University of Heidelberg

Cell and Molecular Biology

 

 

 

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  • Discus throw with cancer signals

     

    The Wnt signaling protein plays an important part in embryonic development and also in the development of diseases such as cancer. It has been unknown until now just how Wnt is carried from cell to cell. Scientists of the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) and Medical Faculty Mannheim of Heidelberg University have now discovered that the protein is shipped on small discus-shaped vesicles called exosomes. The researchers are now investigating whether Wnt exosomes are relevant for the development of cancer.

     

    At the cellular level, fly, frog and man all speak a common language. Cells communicate via signals such as “Wnt” proteins. They use this universal means of communication to transmit important information such as about the basic plan of the body pattern. Transmission errors in the communication of such messages usually have fatal consequences. During embryonic development, they result in severe malformations, while later in life wrong Wnt signaling frequently results in cancer.

     


    Wnt exosomes are being thrown from the cell on the left
    (electrone microscope images) | © Michael Boutros, DKFZ

     

    Cells receive Wnt messages via matching receptor proteins on their surface. However, it has been unknown so far just how these important signaling proteins are carried. Wnt directs body pattern formation and needs to be sent via long distances in an organism. “But since it is a hydrophobic protein, this cannot be done without appropriate packaging”, says Prof. Dr. Michael Boutros, who leads a joint department of DKFZ and Medical Faculty Mannheim of Heidelberg University.

    Scientists in Boutros’ team have now uncovered the secrets of Wnt transport. Inside of Wnt-secreting cells, small bubbles of cell membrane are formed. Their wall, in turn, folds inwards to form what are called exosomes – tiny ferries containing the Wnt protein. These ferries are then sent on their way by being thrown – like a discus – out of the cell.

    However, transport via exosomes is not the only way to mediate the Wnt signal. When the investigators suppressed bubble formation in the cells, the Wnt signal did not subside completely. “We therefore suppose that different ways of signal transmission have different biological functions. Thus, it could make a difference for the target cell whether it receives the biological signal via individual Wnt molecules or whether a whole, fully loaded exosome is incoming,” speculates Dr. Julia Gross, co- author of the publication.

    The Wnt signaling protein has been shown to play an important part in the development of cancer in humans; it has even been discovered in malignant tumors for the first time. Many tumor cells produce too much Wnt, which induces other cells to divide too frequently or to become resistant to chemotherapy. Wnt turns off important tumor suppressor proteins which act as growth brakes. In this way, it fuels tumor growth.

    Scientists have been discovering more and more details about the role of exosomes in many biological processes. Thus, US researchers have recently been able to demonstrate in malignant melanoma that tumor cells release exosomes full of cancer-promoting proteins which make the surrounding tissue susceptible for colonization by tumor metastases. Boutros and his co-workers suspect that Wnt exosomes, too, may play a role in the development of cancer. Furthermore, they intend to investigate whether Wnt exosomes, which are easy to detect in blood, may serve as new tumor markers for medical purposes.

    This research work was carried out as part of the DFG-funded Research Group 1036 and the CellNetworks excellence cluster at the Medical Faculty Mannheim of the University of Heidelberg and the German Cancer Research Center (DKFZ).

     

    Julia Christina Gross, Varun Chaudhary, Kerstin Bartscherer and Michael Boutros: Active Wnt proteins are secreted on exosomes. Nature Cell Biology 2012, DOI: http://dx.doi.org/10.1038/ncb2574

     

    A picture for this press release is available at:

    www.dkfz.de/de/presse/pressemitteilungen/2012/images/Exosomen.jpg

    Caption: Wnt exosomes are being thrown from the cell on the left (electrone microscope images)

    Source: Michael Boutros, DKFZ

     

    » Source: DKFZ press release on 17.09.2012

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  • RAB8B is required for activity and caveolar endocytosis of LRP6

     

    Demir, K., Kirsch, N., Beretta, C.A., Erdmann, G., Ingelfinger, D., Moro, E., Argenton, F., Carl, M., Niehrs, C, Boutros, M. (2013). RAB8B is required for activity and caveolar endocytosis of LRP6. Cell Reports 4:1224-34.

     

    Wnt/β-catenin signaling plays an important role in embryonic development and adult tissue homeostasis. When Wnt ligands bind to the receptor complex, LRP5/6 coreceptors are activated by phosphorylation and concomitantly endocytosed. In vertebrates, Wnt ligands induce caveolin-dependent endocytosis of LRP6 to relay signal downstream, whereas antagonists such as Dickkopf promote clathrin-dependent endocytosis, leading to inhibition. However, little is known about how LRP6 is directed to different internalization mechanisms, and how caveolin-dependent endocytosis is mediated. In an RNAi screen, we identified the Rab GTPase RAB8B as being required for Wnt/β-catenin signaling. RAB8B depletion reduces LRP6 activity, β-catenin accumulation, and induction of Wnt target genes, whereas RAB8B overexpression promotes LRP6 activity and internalization and rescues inhibition of caveolar endocytosis. In Xenopus laevis and Danio rerio, RAB8B morphants show lower Wnt activity during embryonic development. Our results implicate RAB8B as an essential evolutionary conserved component of Wnt/β-catenin signaling through regulation of LRP6 activity and endocytosis.

     

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  •  

  • GenomeRNAi version 11.1 released

     

    8 August 2013

     

    This release gives access to a beta version of our NEW screen comparison tool. We'd really appreciate your feedback very early on in the development of this new feature. TEST IT OUT and LET US KNOW what we should add or change and how you'd ideally like to use this functionality!

    We'd also appreciate if you could take just a few minutes to complete our user survey!

     

    Check it out at: www.genomernai.org

     

    Reference: Schmidt, E.E., Pelz, O., Buhlmann, S., Kerr, G., Horn, T., Boutros, M. (2013). GenomeRNAi: a database for cell-based and in vivo RNAi phenotypes, 2013 update. Nucleic Acids Research 41(Database issue):D1021-6.

     

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  • The Wnt/beta-catenin signaling pathway establishes neuroanatomical asymmetries and their laterality

     

    Hüsken, U., Carl, M. (2013). The Wnt/beta-catenin signaling pathway establishes neuroanatomical asymmetries and their laterality. Mechanisms of Development 130: 330-335.

     

    The vertebrate brain is an immensely complex structure, which exhibits numerous morphological and functional asymmetries. The best described brain asymmetries are found in the diencephalic epithalamus, where the habenulae and the dorso-laterally adjacent pineal complex are lateralized in many species. Research in the past decade has shed light on the establishment of the laterality of these structures as well as their asymmetry per se. In particular work in zebrafish (Danio rerio) has substantially contributed to our understanding, which genetic pathways are involved in these processes. The Wnt/beta-catenin pathway has turned out to play a pivotal role in the regulation of brain laterality and asymmetry and acts reiteratively during embryonic development.

     

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  • GenomeRNAi version 11.0 released

     

    12 June 2013

     

    This release features a new data submission functionality: data submitters are provided with their own login space for uploading and viewing their data, and for sharing them with colleagues/reviewers. Frequent hitters can now be filtered by number or percentage of hits. The database contains 145 RNAi screens in human, and 174 screens in drosophila.

     

    Check it out at: www.genomernai.org

     

    Reference: Schmidt, E.E., Pelz, O., Buhlmann, S., Kerr, G., Horn, T., Boutros, M. (2013). GenomeRNAi: a database for cell-based and in vivo RNAi phenotypes, 2013 update. Nucleic Acids Research 41(Database issue):D1021-6.

     

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  • Tao controls epithelial morphogenesis by promoting Fasciclin 2 endocytosis

     

    Gomez, J.M., Wang, Y., Riechmann, V. (2012). Tao controls epithelial morphogenesis by promoting Fasciclin 2 endocytosis. The Journal of Cell Biology 199:1131-43.

     

    Regulation of epithelial cell shape, for example, changes in relative sizes of apical, basal, and lateral membranes, is a key mechanism driving morphogenesis. However, it is unclear how epithelial cells control the size of their membranes. In the epithelium of the Drosophila melanogaster ovary, cuboidal precursor cells transform into a squamous epithelium through a process that involves lateral membrane shortening coupled to apical membrane extension. In this paper, we report a mutation in the gene Tao, which resulted in the loss of this cuboidal to squamous transition. We show that the inability of Tao mutant cells to shorten their membranes was caused by the accumulation of the cell adhesion molecule Fasciclin 2, the Drosophila N-CAM (neural cell adhesion molecule) homologue. Fasciclin 2 accumulation at the lateral membrane of Tao mutant cells prevented membrane shrinking and thereby inhibited morphogenesis. In wild-type cells, Tao initiated morphogenesis by promoting Fasciclin 2 endocytosis at the lateral membrane. Thus, we identify here a mechanism controlling the morphogenesis of a squamous epithelium.

     

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  • GenomeRNAi version 10.0 released

     

    24 January 2013

     

    GenomeRNAi 10.0 is a data release providing newly curated phenotype data: The database now contains 137 RNAi screens in human, and 173 screens in drosophila. We thank Rebecca Saunders for submitting a genome-wide screen on Hippo pathway regulation!

     

    Check it out at: www.genomernai.org

     

    Reference: Schmidt, E.E., Pelz, O., Buhlmann, S., Kerr, G., Horn, T., Boutros, M. (2013). GenomeRNAi: a database for cell-based and in vivo RNAi phenotypes, 2013 update. Nucleic Acids Research 41(Database issue):D1021-6.

     

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  • GenomeRNAi: a database for cell-based and in vivo RNAi phenotypes, 2013 update

     

    Schmidt, E.E., Pelz, O., Buhlmann, S., Kerr, G., Horn, T., Boutros, M. (2013). GenomeRNAi: a database for cell-based and in vivo RNAi phenotypes, 2013 update. Nucleic Acids Research 41(Database issue):D1021-6.

     

    RNA interference (RNAi) represents a powerful method to systematically study loss-of-function phenotypes on a large scale with a wide variety of biological assays, constituting a rich source for the assignment of gene function. The GenomeRNAi database (http://www.genomernai.org) makes available RNAi phenotype data extracted from the literature for human and Drosophila. It also provides RNAi reagent information, along with an assessment as to their efficiency and specificity. This manuscript describes an update of the database previously featured in the NAR Database Issue. The new version has undergone a complete re-design of the user interface, providing an intuitive, flexible framework for additional functionalities. Screen information and gene-reagent-phenotype associations are now available for download. The integration with other resources has been improved by allowing in-links via GenomeRNAi screen IDs, or external gene or reagent identifiers. A distributed annotation system (DAS) server enables the visualization of the phenotypes and reagents in the context of a genome browser. We have added a page listing 'frequent hitters', i.e. genes that show a phenotype in many screens, which might guide on-going RNAi studies. Structured annotation guidelines have been established to facilitate consistent curation, and a submission template for direct submission by data producers is available for download.

     

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  • Don’t be afraid to set up your fish facility

     

    McNabb, A., Scott, K., von Ochsenstein, E., Seufert, K., Carl, M. (2012). Don’t be afraid to set up your fish facility. Zebrafish 9:120-5.

     

    Most young researchers leaving the safe haven of postdoctoral life for the unchartered territory of a PI position are confronted with a multitude of new tasks. For those using fish as their favorite model system, this often includes the daunting task of setting up and running a fish facility. New PIs are expected to know everything about this, and are even asked in job interviews about the precise details of the facility they anticipate using. Consulting other laboratories, talking to experienced facility managers, and sifting through books and web material will certainly help but can be enormously time consuming and at times frustrating. You will likely receive conflicting advice or encounter information that is overcomplicated and out of date. In this report, we summarize our collective experience of five different fish facilities, and outline what we consider to be the essential steps for setting up and running a successful facility. We also include valuable tips and tricks such as an optimized protocol for brine shrimp hatching. Our aim is to help researchers spend less time worrying about the technical aspects of their facility so that they can focus on their main job-research.

     

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  • GenomeRNAi version 9.0 released

     

    21 September 2012

     

    This release features a dynamic genome browser for each gene, displaying gene, reagent and phenotype data via the DAS technology. Further we have added graphs indicating the score distribution and the position of individual scores on the gene details page. HeLa cell morphology images are available for the results reported by Fuchs et al.

     

    Check it out at: www.genomernai.org

     

    Reference: Gilsdorf, M., Horn, T., Arziman, Z., Pelz, O., Kiner E., Boutros, M. (2010). GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 Update. Nucleic Acids Research 38:D448-52.

     

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  • Habenula circuit development: past, present and future

     

    Beretta, C.A., Dross, N., Gutierrez-Triana, J.A., Ryu, S., Carl, M. (2012). Habenula circuit development: past, present and future. Frontiers in Neuroscience 6:51.

     

    The habenular neural circuit is attracting increasing attention from researchers in fields as diverse as neuroscience, medicine, behavior, development, and evolution. Recent studies have revealed that this part of the limbic system in the dorsal diencephalon is involved in reward, addiction, and other behaviors and its impairment is associated with various neurological conditions and diseases. Since the initial description of the dorsal diencephalic conduction system (DDC) with the habenulae in its center at the end of the nineteenth century, increasingly sophisticated techniques have resolved much of its anatomy and have shown that these pathways relay information from different parts of the forebrain to the tegmentum, midbrain, and hindbrain. The first part of this review gives a brief historical overview on how the improving experimental approaches have allowed the stepwise uncovering much of the architecture of the habenula circuit as we know it today. Our brain distributes tasks differentially between left and right and it has become a paradigm that this functional lateralization is a universal feature of vertebrates. Moreover, task dependent differential brain activities have been linked to anatomical differences across the left-right axis in humans. A good way to further explore this fundamental issue will be to study the functional consequences of subtle changes in neural network formation, which requires that we fully understand DDC system development. As the habenular circuit is evolutionarily highly conserved, researchers have the option to perform such difficult experiments in more experimentally amenable vertebrate systems. Indeed, research in the last decade has shown that the zebrafish is well suited for the study of DDC system development and the phenomenon of functional lateralization. We will critically discuss the advantages of the zebrafish model, available techniques, and others that are needed to fully understand habenular circuit development.

     

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  • GenomeRNAi version 8.0 released

     

    27 June 2012

     

    We are pleased to announce a new release of the GenomeRNAi database! This release (V8.0) features phenotype data from 127 human RNAi screens and 164 screens in D. melanogaster, of which 50 are in vivo screens. The process of updating pre-existing screening data according to the new annotation guidelines is ongoing.

    New features are a "Frequent hitters" page, providing lists of genes sorted by the number of phenotypes they show, and a DAS server, allowing visualisation of GenomeRNAi phenotypes and reagents in the context of a genome browser.

    We encourage you to submit your own data to GenomeRNAi. We provide an Excel template with instructions for straightforward data annotation and submission.

     

    Check it out at: www.genomernai.org

     

    Reference: Gilsdorf, M., Horn, T., Arziman, Z., Pelz, O., Kiner E., Boutros, M. (2010). GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 Update. Nucleic Acids Research 38:D448-52.

     

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  • ERC Advanced Grant for Michael Boutros

     

    "Two ERC grants go to the German Cancer Research Center"

     

    Two DKFZ researchers at a time are awarded a prestigious European Research Council (ERC) Advanced Grant this year. One of the grants is awarded to a project of Prof. Michael Boutros, who endeavors to show, for the first time, the interaction of all genes in cells of a higher organism. Prof. Bruno Kyewski is awarded an ERC grant for his studies of how immune cells learn tolerance of the body’s own structures.

    Launched in 2007, the European Research Council (ERC) supports investigator-initiated frontier research with the aim of promoting visionary projects and opening up new interdisciplinary fields of research. For excellent established researchers, the ERC has yearly calls for proposals for its ERC Advanced Grants, which are awarded in a highly competitive scheme. “It is a tremendous success for DKFZ that two of our scientists at a time have been selected in this year’s round of calls,” said Prof. Dr. Otmar D. Wiestler, Chairman of the Management Board of DKFZ.

     


    Prof. Dr. Michael Boutros | © dkfz.de

     

    One of two awardees is molecular biologist Prof. Dr. Michael Boutros, who leads a joint department at DKFZ and Medical Faculty Mannheim of Heidelberg University. In the project that has been awarded the ERC grant, Boutros aims to generate, for the first time, a genetic interaction map in order to gain a fundamental understanding of how genes interact.

    The links between particular genetic mutations or genetic variants and the development of disease are often very difficult to identify: Many genes can amplify, reduce or even cancel out each others’ effects. Therefore, the effect of individual genetic variants often also depends on what other genes are affected. Michael Boutros and colleagues have developed a new method that uncovers these combined effects of genes. Using a technique called RNA interference, they silenced a set of genes one at a time and two at a time in all pair combinations. By systematically cataloging all interactions among important genes, the scientists obtain a detailed list of interaction partners for each gene, much like a list of friends in a facebook profile. If two facebook users have many friends in common, the odds are that those two people know each other – even if they themselves are not facebook friends. Similarly, by comparing the interaction profiles of genes, the researchers can now predict which genes affect the same cellular processes.

    In cancer cells and cells of the fruit fly Drosophila, the investigators intend to map these interactions across the whole genome. In doing so, they are particularly interested in the interaction of genes that are involved in cell signaling, because defective transmission of growth signals plays an important role in the development of cancer. The ERC will support the project with funds amounting to €2.5 million for a period of five years.

    Michael Boutros studied biology and biochemistry in Aachen and Witten/Herdecke, both in Germany, and New York, U.S.A. He subsequently held research positions at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, and at Harvard Medical School, U.S.A. Since 2003, he has been working at DKFZ as a researcher and head of the Division of Signaling and Functional Genomics.

     


    Prof. Dr. Bruno Kyewski | © dkfz.de

     

    Immunologist Prof. Dr. Bruno Kyewski studies the immune system’s essential feature: its ability to discriminate between self and non-self. If this mechanism becomes dysfunctional, autoimmune diseases may arise. The immune system’s T cells are educated in the thymus to tolerate self proteins. In the past few years, Kyewski has been able to uncover a key aspect of tolerance induction: Body cells normally produce only those proteins which they need for their particular tasks. However, as Kyewski found out, the cells of the thymus epithelium additionally produce a wide array of proteins that they do not need themselves and which were previously thought to be produced exclusively in other body cells. Using this molecular trick, the thymus epithelium can present an enormous range of self proteins to the immune cells.

    In four different autoimmune diseases, Kyewski and colleagues have already shown that tolerance induction is dysfunctional if a particular self protein is not sufficiently presented in the thymus. Kyewski is awarded the ERC grant for taking a closer look at this striking feature of thymic epithelial cells. The immunologist seeks to understand how the production of proteins from other body tissues is regulated in the thymus. At least 10 to 20 percent of all genes of the human genetic material is read by the thymic epithelial cells and translated into proteins. However, each one of these cells has a share of only one to three percent in this. So how does a thymic epithelial cell “know” for which proteins it is responsible? Is there a pattern behind this or is it left to chance? These questions are also of great interest for cancer research because immune cells are also taught tolerance to tumor antigens by the thymus epithelium.

    Bruno Kyewski studied medicine in Bonn, Germany, and Zurich, Switzerland. He subsequently pursued research at the Max Planck Institute of Immunobiology in Freiburg, Germany, and at Stanford University, U.S.A. In 1984 he joined DKFZ, where he has led the Division of Developmental Immunology since 2004.

    The German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) with its more than 2,500 employees is the largest biomedical research institute in Germany. At DKFZ, more than 1,000 scientists investigate how cancer develops, identify cancer risk factors and endeavor to find new strategies to prevent people from getting cancer. They develop novel approaches to make tumor diagnosis more precise and treatment of cancer patients more successful. Jointly with Heidelberg University Hospital, DKFZ has established the National Center for Tumor Diseases (NCT) Heidelberg where promising approaches from cancer research are translated into the clinic. The staff of the Cancer Information Service (KID) offers information about the widespread disease of cancer for patients, their families, and the general public. The center is a member of the Helmholtz Association of National Research Centers. Ninety percent of its funding comes from the German Federal Ministry of Education and Research and the remaining ten percent from the State of Baden-Württemberg.

     

    » Source: DKFZ press release on 06.12.11

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  • “Vacuum-assisted staining”: a simple and efficient method for screening in Drosophila

     

    Berns, N., Woichansky, I., Kraft, N., Hüsken, U., Carl, M., Riechmann, V. (2012). “Vacuum-assisted staining”: a simple and efficient method for screening in Drosophila. Development Genes and Evolution 222:113-118.

     

    The constantly growing number of genetic tools rapidly increases possibilities for various screens in different model organisms and calls for new methods facilitating screen performance. In particular, screening procedures involving fixation and staining of samples are difficult to perform at a genome-wide scale. The time-consuming task to generate these samples makes such screens less attractive. Here, we describe the use of multi-well filter plates for high throughput labellings of different Drosophila organs and zebrafish embryos. Our inexpensive vacuum-assisted staining protocol minimises the risk of sample loss, reduces the amount of staining reagents and drastically decreases labour and repetitive work. The simple handling of the system and the commercial availability of its components makes this method easily applicable to every laboratory.

     

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  • GenomeRNAi version 7.0 released

     

    30 March 2012

     

    GenomeRNAi 7.0 is a data release providing newly curated phenotype data: The database now contains 124 RNAi screens in human, and 158 screens in drosophila, of which 50 have been performed in vivo.

     

    Check it out at: www.genomernai.org

     

    Reference: Gilsdorf, M., Horn, T., Arziman, Z., Pelz, O., Kiner E., Boutros, M. (2010). GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 Update. Nucleic Acids Research 38:D448-52.

     

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  • GenomeRNAi version 6.0 released

     

    12 January 2012

     

    This release features new and updated phenotype data: The database now contains 96 RNAi screens in human, and 150 screens in drosophila (50 in vivo screens). You can link to the GenomeRNAi website using Ensembl, UniProt, Flybase, or CG identifiers; or GenomeRNAi stable IDs. Follow us on Twitter or Facebook. And do consider to submit your own data!

     

    Check it out at: www.genomernai.org

     

    Reference: Gilsdorf, M., Horn, T., Arziman, Z., Pelz, O., Kiner E., Boutros, M. (2010). GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 Update. Nucleic Acids Research 38:D448-52.

     

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  • p24 proteins are required for secretion of Wnt ligands

     

    Buechling, T., Chaudhary, V., Spirohn, K., Weiss, M., Boutros, M. (2011). p24 proteins are required for secretion of Wnt ligands. EMBO reports 12:1265-72.

     

    During development and disease, the exocytosis of signalling molecules, such as Wnt ligands, is essential to orchestrate cellular programs in multicellular organisms. However, it remains a largely unresolved question whether signalling molecules follow specialized transport routes through the exocytic pathway. Here we identify several Drosophila p24 proteins that are required for Wnt signalling. We demonstrate that one of these p24 proteins, namely Opossum, shuttles in the early secretory pathway, and that the Drosophila Wnt proteins are retained in the absence of p24 proteins. Our results indicate that Wnt secretion relies on a specialized anterograde secretion route with p24 proteins functioning as conserved cargo receptors.

     

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  • GenomeRNAi version 5.0 released

     

    15 July 2011

     

    New release of the GenomeRNAi phenotype database!

    GenomeRNAi, a comprehensive database with phenotypes from RNA interference (RNAi) screens in Drosophila melanogaster and Homo sapiens, was considerably upgraded with new data, new functionality and a completely new design for a more intuitive access. The database connects observed phenotypes with annotations of targeted genes and information about RNAi reagents and their predicted quality and also allows to search across species.

    The database now contains phenotype data from 82 RNAi screens in Homo sapiens and 136 screens in Drosophila melanogaster, including 33 in vivo RNAi screens. New annotation standards for better comparison across various datasets have been applied. Furthermore, options for downloading phenotypic data are now available on the website.

     

    Check it out at: http://www.genomernai.org

     

    Reference: Gilsdorf, M., Horn, T., Arziman, Z., Pelz, O., Kiner E., Boutros, M. (2010). GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 Update. Nucleic Acids Research 38:D448-52.

     

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  • Mapping of signaling networks through synthetic genetic interaction analysis by RNAi

     

    Horn, T., Sandmann, T., Fischer, B., Axelsson, E., Huber, W., Boutros, M. (2011). Mapping of signaling networks through synthetic genetic interaction analysis by RNAi. Nature Methods 8:341-6.

     

    The analysis of synthetic genetic interaction networks can reveal how biological systems achieve a high level of complexity with a limited repertoire of components. Studies in yeast and bacteria have taken advantage of collections of deletion strains to construct matrices of quantitative interaction profiles and infer gene function. Yet comparable approaches in higher organisms have been difficult to implement in a robust manner. Here we report a method to identify genetic interactions in tissue culture cells through RNAi. By performing more than 70,000 pairwise perturbations of signaling factors, we identified >600 interactions affecting different quantitative phenotypes of Drosophila melanogaster cells. Computational analysis of this interaction matrix allowed us to reconstruct signaling pathways and identify a conserved regulator of Ras-MAPK signaling. Large-scale genetic interaction mapping by RNAi is a versatile, scalable approach for revealing gene function and the connectivity of cellular networks.

     

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  • An RNAi screen identifies USP2 as a factor required for TNF-α induced NF-κB signaling

     

    Metzig, M., Nickles, D., Falschlehner, C., Lehmann-Koch, J., Straub, B.K., Roth, W., Boutros, M. (2011). An RNAi screen identifies USP2 as a factor required for TNF-α induced NF-κB signaling. International Journal of Cancer 129(3):607-18.

     

    Tumor necrosis factor α (TNF-α) signaling pathways play important roles during tumorigenesis and inflammation. Ubiquitin-dependent processes are central to the regulation of TNF-α and nuclear factor κB (NF-κB) signaling. We performed a targeted siRNA screen for Ubiquitin-specific proteases (USP) and identified USP2 as a modulator of TNF-α-induced NF-κB signaling. We showed that USP2 is required for the phosphorylation of IκB, nuclear translocation of NF-κB and expression of NF-κB dependent target genes and IL-8 secretion. Our study also provides evidence for isoform-specific functions of USP2. The immunohistochemical analysis of breast carcinomas revealed that USP2 expression is frequently downregulated. Together our results implicate USP2 as a novel positive regulator of TNF-α-induced NF-κB signaling and show that its expression is altered in tumor cells.

     

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  • All four zebrafish Wnt7 genes are expressed during early brain development

     

    Beretta, C.A., Brinkmann, I., Carl, M. (2011). All four zebrafish Wnt7 genes are expressed during early brain development. Gene Expression Patterns 11, 277-284.

     

    Wnt-signalling is involved in a number of biological processes in the course of embryonic development, cell fate determination, proliferation, stem cell maintenance and oncogenesis. Wnt ligands are secreted glycoproteins and the number of Wnt isoforms varies between five in nematodes and twenty-seven in fish.

    The highly conserved group of Wnt7 genes has been found to signal via at least three Wnt-signalling pathways dependent on the developmental context. These ligands have been identified as important regulators in a number of processes ranging from formation of bones, lungs, kidneys, reproductive organs and placenta to vasculogenesis and synaptogenesis in the brain. The importance of Wnt7 function is underscored by their implication in disease syndromes in man.

    Unlike the single Wnt7a and Wnt7b mammalian genes we find that the zebrafish genome contains two paralogues genes for each Wnt7 ligand. Here we compare these four Wnt7 genes evolutionarily and analyse their expression during the first two days of embryonic development. We find Wnt7 genes mainly expressed in a number of CNS structures at developmental stages at which patterning and neural specification takes place. The timely and spatially overlapping as well as complementary gene expression suggests diverse as well as redundant involvements during brain development.

     

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  • Drosophila Ras/MAPK signalling regulates innate immune responses in immune and intestinal stem cells

     

    Ragab, A., Buechling, T., Gesellchen, V., Spirohn, K., Boettcher, A.L., Boutros, M. (2011). Drosophila Ras/MAPK signalling regulates innate immune responses in immune and intestinal stem cells. EMBO Journal 30:1123-36.

     

    Immune signalling pathways need to be tightly regulated as overactivation of these pathways can result in chronic inflammatory diseases and cancer. NF-κB signalling and associated innate immune pathways are crucial in the first line of defense against infection in all animals. In a genome-wide RNAi screen for modulators of Drosophila immune deficiency (IMD)/NF-κB signalling, we identified components of the Ras/MAPK pathway as essential for suppression of IMD pathway activity, even in the absence of an immune challenge. Downregulation of Ras/MAPK activity mimics the induction of innate immune responses by microbial patterns. Conversely, ectopic Ras/MAPK pathway activation results in the suppression of Drosophila IMD/NF-κB signalling. Mechanistically, we show that the Ras/MAPK pathway acts by inducing transcription of the IMD pathway inhibitor Pirk/Rudra/PIMS. Finally, in vivo experiments demonstrate a requirement for Ras/MAPK signalling in restricting innate immune responses in haemocytes, fat body and adult intestinal stem cells. Our observations provide an example of a pathway that promotes cell proliferation and has simultaneously been utilized to limit the immune response.

     

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  • Wnt/Frizzled signaling requires dPRR, the Drosophila homolog of the prorenin receptor

     

    Buechling, T., Bartscherer, K., Ohkawara, B., Chaudhary, V., Spirohn, K., Niehrs, C., Boutros., M. (2010). Wnt/Frizzled signaling requires dPRR, the Drosophila homolog of the prorenin receptor. Current Biology 20:1263-1268.

     

    Wnt/Wg signaling pathways are of key importance during development and disease. Canonical and noncanonical Wnt/Frizzled (Fz) pathways share a limited number of signaling components that are part of the membrane proximal signaling complex. In Drosophila, Fz and Dishevelled (Dsh) are the only two components known to be involved in both Wnt/beta-catenin and planar cell polarity (PCP) signaling. PCP signaling is required for the planar polarization of epithelial cells, which occurs, for instance, during hair orientation and gastrulation in vertebrates. Both pathways have been studied intensively in the past years. However, it still remains unresolved whether additional components are required at the receptor complex. Here we identify the Drosophila homolog of the mammalian prorenin receptor (dPRR) as a conserved modulator of canonical Wnt/beta-cat and Fz/PCP signaling. We show that dPRR depletion affects Wg target genes in cultured cells and in vivo. PRR is required for epithelial planar polarity in Drosophila and for convergent extension movements in Xenopus gastrulae. Furthermore, dPRR binds to Fz and Fz2 receptors. In summary, our data suggest that dPRR has an evolutionarily conserved role at the receptor level for activation of canonical and noncanonical Wnt/Fz signaling pathways.

     

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  • Stepwise polarisation of the Drosophila follicular epithelium

     

    Franz, A., Riechmann, V. (2010) Stepwise polarisation of the Drosophila follicular epithelium. Developmental Biology 338:136-147.

     

    The function of epithelial tissues is dependent on their polarised architecture, and loss of cell polarity is a hallmark of various diseases. Here we analyse cell polarisation in the follicular epithelium of Drosophila, an epithelium that arises by a mesenchymal-epithelial transition. Although many epithelia are formed by mesenchymal precursors, it is unclear how they polarise. Here we show how lateral, apical, and adherens junction proteins act stepwise to establish polarity in the follicular epithelium. Polarisation starts with the formation of adherens junctions, whose positioning is controlled by combined activities of Par-3, beta-catenin, and Discs large. Subsequently, Par-6 and aPKC localise to the apical membrane in a Par-3-dependent manner. Apical membrane specification continues by the accumulation of the Crumbs complex, which is controlled by Par-3, Par-6, and aPKC. Thus, our data elucidate the genetic mechanisms leading to the stepwise polarisation of an epithelium with a mesenchymal origin.

     

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  • No Relaxing for Cancer Cells

     

    Many tumor cells would not be viable due to aberrant chromosome distribution if they had not developed a special trick. A research team led by scientists from the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) has investigated which genes are responsible for this survival strategy of cancer cells. To this end, they systematically switched off each individual gene of cancer cells using the RNAi method. The researchers have now published an article in Science Translational Medicine revealing that cancer cells rely on the tension of specific protein fibers to be able to multiply. Thus, proteins which maintain this tension are promising targets for new, target-specific anticancer drugs: If they are switched off, cancer cells die.
    Joint Press Release of the German Cancer Research Center and Heidelberg University Hospitals

    The two centrosomes of a cell are responsible for cell division to proceed correctly. From these polar bodies in the cytoplasm protein fibers form which correctly distribute the duplicated chromosome set to the newly forming daughter cells. Seen under the microscope, these fibers have the shape of a spindle. Cancer cells, however, often have more than two centrosomes. As a result, their spindle fibers do not necessarily assume the normal shape of a spindle with two poles; instead, they can have a dysfunctional, multipolar shape. Such malformed spindles distribute the chromosomes unevenly among the daughter cells, which are then no longer viable.

    Hence, tumor cells only survive if they manage to partition their chromosomes correctly in spite of extra centrosomes. To do so, many cancer cells have developed a special trick: They form clusters of centrosomes. Two clusters are formed per cell and a functioning bipolar spindle can develop between these two. Professor Dr. Alwin Krämer, head of a Clinical Cooperation Unit of DKFZ and Heidelberg University Hospitals has recognized this trick as a previously underrated Achilles’ heel of cancer cells, which might be used for destroying them. Jointly with colleagues from DKFZ, Heidelberg University Hospitals, Mannheim Medical Faculty and Mayo Clinic in the U.S., he systematically investigated the question of which genes enable cancer cells to form centrosome clusters and, thus, to escape cell death.

    With the support of researchers from the division of Professor Dr. Michael Boutros, DKFZ and Mannheim Medical Faculty, the investigators switched off each individual gene of the cancer cells. Then they searched under the microscope for multipolar, malformed spindles. They found 82 genes which play a role in centrosome clustering. The team took a closer look at 22 of these and investigated their particular role in clustering. In the process, the scientists discovered a key mechanism: For the centrosomes to be bundled into clusters, the spindle fibers need to be under tension. Only tightly stretched spindle fibers will position the centrosomes close enough to each other for clusters to form. A whole range of proteins are responsible for this tension. If their genes are silenced, multipolar spindles form and the cancer cells die. This mechanism might be used for developing new cancer therapies.

    “Such a therapy would hit the cancer very specifically, because only tumor cells have extra centrosomes and depend on the survival trick of clustering,” study head Alwin Krämer explains. In the framework of a strategic alliance of DKFZ with Bayer-Schering, the researchers in Krämer’s team are now planning to look among the identified genes for suitable targets for a targeted cancer therapy.

    Blanka Leber, Bettina Maier, Florian Fuchs, Jing Chi, Phillip Riffel, Simon Anderhub, Ludmila Wagner, Anthony D. Ho, Jeffrey L. Salisbury, Michael Boutros und Alwin Krämer: Proteins Required for Centrosome Clustering in Cancer Cells. Science Translational Medicine, 2010

    A picture for this press release is available on the Internet at:
    www.dkfz.de/de/presse/pressemitteilungen/2010/images/PM_30_Kraemer.jpg

    Picture caption: Multipolar, malformed spindle of a cancer cell
    Picture source: German Cancer Research Center

     


    Multipolar, malformed spindle of a cancer cell

     

    » Source: DKFZ press release on 28.05.10

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  • Molecular network of "death receptors"

     

    Research on apoptosis signaling networks coordinated by the University of Heidelberg

    The Medical Faculty Mannheim of the University of Heidelberg coordinates the international ApoNET research project as a part of an EU-wide initiative on systems biology. The project aims a better understanding of apoptosis networks in liver cells with the help of modern genome sequencing methods and computer models. The project is funded through the ERASysBio+ program with 1.7 million Euro by the Federal Ministry for Education and Research and the European Commission.
    Professor Dr. Michael Boutros, Head of the Department of Cell and Molecular Biology at the Medical Faculty Mannheim of the University of Heidelberg and Head of the Department of Signaling and Functional Genomics at the German Cancer Research Center, coordinates the interdisciplinary European consortium ApoNET. Project partners are Professor Dr. Rainer Spang (University of Regensburg) and Professor Dr. Henning Walczak (Imperial College London, UK).

     

    » Read the whole press release (in German)

     

    Das molekulare Netzwerk der „Todesrezeptoren“ auf dem Prüfstand

    Forschungsprojekt zu Apoptose-Signalnetzwerken von der Universität Heidelberg aus koordiniert

     

    Im Rahmen einer EU-weiten Initiative zur Systembiologie wird von der Medizinischen Fakultät Mannheim der Universität Heidelberg das internationale ApoNET Forschungsprojekt koordiniert, das mit modernen Genom-Sequenziermethoden und Computermodellen zu einem besseren Verständnis von Apoptose-Netzwerken in Leberzellen beitragen soll. Das Projekt wird im Rahmen des EraSysBio+ Programms mit 1.7 Millionen Euro vom Bundesministerium für Bildung und Forschung und von der Europäischen Kommission gefördert.

    Professor Dr. Michael Boutros, Inhaber des Lehrstuhls für Zell- und Molekularbiologie an der Medizinischen Fakultät Mannheim der Universität Heidelberg und Leiter der Abteilung Signalwege und Funktionelle Genomik am Deutschen Krebsforschungszentrum, koordiniert das interdisziplinäre EU-Konsortium ApoNET. Projektpartner sind Professor Dr. Rainer Spang (Universität Regensburg) und Professor Dr. Henning Walczak (Imperial College London, UK).

    Neuartige Krebstherapien sind darauf ausgerichtet, gezielt den Zelltod von Krebszellen herbeizuführen ohne normale Zellen zu zerstören. Einige Krebsarten sind jedoch häufig resistent gegenüber diesen Therapien, weil die Krebszellen den programmierten Zelltod (Apoptose) nicht einleiten können. Die Apoptose ist eine Art Selbstmordprogramm, das die betroffenen Zellen aktiv und streng kontrolliert durchführen. In diesem Prozess spielen die so genannten „Todesrezeptoren“ eine entscheidende Rolle.

    Eine weltweit besonders häufig vorkommende Krebsart ist der Leberzellkrebs. Die Behandlung der Erkrankung scheitert oft an einem blockierten Apoptose-Signalweg. Um effektive Therapien für Leberzellkrebs entwickeln zu können ist es wichtig zu verstehen, wie die Apoptose-Signalnetzwerke in normalen Leberzellen reguliert und in Krebszellen dereguliert sind.

    Das ERASysBio+ Konsortium um Professor Boutros hat sich zum Ziel gesetzt, die Funktion der Signalnetzwerke von „Todesrezeptoren“ bei Leberzellkrebs systematisch zu analysieren. Die interdisziplinären Arbeiten im Konsortium laufen dabei eng mit den Projektpartnern Professor Spang und Professor Walczak zusammen. Das transnationale Projekt zielt darauf ab, das grundlegende biologische System zu verstehen, welches die Signale in normalen gegenüber veränderten Leberzellen steuert, und damit Vorhersagen in dem System möglich zu machen.

    Basierend auf den experimentellen Hochdurchsatz Sequenzierungs-Daten, die die Arbeitsgruppen von Professor Boutros und Professor Walczak erarbeiten, wird die Gruppe um Professor Spang Computer-basierte Modelle generieren, um die kritischen Punkte in der Regulation dieser Signalwege zu finden. Diese statistischen Modelle werden die Wissenschaftler nutzen, um die Aktivitäten dieser Signalwege in Leberzellen zu rekonstruieren und mögliche neue Angriffspunkte für Therapien zu finden.

    Zusätzlich zu neuen Erkenntnissen in der Signalweiterleitung durch „Todesrezeptoren“ in normalen und veränderten Zellen auf der Systemebene erwarten die Wissenschaftler, dass die Studie auch zu neuen Einsichten der prinzipiellen Mechanismen in der Tumorentstehung und der Therapie von resistenten Tumoren führt.

    ERA-NET ERASysBio+
    ERASysBio+ ist ein Programm, das gezielt die Anwendung systembiologischer Forschungsansätze in der Biomedizin von EU-Partnerländern fördert. Ziel ist es, transnationale Forschungs- und Entwicklungsprojekte im Bereich der aufstrebenden inter-disziplinären Wissenschaft der Systembiologie zu etablieren.
    ERA steht für "European Research Area" und damit für die Koordinierung von Forschungs- oder technologischen Entwicklungstätigkeiten in Europa und auf nationalen Ebenen.

    Kick-Off Meeting der transnationalen Forschungsprojekte ERASysBio+
    am 17. und 18. Mai 2010 in Paris

     


    Fluoreszenz-gefärbte Leberkrebszellen
    [blau: Zellkerne, grün: Zytoskelett (Aktin),
    rot: Zytoskelett an den Verbindungen
    zwischen den Zellen (Färbung mit Phalloidin)]

     

     

    » Source (in German): Press release of the Medical Faculty Mannheim on 17.05.10

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  • High-throughput sequencing is driving research

     

    First next-generation sequencer at the University of Heidelberg

    Scientists at the Department of Cell and Molecular Biology at the Medical Faculty Mannheim, University of Heidelberg, are now supported in their research by modern sequencing technology. The staff at the Department, established in fall 2008 and headed by Professor Dr. Michael Boutros, analyzes signaling processes that play important roles in the development of organisms and human diseases.
    The next-generation sequencer (SOLiD, Applied Biosystems/Life Technologies) is the first device of its kind at the University of Heidelberg. It allows the decoding of the genetic material (genome) in a few days and is, for example, used by scientists for identifying disease-causing mutations in the human genome.

     

    » Read the whole press release (in German)

     

    Hochleistungs-Sequenzierung treibt Forschung voran

    Erstes Sequenziergerät der neuesten Generation der Universität Heidelberg

     

    Wissenschaftler des Lehrstuhls für Zell- und Molekularbiologie an der Medizinischen Fakultät Mannheim der Universität Heidelberg werden ab sofort in ihrer Forschung durch modernste Sequenzierungstechnik unterstützt. Die Mitarbeiter des im Herbst 2008 neu gegründeten Lehrstuhls beschäftigen sich unter der Leitung des Lehrstuhlinhabers Professor Dr. Michael Boutros mit der Analyse von Signalprozessen, die während der Entstehung von Krankheiten und auch bei der Entwicklung eines Organismus eine wichtige Rolle spielen.

    Der Hochleistungs-Sequenzierer der neuesten Generation (SOLiD, Applied Biosystems/Life Technologies) ist das erste Gerät dieser Art an der Universität Heidelberg. Er ermöglicht die Entschlüsselung des Erbguts (Genom) in wenigen Tagen und wird von den Wissenschaftlern beispielsweise dafür eingesetzt, im menschlichen Genom krankheitsauslösende Mutationen ausfindig zu machen.

    Die Wissenschaftler des Mannheimer Lehrstuhls beschäftigen sich insbesondere mit der zellulären Übertragung von Informationen und deren Fehlleitung bei Krankheiten. Fortwährend erhalten Zellen aus ihrer Umgebung Reize, die ihr Verhalten bestimmen. In der Zelle lösen diese Reize Signale aus, die über so genannte Signalkaskaden in das Innere der Zelle weitergeleitet werden. Im Zellinneren löst das Signal eine Reaktion aus, wie beispielsweise die Teilung oder Differenzierung der Zelle. Die zelluläre Signalübertragung ist von entscheidender Bedeutung bei der Entstehung von Krebs, bei der Regulation von Stammzellen und vielen weiteren Prozessen. Die Wissenschaftler am Lehrstuhl für Zell- und Molekularbiologie verwenden moderne genomische und bioinformatische Ansätze, um neue Signalfaktoren zu identifizieren und deren Funktion aufzuklären. Auf der Basis der neuen Sequenzierungstechnologie werden sie außerdem neue, spezifische Methoden für Ihre Forschungsansätze entwickeln.

    Ein wichtiger wissenschaftlicher Ansatz, Krankheiten zu verstehen und neue therapeutische Angriffspunkte zu finden liegt darin, die zelluläre Signalübertragung im normalen und erkrankten Gewebe zu vergleichen. Die Signale, die eine Zelle erhält, hinterlassen Spuren - auch Signaturen genannt - die sich mittels RNA-Sequenzierung erfassen lassen. Die Wissenschaftler nutzen die Hochleistungs-Sequenzierung um zu untersuchen, wie sich die Signaturen in Zellen durch die erhaltenen Signale ändern.

    Der SOLiD Hochleistungs-Sequenzierer wurde als von der Deutschen Forschungsgemeinschaft (DFG) gefördertes Gerät angeschafft. Er gehört zu den wichtigsten Sequenzier-Systemen auf dem derzeitigen Markt. Die damit entwickelten neuen Methoden werden als „Next Generation Sequencing“ (NGS) bezeichnet. Forscher weltweit setzen NGS-Technologien ein, um das Erbgut auf Fehler zu untersuchen.

     


    Der SOLiD Hochleistungs-Sequenzierer
    am Lehrstuhl für Zell- und Molekularbiologie

     

     

    » Source (in German): Press release of the Medical Faculty Mannheim on 24.03.10

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  • Description of our research in the Annual Report 2008,
    Helmholtz Association of German Research Centres

 

 

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Last updated: 15.05.2014