Expression Of Dominant-negative Mutant Research Articles

VE-CADHERIN is expressed transiently in early ISL1+ cardiovascular progenitor cells and facilitates cardiac differentiation.

Adherens junctions (AJs) provide adhesive properties through cadherins and associated cytoplasmic catenins and participate in morphogenetic processes. We examined AJs formed between ISL1+ cardiovascular progenitor cells during differentiation of embryonic stem cells (ESCs) invitro and in mouse embryogenesis invivo. We found that, in addition to N-CADHERIN, a percentage of ISL1+ cells transiently formed vascular endothelial (VE)-CADHERIN-mediated AJs during invitro differentiation on days 4 and 5, and the same pattern was observed invivo. Fluorescence-activated cell sorting (FACS) analysis extended morphological data showing that VE-CADHERIN+/ISL1+ cells constitute a significant percentage of cardiac progenitors on days 4 and 5. The VE-CADHERIN+/ISL1+ cell population represented one-third of the emerging FLK1+/PDGFRa+ cardiac progenitor cells (CPCs) for a restricted time window (days 4-6). Ablation of VE-CADHERIN during ESC differentiation results in severe inhibition of cardiac differentiation. Disruption of all classic cadherins in the VE-CADHERIN+ population via a cadherin dominant-negative mutant's expression resulted in a dramatic decrease in the ISL1+ population and inhibition of cardiac differentiation.

H3K4 Methylation Promotes Expression of Mitochondrial Dynamics Regulators to Ensure Oocyte Quality in Mice.

Significantly decreased H3K4 methylation in oocytes from aged mice indicates the important roles of H3K4 methylation in female reproduction. However, how H3K4 methylation regulates oocyte development remains largely unexplored. In this study, it is demonstrated that oocyte-specific expression of dominant negative mutant H3.3-K4M led to a decrease of the level of H3K4 methylation in mouse oocytes, resulting in reduced transcriptional activity and increased DNA methylation in oocytes, disturbed oocyte developmental potency, and fertility of female mice. The impaired expression of genes regulating mitochondrial functions in H3.3-K4M oocytes, accompanied by mitochondrial abnormalities, is further noticed. Moreover, early embryos from H3.3-K4M oocytes show developmental arrest and reduced zygotic genome activation. Collectively, these results show that H3K4 methylation in oocytes is critical to orchestrating gene expression profile, driving the oocyte developmental program, and ensuring oocyte quality. This study also improves understanding of how histone modifications regulate organelle dynamics in oocytes.

Combined role for YAP-TEAD and YAP-RUNX2 signalling in substrate-stiffness regulation of cardiac fibroblast proliferation

Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM) in part mediated by increased cardiac fibroblast proliferation However, our understanding of the mechanisms regulating cardiac fibroblast proliferation are incomplete. Here we characterise a novel mechanism involving a combined activation of Yes-associated protein (YAP) targets RUNX Family Transcription Factor 2 (RUNX2) and TEA Domain Transcription Factor (TEAD).We demonstrate that cardiac fibroblast proliferation is enhanced by interaction with a stiff ECM compared to a soft ECM. This is associated with activation of the transcriptional co-factor, YAP. We demonstrate that this stiffness induced activation of YAP enhances the transcriptional activity of both TEAD and RUNX2 transcription factors. Inhibition of either TEAD or RUNX2, using gene silencing, expression of dominant-negative mutants or pharmacological inhibition, reduces cardiac fibroblast proliferation. Using mutants of YAP, defective in TEAD or RUNX2 activation ability, we demonstrate a dual role of YAP-mediated activation of TEAD and RUNX2 for substrate stiffness induced cardiac fibroblast proliferation.Our data highlights a previously unrecognised role of YAP mediated RUNX2 activation for cardiac fibroblast proliferation in response to increased ECM stiffness.

MRCK-Alpha and Its Effector Myosin II Regulatory Light Chain Bind ABCB4 and Regulate Its Membrane Expression.

ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.

The Rab5-Rab11 Endosomal Pathway is Required for BDNF-Induced CREB Transcriptional Regulation in Hippocampal Neurons.

Brain-derived neurotrophic factor (BDNF) is a key regulator of the morphology and connectivity of central neurons. We have previously shown that BDNF/TrkB signaling regulates the activity and mobility of the GTPases Rab5 and Rab11, which in turn determine the postendocytic sorting of signaling TrkB receptors. Moreover, decreased Rab5 or Rab11 activity inhibits BDNF-induced dendritic branching. Whether Rab5 or Rab11 activity is important for local events only or for regulating nuclear signaling and gene expression is unknown. Here, we investigated, in rat hippocampal neuronal cultures derived from embryos of unknown sex, whether BDNF-induced signaling cascades are altered when early and recycling endosomes are disrupted by the expression of dominant-negative mutants of Rab5 and Rab11. The activity of both Rab5 and Rab11 was required for sustained activity of Erk1/2 and nuclear CREB phosphorylation, and increased transcription of a BDNF-dependent program of gene expression containing CRE binding sites, which includes activity-regulated genes such as Arc, Dusp1, c-fos, Egr1, and Egr2, and growth and survival genes such as Atf3 and Gem Based on our results, we propose that early and recycling endosomes provide a platform for the integration of neurotrophic signaling from the plasma membrane to the nucleus in neurons, and that this mechanism is likely to regulate neuronal plasticity and survival.SIGNIFICANCE STATEMENT BDNF is a neurotrophic factor that regulates plastic changes in the brain, including dendritic growth. The cellular and molecular mechanisms underlying this process are not completely understood. Our results uncover the cellular requirements that central neurons possess to integrate the plasma membrane into nuclear signaling in neurons. Our results indicate that the endosomal pathway is required for the signaling cascade initiated by BDNF and its receptors at the plasma membrane to modulate BDNF-dependent gene expression and neuronal dendritic growth mediated by the CREB transcription factor. CREB is a key transcription factor regulating circuit development and learning and memory.

Valosin-containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress.

Valosin-containing protein (VCP)/p97/Cdc48 is one of the best-characterised type II cytosolic AAA+ ATPases most known for their role in ubiquitin-dependent protein quality control. Here, we provide functional insights into the role of the Leishmania VCP/p97 homologue (LiVCP) in the parasite intracellular development. We demonstrate that although LiVCP is an essential gene, Leishmania infantum promastigotes can grow with less VCP. In contrast, growth of axenic and intracellular amastigotes is dramatically affected upon decreased LiVCP levels in heterozygous and temperature sensitive (ts) LiVCP mutants or the expression of dominant negative mutants known to specifically target the second conserved VCP ATPase domain, a major contributor of the VCP overall ATPase activity. Interestingly, these VCP mutants are also unable to survive heat stress, and a ts VCP mutant is defective in amastigote growth. Consistent with LiVCP's essential function in amastigotes, LiVCP messenger ribonucleic acid undergoes 3'Untranslated Region (UTR)-mediated developmental regulation, resulting in higher VCP expression in amastigotes. Furthermore, we show that parasite mutant lines expressing lower VCP levels or dominant negative VCP forms exhibit high accumulation of polyubiquitinated proteins and increased sensitivity to proteotoxic stress, supporting the ubiquitin-selective chaperone function of LiVCP. Together, these results emphasise the crucial role LiVCP plays under heat stress and during the parasite intracellular development.

Abstract 93: Transcriptional control of glucocorticoid responses in leukemia

Abstract Glucocorticoids (GCs) are central to all major therapy regimens for pre-B cell-derived acute lymphoblastic leukemia (ALL), but have no activity in myeloid leukemia. Such divergent responses represent an empirically established clinical standard; however, neither the mechanism by which GCs induce cell death nor the biological basis for the distinct responses in B-cell and myeloid leukemias is clear. Studying patient-derived samples revealed that NR3C1 (glucocorticoid receptor) levels were 6- to 20-fold higher in pre-B ALL compared to chronic myeloid leukemia (CML). High levels of Nr3c1 were reduced upon B- to myeloid-lineage conversion, suggesting that regulation of NR3C1 expression and GC responsiveness depend on a B-cell transcriptional program. B-cell transcription factors (e.g. PAX5, IKZF1) are critical for B-cell development, yet they are genetically lesioned in more than 80% of pre-B ALL cases. Despite such high frequency, the significance of these inactivating lesions remains elusive. Combining ChIP-seq and RNA-seq analyses, we identified a novel B-cell transcriptional program for activation of NR3C1 and its transcriptional target TXNIP (a negative regulator of glucose uptake). Reconstitution of PAX5 or IKZF1 expression in haploinsufficient patient-derived pre-B ALL cells increased NR3C1 and TXNIP levels. Conversely, expression of dominant negative mutant of PAX5 or IKZF1 abolished NR3C1 expression. Loss of Nr3c1 or Txnip in murine BCR-ABL1-driven pre-B ALL cells resulted in survival advantage in competitive growth assays. Importantly, loss of Nr3c1 or Txnip significantly elevated glucose uptake, lactate production and cellular ATP levels. These findings suggest that GCs induce cell death by exacerbating glucose and energy depletion. Notably, reconstitution of PAX5 or IKZF1 rendered haploinsufficient patient-derived pre-B ALL cells more sensitive to dexamethasone (dex) treatment. In contrast, dominant-negative PAX5 or IKZF1 largely de-sensitized pre-B ALL cells expressing wildtype PAX5 or IKZF1. These findings suggest that B-cell transcription factors set the threshold for GC responsiveness in pre-B ALL. Since relapsed ALL cells often acquire GC resistance, drug-combinations may be useful to prevent GC-resistance. As expected, loss of Nr3c1 abrogated responses to GCs. Interestingly, loss of Txnip also largely rescued GC-induced cell death in pre-B ALL cells. On this basis, we tested drug interactions between GCs and TXNIP agonists, 3-O-methylglucose (3-OMG) and D-allose. Treating patient-derived GC-refractory pre-B ALL cells with 3-OMG or D-allose strongly synergized with GC-treatment. Collectively, our findings provide a mechanistic explanation for the empiric finding that GCs are effective in the treatment of B-cell but not myeloid malignancies, and identify TXNIP as a novel therapeutic target in pre-B ALL. Note: This abstract was not presented at the meeting. Citation Format: Lai N. Chan, Zhengshan Chen, Gang Xiao, Jae Woong Lee, Kadriye Nehir Cosgun, Huimin Geng, Valeria Cazzaniga, Hilde Schjerven, Ross A. Dickins, Markus Muschen. Transcriptional control of glucocorticoid responses in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 93. doi:10.1158/1538-7445.AM2017-93

Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus.

Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

Oxidation of heat shock protein 60 and protein disulfide isomerase activates ERK and migration of human hepatocellular carcinoma HepG2.

Hepatocyte growth factor (HGF) and its receptor c-Met were frequently deregulated in hepatocellular carcinoma (HCC). Signaling pathways activated by HGF-c-Met are promising targets for preventing HCC progression. HGF can induce the reactive oxygen species (ROS) signaling for cell adhesion, migration and invasion of tumors including HCC. On the other hand, extracellular signal-regulated kinases (ERK), member of mitogen activated kinase, can be activated by ROS for a lot of cellular processes. As expected, HGF-induced phosphorylation of ERK and progression of HCC cell HepG2 were suppressed by ROS scavengers. By N-(biotinoyl)-N′-(iodoacetyl)-ethylenediamine (BIAM) labeling method, a lot of cysteine (−SH)-containing proteins with M.W. 50–75 kD were decreased in HepG2 treated with HGF or two other ROS generators, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phenazine methosulfate. These redox sensitive proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among them, two chaperones, heat shock protein 60 (HSP60) and protein disulfide isomerase (PDI), were found to be the most common redox sensitive proteins in responding to all three agonists. Affinity blot of BIAM-labeled, immunoprecipitated HSP60 and PDI verified that HGF can decrease the cysteine (−SH) containing HSP60 and PDI. On the other hand, HGF and TPA increased cysteinyl glutathione-containing HSP60, consistent with the decrease of cysteine (−SH)-containing HSP60. Moreover, depletion of HSP60 and PDI or expression of dominant negative mutant of HSP60 with alteration of Cys, effectively prevented HGF-induced ERK phosphorylation and HepG2 migration.In conclusion, the redox sensitive HSP60 and PDI are required for HGF-induced ROS signaling and potential targets for preventing HCC progressions.

Post-training activation of Rac1 in the basolateral amygdala is required for the formation of both short-term and long-term auditory fear memory.

Rac1, a member of the Rho family of small GTPases, is crucial for morphological changes of the mature neuronal synapse including spine formation and activity-dependent spine enlargement, while its role in the formation of associated memories, such as conditioned fear memory, is not clear. Here, we report that selective deletion of Rac1 in excitatory neurons, but not in parvalbumin inhibitory neurons, impaired short- and long-term memories (STM and LTM) of fear conditioning. Conditional knockout of Rac1 before associative fear training in the basolateral amygdala (BLA), a key area for fear memory acquisition and storage, impaired fear memory. The expression of dominant-negative mutant of Rac1, or infusion of Rac1 inhibitor NSC23766 into BLA blocked both STM and LTM of fear conditioning. Furthermore, selective inhibition of Rac1 activation in BLA immediately following fear conditioning impaired STM and LTM, demonstrating that fear conditioning-induced Rac1 activation in BLA plays a critical role in the formation of both STM and LTM of conditioned fear.

Repurposing statins for Alzheimer’s disease

By inducing the abundance of neurotrophins, statins enhance memory in Alzheimer’s disease.

Rab32 is essential for maintaining functional acidocalcisomes, and for growth and infectivity of Trypanosoma cruzi.

The contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease, collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress; it also has a role in cell shrinking after hyperosmotic stress. Here, we report that, in addition to its role in osmoregulation, the CVC of T. cruzi has a role in the biogenesis of acidocalcisomes. Expression of dominant-negative mutants of the CVC-located small GTPase Rab32 (TcCLB.506289.80) results in lower numbers of less-electron-dense acidocalcisomes, lower content of polyphosphate, lower capacity for acidocalcisome acidification and Ca(2+) uptake that is driven by the vacuolar proton pyrophosphatase and the Ca(2+)-ATPase, respectively, as well as less-infective parasites, revealing the role of this organelle in parasite infectivity. By using fluorescence, electron microscopy and electron tomography analyses, we provide further evidence of the active contact of acidocalcisomes with the CVC, indicating an active exchange of proteins between the two organelles.

The ARL2 GTPase is required for mitochondrial morphology, motility, and maintenance of ATP levels.

ARF-like 2 (ARL2) is a member of the ARF family and RAS superfamily of regulatory GTPases, predicted to be present in the last eukaryotic common ancestor, and essential in a number of model genetic systems. Though best studied as a regulator of tubulin folding, we previously demonstrated that ARL2 partially localizes to mitochondria. Here, we show that ARL2 is essential to a number of mitochondrial functions, including mitochondrial morphology, motility, and maintenance of ATP levels. We compare phenotypes resulting from ARL2 depletion and expression of dominant negative mutants and use these to demonstrate that the mitochondrial roles of ARL2 are distinct from its roles in tubulin folding. Testing of current models for ARL2 actions at mitochondria failed to support them. Rather, we found that knockdown of the ARL2 GTPase activating protein (GAP) ELMOD2 phenocopies two of three phenotypes of ARL2 siRNA, making it a likely effector for these actions. These results add new layers of complexity to ARL2 signaling, highlighting the need to deconvolve these different cell functions. We hypothesize that ARL2 plays essential roles inside mitochondria along with other cellular functions, at least in part to provide coupling of regulation between these essential cell processes.

Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5

BackgroundAustralian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined.MethodsABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol.ResultsHere we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry.ConclusionsThe predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization

The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P1) phosphorylation was studied. Activation of S1P1 receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P1 phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P1 phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P1 phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and β isoforms in S1P1 immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or β and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P1 phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P1.

ADP Ribosylation Factor 1 Plays an Essential Role in the Replication of a Plant RNA Virus

Eukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery.

Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

SummaryThe respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.

The ER/Golgi Interface - Is There Anything in-between?

OPINION article Front. Plant Sci., 18 April 2012Sec. Plant Cell Biology Volume 3 - 2012 | https://doi.org/10.3389/fpls.2012.00073

Baculovirus GP64-mediated entry into mammalian cells.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

Activation of Ras Requires the ERM-Dependent Link of Actin to the Plasma Membrane

BackgroundReceptor tyrosine kinases (RTKs) participate in a multitude of signaling pathways, some of them via the small G-protein Ras. An important component in the activation of Ras is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras.Principal FindingsWe can now demonstrate that the activation of Ras requires, in addition, the essential participation of ezrin, radixin and/or moesin (ERM), a family of actin-binding proteins, and of actin. Disrupting either the interaction of the ERM proteins with co-receptors, down-regulation of ERM proteins by siRNA, expression of dominant-negative mutants of the ERM proteins or disruption of F-actin, abolishes growth factor-induced Ras activation. Ezrin/actin catalyzes the formation of a multiprotein complex consisting of RTK, co-receptor, Grb2, SOS and Ras. We also identify binding sites for both Ras and SOS on ezrin; mutations of these binding sites destroy the interactions and inhibit Ras activation. Finally, we show that the formation of the ezrin-dependent complex is necessary to enhance the catalytic activity of SOS and thereby Ras activation.ConclusionsTaking these findings together, we propose that the ERM proteins are novel scaffolds at the level of SOS activity control, which is relevant for both normal Ras function and dysfunction known to occur in several human cancers.