Arf GTPases Research Articles

ASAP3 regulates microvilli structure in parietal cells and presents intervention target for gastric acidity

Gastric acidity-associated disorders such as peptic ulcer and reflux diseases are widespread, and the reported resistance and side effects of currently used medicines suggest an urgent requirement for alternative therapeutic approaches. Here we demonstrate a critical role of ASAP3 in regulating the microvilli structure of parietal cells in vivo, and reveal the feasibility of controlling gastric acidity by targeting ASAP3. Conditional knockout of ASAP3 in mice caused elongation and stacking of microvilli in parietal cells, and substantially decreased gastric acid secretion. These were associated with active assembly of F-actin caused by a higher level of GTP-bound Arf6 GTPase. Consistently, a small molecular compound QS11 inhibited ASAP3 function and significantly reduced gastric acidity in vivo. Of note, the expression of ASAP3 was positively correlated with gastric acid secretion in 90 human cases, and high expression of ASAP3 was associated with reflux disease and peptic ulcer. These results reveal for the first time that ASAP3 regulates the microvilli structures in parietal cells. Our data also suggest ASAP3 as a feasible and drugable therapeutic target for gastric acidity-associated diseases.

Role of Arf GTPases in fungal morphogenesis and virulence.

Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth, which requires sustained membrane traffic and polarized growth. In many organisms, small GTPases of the Arf (ADP-ribosylation factor) family regulate membrane/protein trafficking, yet little is known about their role in fungal filamentous growth. To investigate these GTPases in C. albicans, we generated loss of function mutants in all 3 Arf proteins, Arf1-Arf3, and 2 Arf-like proteins, Arl1 and Arl3. Our results indicate that of these proteins, Arf2 is required for viability and sensitivity to antifungal drugs. Repressible ARF2 expression results in defects in filamentous growth, cell wall integrity and virulence, likely due to alteration of the Golgi. Arl1 is also required for invasive filamentous growth and, although arl1/arl1 cells can initiate hyphal growth, hyphae are substantially shorter than that of the wild-type, due to the inability of this mutant to maintain hyphal growth at a single site. We show that this defect does not result from an alteration of phospholipid distribution and is unlikely to result from the sole Golgin Imh1 mislocalization, as Imh1 is not required for invasive filamentous growth. Rather, our results suggest that the arl1/arl1 hyphal growth defect results from increased secretion in this mutant. Strikingly, the arl1/arl1 mutant is drastically reduced in virulence during oropharyngeal candidiasis. Together, our results highlight the importance of Arl1 and Arf2 as key regulators of hyphal growth and virulence in C. albicans and identify a unique function of Arl1 in secretion.

The small GTPase Arf6 regulates sea urchin morphogenesis

The small GTPase Arf6 is a conserved protein that is expressed in all metazoans. Arf6 remodels cytoskeletal actin and mediates membrane protein trafficking between the plasma membrane in its active form and endosomal compartments in its inactive form. While a rich knowledge exists for the cellular functions of Arf6, relatively little is known about its physiological role in development. This study examines the function of Arf6 in mediating cellular morphogenesis in early development. We dissect the function of Arf6 with a loss-of-function morpholino and constitutively active Arf6-Q67L construct. We focus on the two cell types that undergo active directed migration: the primary mesenchyme cells (PMCs) that give rise to the sea urchin skeleton and endodermal cells that form the gut. Our results indicate that Arf6 plays an important role in skeleton formation and PMC migration, in part due to its ability to remodel actin. We also found that embryos injected with Arf6 morpholino have gastrulation defects and embryos injected with constitutively active Arf6 have endodermal cells detached from the gut epithelium with decreased junctional cadherin staining, indicating that Arf6 may mediate the recycling of cadherin. Thus, Arf6 impacts cells that undergo coordinated movement to form embryonic structures in the developing embryo.

Membrane Binding and Remodelling by the COPII Coat

Dynamic remodelling of cellular membranes in conjunction with protein complex assembly and disassembly constitutes a key component of intracellular trafficking pathways. Reconstitution of selected steps outside the cell using artificial membranes facilitates the use of biophysical techniques for investigating structure and dynamics of protein-lipid assemblies. COPII-coated membrane vesicles are responsible for transporting cargo from the ER towards the Golgi apparatus in the secretory pathway. The small GTPase Sar1, which belongs to the Sar1/Arf family of GTPases and the Ras-superfamily, is an essential component in COPII-vesicle formation. Upon activation by GTP, Sar1 binds to membranes, embedding an amphipathic helix into the proximal leaflet of the bilayer. The role of GTP presence versus GTP hydrolysis in the fission of COPII vesicles is not completely clear. We study COPII vesicle formation in a bottom-up fashion on baker's yeast proteins and model membranes using cryo-electron microscopy techniques (cryo-EM), confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS). Cryo-EM shows strongly different COPII coated vesicle morphologies under GTP hydrolyzing versus non-hydrolyzing conditions. More subtle differences are observed by cryo-EM among reconstituted COPII samples under non-hydrolyzing conditions. Membrane binding of the small GTPases Sar1 and Arf1 is studied with biophysical techniques: By combining FCS with a Langmuir film balance, the protein's footprint in the proximal membrane leaflet is revealed. Dual-color fluorescence cross-correlation spectroscopy (FCCS) analysis has been developed into a method for obtaining binding curves and testing binding models of protein binding to freely diffusing vesicles. The higher specificity makes fluorescence cross-correlation advantageous compared to previous fluorescence autocorrelation analysis also for small vesicle binding studies.

Sec71 functions as a GEF for the small GTPase Arf1 to govern dendrite pruning of Drosophila sensory neurons.

Pruning, whereby neurons eliminate their excess neurites, is central for the maturation of the nervous system. In Drosophila, sensory neurons, ddaCs, selectively prune their larval dendrites without affecting their axons during metamorphosis. However, it is unknown whether the secretory pathway plays a role in dendrite pruning. Here, we show that the small GTPase Arf1, an important regulator of the secretory pathway, is specifically required for dendrite pruning of ddaC/D/E sensory neurons but dispensable for apoptosis of ddaF neurons. Analyses of the GTP- and GDP-locked forms of Arf1 indicate that the cycling of Arf1 between GDP-bound and GTP-bound forms is essential for dendrite pruning. We further identified Sec71 as a guanine nucleotide exchange factor for Arf1 that preferentially interacts with its GDP-bound form. Like Arf1, Sec71 is also important for dendrite pruning, but not for apoptosis, of sensory neurons. Arf1 and Sec71 are interdependent for their localizations on Golgi. Finally, we show that the Sec71/Arf1-mediated trafficking process is a prerequisite for Rab5-dependent endocytosis to facilitate endocytosis and degradation of the cell-adhesion molecule Neuroglian (Nrg).

Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO.

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.

Proangiogenic TF-FVIIa-PAR2 Signaling Requires Matriptase-Independent Integrin Interaction

The close link between coagulation activation and cancer progression is supported by clinical and experimental studies. A central molecular pathways by which tumor cells interact with the hemostatic system is through the expression of the cell surface receptor tissue factor (TF) that in complex with coagulation factor VIIa (FVIIa) triggers the extrinsic pathway of blood coagulation, contributes to cancer associated thrombosis, and promotes direct tumor cell signaling through protease-activated receptors (PARs). Genetic and pharmacological evidence shows that epithelial and tumor cell TF-FVIIa signaling induces a diverse set of proangiogenic and immune modulatory cytokines, chemokines and growth factors. However, the mechanistic details of this signaling pathway remain incompletely understood. Furthermore, a recent study has suggested that the TF-FVIIa complex does not directly cleave PAR2, but rather induces PAR signaling through initiating a cell surface protease cascade involving the transmembrane serine protease matriptase. Here, we employ mutants of FVIIa to characterize the roles of cell surface proteolysis and endosomal signaling by TF in keratinocytes and cancer cells.We studied TF-expressing MDA-MB-231 breast cancer cells, A7 melanoma cells transduced with TF and PAR2, and non-tumorigenic HaCaT keratinocytes. Stimulation with 10 nM FVIIa or 5 nM recombinant matriptase promoted productive PAR2 signaling in all three cell types as assessed by quantification of cytokine IL-8 mRNA up-regulation. Addition of the serine protease inhibitor aprotinin abolished IL-8 upregulation by recombinant matriptase, but not by FVIIa. Importantly, matriptase was expressed by HaCaT and A7 melanoma cells, whereas MDA-MB-231 cells did not express matriptase, while responding to FVIIa with efficient proangiogenic signaling. HaCaT and A7 cells showed shedding of the matriptase protease domain following incubation with FVIIa, suggesting activation of the serine protease. However, the signaling-defective FVIIa Q40A mutant also promoted matriptase shedding without inducing IL-8. We had previously shown that FVIIa induces association of TF with integrin β1 hetorodimers. We characterized an integrin-binding deficient FVIIa mutant, E26A, rendered incapable to interact with active integrin β1, resulting in defective TF-FVIIa internalization, TF-FVIIa stimulated cell migration and induction of IL-8. FVIIa E26A promoted matriptase shedding as observed with wild-type FVIIa. We found that inhibition of the small rho GTPase arf6, which controls cellular trafficking of β1 integrins, by pharmacological inhibitors or expression of a dominant negative arf6 mutant prevented IL-8 induction by TF-FVIIa, but not by direct stimulation of PAR2 with agonist peptide. Pharmacological activation of arf6 or expression of arf6 constitutive active mutant on the other hand promoted translocation of TF-FVIIa, which unlike PAR2 agonist peptide induced delayed and prolonged MAPK activation downstream of PAR2. This data show that TF-FVIIa triggers PAR2 signaling dependent on integrin-arf6-regulated trafficking and prolonged activation of endosomal signaling.Our data support the conclusion that the FVIIa induced interaction of TF with integrins and endocytosis of the TF-FVIIa-integrin complex plays a pivotal role in inducing proangiogenic cytokines downstream of proteolytic cleavage of PAR2. Mutants of FVIIa that are defective in supporting this pathway fully promote cell surface matriptase activation, but fail to upregulate tumor cell proangiogenic responses or tumor cell migration. These results argue against a role for matriptase as an intermediary in TF-FVIIa induced PAR2 activation, but do not exclude that matriptase modulates homeostatic epithelial responses through PAR2 signaling in non-malignant cells. These data furthermore suggest that matriptase inhibitors cannot substitute for targeting TF as potential therapeutic strategy to attenuate cancer proangiogenic signaling of the hemostatic system DisclosuresOstergaard:Novo Nordisk A/S: Employment. Ruf:Iconic Therapeutics: Consultancy.

Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice.

Upon vessel injury, platelets become activated and rapidly reorganize their actin cytoskeleton to adhere to the site of endothelial damage, triggering the formation of a fibrin-rich plug to prevent further blood loss. Inactivation of Pdlim7 provides the new perspective that regulation of actin cytoskeletal changes in platelets is dependent on the encoded PDZ-LIM protein. Loss-of-function of Pdlim7 triggers hypercoagulopathy and causes significant perinatal lethality in mice. Our in vivo and in vitro studies reveal that Pdlim7 is dynamically distributed along actin fibers, and lack of Pdlim7 leads to a marked inability to rearrange the actin cytoskeleton. Specifically, the absence of Pdlim7 prevents platelets from bundling actin fibers into a concentric ring that defines the round spread shape of activated platelets. Similarly, in mouse embryonic fibroblasts, loss of Pdlim7 abolishes the formation of stress fibers needed to adopt the typical elongated fibroblast shape. In addition to revealing a fundamental cell biological role in actin cytoskeletal organization, we also demonstrate a function of Pdlim7 in regulating the cycling between the GTP/GDP-bound states of Arf6. The small GTPase Arf6 is an essential factor required for actin dynamics, cytoskeletal rearrangements, and platelet activation. Consistent with our findings of significantly elevated initial F-actin ratios and subsequent morphological aberrations, loss of Pdlim7 causes a shift in balance towards an increased Arf6-GTP level in resting platelets. These findings identify a new Pdlim7-Arf6 axis controlling actin dynamics and implicate Pdlim7 as a primary endogenous regulator of platelet-dependent hemostasis.

Machineries regulating the activity of the small GTPase Arf6 in cancer cells are potential targets for developing innovative anti-cancer drugs.

The Small GTPase ADP-ribosylation factor 6 (Arf6) functions as the molecular switch in cellular signaling pathways by cycling between GDP-bound inactive and GTP-bound active form, which is precisely regulated by two regulators, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Numerous studies have shown that these machineries play critical roles in tumor angiogenesis/growth and cancer cell invasion/metastasis through regulating the cycling of Arf6. Here, we summarize accumulating knowledge for involvement of Arf6 GEFs/GAPs and small molecule inhibitors of Arf6 signaling/cycling in cancer progression, and discuss possible strategies for developing innovative anti-cancer drugs targeting Arf6 signaling/cycling.

Study of Ethanol-Induced Golgi Disorganization Reveals the Potential Mechanism of Alcohol-Impaired N-Glycosylation.

It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi; however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation. HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35mM EtOH for 72hours. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8weeks. Characterization of Golgi-associated mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), α-1,2-mannosidase (Man-I), and α-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by three-dimensional structured illumination microscopy (3D SIM). First, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor; however, the high-mannose-type N-glycans are increased. Further analysis by 3D SIM revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of ADP-ribosylation factor 1 (Arf1) GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (i) EtOH specifically blocks activation of Arf1, and (ii) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man-II, is giantin sensitive. Thus, we provide the mechanism by which EtOH-induced Golgi remodeling may significantly modify formation of N-glycans.

C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons.

Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell-derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics.

Small GTPase ARF6 Regulates Endocytic Pathway Leading to Degradation of ATP-Binding Cassette Transporter A1.

ABCA1 (ATP-binding cassette transporter A1) is the principal protein responsible for cellular cholesterol efflux. Abundance and functionality of ABCA1 is regulated both transcriptionally and post-translationally, with endocytosis of ABCA1 being an important element of post-translational regulation. Functional ABCA1 resides on the plasma membrane but can be internalized and either degraded or recycled back to the plasma membrane. The interaction between the degradative and recycling pathways determines the abundance of ABCA1 and may contribute to the efflux of intracellular cholesterol. Here, we show that the principal pathway responsible for the internalization of ABCA1 leading to its degradation in macrophages is ARF6-dependent endocytic pathway. This pathway was predominant in the regulation of ABCA1 abundance and efflux of plasma membrane cholesterol. Conversely, the efflux of intracellular cholesterol was predominantly controlled by ARF6-independent pathways, and inhibition of ARF6 shifted ABCA1 into recycling endosomes enhancing efflux of intracellular cholesterol. We conclude that ARF6-dependent pathway is the predominant route responsible for the ABCA1 internalization and degradation, whereas ARF6-independent endocytic pathways may contribute to ABCA1 recycling and efflux of intracellular cholesterol.

Inhibition of WAVE Regulatory Complex Activation by a Bacterial Virulence Effector Counteracts Pathogen Phagocytosis

SummaryTo establish pathogenicity, bacteria must evade phagocytosis directed by remodeling of the actin cytoskeleton. We show that macrophages facilitate pathogen phagocytosis through actin polymerization mediated by the WAVE regulatory complex (WRC), small GTPases Arf and Rac1, and the Arf1 activator ARNO. To establish extracellular infections, enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli hijack the actin cytoskeleton by injecting virulence effectors into the host cell. Here, we find that the virulence effector EspG counteracts WRC-dependent phagocytosis, enabling EPEC and EHEC to remain extracellular. By reconstituting membrane-associated actin polymerization, we find that EspG disabled WRC activation through two mechanisms: EspG interaction with Arf6 blocked signaling to ARNO while EspG binding of Arf1 impeded collaboration with Rac1, thereby inhibiting WRC recruitment and activation. Investigating the mode of EspG interference revealed sites in Arf1 required for WRC activation and a mechanism facilitating pathogen evasion of innate host defenses.

The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin.

AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3) and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1). Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD) and schizophrenia (SZ); yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse ortholog of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function.

ACAP3 regulates neurite outgrowth through its GAP activity specific to Arf6in mouse hippocampal neurons.

ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 3) belongs to the ACAP family of GAPs (GTPase-activating proteins) for the small GTPase Arf (ADP-ribosylation factor). However, its specificity to Arf isoforms and physiological functions remain unclear. In the present study, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild-type ACAP3, but not by its GAP activity-deficient mutant. Ectopically expressed ACAP3in HEK (human embryonic kidney)-293T cells showed the GAP activity specific to Arf6. In support of this observation, the level of GTP-bound Arf6 was significantly increased by knockdown of ACAP3in hippocampal neurons. In addition, knockdown and knockout of Arf6in mouse hippocampal neurons suppressed neurite outgrowth. These results demonstrate that ACAP3 positively regulates neurite outgrowth through its GAP activity specific to Arf6. Furthermore, neurite outgrowth suppressed by ACAP3 knockdown was rescued by expression of a fast cycle mutant of Arf6 that spontaneously exchanges guanine nucleotides on Arf6, but not by that of wild-type, GTP- or GDP-locked mutant Arf6. Thus cycling between active and inactive forms of Arf6, which is precisely regulated by ACAP3in concert with a guanine-nucleotide-exchange factor(s), seems to be required for neurite outgrowth of hippocampal neurons.

Structural Insights into Arl1-Mediated Targeting of the Arf-GEF BIG1 to the trans-Golgi

SummaryThe GTPase Arf1 is the major regulator of vesicle traffic at both the cis- and trans-Golgi. Arf1 is activated at the cis-Golgi by the guanine nucleotide exchange factor (GEF) GBF1 and at the trans-Golgi by the related GEF BIG1 or its paralog, BIG2. The trans-Golgi-specific targeting of BIG1 and BIG2 depends on the Arf-like GTPase Arl1. We find that Arl1 binds to the dimerization and cyclophilin binding (DCB) domain in BIG1 and report a crystal structure of human Arl1 bound to this domain. Residues in the DCB domain that bind Arl1 are required for BIG1 to locate to the Golgi in vivo. DCB domain-binding residues in Arl1 have a distinct conformation from those in known Arl1-effector complexes, and this plasticity allows Arl1 to interact with different effectors of unrelated structure. The findings provide structural insight into how Arf1 GEFs, and hence active Arf1, achieve their correct subcellular distribution.

ARF6 Is an Actionable Node that Orchestrates Oncogenic GNAQ Signaling in Uveal Melanoma

Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as β-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and β-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.

Enterohaemorrhagic E. coli modulates an ARF6:Rab35 signaling axis to prevent recycling endosome maturation during infection

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) manipulate a plethora of host cell processes to establish infection of the gut mucosa. This manipulation is achieved via the injection of bacterial effector proteins into host cells using a Type III secretion system. We have previously reported that the conserved EHEC and EPEC effector EspG disrupts recycling endosome function, reducing cell surface levels of host receptors through accumulation of recycling cargo within the host cell. Here we report that EspG interacts specifically with the small GTPases ARF6 and Rab35 during infection. These interactions target EspG to endosomes and prevent Rab35-mediated recycling of cargo to the host cell surface. Furthermore, we show that EspG has no effect on Rab35-mediated uncoating of newly formed endosomes, and instead leads to the formation of enlarged EspG/TfR/Rab11 positive, EEA1/Clathrin negative stalled recycling structures. Thus, this paper provides a molecular framework to explain how EspG disrupts recycling whilst also reporting the first known simultaneous targeting of ARF6 and Rab35 by a bacterial pathogen.

P53- and mevalonate pathway-driven malignancies require Arf6 for metastasis and drug resistance.

Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial-mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program. The MVP enzyme geranylgeranyl transferase II (GGT-II) and its substrate Rab11b are critical for Arf6 trafficking to the plasma membrane, where it is activated by receptor tyrosine kinases. Consistently, mutant p53, which is known to support tumorigenesis via MVP, promotes Arf6 activation via GGT-II and Rab11b. Inhibition of MVP and GGT-II blocked invasion and metastasis and reduced cancer cell resistance against chemotherapy agents, but only in cells overexpressing Arf6 and components of the mesenchymal program. Overexpression of Arf6 and mesenchymal proteins as well as enhanced MVP activity correlated with poor patient survival. These results provide insights into the molecular basis of MVP-driven malignancy.

Unfolded protein response regulates yeast small GTPase Arl1p activation at late Golgi via phosphorylation of Arf GEF Syt1p

ADP ribosylation factor (Arf) GTPases are key regulators of membrane traffic at the Golgi complex. In yeast, Arf guanine nucleotide-exchange factor (GEF) Syt1p activates Arf-like protein Arl1p, which was accompanied by accumulation of golgin Imh1p at late Golgi, but whether and how this function of Syt1p is regulated remains unclear. Here, we report that the inositol-requiring kinase 1 (Ire1p)-mediated unfolded protein response (UPR) modulated Arl1p activation at late Golgi. Arl1p activation was dependent on both kinase and endo-RNase activities of Ire1p. Moreover, constitutively active transcription factor Hac1p restored the Golgi localization of Arl1p and Imh1p inIRE1-deleted cells. Elucidating the mechanism of Ire1p-Hac1p axis actions, we found that it regulated phosphorylation of Syt1p, which enhances Arl1p activation, recruitment of Imh1p to the Golgi, and Syt1p interaction with Arl1p. Consistent with these findings, the induction of UPR by tunicamycin treatment increases phosphorylation of Syt1p, resulting in Arl1p activation. Thus, these findings clarify how the UPR influences the roles of Syt1p, Arl1p, and Imh1p in Golgi transport.