dictyNews
Electronic Edition
Volume 38, number 26
October 5, 2012
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This edition also contains the contents of the upcoming
DICTYOSTELIUM DISCOIDEUM PROTOCOLS II
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Abstracts
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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 [[log in to unmask]]
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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 [[log in to unmask]]
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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. O’Day(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 [[log in to unmask]]]
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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 Reimegård1, 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,
Pettenkoferstraße 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 [[log in to unmask]]
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[End dictyNews, volume 38, number 26]
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