dictyNews Electronic Edition Volume 38, number 26 October 5, 2012 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase This edition also contains the contents of the upcoming DICTYOSTELIUM DISCOIDEUM PROTOCOLS II ========= Abstracts ========= Malleilactone, a Polyketide Synthase-Derived Virulence Factor Encoded by the Cryptic Secondary Metabolome of Burkholderia pseudomallei Group Pathogens. Biggins JB, Ternei MA, Brady SF. Laboratory of Genetically Encoded Small Molecules, The Rockefeller University and Howard Hughes Medical Institute , 1230 York Avenue, New York, New York 10068, United States. J Am Chem Soc. 2012 Aug 15;134(32):13192-5. Sequenced bacterial genomes are routinely found to contain gene clusters that are predicted to encode metabolites not seen in fermentation-based studies. Pseudomallei group Burkholderia are emerging pathogens whose genomes are particularly rich in cryptic natural product biosynthetic gene clusters. We systematically probed the influence of the cryptic secondary metabolome on the virulence of these bacteria and found that disruption of the MAL gene cluster, which is natively silent in laboratory fermentation experiments and conserved across this group of pathogens, attenuates virulence in animal models. Using a promoter exchange strategy to activate the MAL cluster, we identified malleilactone, a polyketide synthase- derived cytotoxic siderophore encoded by this gene cluster. Small molecules targeting malleilactone biosynthesis either alone or in conjunction with antibiotics could prove useful as therapeutics to combat melioidosis and glanders. Submitted by John Biggins [jbiggins@rockefeller.edu] --------------------------------------------------------------------------- Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium. Macro L, Jaiswal JK, Simon SM. J Cell Sci. 2012 Sep 19. [Epub ahead of print] The protein clathrin mediates one of the major pathways of endocytosis from the extracellular milieu and plasma membrane. In single cell eukaryotes, such as S.cerevisiae, clathrin is not an essential gene raising the question whether clathrin conveys specific advantages for multicellularity. Furthermore, in contrast to mammalian cells, endocytosis in S.cerevisiae is not dependent on clathrin or the endocytic adaptor molecule AP2. We therefore sought to study the requirement for components of clathrin- mediated endocytosis (CME) in another unicellular system, the organism Dictyostelium. We have identified a heterotetrameric AP2 complex in Dictyostelium similar to that in higher eukaryotes. By simultaneously imaging fluorescently tagged clathrin and AP2 we find that, similar to higher eukaryotes, these proteins co-localize to membrane puncta that move into the cell together. We show that the contractile vacuole marker protein, dajumin-GFP, is trafficked via the cell membrane and identify it as a cargo that is internalized by CME in a clathrin-dependent, AP2 independent mechanism. This pathway is distinct from other endocytic mechanisms in Dictyostelium. Our finding that CME is required for the internalization of contractile vacuole proteins from the cell membrane explains the contractile vacuole biogenesis defect in Dictyostelium cells lacking clathrin. Our results lead to the implication that the machinery for CME and its role in organelle maintenance appeared early in eukaryotic evolution. We suggest that dependence of endocytosis on specific components of the CME pathway may have evolved later as demonstrated by internalization independent of AP2 function. Submitted by Laura Macro [macro.laura@gmail.com] --------------------------------------------------------------------------- Cyclin-dependent kinase 5 is a calmodulin-binding protein that associates with puromycin-sensitive aminopeptidase in the nucleus of Dictyostelium Robert J. Huber(a),(1), Andrew Catalano(a), and Danton H. OĠDay(a,b) (a) University of Toronto, Department of Cell & Systems Biology, 25 Harbord Street, Toronto, Ontario, Canada M5S 3G5 (b) University of Toronto Mississauga, Department of Biology, 3359 Mississauga Road North, Mississauga, Ontario, Canada L5L 1C6 (1)Present Address: Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Richard B. Simches Research Center, 185 Cambridge Street, Boston, Massachusetts, USA 02114 BBA - Molecular Cell Research, in press Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that has been implicated in a number of cellular processes. In Dictyostelium, Cdk5 localizes to the nucleus and cytoplasm, interacts with puromycin-sensitive aminopeptidase A (PsaA), and regulates endocytosis, secretion, growth, and multicellular development. Here we show that Cdk5 is a calmodulin (CaM)-binding protein (CaMBP) in Dictyostelium. Cdk5, PsaA, and CaM were all present in isolated nuclei and Cdk5 and PsaA co-immunoprecipitated with nuclear CaM. Although nuclear CaMBPs have previously been identified in Dictyostelium, the detection of CaM in purified nuclear fractions had not previously been shown. Putative CaM-binding domains (CaMBDs) were identified in Cdk5 and PsaA. Deletion of one of the two putative CaMBDs in Cdk5 (132LLINRKGELKLADFGLARAFGIP154) prevented CaM-binding indicating that this region encompasses a functional CaMBD. This deletion also increased the nuclear distribution of Cdk5 suggesting that CaM regulates the nucleocytoplasmic transport of Cdk5. A direct binding between CaM and PsaA could not be determined since deletion of the one putative CaMBD in PsaA prevented the nuclear localization of the deletion protein. Together, this study provides the first direct evidence for nuclear CaM in Dictyostelium and the first evidence in any system for Cdk5 being a CaMBP. Submitted by Robert Huber [rhuber@chgr.mgh.harvard.edu]] --------------------------------------------------------------------------- Dictyostelium discoideum Protocols II to be published in the "METHODS IN MOLECULAR BIOLOGY" series of Humana Press (now Springer) Editors: Ludwig Eichinger and Francisco Rivero Part I. The Amoebozoa: Basics and Community Resources. 1. The Amoebozoa Christina Schilde and Pauline Schaap* College of Life Sciences, University of Dundee, Dundee DD15EH, UK. 2. The model organism Dictyostelium discoideum Salvatore Bozzaro Department of Clinical and Biological Sciences, University of Turin, AOU S. Luigi, 10043 Orbassano (Torino), Italy. 3. Comparative genomics of the Dictyostelids William F. Loomis Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0116, USA. 4. One stop shop for everything Dictyostelium: dictyBase and the Dicty Stock Center in 2012 Petra Fey, Robert J. Dodson, Siddhartha Basu and Rex L. Chisholm dictyBase and the Dicty Stock Center, Northwestern University, Center for Genetic Medicine, Lurie 7-125, 303 E Superior Street, Chicago, IL 60611, USA. 5. Fluorescent reporters and methods to analyze fluorescent signals Annette Mueller-Taubenberger and Hellen C. Ishikawa-Ankerhold Ludwig-Maximilians-Universitaet Muenchen, Institut fuer Anatomie und Zellbiologie, Schillerstr. 42, 80336 Muenchen, Germany. 6. Collection and cultivation of Dictyostelids from the wild Tracy E. Douglas, Debra A. Brock, Boahemaa Adu-Oppong, David C. Queller and Joan E. Strassmann Department of Biology, Washington University, St Louis MO 63130, USA. Part II. Large Scale Analysis 7. Identification and verification of microRNAs by high-throughput sequencing Jimmie Haellman1, Lotta Avesson2, Johan ReimegŒrd1, Max Kaeller1 and Fredrik Soederbom2 1KTH Royal Institute of Technology, Science for Life Laboratory (SciLifeLab Stockholm), School of Biotechnology, Division of Gene Technology, SE-171 65, Solna, Sweden. 2Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, Box 590, S-75124 Uppsala, Sweden. 8. Transcriptional profiling of Dictyostelium with RNA sequencing Edward Roshan Miranda1,2, Gregor Rot3, Marko Toplak3, Balaji Santhanam1,4, Tomaz Curk3, Gad Shaulsky1,2,4 and Blaz Zupan1,3 1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA. 2 Graduate Program in Developmental Biology, Baylor College of Medicine, Houston, TX, 77030, USA. 3 Faculty of Computer and Information Science, University of Ljubljana, SI-1000 Ljubljana, Slovenia. 4 Graduate Program in Structural Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX, 77030, USA. 9. Analysis of chromatin organization by deep sequencing technologies James L. Platt1,2, Nick A. Kent1, Adrian J. Harwood1, and Alan R. Kimmel2 1School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK. 2Laboratory of Cellular and Developmental Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. 10. Pharmacogenetics of resistance to cisplatin and other anti-cancer drugs and the role of sphingolipid metabolism. Stephen Alexander, William S. Swatson, and Hannah Alexander Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA. 11. N-glycomic and -glycoproteomic studies in the social amoebae Christa L. Feasley1, Alba Hykollari2, Katharina Paschinger2, Iain B. H. Wilson2, and Christopher M. West1 1Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 USA. 2Department fuer Chemie, Universitaet fuer Bodenkultur, Muthgasse 18, A-1190, Vienna, Austria. Part III. Molecular Biology, Cell Biology, Biochemistry, Biophysics 12. Measuring cheating, fitness, and segregation in D. discoideum Neil J. Buttery, Jeff Smith, David C. Queller and Joan E. Strassmann Department of Biology, Campus Box 1137, Washington University in Saint Louis, One Brookings Drive, Saint Louis, Missouri, USA. 13. The application of the Cre-loxP system for generating multiple knock-out and knock-in targeted loci Jan Faix1, Joern Linkner1, Benjamin Nordholz1, James L. Platt2,3, Xin-Hua Liao3, and Alan R. Kimmel3 1Institute for Biophysical Chemistry, Hannover Medical School,D-30623 Hannover, Germany. 2School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3AX, UK. 3Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA. 14. Extrachromosomal inducible expression Douwe M. Veltman1 and Peter J.M. Van Haastert2 1Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, UK. 2Department of Cell Biochemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. 15. Isolation of Dictyostelium nuclei for light and electron microscopy Petros Batsios1, Otto Baumann2, Ralph Graef1 and Irene Meyer1 1Institute for Biochemistry and Biology, Dept. of Cell Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany. 2Institute for Biochemistry and Biology, Dept. of Animal Physiology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany. 16. Investigation of DNA Repair Anne-Marie C. Couto, Nicholas D. Lakin and Catherine Pears Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK. 17. Transcript localization in D. discoideum cells by RNA FISH Patrick Hofmann1, Janis Kruse1,2 and Christian Hammann1,2 1Heisenberg Research Group Ribogenetics; Technical University of Darmstadt; 64287 Darmstadt, Germany. 2present address: Ribogenetics@Biochemistry Laboratory, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany. 18. Analysis of mitochondrial gene expression Jessica E. Accari, Sam Manna, Paul R. Fisher, and Christian Barth Department of Microbiology, La Trobe University, Victoria, Australia. 19. Mitochondrial respiratory complex function and the phenotypic consequences of dysfunction Sarah J. Annesley1, Sergio Carilla-Latorre2, Ricardo Escalante2 and Paul R. Fisher1 1Department of Microbiology, La Trobe University, Victoria, VIC 3086, Australia. 2Instituto de Investigaciones Biomedicas Alberto Sols, CSIC-UAM, Arturo Duperier 4, 28029 Madrid, Spain. 20. Micropipette aspiration for studying cellular mechanosensory responses and mechanics Yee-Seir Kee1,4 and Douglas N. Robinson1,2,3 Departments of 1Cell Biology, 2Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205 USA, 3Chemical and Biomolecular Engineering, 4Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218 USA. Part IV Vesicle formation, trafficking and infection with bacterial pathogens 21. Quantitative analysis of phagocytosis and phagosome maturation Natascha Sattler, Roger Monroy and Thierry Soldati Department de Biochimie, Faculte des Sciences, Universite de Geneve, Sciences II, 30 quai Ernest Ansermet, CH-1211, Switzerland. 22. Setting up and monitoring an infection of Dictyostelium discoideum with mycobacteria Sonia Arafah1, Sebastien Kicka1, Valentin Trofimov1, Monica Hagedorn1,2, Nuria Andreu3, Siouxsie Wiles4, Brian Robertson3, and Thierry Soldati1 1Department de Biochimie, Faculte des Sciences, Universite de Geneve, Sciences II, 30 quai Ernest Ansermet, CH-1211 Geneve-4, Switzerland. 2present address: Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany. 3Centre for Molecular Microbiology and Infection, Department of Medicine, Imperial College London, Flowers Building, London, SW7 2AZ, UK. 4Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, 85 Park Road, Auckland, 1142, New Zealand. 23. Isolation of pathogen-containing vacuoles Olga Shevchuk and Michael Steinert Institut fuer Mikrobiologie, Technische Universitaet Braunschweig, Spielmannstr. 7, D-38106 Braunschweig, Germany. 24. Immuno-magnetic purification of fluorescent Legionella-containing vacuoles Ivo Finsel, Christine Hoffmann and Hubert Hilbi Max von Pettenkofer Institute, Ludwig-Maximilians University, Pettenkoferstra§e 9a, 80336 Munich, Germany. 25. Secretory lysosomes in Dictyostelium: visualization, characterization and dynamics Wanessa C. Lima and Pierre Cosson Cell Physiology and Metabolism Department, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland. 26. Monitoring autophagy in Dictyostelium Ana Mesquita, Javier Calvo-Garrido, Sergio Carilla-Latorre and Ricardo Escalante Instituto de Investigaciones Biomedicas Alberto Sols, Arturo Duperier 4, 28029-Madrid, Spain. Submitted by Ludwig Eichinger [ludwig.eichinger@uni-koeln.de] ============================================================== [End dictyNews, volume 38, number 26]