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Recent Submissions

  • Integrative analyses for omics data: a Bayesian mixture model to assess the concordance of ChIP-chip and ChIP-seq measurements.

    Schäfer, Martin; Lkhagvasuren, Otgonzul; Klein, Hans-Ulrich; Elling, Christian; Wüstefeld, Torsten; Müller-Tidow, Carsten; Zender, Lars; Koschmieder, Steffen; Dugas, Martin; Ickstadt, Katja; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2012)
    The analysis of different variations in genomics, transcriptomics, epigenomics, and proteomics has increased considerably in recent years. This is especially due to the success of microarray and, more recently, sequencing technology. Apart from understanding mechanisms of disease pathogenesis on a molecular basis, for example in cancer research, the challenge of analyzing such different data types in an integrated way has become increasingly important also for the validation of new sequencing technologies with maximum resolution. For this purpose, a methodological framework for their comparison with microarray techniques in the context of smallest sample sizes, which result from the high costs of experiments, is proposed in this contribution. Based on an adaptation of the externally centered correlation coefficient ( Schäfer et al. 2009 ), it is demonstrated how a Bayesian mixture model can be applied to compare and classify measurements of histone acetylation that stem from chromatin immunoprecipitation combined with either microarray (ChIP-chip) or sequencing techniques (ChIP-seq) for the identification of DNA fragments. Here, the murine hematopoietic cell line 32D, which was transduced with the oncogene BCR-ABL, the hallmark of chronic myeloid leukemia, was characterized. Cells were compared to mock-transduced cells as control. Activation or inhibition of other genes by histone modifications induced by the oncogene is considered critical in such a context for the understanding of the disease.
  • The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages.

    Altun, Mikael; Walter, Thomas S; Kramer, Holger B; Herr, Patrick; Iphöfer, Alexander; Boström, Johan; David, Yael; Komsany, Alia; Ternette, Nicola; Navon, Ami; Stuart, David I; Ren, Jingshan; Kessler, Benedikt M; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015)
    Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.
  • Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation.

    Steffen, Anika; Ladwein, Markus; Dimchev, Georgi A; Hein, Anke; Schwenkmezger, Lisa; Arens, Stefan; Ladwein, Kathrin I; Margit Holleboom, J; Schur, Florian; Victor Small, J; Schwarz, Janett; Gerhard, Ralf; Faix, Jan; Stradal, Theresia E B; Brakebusch, Cord; Rottner, Klemens; Institute of Genetics, University of Bonn, Karlrobert-Kreiten Strasse 13, D-53115 Bonn, Germany. (2013-10-15)
    Cell migration is commonly accompanied by protrusion of membrane ruffles and lamellipodia. In two-dimensional migration, protrusion of these thin sheets of cytoplasm is considered relevant to both exploration of new space and initiation of nascent adhesion to the substratum. Lamellipodium formation can be potently stimulated by Rho GTPases of the Rac subfamily, but also by RhoG or Cdc42. Here we describe viable fibroblast cell lines genetically deficient for Rac1 that lack detectable levels of Rac2 and Rac3. Rac-deficient cells were devoid of apparent lamellipodia, but these structures were restored by expression of either Rac subfamily member, but not by Cdc42 or RhoG. Cells deficient in Rac showed strong reduction in wound closure and random cell migration and a notable loss of sensitivity to a chemotactic gradient. Despite these defects, Rac-deficient cells were able to spread, formed filopodia and established focal adhesions. Spreading in these cells was achieved by the extension of filopodia followed by the advancement of cytoplasmic veils between them. The number and size of focal adhesions as well as their intensity were largely unaffected by genetic removal of Rac1. However, Rac deficiency increased the mobility of different components in focal adhesions, potentially explaining how Rac - although not essential - can contribute to focal adhesion assembly. Together, our data demonstrate that Rac signaling is essential for lamellipodium protrusion and for efficient cell migration, but not for spreading or filopodium formation. Our findings also suggest that Rac GTPases are crucial to the establishment or maintenance of polarity in chemotactic migration.
  • Arp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin.

    Koestler, Stefan A; Steffen, Anika; Nemethova, Maria; Winterhoff, Moritz; Luo, Ningning; Holleboom, J Margit; Krupp, Jessica; Jacob, Sonja; Vinzenz, Marlene; Schur, Florian; Schlüter, Kai; Gunning, Peter W; Winkler, Christoph; Schmeiser, Christian; Faix, Jan; Stradal, Theresia E B; Small, J Victor; Rottner, Klemens; Institute of Genetics, University of Bonn, D-53115 Bonn, Germany Institute of Molecular Biotechnology, Austrian Academy of Sciences, A-1030 Vienna, Austria Johann Radon Institute for Computational and Applied Mathematics, Austrian Academy of Sciences, A-1030 Vienna, Austria Institute for Biophysical Chemistry, Hannover Medical School, D-30625 Hannover, Germany Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany Institute for Molecular Cell Biology, University of Münster, D-48149 Münster, Germany Oncology Research Unit, School of Medical Sciences, University of New South Wales, Sydney 2052, Australia Faculty of Mathematics, University of Vienna, A-1090 Vienna, Austria. (2013-09)
    Lamellipodia are sheet-like protrusions formed during migration or phagocytosis and comprise a network of actin filaments. Filament formation in this network is initiated by nucleation/branching through the actin-related protein 2/3 (Arp2/3) complex downstream of its activator, suppressor of cAMP receptor/WASP-family verprolin homologous (Scar/WAVE), but the relative relevance of Arp2/3-mediated branching versus actin filament elongation is unknown. Here we use instantaneous interference with Arp2/3 complex function in live fibroblasts with established lamellipodia. This allows direct examination of both the fate of elongating filaments upon instantaneous suppression of Arp2/3 complex activity and the consequences of this treatment on the dynamics of other lamellipodial regulators. We show that Arp2/3 complex is an essential organizer of treadmilling actin filament arrays but has little effect on the net rate of actin filament turnover at the cell periphery. In addition, Arp2/3 complex serves as key upstream factor for the recruitment of modulators of lamellipodia formation such as capping protein or cofilin. Arp2/3 complex is thus decisive for filament organization and geometry within the network not only by generating branches and novel filament ends, but also by directing capping or severing activities to the lamellipodium. Arp2/3 complex is also crucial to lamellipodia-based migration of keratocytes.
  • Cytotoxic necrotizing factor-y boosts yersinia effector translocation by activating rac protein.

    Wolters, Manuel; Boyle, Erin C; Lardong, Kerstin; Trülzsch, Konrad; Steffen, Anika; Rottner, Klemens; Ruckdeschel, Klaus; Aepfelbacher, Martin; From the Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. (2013-08-09)
    Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac.
  • Minimal increase in genetic diversity enhances predation resistance.

    Koh, Kai S; Matz, Carsten; Tan, Chuan H; LE, Hoang L; Rice, Scott A; Marshall, Dustin J; Steinberg, Peter D; Kjelleberg, Staffan; Centre for Marine Bio-Innovation, University of New South Wales, Sydney, NSW, Australia School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia. (2012-04)
    The importance of species diversity to emergent, ecological properties of communities is increasingly appreciated, but the importance of within-species genetic diversity for analogous emergent properties of populations is only just becoming apparent. Here, the properties and effects of genetic variation on predation resistance in populations were assessed and the molecular mechanism underlying these emergent effects was investigated. Using biofilms of the ubiquitous bacterium Serratia marcescens, we tested the importance of genetic diversity in defending biofilms against protozoan grazing, a main source of mortality for bacteria in all natural ecosystems. S. marcescens biofilms established from wild-type cells produce heritable, stable variants, which when experimentally combined, persist as a diverse assemblage and are significantly more resistant to grazing than either wild type or variant biofilms grown in monoculture. This diversity effect is biofilm-specific, a result of either facilitation or resource partitioning among variants, with equivalent experiments using planktonic cultures and grazers resulting in dominance by a single resistant strain. The variants studied are all the result of single nucleotide polymorphisms in one regulatory gene suggesting that the benefits of genetic diversity in clonal biofilms can occur through remarkably minimal genetic change. The findings presented here provide a new insight on the integration of genetics and population ecology, in which diversity arising through minimal changes in genotype can have major ecological implications for natural populations.
  • A cluster of cooperating tumor-suppressor gene candidates in chromosomal deletions.

    Xue, Wen; Kitzing, Thomas; Roessler, Stephanie; Zuber, Johannes; Krasnitz, Alexander; Schultz, Nikolaus; Revill, Kate; Weissmueller, Susann; Rappaport, Amy R; Simon, Janelle; Zhang, Jack; Luo, Weijun; Hicks, James; Zender, Lars; Wang, Xin Wei; Powers, Scott; Wigler, Michael; Lowe, Scott W; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. (2012-05-22)
    The large chromosomal deletions frequently observed in cancer genomes are often thought to arise as a "two-hit" mechanism in the process of tumor-suppressor gene (TSG) inactivation. Using a murine model system of hepatocellular carcinoma (HCC) and in vivo RNAi, we test an alternative hypothesis, that such deletions can arise from selective pressure to attenuate the activity of multiple genes. By targeting the mouse orthologs of genes frequently deleted on human 8p22 and adjacent regions, which are lost in approximately half of several other major epithelial cancers, we provide evidence suggesting that multiple genes on chromosome 8p can cooperatively inhibit tumorigenesis in mice, and that their cosuppression can synergistically promote tumor growth. In addition, in human HCC patients, the combined down-regulation of functionally validated 8p TSGs is associated with poor survival, in contrast to the down-regulation of any individual gene. Our data imply that large cancer-associated deletions can produce phenotypes distinct from those arising through loss of a single TSG, and as such should be considered and studied as distinct mutational events.
  • Immune surveillance of senescent cells--biological significance in cancer- and non-cancer pathologies.

    Hoenicke, Lisa; Zender, Lars; Helmholtz Centre for Infection Research, Braunschweig, Germany. (2012-06)
    Cellular senescence, a state of stable growth arrest, can occur in response to various stress stimuli such as telomere shortening, treatment with chemotherapeutic drugs or the aberrant activation of oncogenes. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it has become clear that this 'secretory phenotype' can have pro- as well as anti-tumorigenic effects. Recent work from our laboratory showed that premalignant, senescent hepatocytes are recognized and cleared through an antigen-specific immune response and that this immune response, designated as 'senescence surveillance' is crucial for tumor suppression in the liver [(Kang,T.W. et al. (2011) Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature, 479, 547-551]. It is an emerging concept that immune responses against senescent cells have a broader biological significance in cancer- as well as non-cancer pathologies and current data suggest that distinct immune responses are engaged to clear senescent cells in different disease settings. In this review article, we will discuss different examples how immune responses against senescent cells are involved to restrict disease progression in cancer- and non-cancer pathologies.
  • Essential role for Abi1 in embryonic survival and WAVE2 complex integrity.

    Dubielecka, Patrycja M; Ladwein, Kathrin I; Xiong, Xiaoling; Migeotte, Isabelle; Chorzalska, Anna; Anderson, Kathryn V; Sawicki, Janet A; Rottner, Klemens; Stradal, Theresia E; Kotula, Leszek; Laboratory of Cell Signaling, New York Blood Center, New York, NY 10065, USA. (2011-04-26)
    Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.
  • RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes.

    Jackson, Ben; Peyrollier, Karine; Pedersen, Esben; Basse, Astrid; Karlsson, Richard; Wang, Zhipeng; Lefever, Tine; Ochsenbein, Alexandra M; Schmidt, Gudula; Aktories, Klaus; Stanley, Alanna; Quondamatteo, Fabio; Ladwein, Markus; Rottner, Klemens; van Hengel, Jolanda; Brakebusch, Cord; Biomedical Institute, BRIC, University of Copenhagen, 2200 Copenhagen, Denmark. (2011-03)
    RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.
  • The promoter of human telomerase reverse transcriptase is activated during liver regeneration and hepatocyte proliferation.

    Sirma, Hüseyin; Kumar, Mukesh; Meena, Jitendra K; Witt, Britta; Weise, Julia M; Lechel, Andre; Ande, Satyanarayana; Sakk, Vadim; Guguen-Guillouzo, Christiane; Zender, Lars; Rudolph, Karl-Lenhard; Günes, Cagatay; Heinrich-Pette-Institute, Hamburg, Germany. (2011-07)
    Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro.
  • Cortactin deficiency is associated with reduced neutrophil recruitment but increased vascular permeability in vivo.

    Schnoor, Michael; Lai, Frank P L; Zarbock, Alexander; Kläver, Ruth; Polaschegg, Christian; Schulte, Dörte; Weich, Herbert A; Oelkers, J Margit; Rottner, Klemens; Vestweber, Dietmar; Max Planck Institute for Molecular Biomedicine, D 48149 Münster, Germany. (2011-08-01)
    Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of β(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.
  • Theoretical model for cellular shapes driven by protrusive and adhesive forces.

    Kabaso, Doron; Shlomovitz, Roie; Schloen, Kathrin; Stradal, Theresia; Gov, Nir S; Department of Chemical Physics, The Weizmann Institute of Science, Rehovot, Israel. (2011-05)
    The forces that arise from the actin cytoskeleton play a crucial role in determining the cell shape. These include protrusive forces due to actin polymerization and adhesion to the external matrix. We present here a theoretical model for the cellular shapes resulting from the feedback between the membrane shape and the forces acting on the membrane, mediated by curvature-sensitive membrane complexes of a convex shape. In previous theoretical studies we have investigated the regimes of linear instability where spontaneous formation of cellular protrusions is initiated. Here we calculate the evolution of a two dimensional cell contour beyond the linear regime and determine the final steady-state shapes arising within the model. We find that shapes driven by adhesion or by actin polymerization (lamellipodia) have very different morphologies, as observed in cells. Furthermore, we find that as the strength of the protrusive forces diminish, the system approaches a stabilization of a periodic pattern of protrusions. This result can provide an explanation for a number of puzzling experimental observations regarding cellular shape dependence on the properties of the extra-cellular matrix.
  • High-resolution X-ray structure of the trimeric Scar/WAVE-complex precursor Brk1.

    Linkner, Joern; Witte, Gregor; Stradal, Theresia; Curth, Ute; Faix, Jan; Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany. (2011)
    The Scar/WAVE-complex links upstream Rho-GTPase signaling to the activation of the conserved Arp2/3-complex. Scar/WAVE-induced and Arp2/3-complex-mediated actin nucleation is crucial for actin assembly in protruding lamellipodia to drive cell migration. The heteropentameric Scar/WAVE-complex is composed of Scar/WAVE, Abi, Nap, Pir and a small polypeptide Brk1/HSPC300, and recent work suggested that free Brk1 serves as a homooligomeric precursor in the assembly of this complex. Here we characterized the Brk1 trimer from Dictyostelium by analytical ultracentrifugation and gelfiltration. We show for the first time its dissociation at concentrations in the nanomolar range as well as an exchange of subunits within different DdBrk1 containing complexes. Moreover, we determined the three-dimensional structure of DdBrk1 at 1.5 Å resolution by X-ray crystallography. Three chains of DdBrk1 are associated with each other forming a parallel triple coiled-coil bundle. Notably, this structure is highly similar to the heterotrimeric α-helical bundle of HSPC300/WAVE1/Abi2 within the human Scar/WAVE-complex. This finding, together with the fact that Brk1 is collectively sandwiched by the remaining subunits and also constitutes the main subunit connecting the triple-coil domain of the HSPC300/WAVE1/Abi2/ heterotrimer to Sra1(Pir1), implies a critical function of this subunit in the assembly process of the entire Scar/WAVE-complex.
  • Microtubules as platforms for assaying actin polymerization in vivo.

    Oelkers, J Margit; Vinzenz, Marlene; Nemethova, Maria; Jacob, Sonja; Lai, Frank P L; Block, Jennifer; Szczodrak, Malgorzata; Kerkhoff, Eugen; Backert, Steffen; Schlüter, Kai; Stradal, Theresia E B; Small, J Victor; Koestler, Stefan A; Rottner, Klemens; Helmholtz Centre for Infection Research, Braunschweig, Germany. (2011)
    The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process.
  • RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes.

    Jackson, Ben; Peyrollier, Karine; Pedersen, Esben; Basse, Astrid; Karlsson, Richard; Wang, Zhipeng; Lefever, Tine; Ochsenbein, Alexandra M; Schmidt, Gudula; Aktories, Klaus; Stanley, Alanna; Quondamatteo, Fabio; Ladwein, Markus; Rottner, Klemens; van Hengel, Jolanda; Brakebusch, Cord; Biomedical Institute, BRIC, University of Copenhagen, 2200 Copenhagen, Denmark. (2011-03)
    RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.
  • Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines.

    Emadi Baygi, Modjtaba; Soheili, Zahra Soheila; Schmitz, Ingo; Sameie, Shahram; Schulz, Wolfgang A; Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. (2010-12)
    The epithelial-mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.
  • In vitro field exposition of skin cells between 100 GHz and 2.52 THz

    KLeine-Ostmann, Thomas; Jastrow, Christian; Salhi, Mohamed Amine; Schrader, Thorsten; Hintzsche, Henning; Stopper, Helga; Kärst, Uwe; Heinen, B.; Baaske, Kai; Koch, M.; Helmholtz Center for Infection Research (HZI), 38124 Braunschweig, Germany. (IEEE, 2010)
  • Introduction to Small GTPases

    Williams, Michael J.; Rottner, Klemens; Helmholtz Center for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany (2010-07)
  • Urinary collagen fragments are significantly altered in diabetes: a link to pathophysiology.

    Maahs, David M; Siwy, Justyna; Argilés, Angel; Cerna, Marie; Delles, Christian; Dominiczak, Anna F; Gayrard, Nathalie; Iphöfer, Alexander; Jänsch, Lothar; Jerums, George; Medek, Karel; Mischak, Harald; Navis, Gerjan J; Roob, Johannes M; Rossing, Kasper; Rossing, Peter; Rychlík, Ivan; Schiffer, Eric; Schmieder, Roland E; Wascher, Thomas C; Winklhofer-Roob, Brigitte M; Zimmerli, Lukas U; Zürbig, Petra; Snell-Bergeon, Janet K; Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, Colorado, United States of America. David.Maahs@ucdenver.edu (2010)
    The pathogenesis of diabetes mellitus (DM) is variable, comprising different inflammatory and immune responses. Proteome analysis holds the promise of delivering insight into the pathophysiological changes associated with diabetes. Recently, we identified and validated urinary proteomics biomarkers for diabetes. Based on these initial findings, we aimed to further validate urinary proteomics biomarkers specific for diabetes in general, and particularity associated with either type 1 (T1D) or type 2 diabetes (T2D).

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