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Search Results: 1 - 10 of 8979 matches for " Roland Gerhard Heym equal contributor "
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A Cytoplasmic Complex Mediates Specific mRNA Recognition and Localization in Yeast
Marisa Müller equal contributor,Roland Gerhard Heym equal contributor,Andreas Mayer,Katharina Kramer,Maria Schmid,Patrick Cramer,Henning Urlaub,Ralf-Peter Jansen,Dierk Niessing
PLOS Biology , 2011, DOI: 10.1371/journal.pbio.1000611
Abstract: In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles (mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity remain unresolved. We used ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro–interaction and reconstitution assays, we found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNA-binding proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step forward in our mechanistic understanding of mRNA localization in general.
A Cytoplasmic Complex Mediates Specific mRNA Recognition and Localization in Yeast
Marisa Müller,Roland Gerhard Heym,Andreas Mayer,Katharina Kramer,Maria Schmid,Patrick Cramer,Henning Urlaub,Ralf-Peter Jansen,Dierk Niessing
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1000611
Abstract: In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles (mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity remain unresolved. We used ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro–interaction and reconstitution assays, we found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNA-binding proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step forward in our mechanistic understanding of mRNA localization in general.
The Roles of APC and Axin Derived from Experimental and Theoretical Analysis of the Wnt Pathway
Ethan Lee equal contributor,Adrian Salic equal contributor,Roland Krüger equal contributor,Reinhart Heinrich ,Marc W Kirschner
PLOS Biology , 2003, DOI: 10.1371/journal.pbio.0000010
Abstract: Wnt signaling plays an important role in both oncogenesis and development. Activation of the Wnt pathway results in stabilization of the transcriptional coactivator β-catenin. Recent studies have demonstrated that axin, which coordinates β-catenin degradation, is itself degraded. Although the key molecules required for transducing a Wnt signal have been identified, a quantitative understanding of this pathway has been lacking. We have developed a mathematical model for the canonical Wnt pathway that describes the interactions among the core components: Wnt, Frizzled, Dishevelled, GSK3β, APC, axin, β-catenin, and TCF. Using a system of differential equations, the model incorporates the kinetics of protein–protein interactions, protein synthesis/degradation, and phosphorylation/dephosphorylation. We initially defined a reference state of kinetic, thermodynamic, and flux data from experiments using Xenopus extracts. Predictions based on the analysis of the reference state were used iteratively to develop a more refined model from which we analyzed the effects of prolonged and transient Wnt stimulation on β-catenin and axin turnover. We predict several unusual features of the Wnt pathway, some of which we tested experimentally. An insight from our model, which we confirmed experimentally, is that the two scaffold proteins axin and APC promote the formation of degradation complexes in very different ways. We can also explain the importance of axin degradation in amplifying and sharpening the Wnt signal, and we show that the dependence of axin degradation on APC is an essential part of an unappreciated regulatory loop that prevents the accumulation of β-catenin at decreased APC concentrations. By applying control analysis to our mathematical model, we demonstrate the modular design, sensitivity, and robustness of the Wnt pathway and derive an explicit expression for tumor suppression and oncogenicity.
Integrating Computational Biology and Forward Genetics in Drosophila
Stein Aerts equal contributor,Sven Vilain equal contributor,Shu Hu equal contributor,Leon-Charles Tranchevent equal contributor,Roland Barriot,Jiekun Yan,Yves Moreau,Bassem A. Hassan ,Xiao-Jiang Quan equal contributor
PLOS Genetics , 2009, DOI: 10.1371/journal.pgen.1000351
Abstract: Genetic screens are powerful methods for the discovery of gene–phenotype associations. However, a systems biology approach to genetics must leverage the massive amount of “omics” data to enhance the power and speed of functional gene discovery in vivo. Thus far, few computational methods for gene function prediction have been rigorously tested for their performance on a genome-wide scale in vivo. In this work, we demonstrate that integrating genome-wide computational gene prioritization with large-scale genetic screening is a powerful tool for functional gene discovery. To discover genes involved in neural development in Drosophila, we extend our strategy for the prioritization of human candidate disease genes to functional prioritization in Drosophila. We then integrate this prioritization strategy with a large-scale genetic screen for interactors of the proneural transcription factor Atonal using genomic deficiencies and mutant and RNAi collections. Using the prioritized genes validated in our genetic screen, we describe a novel genetic interaction network for Atonal. Lastly, we prioritize the whole Drosophila genome and identify candidate gene associations for ten receptor-signaling pathways. This novel database of prioritized pathway candidates, as well as a web application for functional prioritization in Drosophila, called Endeavour-HighFly, and the Atonal network, are publicly available resources. A systems genetics approach that combines the power of computational predictions with in vivo genetic screens strongly enhances the process of gene function and gene–gene association discovery.
Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death
Roxane Simeone equal contributor,Alexandre Bobard equal contributor,Juliane Lippmann,Wilbert Bitter,Laleh Majlessi,Roland Brosch ,Jost Enninga
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002507
Abstract: Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective.
Tre1, a G Protein-Coupled Receptor, Directs Transepithelial Migration of Drosophila Germ Cells
Prabhat S Kunwar equal contributor,Michelle Starz-Gaiano equal contributor,Roland J Bainton,Ulrike Heberlein,Ruth Lehmann
PLOS Biology , 2003, DOI: 10.1371/journal.pbio.0000080
Abstract: In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG) is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR), Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.
Souffle/Spastizin Controls Secretory Vesicle Maturation during Zebrafish Oogenesis
Palsamy Kanagaraj equal contributor,Amandine Gautier-Stein equal contributor,Dietmar Riedel,Christoph Schomburg,Joan Cerdà,Nadine Vollack,Roland Dosch
PLOS Genetics , 2014, DOI: doi/10.1371/journal.pgen.1004449
Abstract: During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.
The HIV-1 Envelope Transmembrane Domain Binds TLR2 through a Distinct Dimerization Motif and Inhibits TLR2-Mediated Responses
Eliran Moshe Reuven equal contributor,Mohammad Ali equal contributor,Etai Rotem,Roland Schwarzter,Andrea Gramatica,Anthony H. Futerman,Yechiel Shai
PLOS Pathogens , 2014, DOI: doi/10.1371/journal.ppat.1004248
Abstract: HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.
High Seroprevalence of Rift Valley Fever and Evidence for Endemic Circulation in Mbeya Region, Tanzania, in a Cross-Sectional Study
Norbert Heinrich equal contributor ,Elmar Saathoff equal contributor,Nina Weller,Petra Clowes,Inge Kroidl,Elias Ntinginya,Harun Machibya,Leonard Maboko,Thomas L?scher,Gerhard Dobler,Michael Hoelscher
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001557
Abstract: Background The Rift Valley fever virus (RVFV) is an arthropod-borne phlebovirus. RVFV mostly causes outbreaks among domestic ruminants with a major economic impact. Human infections are associated with these events, with a fatality rate of 0.5–2%. Since the virus is able to use many mosquito species of temperate climates as vectors, it has a high potential to spread to outside Africa. Methodology/Principal Findings We conducted a stratified, cross-sectional sero-prevalence survey in 1228 participants from Mbeya region, southwestern Tanzania. Samples were selected from 17,872 persons who took part in a cohort study in 2007 and 2008. RVFV IgG status was determined by indirect immunofluorescence. Possible risk factors were analyzed using uni- and multi-variable Poisson regression models. We found a unique local maximum of RVFV IgG prevalence of 29.3% in a study site close to Lake Malawi (N = 150). The overall seroprevalence was 5.2%. Seropositivity was significantly associated with higher age, lower socio-economic status, ownership of cattle and decreased with distance to Lake Malawi. A high vegetation density, higher minimum and lower maximum temperatures were found to be associated with RVFV IgG positivity. Altitude of residence, especially on a small scale in the high-prevalence area was strongly correlated (PR 0.87 per meter, 95% CI = 0.80–0.94). Abundant surface water collections are present in the lower areas of the high-prevalence site. RVF has not been diagnosed clinically, nor an outbreak detected in the high-prevalence area. Conclusions RVFV is probably circulating endemically in the region. The presence of cattle, dense vegetation and temperate conditions favour mosquito propagation and virus replication in the vector and seem to play major roles in virus transmission and circulation. The environmental risk-factors that we identified could serve to more exactly determine areas at risk for RVFV endemicity.
Seroprevalence of Alphavirus Antibodies in a Cross-Sectional Study in Southwestern Tanzania Suggests Endemic Circulation of Chikungunya
Nina Weller equal contributor,Petra Clowes equal contributor,Gerhard Dobler,Elmar Saathoff,Inge Kroidl,Nyanda Elias Ntinginya,Leonard Maboko,Thomas L?scher,Michael Hoelscher,Norbert Heinrich
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0002979
Abstract: Background To date, Alphavirus infections and their most prominent member, chikungunya fever, a viral disease which first became apparent in Tanzania in 1953, have been very little investigated in regions without epidemic occurrence. Few data exist on burden of disease and socio-economic and environmental covariates disposing to infection. Methods A cross-sectional seroprevalence study was undertaken in 1,215 persons from Mbeya region, South-Western Tanzania, to determine the seroprevalence of anti-Alphavirus IgG antibodies, and to investigate associated risk factors. Results 18% of 1,215 samples were positive for Alphavirus IgG. Seropositivity was associated with participant age, low to intermediate elevation, flat terrain and with IgG positivity for Rift Valley fever, Flaviviridae, and rickettsiae of the spotted fever group. When comparing the geographical distribution of Alphavirus seropositivity to that of Rift Valley fever, it was obvious that Alphaviruses had spread more widely throughout the study area, while Rift Valley fever was concentrated along the shore of Lake Malawi. Conclusion Alphavirus infections may contribute significantly to the febrile disease burden in the study area, and are associated with several arthropod-borne infections. Their spread seems only limited by factors affecting mosquitoes, and seems less restricted than that of Rift Valley fever.
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