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Mast cells are one of major players in allergic responses. Mast cell activation via the high affinity IgE receptor (FcεRI) causes degranulation and release of de novo synthesized proinflammatory cytokines in a process that involves vesicle trafficking. Considering that the GTPase ADP-ribosylation factor 1 (Arf1) orchestrates and maintains membrane traffic and organelle structure, it seems likely that Arf1 contributes to mast cell activation. Actually, it has been reported that pharmaceutical blockade of the Arf1 pathway suppresses cytokine secretion and mast cell degranulation. However, physiological roles of Arf1 in mast cells remain elusive. Here, by using a genetic approach, we demonstrate that Arf1 is required for optimal mTORC1 activation upon IL-3 and facilitates mast cell proliferation. On the other hand, contrary to our expectation, Arf1-deficiency had little impact on FcεRI-induced degranulation nor cytokine secretion. Our findings reveal an unexpected role of Arf1 in mast cell expansion and its potential as a therapeutic target in the mast cell proliferative disorders., (© 2022. The Author(s).)

The spatial organization of cells depends on coordination between cytoskeletal systems and intracellular organelles. The Arf1 small G protein and its activator GBF1 are important regulators of Golgi organization, maintaining its morphology and function. Here we show that GBF1 and its substrate Arf1 regulate the spatial organization of mitochondria in a microtubule-dependent manner. Miro is a mitochondrial membrane protein that interacts through adaptors with microtubule motor proteins such as cytoplasmic dynein, the major microtubule minus end directed motor. We demonstrate a physical interaction between GBF1 and Miro, and also between the active GTP-bound form of Arf1 and Miro. Inhibition of GBF1, inhibition of Arf1 activation, or overexpression of Miro, caused a collapse of the mitochondrial network towards the centrosome. The change in mitochondrial morphology upon GBF1 inhibition was due to a two-fold increase in the time engaged in retrograde movement compared to control conditions. Electron tomography revealed that GBF1 inhibition also resulted in larger mitochondria with more complex morphology. Miro silencing or drug inhibition of cytoplasmic dynein activity blocked the GBF1-dependent repositioning of mitochondria. Our results show that blocking GBF1 function promotes dynein- and Miro-dependent retrograde mitochondrial transport along microtubules towards the microtubule-organizing center, where they form an interconnected network.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is a ligand for the EGF receptor (EGFR), one of the most commonly amplified receptor tyrosine kinases (RTKs) in glioblastoma (GBM). While HBEGF has been found to be expressed in a subset of malignant gliomas, its sufficiency for glioma initiation has not been evaluated. In this study, we demonstrate that HBEGF can initiate GBM in mice in the context of Ink4a/Arf and Pten loss, and that these tumors are similar to the classical GBM subtype observed in patients. Isogenic astrocytes from these mice showed activation not only of Egfr but also the RTK Axl in response to HBEGF stimulation. Deletion of either Egfr or Axl decreased the tumorigenic properties of HBEGF-transformed cells; however, only EGFR was able to rescue the phenotype in cells lacking both RTKs indicating that Egfr is required for activation of Axl in this context. Silencing of HBEGF in vivo resulted in tumor regression and significantly increased survival, suggesting that HBEGF may be a clinically relevant target.

AP-1 is a clathrin adaptor recruited to the trans-Golgi Network where it can interact with specific signals found in the cytosolic tail of cargo proteins to incorporate them into clathrin-coated vesicles for trafficking. The small G protein Arf1 regulates the spatiotemporal recruitment of AP-1 and also drives a conformational change favoring an interaction with cargo proteins. A recent crystal structure and in vitro experiments highlighted potential residues mediating the AP-1/Arf1 interaction and the unlocking of the complex. We have used bioluminescence resonance energy transfer (BRET) to study the Arf1/AP-1 interaction and AP-1 conformational changes in vivo. We identified novel residues required for this interaction in addition to those predicted in the crystal structure. We also studied the conformational changes in AP-1 driven by Arf1 in live cells and found that opening of the complex is prerequisite for oligomerization. Using Arf1 knockout cells generated by CRISPR/Cas9, we demonstrated that residue 172 in Arf1 is necessary for AP-1 activation and is required for the efficient sorting of the lysosomal protein prosaposin. We have used BRET to study the in vivo activation of AP-1. The advantages of BRET include expressing full-length proteins in their native environment that have been fully post-translationally modified.

Enterovirus 71 (EV71) is a human pathogen that causes hand, foot, mouth disease and neurological complications. Although EV71, as well as other enteroviruses, initiates a remodeling of intracellular membrane for genomic replication, the regulatory mechanism remains elusive. By screening human cDNA library, we uncover that the Golgi resident protein acyl-coenzyme A binding domain-containing 3 (ACBD3) serves as a target of the 3A protein of EV71. This interaction occurs in cells expressing 3A or infected with EV71. Genetic inhibition or deletion of ACBD3 drastically impairs viral RNA replication and plaque formation. Such defects are corrected upon restoration of ACBD3. In infected cells, EV71 3A redirects ACBD3, to the replication sites. I44A or H54Y substitution in 3A interrupts the binding to ACBD3. As such, viral replication is impeded. These results reveal a mechanism of EV71 replication that involves host ACBD3 for viral replication.

How multicellular organisms maintain immune homeostasis across various organs and cell types is an outstanding question in immune biology and cell signaling. In Drosophila, blood cells (hemocytes) respond to local and systemic cues to mount an immune response. While endosomal regulation of Drosophila hematopoiesis is reported, the role of endosomal proteins in cellular and humoral immunity is not well-studied. Here we demonstrate a functional role for endosomal proteins in immune homeostasis. We show that the ubiquitous trafficking protein ADP Ribosylation Factor 1 (ARF1) and the hemocyte-specific endosomal regulator Asrij differentially regulate humoral immunity. Asrij and ARF1 play an important role in regulating the cellular immune response by controlling the crystal cell melanization and phenoloxidase activity. ARF1 and Asrij mutants show reduced survival and lifespan upon infection, indicating perturbed immune homeostasis. The ARF1-Asrij axis suppresses the Toll pathway anti-microbial peptides (AMPs) by regulating ubiquitination of the inhibitor Cactus. The Imd pathway is inversely regulated- while ARF1 suppresses AMPs, Asrij is essential for AMP production. Several immune mutants have reduced Asrij expression, suggesting that Asrij co-ordinates with these pathways to regulate the immune response. Our study highlights the role of endosomal proteins in modulating the immune response by maintaining the balance of AMP production. Similar mechanisms can now be tested in mammalian hematopoiesis and immunity.

Neuronal calcium sensor-1 (NCS-1) mediates changes in cellular function by regulating various target proteins. Many potential targets have been identified but the physiological significance of only a few has been established. Upon temperature elevation, Caenorhabditis elegans exhibits reversible paralysis. In the absence of NCS-1, worms show delayed onset and a shorter duration of paralysis. This phenotype can be rescued by re-expression of ncs-1 in AIY neurons. Mutants with defects in four potential NCS-1 targets (arf-1.1, pifk-1, trp-1 and trp-2) showed qualitatively similar phenotypes to ncs-1 null worms, although the effect of pifk-1 mutation on time to paralysis was considerably delayed. Inhibition of pifk-1 also resulted in a locomotion phenotype. Analysis of double mutants showed no additive effects between mutations in ncs-1 and trp-1 or trp-2. In contrast, double mutants of arf-1.1 and ncs-1 had an intermediate phenotype, consistent with NCS-1 and ARF-1.1 acting in the same pathway. Over-expression of arf-1.1 in the AIY neurons was sufficient to rescue partially the phenotype of both the arf-1.1 and the ncs-1 null worms. These findings suggest that ARF-1.1 interacts with NCS-1 in AIY neurons and potentially pifk-1 in the Ca(2+) signaling pathway that leads to inhibited locomotion at an elevated temperature.

It has been shown that inhibition of GTPase-activating protein of ADP-ribosylation factor (Arf), ArfGAP, with a small molecule (QS11) results in synergistic activation of Wnt/β-catenin signaling. However, the role of Arf in Wnt/β-catenin signaling has not yet been elucidated. Here, we show that activation of Arf is essential for Wnt/β-catenin signaling. The level of the active form of Arf (Arf-GTP) transiently increased in the presence of Wnt, and this induction event was abrogated by blocking the interaction between Wnt and Frizzled (Fzd). In addition, knockdown of Fzds, Dvls or LRP6 blocked the Wnt-mediated activation of Arf. Consistently, depletion of Arf led to inhibition of Wnt-mediated membrane PtdIns (4,5)P2 (phosphatidylinositol 4, 5-bisphosphate) synthesis and LRP6 phosphorylation. Overall, our data suggest that transient activation of Arf modulates LRP6 phosphorylation for the transduction of Wnt/β-catenin signaling.

Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome. The ability of the myristoyl group to facilitate dynamic protein-protein and protein-membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.

In Saccharomyces cerevisiae, the synthesis of chitin is temporally and spatially regulated through the transport of Chs3p (chitin synthase III) to the plasma membrane in the bud neck region. Traffic of Chs3p from the trans-Golgi network (TGN)/early endosome to the plasma membrane requires the function of Chs5p and Chs6p. Chs6p belongs to a family of four proteins that we have named ChAPs for Chs5p-Arf1p-binding Proteins. Here, we show that all ChAPs physically interact not only with Chs5p but also with the small GTPase Arf1p. A short sequence at the C-terminus of the ChAPs is required for protein function and the ability to bind to Chs5p. Simultaneous disruption of two members, Deltabud7 and Deltabch1, phenocopies a Deltachs6 or Deltachs5 deletion with respect to Chs3p transport. Moreover, the ChAPs interact with each other and can form complexes. In addition, they are all at least partially localized to the TGN in a Chs5p-dependent manner. Most importantly, several ChAPs can interact physically with Chs3p. We propose that the ChAPs facilitate export of cargo out of the Golgi.

The tumor suppressor p19ARF inhibits Mdm2, which restricts the activity of p53. Complicated feedback and control mechanisms regulate ARF, Mdm2, and p53 interactions. Here we report that ARF haploinsufficiency completely rescued the p53-dependent effects of Mdm2 haploinsufficiency on B-cell development, survival, and transformation. In contrast to Mdm2+/- B cells, Mdm2+/- B cells deficient in ARF were similar to wild-type B cells in their rates of growth and apoptosis and activation of p53. Consequently, the profoundly reduced numbers of B cells in Mdm2+/-Emu-myc transgenic mice were restored to normal levels in ARF+/-Mdm2+/-Emu-myc transgenics. Additionally, ARF+/-Mdm2+/-Emu-myc transgenics developed lymphomas at rates analogous to those observed for wild-type Emu-myc transgenics, demonstrating that loss of one allele of ARF rescued the protracted lymphoma latency in Mdm2+/-Emu-myc transgenics. Importantly, in ARF+/-Mdm2+/-Emu-myc transgenic lymphomas, p53 was inactivated at the frequency observed in lymphomas of wild-type Emu-myc transgenics. Collectively, these results support a model whereby the stoichiometry of Mdm2 and ARF controls apoptosis and tumor development, which should have significant implications in the treatment of malignancies that have inactivated ARF.

CD43 or leukosialin is a transmembrane sialoglycoprotein, whose extracellular domain participates in cell adhesiveness and the cytoplasmic tail regulates a variety of intracellular signal transduction pathways involved in cell proliferation. CD43 is abundantly expressed on the surface of hematopoietic cells, but CD43 expression is also frequently found in the tumor cells of nonhematopoietic origin. In the early stages of some tumors, the accumulation of tumor suppressor protein p53 has been described. Here, we show that the expression of CD43 causes the induction of functionally active p53 protein. Moreover, we found that the activation of p53 by CD43 is mediated by tumor suppressor protein ARF. The coexpression of CD43 and ARF in ARF-null mouse embryonic fibroblasts resulted in programmed cell death, but that was not the case when CD43 alone was expressed in these cells. These data provide the first evidence of the connection between p53- and CD43-dependent pathways.

Small GTP-binding (G) proteins are activated by GDP/GTP nucleotide exchange stimulated by guanine nucleotide exchange factors (GEFs). Nucleotide dissociation from small G protein-GEF complexes involves transient GDP-bound intermediates whose structures have never been described. In the case of Arf proteins, small G proteins that regulate membrane traffic in eukaryotic cells, such intermediates can be trapped either by the natural inhibitor brefeldin A or by charge reversal at the catalytic glutamate of the Sec7 domain of their GEFs. Here we report the crystal structures of these intermediates that show that membrane recruitment of Arf and nucleotide dissociation are separate reactions stimulated by Sec7. The reactions proceed through sequential rotations of the Arf.GDP core towards the Sec7 catalytic site, and are blocked by interfacial binding of brefeldin A and unproductive stabilization of GDP by charge reversal. The structural characteristics of the reaction and its modes of inhibition reveal unexplored ways in which to inhibit the activation of small G proteins.

Cytosolic coat proteins that bind reversibly to membranes have a central function in membrane transport within the secretory pathway. One well-studied example is COPI or coatomer, a heptameric protein complex that is recruited to membranes by the GTP-binding protein Arf1. Assembly into an electron-dense coat then helps in budding off membrane to be transported between the endoplasmic reticulum (ER) and Golgi apparatus. Here we propose and corroborate a simple model for coatomer and Arf1 activity based on results analysing the distribution and lifetime of fluorescently labelled coatomer and Arf1 on Golgi membranes of living cells. We find that activated Arf1 brings coatomer to membranes. However, once associated with membranes, Arf1 and coatomer have different residence times: coatomer remains on membranes after Arf1-GTP has been hydrolysed and dissociated. Rapid membrane binding and dissociation of coatomer and Arf1 occur stochastically, even without vesicle budding. We propose that this continuous activity of coatomer and Arf1 generates kinetically stable membrane domains that are connected to the formation of COPI-containing transport intermediates. This role for Arf1/coatomer might provide a model for investigating the behaviour of other coat protein systems within cells.

Polyoma virus (Py) differs from other small DNA tumor viruses in not encoding a protein that inactivates p53. The complete Py early region encoding the large T-antigen (PyLT), middle T-antigen (PyMT) and small T-antigen (PyST) will transform primary rodent cells and REF52 cells, but PyMT, the main Py oncogene, by itself will only transform these cells when p53 or ARF is inactivated. We have related Py oncogene cooperation with the effects of the Py T-antigens on the ARF-p53 signaling pathway. PyMT activates an ARF-induced p53-mediated block to cell division explaining the inability of PyMT alone to generate dividing transformed cells. In contrast, in REF52 cells transformed by the whole Py early region (PyREF52), ARF is upregulated but p53 is not activated. Thus PyLT and/or PyST negates the PyMT-induced ARF-mediated block to cell division by disrupting the signaling pathway from ARF to p53. Although there is no detectable interaction or co-localization of endogenous ARF (nucleoli) and MDM2 (nucleoplasm) in PyREF52 cells, expression of transfected ectopic ARF results in an MDM2/ARF interaction and sequestration of MDM2 into the nucleoli. Sequestration of MDM2 by ARF in the nucleoli is not essential for a p53 response in REF52 cells as activation of Raf in REF52Raf-ER cells results in an ARF-induced p53-mediated cell cycle block in the absence of a detectable ARF-MDM2 interaction. Py may provide new insights into the cellular ARF-p53 signaling pathway.

Small G proteins are GTP-dependent molecular switches that regulate numerous cellular functions. They can be classified into homologous subfamilies that are broadly associated with specific biological processes. Cross-talk between small G-protein families has an important role in signalling, but the mechanism by which it occurs is poorly understood. The coordinated action of Arf and Rho family GTPases is required to regulate many cellular processes including lipid signalling, cell motility and Golgi function. Arfaptin is a ubiquitously expressed protein implicated in mediating cross-talk between Rac (a member of the Rho family) and Arf small GTPases. Here we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, but binds to Rac.GTP and Rac.GDP with similar affinities. The X-ray structure of Arfaptin reveals an elongated, crescent-shaped dimer of three-helix coiled-coils. Structures of Arfaptin with Rac bound to either GDP or the slowly hydrolysable analogue GMPPNP show that the switch regions adopt similar conformations in both complexes. Our data highlight fundamental differences between the molecular mechanisms of Rho and Ras family signalling, and suggest a model of Arfaptin-mediated synergy between the Arf and Rho family signalling pathways.