@article{GroenewegvanRoyenFenzetal.2014, author = {Groeneweg, Femke L. and van Royen, Martin E. and Fenz, Susanne and Keizer, Veer I. P. and Geverts, Bart and Prins, Jurrien and de Kloet, E. Ron and Houtsmuller, Adriaan B. and Schmidt, Thomas S. and Schaaf, Marcel J. M.}, title = {Quantitation of Glucocorticoid Receptor DNA-Binding Dynamics by Single-Molecule Microscopy and FRAP}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {3}, doi = {10.1371/journal.pone.0090532}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117085}, pages = {e90532}, year = {2014}, abstract = {Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (similar to 0.7 s) and the other half for longer time periods (similar to 2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (<= 1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.}, language = {en} } @article{PiteauPapatheodorouSchwanetal.2014, author = {Piteau, Marianne and Papatheodorou, Panagiotis and Schwan, Carsten and Schlosser, Andreas and Aktories, Klaus and Schmidt, Gudula}, title = {Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)}, series = {PLoS Pathogens}, volume = {10}, journal = {PLoS Pathogens}, number = {1}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1003884}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117987}, pages = {e1003884}, year = {2014}, abstract = {The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin's action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions. Author Summary We study a crucial virulence factor produced by pathogenic Escherichia coli strains, the Cytotoxic Necrotizing Factor 1 (CNF1). More than 80\% of urinary tract infections (UTIs), which are counted among the most common bacterial infections of humans, are caused by Uropathogenic Escherichia coli (UPEC) strains. We and others elucidated the molecular mechanism of the E. coli toxin CNF1. It constitutively activates Rho GTPases by a direct covalent modification. The toxin enters mammalian cells by receptor-mediated endocytosis. Here, we identified the protein receptor for CNF1 by co-precipitation of cell surface molecules with the tagged toxin and subsequent Maldi-TOF analysis. We identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as receptor for CNF1 and located its interaction site to the C-terminal part of the toxin. We performed direct protein-protein interaction analysis and competition studies. Moreover, cells deficient in Lu/BCAM could not bind labeled CNF1. The identification of a toxin's cellular receptor and receptor binding region is an important task for understanding the pathogenic function of the toxin and, moreover, to make the toxin accessible for its use as a cellbiological and pharmacological tool, for example for the generation of immunotoxins.}, language = {en} } @article{NorrmenFigliaLebrunJulienetal.2014, author = {Norrmen, Camilla and Figlia, Gianluca and Lebrun-Julien, Frederic and Pereira, Jorge A. and Tr{\"o}tzm{\"u}ller, Martin and K{\"o}feler, Harald C. and Rantanen, Ville and Wessig, Carsten and van Deijk, Anne-Lieke F. and Smit, August B. and Verheijen, Mark H. G. and R{\"u}egg, Markus A. and Hall, Michael N. and Suter, Ueli}, title = {mTORC1 Controls PNS Myelination along the mTORC1-RXR gamma-SREBP-Lipid Biosynthesis Axis in Schwann Cells}, series = {Cell Reports}, volume = {9}, journal = {Cell Reports}, number = {2}, issn = {2211-1247}, doi = {10.1016/j.celrep.2014.09.001}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114847}, pages = {646-660}, year = {2014}, abstract = {Myelin formation during peripheral nervous system (PNS) development, and reformation after injury and in disease, requires multiple intrinsic and extrinsic signals. Akt/mTOR signaling has emerged as a major player involved, but the molecular mechanisms and downstream effectors are virtually unknown. Here, we have used Schwann-cell-specific conditional gene ablation of raptor and rictor, which encode essential components of the mTOR complexes 1 (mTORC1) and 2 (mTORC2), respectively, to demonstrate that mTORC1 controls PNS myelination during development. In this process, mTORC1 regulates lipid biosynthesis via sterol regulatory element-binding proteins (SREBPs). This course of action is mediated by the nuclear receptor RXRg, which transcriptionally regulates SREBP1c downstream of mTORC1. Absence of mTORC1 causes delayed myelination initiation as well as hypomyelination, together with abnormal lipid composition and decreased nerve conduction velocity. Thus, we have identified the mTORC1-RXR gamma-SREBP axis controlling lipid biosynthesis as a major contributor to proper peripheral nerve function.}, language = {en} } @article{FaganDollarLuetal.2014, author = {Fagan, Jeremy K. and Dollar, Gretchen and Lu, Qiuheng and Barnett, Austen and Jorge, Joaquin Pechuan and Schlosser, Andreas and Pfleger, Cathie and Adler, Paul and Jenny, Andreas}, title = {Combover/CG10732, a Novel PCP Effector for Drosophila Wing Hair Formation}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {9}, issn = {1932-6203}, doi = {10.1371/journal.pone.0107311}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115394}, pages = {e107311}, year = {2014}, abstract = {The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho Kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, we identified Combover (Cmb)/CG10732 as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, we show that Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. Our data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh.}, language = {en} } @article{SenecalIsabelleFritzleretal.2014, author = {Senecal, Jean-Luc and Isabelle, Catherine and Fritzler, Marvin J. and Targoff, Ira N. and Goldstein, Rose and Gagne, Michel and Raynauld, Jean-Pierre and Joyal, France and Troyanov, Yves and Dabauvalle, Marie-Christine}, title = {An Autoimmune Myositis-Overlap Syndrome Associated With Autoantibodies to Nuclear Pore Complexes Description and Long-Term Follow-up of the Anti-Nup Syndrome}, series = {Medicine}, volume = {93}, journal = {Medicine}, number = {24}, issn = {0025-7974}, doi = {10.1097/MD.0000000000000223}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114829}, pages = {361-372}, year = {2014}, abstract = {Autoimmune myositis encompasses various myositis-overlap syndromes, each being identified by the presence of serum marker autoantibodies. We describe a novel myositis-overlap syndrome in 4 patients characterized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes. The clinical phenotype was characterized by prominent myositis in association with erosive, anti-CCP, and rheumatoid factor-positive arthritis, trigeminal neuralgia, mild interstitial lung disease, Raynaud phenomenon, and weight loss. The myositis was typically chronic, relapsing, and refractory to corticosteroids alone, but remitted with the addition of a second immuno-modulating drug. There was no clinical or laboratory evidence for liver disease. The prognosis was good with 100\% long-term survival (mean follow-up 19.5 yr). By indirect immunofluorescence on HEp-2 cells, sera from all 4 patients displayed a high titer of antinuclear autoantibodies (ANA) with a distinct punctate peripheral (rim) fluorescent pattern of the nuclear envelope characteristic of nuclear pore complexes. Reactivity with nuclear pore complexes was confirmed by immunoelectron microscopy. In a cohort of 100 French Canadian patients with autoimmune myositis, the nuclear pore complex fluorescent ANA pattern was restricted to these 4 patients (4\%). It was not observed in sera from 393 adult patients with systemic sclerosis (n = 112), mixed connective tissue disease (n = 35), systemic lupus (n = 94), rheumatoid arthritis (n = 45), or other rheumatic diseases (n = 107), nor was it observed in 62 normal adults. Autoantibodies to nuclear pore complexes were predominantly of IgG isotype. No other IgG autoantibody markers for defined connective tissue diseases or overlap syndromes were present, indicating a selective and highly focused immune response. In 3 patients, anti-nuclear pore complex autoantibody titers varied in parallel with myositis activity, suggesting a pathogenic link to pathophysiology. The nuclear pore complex proteins, that is, nucleoporins (nup), recognized by these sera were heterogeneous and included Nup358/RanBP2 (n = 2 patients), Nup90 (n = 1), Nup62 (n = 1), and gp210 (n = 1). Taken together the data suggest that nup autoantigens themselves drive the anti-nup autoimmune response. Immunogenetically, the 4 patients shared the DQA1*0501 allele associated with an increased risk for autoimmune myositis. In conclusion, we report an apparent novel subset of autoimmune myositis in our population of French Canadian patients with connective tissue diseases. This syndrome is recognized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes that react with nups, consistent with an "anti-nupsyndrome.''}, language = {en} }