how the trafficking via these endosomes is regulated and how regulatory proteins such as Rab35, Rab8, Arf6 and EHD1 control this trafficking. Recent studies point to the protein MICAL-L1 as a major scaffold for these regulators. MICAL-L1 not only localizes to these tubular recycling endosomes and regulates trafficking, but it also controls the localization of EHD1 and Rab8 to these structures. It also connects its associated membranes to the motor proteins dynein and kinesin through its binding partner, CRMP2. Our recent study promotes MICAL-L1 as a Rab35 effector, where Rab35, both directly and indirectly through Arf6, controls the localization of MICAL-L1 and Rab8 to tubular membranes. We find that MICAL-L1 is a multi-tasking scaffold connecting various proteins to recycling endosomes for efficient trafficking.

Mono-ADP-ribosylation is a reversible post-translational modification that can modulate the functions of target proteins. We have previously demonstrated that the beta subunit of heterotrimeric G proteins is endogenously mono-ADP-ribosylated, and once modified, the beta gamma dimer is inactive toward its effector enzymes. To better understand the physiological relevance of this post-translational modification, we have studied its hormonal regulation. Here, we report that G beta subunit mono-ADP-ribosylation is differentially modulated by G protein-coupled receptors. In intact cells, hormone stimulation of the thrombin receptor induces G beta subunit mono-ADP-ribosylation, which can affect G protein signaling. Conversely, hormone stimulation of the gonadotropin-releasing hormone receptor (GnRHR) inhibits G beta subunit mono-ADP-ribosylation. We also provide the first demonstration that activation of the GnRHR can activate the ADP-ribosylation factor Arf6, which in turn inhibits G beta subunit mono-ADP-ribosylation. Indeed, removal of Arf6 from purified plasma membranes results in loss of GnRHR-mediated inhibition of G beta subunit mono-ADP-ribosylation, which is fully restored by re-addition of purified, myristoylated Arf6. We show that Arf6 acts as a competitive inhibitor of the endogenous ADP-ribosyltransferase and is itself modified by this enzyme. These data provide further understanding of the mechanisms that regulate endogenous ADP-ribosylation of the G beta subunit, and they demonstrate a novel role for Arf6 in hormone regulation of G beta subunit mono-ADP-ribosylation.

The small GTPase ADP-ribosylation factor 6 (ARF6) plays crucial roles in a wide variety of cell functions. To better understand the molecular mechanisms of ARF6-mediated signaling and cellular functions, we sought new ARF6-binding proteins in the mouse brain. We identified the signaling scaffold protein JNK-interacting protein 3 (JIP3), which is exclusively expressed in neurons, as a downstream effector of ARF6. Overexpression of a unique dominant negative mutant of ARF6, which was unable to interact with JIP3, and knockdown of JIP3 in mouse cortical neurons stimulated the elongation and branching of neurites. These results provide evidence that ARF6/JIP3 signaling regulates neurite morphogenesis.Structured summaryMINT-7892698: PIP5K gamma 661 (uniprotkb:O70161) physically interacts (MI:0915) with Arf6 (uniprotkb:P62331) by anti tag coimmunoprecipitation (MI:0007)MINT-7892333, MINT-7892573, MINT-7892594, MINT-7892629, MINT-7892644, MINT-7892522, MINT-7892716: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JLP (uniprotkb:Q58A65) by anti tag coimmunoprecipitation (MI:0007)MINT-7892509: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by pull down (MI:0096)MINT-7892770: Arf6 (uniprotkb:P62331) binds (MI:0407) to JIP3 (uniprotkb:Q9ESN9) by pull down (MI:0096)MINT-7892755: Arf6 (uniprotkb:P62331) binds (MI:0407) to JLP (uniprotkb:Q58A65) by pull down (MI:0096)MINT-7892289, MINT-7892314: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JLP (uniprotkb:Q58A65) by pull down (MI:0096)MINT-7892353, MINT-7892615, MINT-7892657, MINT-7892672, MINT-7892549, MINT-7892738: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by anti tag coimmunoprecipitation (MI:0007)

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglyc-erol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5″-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium.Weexplore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion.Wepropose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance ofARF6as a key factor for human sperm exocytosis and fertilization. Fil: Pelletán, Leonardo Enuar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Suhaiman, Laila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Vaquer, Cintia Celina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Bustos, Matias Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: de Blas, Gerardo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Vitale, Nicolas. Centre National de la Recherche Scientifique; Francia. Université de Strasbourg; Francia Fil: Mayorga, Luis Segundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Belmonte, Silvia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina

Integrins are adhesion and survival molecules involved in axon growth during CNS development, as well as axon regeneration after injury in the peripheral nervous system (PNS). Adult CNS axons do not regenerate after injury, partly due to a low intrinsic growth capacity. We have previously studied the role of integrins in axon growth in PNS axons; in the present study, we investigate whether integrin mechanisms involved in PNS regeneration may be altered or lacking from mature CNS axons by studying maturing CNS neuronsin vitro. In rat cortical neurons, we find that integrins are present in axons during initial growth but later become restricted to the somato-dendritic domain. We investigated how this occurs and whether it can be altered to enhance axonal growth potential. We find a developmental change in integrin trafficking; transport becomes predominantly retrograde throughout axons, but not dendrites, as neurons mature. The directionality of transport is controlled through the activation state of ARF6, with developmental upregulation of the ARF6 GEF ARNO enhancing retrograde transport. Lowering ARF6 activity in mature neurons restores anterograde integrin flow, allows transport into axons, and increases axon growth. In addition, we found that the axon initial segment is partly responsible for exclusion of integrins and removal of this structure allows integrins into axons. Changing posttranslational modifications of tubulin with taxol also allows integrins into the proximal axon. The experiments suggest that the developmental loss of regenerative ability in CNS axons is due to exclusion of growth-related molecules due to changes in trafficking.

HERC1 is a very large protein involved in membrane traffic through both its ability to bind clathrin and its guanine nucleotide exchange factor (GEF) activity over ARF and Rab family GTPases. Herein, we show that HERC1 is recruited onto actin-rich surface protrusions in ARF6-transfected HeLa cells upon aluminum fluoride (AlF4−) treatment. Moreover, the fact that HERC1 overexpression does not stimulate protrusion formation in the absence of AlF4−, in conditions where ARNO does, indicates that HERC1 is not acting as an ARF6-GEF in this system, but that instead its recruitment takes place downstream of ARF6 activation. Finally, we suggest a phosphoinositide-binding mechanism whereby HERC1 may translocate to these protrusions.

Efficient entry of the bacterium Yersinia pseudotuberculosis into mammalian cells requires the binding of the bacterial invasin protein to β1 integrin receptors and the activation of the small GTPase Rac1. We report here that this Rac1-dependent pathway involves recruitment of phosphoinositol-4-phosphate-5-kinase (PIP5K) to form phosphoinositol-4,5-bisphosphate (PIP2) at the phagocytic cup. Reducing the concentration of PIP2 in the target cell by using a membrane-targeted PIP2-specific phosphatase lowered bacterial uptake proportionately. PIP2 formation is regulated by Arf6. An Arf6 derivative defective for nucleotide binding (Arf6N122I) interfered with uptake and decreased the level of PIP2 around extracellular bacteria bound to host cells. This reduction in PIP2 occurred in spite of fact that PIP5K appeared to be recruited efficiently to the site of bacterial binding, indicating a role for Arf6 in activation of the kinase. The elimination of the Rac1-GTP–bound form from the cell by the introduction of the Y. pseudotuberculosis YopE RhoGAP protein could be bypassed by the overproduction of either PIP5K or Arf6, although the degree of bypass was greater for Arf6 transfectants. These results indicate that both Arf6 and PIP5K are involved in integrin-dependent uptake, and that Arf6 participates in both activation of PIP5K as well as in other events associated with bacterial uptake.

Engulfment of particles by phagocytes is induced by their interaction with specific receptors on the cell surface, which leads to actin polymerization and the extension of membrane protrusions to form a closed phagosome. Membrane delivery from internal pools is considered to play an important role in pseudopod extension during phagocytosis. Here, we report that endogenous ADP ribosylation factor 6 (ARF6), a small GTP-binding protein, undergoes a sharp and transient activation in macrophages when phagocytosis was initiated via receptors for the Fc portion of immunoglobulins (FcRs). A dominant-negative mutant of ARF6 (T27N mutation) dramatically affected FcR-mediated phagocytosis. Expression of ARF6-T27N lead to a reduction in the focal delivery of vesicle-associated membrane protein 3+ endosomal recycling membranes at phagocytosis sites, whereas actin polymerization was unimpaired. This resulted in an early blockade in pseudopod extension and accumulation of intracellular vesicles, as observed by electron microscopy. We conclude that ARF6 is a major regulator of membrane recycling during phagocytosis.

Summary Investigations into the role of tomato ARF6 and ARF8 reveal that they are critical components in floral and gynoecium development before anthesis., Auxin regulates the expression of diverse genes that affect plant growth and development. This regulation requires AUXIN RESPONSE FACTORS (ARFs) that bind to the promoter regions of these genes. ARF6 and ARF8 in Arabidopsis thaliana are required to promote inflorescence stem elongation and late stages of petal, stamen, and gynoecium development. All seed plants studied thus far have ARF6 and ARF8 orthologues as well as the microRNA miR167, which targets ARF6 and ARF8. Whether these genes have broadly conserved roles in flower development is not known. To address this question, the effects of down-regulation of ARF6 and ARF8 were investigated through transgenic expression of Arabidopsis MIR167a in tomato, which diverged from Arabidopsis before the radiation of dicotyledonous plants approximately 90–112 million years ago. The transgenic tomato plants overexpressing MIR167a exhibited reductions in leaf size and internode length as well as shortened petals, stamens, and styles. More significantly, the transgenic plants were female-sterile as a result of failure of wild-type pollen to germinate on the stigma surface and/or to grow through the style. RNA-Seq analysis identified many genes with significantly altered expression patterns, including those encoding products with functions in ‘transcription regulation’, ‘cell wall’ and ‘lipid metabolism’ categories. Putative orthologues of a subset of these genes were also differentially expressed in Arabidopsis arf6 arf8 mutant flowers. These results thus suggest that ARF6 and ARF8 have conserved roles in controlling growth and development of vegetative and flower organs in dicots.

ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment and activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show that PIP2 visualized by expressing a fusion protein of the pleckstrin homology domain from PLCδ and green fluorescent protein (PH-GFP), colocalized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion formation, the uptake of PM into macropinosomes enriched in PIP2, and recycling of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as β1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment. Overexpression of human PIP 5-kinase α mimicked the effects seen with Arf6 Q67L. These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.

During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell–cell and cell–extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-functionArf6mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but notFIP3mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking.

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.

GEP100, a guanine nucleotide exchanging factor (GEF) for Arf6, plays a pivotal role in promoting breast cancer cell invasion both in vitro and in vivo. However, the precise mechanism for GEP100-mediated cell invasion is still poorly understood. In this study, we found that down-regulation of endogenous GEP100 in MDA-MB-231 cells significantly inhibited EGF-induced cell invasion, which was rescued by over-expression of ectopic GEP100. EGF increased Arf6 activity, ERK phosphorylation, and uPAR expression in a time dependent manner. Additionally, blocking Arf6 with Arf6 siRNA largely abolished EGF-induced cell invasion. GEP100 siRNA or Arf6 siRNA suppressed EGF-induced ERK activity and uPAR expression. Furthermore, blocking ERK signaling with U0126, a specific inhibitor for MEK, markedly inhibited EGF-induced uPAR expression and consequently cell invasion. Inhibition of uPAR expression by uPAR siRNA also significantly abolished EGF-induced cell invasion. Taken together, this study illustrates that GEP100 regulates an Arf6/ERK/uPAR signaling cascade in EGF-induced breast cancer cell invasion. These findings could provide a rationale for designing new therapies based on inhibition of breast cancer metastasis.

Phospholipase D1 and D2 (PLD1, PLD2) both have PX and PH domains in their N-terminal regions with these inositol lipid binding domains playing key roles in regulating PLD activity and localisation. The activity of PLD1 is also regulated by protein kinase C and members of the Rho and Arf families of GTPases. Each of these proteins binds to unique sites; however, there appears to be little in vitro discrimination between individual family members. In agonist-stimulated cells, however, there is specificity, with, for example in RBL-2H3 cells, antigen stimulating the activation of PLD1 by association with Arf6, Rac1 and protein kinase Cα. PLD2 appears to be less directly regulated by GTPases and rather is primarily controlled through interaction with phosphatidylinositol 4-phosphate 5-kinase that generates the activating phosphatidylinositol 4,5-bisphosphate.

Aneurysmal bone cyst (ABC) is an aggressive, pediatric bone tumor characterized by extensive destruction of the surrounding bone. Although first described over 60 years ago, its molecular etiology remains poorly understood. Recent work revealed that ABCs harbor translocation of TRE17/USP6, leading to its transcriptional upregulation. TRE17 encodes a ubiquitin-specific protease (USP), and a TBC domain that mediates binding to the Arf6 GTPase. However, the mechanisms by which TRE17 overexpression contributes to tumor pathogenesis, and the role of its USP and TBC domains, are unknown. ABCs are characterized by osteolysis, inflammatory recruitment and extensive vascularization, the processes in which matrix proteases have a prominent role. This led us to explore whether TRE17 regulates the production of matrix metalloproteinases ( MMPs). In this study we show that TRE17 is sufficient to induce expression of MMP-9 and MMP-10, in a manner requiring its USP activity, but not its ability to bind Arf6. TRE17 induces transcription of MMP-9 through activation of nuclear factor-kappa B (NF-kappa B), mediated in part by the GTPase RhoA and its effector kinase, ROCK. Furthermore, xenograft studies show that TRE17 induces formation of tumors that reproduce multiple features of ABC, including a high degree of vascularization, with an essential role for the USP domain. In sum, these studies reveal that TRE17 is sufficient to initiate tumorigenesis, identify MMPs as novel TRE17 effectors that likely contribute to ABC pathogenesis and define the underlying signaling mechanism of their induction. Oncogene ( 2010) 29, 3619-3629; doi: 10.1038/onc.2010.116; published online 26 April 2010

The extended carboxyl terminus of the A2A-adenosine receptor is known to engage several proteins other than those canonically involved in signalling by GPCRs (i.e., G proteins, G protein-coupled receptor kinases/GRKs, arrestins). The list includes the deubiquinating enzyme USP4, α-actinin, the guanine nucleotide exchange factor for ARF6 ARNO, translin-X-associated protein, calmodulin, the neuronal calcium binding protein NECAB2 and the synapse associated protein SAP102. However, if the fate of the A2A-receptor is taken into account — from its birthplace in the endoplasmic reticulum to its presumed site of disposal in the lysosome, it is evident that many more proteins must interact with the A2A-adenosine receptor. There are several arguments that support the conjecture that these interactions will preferentially occur with the carboxyl terminus of the A2A-adeonsine receptor: (i) the extended carboxyl terminus (of 122 residues=) offers the required space to accommodate companions; (ii) analogies can be drawn with other receptors, which engage several of these binding partners with their C-termini. This approach allows for defining the nature of the unknown territory. As an example, we posit a chaperone/coat protein complex-II (COPII) exchange model that must occur on the carboxyl terminus of the receptor. This model accounts for the observation that a minimum size of the C-terminus is required for correct folding of the receptor. It also precludes premature recruitment of the COPII-coat to a partially folded receptor. This article is part of a Special Issue entitled: “Adenosine Receptors”.

Pleckstrin homology (PH) domains are modules characterised by a conserved three-dimensional protein fold. Several PH domains bind phosphoinositides with high affinity and specificity whilst most others do not. ARAP3 is a dual GTPase activating protein for Arf6 and RhoA which was identified in a screen for phosphatidylinositol-(3,4,5)-trisphophate (PtdIns(3,4,5)P(3)) binding proteins. It is a regulator of cell shape and adhesion, and is itself regulated by PtdIns(3,4,5)P(3,) which acts to recruit ARAP3 to the plasma membrane and to catalytically activate it. We show here that ARAP3 binds to PtdIns(3,4,5)P(3) in an unusual, PH domain-dependent manner. None of the five PH domains are sufficient to bind PtdIns(3,4,5)P(3) in isolation. Instead, the minimal PtdIns(3,4,5)P(3) binding fragment comprises ARAP3's N-terminal tandem PH domains, and an N-terminal linker region. For substantial binding, the N-terminal sterile alpha motif (SAM) domain is also required. Site-directed mutagenesis of either of the two N-terminal PH domains within the fragment greatly reduces binding to PtdIns(3,4,5)P(3), however, in the context of the full-length protein, point mutations in the second PH domain have a lesser effect on binding, whilst deletion of any one of the five PH domains abolishes PtdIns(3,4,5)P(3) binding. We propose a mechanism by which basic residues from the N-terminal tandem PH domains, and from elsewhere in the protein synergise to mediate strong, specific PtdIns(3,4,5)P(3) binding.

International audience; The JNK-interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP-binding protein ARF6. The interaction of ARF6-GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin-1 and dynactin. Here, we report the crystal structure of ARF6-GTP bound to the JIP4-LZII at 1.9 A resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6-(JIP4)(2)-ARF6 configuration. Comparison of the ARF6-JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site-directed mutagenesis and surface plasmon resonance, we further show that non-conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure-derived model of the association of the ARF6-JIP3/JIP4 complex with membranes shows that the JIP4-LZII coiled-coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6-mediated motor switch regulatory function.

Tumors are tissue-specific diseases, and their mechanisms of invasion and metastasis are highly diverse. In breast cancer, biomarkers that specifically correlate with the invasive phenotypes have not been clearly identified. A small GTPase Arf6 primarily regulates recycling of plasma membrane components. We have shown that Arf6 and its effector AMAP1 (DDEF1, DEF1, ASAP1 and centaurin beta4) are abnormally overexpressed in some breast cancers and used for their invasion and metastasis. Overexpression of these proteins is independent of the transcriptional upregulation of their genes, and occurs only in highly malignant breast cancer cells. We recently identified GEP100 (BRAG2) to be responsible for the Arf6 activation to induce invasion and metastasis, by directly binding to ligand-activated epidermal growth factor receptor (EGFR). A series of our studies revealed that for activation of the invasion pathway of EGFR, it is prerequisite that Arf6 and AMAP1 both are highly overexpressed, and that EGFR is activated by ligands. Pathological analyses indicate that a significant large population of human ductal cancers may utilize the EGFR-GEP100-Arf6-AMAP1 pathway for their malignancy. Microenvironments have been highly implicated in the malignancy of mammary tumors. Our results reveal an aspect of the precise molecular mechanisms of some breast cancers, in which full invasiveness is not acquired just by intracellular alterations of cancer cells, but extracellular factors from microenvironments may also be necessary. Possible translation of our knowledge to cancer therapeutics will also be discussed.

G-protein-coupled receptors (GPCRs) transduce the binding of extracellular stimuli into intracellular signalling cascades that can lead to morphological changes. Here, we demonstrate that stimulation of the calcium-sensing receptor (CaSR), a GPCR that promotes chemotaxis by detecting increases in extracellular calcium, triggers plasma membrane (PM) ruffling via a pathway that involves β-arrestin 1, Arf nucleotide binding site opener (ARNO), ADP-ribosylating factor 6 (ARF6) and engulfment and cell motility. protein (ELMO). Expression of dominant negative β-arrestin 1 or its knockdown with siRNA impaired the CaSR-induced PM ruffling response. Expression of a catalytically inactive ARNO also reduced CaSR-induced PM ruffling. Furthermore, β-arrestin 1 co-immunoprecipitated with the CaSR and ARNO under resting conditions. Agonist treatment did not markedly alter β-arrestin 1 binding to the CaSR or to ARNO but it did elicit the translocation and colocalisation of the CaSR, β-arrestin 1 and ARNO to membrane protrusions. Furthermore, ARF6 and ELMO, two proteins known to couple ARNO to the cytoskeleton, were required for CaSR-dependent morphological changes and translocated to the PM ruffles. These data suggest that cells ruffle upon CaSR stimulation via a mechanism that involves translocation of β-arrestin 1 pre-assembled with the CaSR or ARNO, and that ELMO plays an essential role in this CaSR-signalling-induced cytoskeletal reorganisation., SCOPUS: ar.j, info:eu-repo/semantics/published

The lack of current treatment and preventable measures for acute kidney injury (AKI) in hospitalized patients results in an increased mortality rate of up to 80% and elevated health costs. Additionally, if not properly repaired, those who survive AKI may develop fibrosis and long-term kidney damage. The molecular aspects of kidney injury and repair are still uncertain. Hepatocyte growth factor (HGF) promotes recovery of the injured kidney by inducing survival and migration of tubular epithelial cells to repopulate bare tubule areas. HGF-stimulated kidney epithelial cell migration requires the activation of ADP-ribosylation factor 6 (Arf6) and Rac1 via the cytohesin family of Arf-guanine-nucleotide exchange factors (GEFs), in vitro. We used an ischemia and reperfusion injury (IRI) mouse model to analyze the effects of modulating this signaling pathway on kidney recovery. We treated IRI mice with either HGF, the cytohesin inhibitor SecinH3, or a combination of both. As previously reported, HGF treatment promoted rapid improvement of kidney function as evidenced by creatinine (Cre) and blood urea nitrogen (BUN) levels. In contrast, simultaneous treatment with SecinH3 and HGF blocks the ability of HGF to promote kidney recovery. Immunohistochemistry showed that HGF treatment promoted recovery of tubule structure, and had enhanced levels of active, GTP-bound Arf6 and GTP-Rac1. SecinH3 treatment, however, caused a dramatic decrease in GTP-Arf6 and GTP-Rac1 levels when compared to kidney sections from HGF-treated IRI mice. Additionally, SecinH3 counteracted the renal reparative effects of HGF. Our results support the conclusion that cytohesin function is required for HGF-stimulated renal IRI repair.

The small guanosine triphosphate (GTP)–binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP–bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.