Identification of a Guanine Nucleotide Exchange Factor for Arf3, the Yeast Orthologue of Mammalian Arf6

Small G proteins of the Arf and Rab families are fundamental to the organisation and activity of intracellular membranes. One of the most well characterised of these G proteins is mammalian Arf6, a protein that participates in many cellular processes including endocytosis, actin remodelling and cell adhesion. Exchange of GDP for GTP on Arf6 is performed by a variety of guanine nucleotide exchange factors (GEFs), principally of the cytohesin (PSCD) and EFA6 (PSD) families. In this paper we describe the characterisation of a GEF for the yeast orthologue of Arf6, Arf3, which we have named Yel1 (yeast EFA6-like-1) using yeast genetics, fluorescence microscopy and in vitro nucleotide exchange assays. Yel1 appears structurally related to the EFA6 family of GEFs, having an N-terminal Sec7 domain and C-terminal PH and coiled-coil domains. We find that Yel1 is constitutively targeted to regions of polarised growth in yeast, where it co-localises with Arf3. Moreover the Sec7 domain of Yel1 is required for its membrane targeting and for that of Arf3. Finally we show that the isolated Yel1 Sec7 domain strongly stimulates nucleotide exchange activity specifically on Arf3 in vitro

Confining Domains Lead to Reaction Bursts: Reaction Kinetics in the Plasma Membrane

Confinement of molecules in specific small volumes and areas within a cell is likely to be a general strategy that is developed during evolution for regulating the interactions and functions of biomolecules. The cellular plasma membrane, which is the outermost membrane that surrounds the entire cell, was considered to be a continuous two-dimensional liquid, but it is becoming clear that it consists of numerous nano-meso-scale domains with various lifetimes, such as raft domains and cytoskeleton-induced compartments, and membrane molecules are dynamically trapped in these domains. In this article, we give a theoretical account on the effects of molecular confinement on reversible bimolecular reactions in a partitioned surface such as the plasma membrane. By performing simulations based on a lattice-based model of diffusion and reaction, we found that in the presence of membrane partitioning, bimolecular reactions that occur in each compartment proceed in bursts during which the reaction rate is sharply and briefly increased even though the asymptotic reaction rate remains the same. We characterized the time between reaction bursts and the burst amplitude as a function of the model parameters, and discussed the biological significance of the reaction bursts in the presence of strong inhibitor activity

Prostate Cancer-Specific and Potent Antitumor Effect of a DD3-Controlled Oncolytic Virus Harboring the PTEN Gene

Prostate cancer is a major health problem for men in Western societies. Here we report a Prostate Cancer-Specific Targeting Gene-Viro-Therapy (CTGVT-PCa), in which PTEN was inserted into a DD3-controlled oncolytic viral vector (OV) to form Ad.DD3.E1A.E1B(Δ55)-(PTEN) or, briefly, Ad.DD3.D55-PTEN. The woodchuck post-transcriptional element (WPRE) was also introduced at the downstream of the E1A coding sequence, resulting in much higher expression of the E1A gene. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical bio-diagnostic marker. PTEN is frequently inactivated in primary prostate cancers, which is crucial for prostate cancer progression. Therefore, the Ad.DD3.D55-PTEN has prostate cancer specific and potent antitumor effect. The tumor growth rate was almost completely inhibited with the final tumor volume after Ad.DD3.D55-PTEN treatment less than the initial volume at the beginning of Ad.DD3.D55-PTEN treatment, which shows the powerful antitumor effect of Ad.DD3.D55-PTEN on prostate cancer tumor growth. The CTGVT-PCa construct reported here killed all of the prostate cancer cell lines tested, such as DU145, 22RV1 and CL1, but had a reduced or no killing effect on all the non-prostate cancer cell lines tested. The mechanism of action of Ad.DD3.D55-PTEN was due to the induction of apoptosis, as detected by TUNEL assays and flow cytometry. The apoptosis was mediated by mitochondria-dependent and -independent pathways, as determined by caspase assays and mitochondrial membrane potential

ADP-Ribosylation Factor 6 Expression and Activation Are Reduced in Myometrium in Complicated Pregnancies

ARF6 (ADP-ribosylation factor 6) small GTP binding protein plays critical roles in actin cytoskeleton rearrangements and membrane trafficking, including internalisation of G protein coupled receptors (GPCR). ARF6 operates by cycling between GDP-bound (inactive) and GTP-bound (active) forms and is a potential regulator of GPCR-mediated uterine activity during pregnancy and labour. ARF6 contains very low intrinsic GTP binding activity and depends on GEFs (guanine nucleotide exchange factors) such as CYTH3 (cytohesin 3) to bind GTP. ARF6 and CYTH3 were originally cloned from human placenta, but there is no information on their expression in other reproductive tissues.The expression of ARF6, ARF1, and CYTH1-4 was investigated by measuring mRNA (using RT-PCR) and protein levels (using immunoblotting) in samples of myometrium obtained from non-pregnant women, and women with normal pregnancies, before or after the spontaneous onset of labour. We also analysed myometrial samples from women with spontaneous preterm labour and from women with complicated pregnancies requiring emergency preterm delivery. The GST)-effector pull down assay was used to study the presence of active ARF6 and ARF1 in all myometrial extracts.ARF6, ARF1 and CYTH3 but not CYTH1, CYTH2 and CYTH4 were expressed in all samples and the levels did not change with pregnancy or labour. However, ARF6 and CYTH3 but not ARF1 levels were significantly reduced in complicated pregnancies. The alterations in the expression of ARF6 and its GEF in human myometrium indicate a potential involvement of this signalling system in modulating the response of myometrial smooth muscle in complicated pregnancies. The levels of ARF6-GTP or ARF1-GTP did not change with pregnancy or labour but ARF6-GTP levels were significantly decreased in women with severe complications of pregnancy.We have demonstrated a functional ARF6 system in human myometrium and a correlation between ARF6 level and activity in uterine and abnormal pregnancy

The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1

The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein

Phenotypic characterisation of regulatory T cells in dogs reveals signature transcripts conserved in humans and mice

Regulatory T cells (Tregs) are a double-edged regulator of the immune system. Aberrations of Tregs correlate with pathogenesis of inflammatory, autoimmune and neoplastic disorders. Phenotypically and functionally distinct subsets of Tregs have been identified in humans and mice on the basis of their extensive portfolios of monoclonal antibodies (mAb) against Treg surface antigens. As an important veterinary species, dogs are increasingly recognised as an excellent model for many human diseases. However, insightful study of canine Tregs has been restrained by the limited availability of mAb. We therefore set out to characterise CD4+CD25high T cells isolated ex vivo from healthy dogs and showed that they possess a regulatory phenotype, function, and transcriptomic signature that resembles those of human and murine Tregs. By launching a cross-species comparison, we unveiled a conserved transcriptomic signature of Tregs and identified that transcript hip1 may have implications in Treg function

Phospholipase D signaling: orchestration by PIP2 and small GTPases

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions

PIP3 controls synaptic function by maintaining AMPA receptor clustering at the postsynaptic membrane

Supplementary information is available on the Nature Neuroscience website.Despite their low abundance, phosphoinositides are critical regulators of intracellular signaling and membrane compartmentalization. However, little is known of phosphoinositide function at the postsynaptic membrane. Here we show that continuous synthesis and availability of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) at the postsynaptic terminal is necessary for sustaining synaptic function in rat hippocampal neurons. This requirement was specific for synaptic, but not extrasynaptic, AMPA receptors, nor for NMDA receptors. PIP3 downregulation impaired PSD-95 accumulation in spines. Concomitantly, AMPA receptors became more mobile and migrated from the postsynaptic density toward the perisynaptic membrane within the spine, leading to synaptic depression. Notably, these effects were only revealed after prolonged inhibition of PIP3 synthesis or by direct quenching of this phosphoinositide at the postsynaptic cell. Therefore, we conclude that a slow, but constant, turnover of PIP3 at synapses is required for maintaining AMPA receptor clustering and synaptic strength under basal conditions.This work was supported by grants from the US National Institute of Mental Health (J.A.E. and J.R.M.), the Dana Foundation (J.A.E.) and the Spanish Ministry of Science and Innovation (J.A.E.). M.F.-M. and S.K. are supported by postdoctoral contracts, and M.R. by a predoctoral fellowship, from the Spanish Ministry of Science and Innovation.Peer reviewe