dictyNews Electronic Edition Volume 39, number 20 July 12, 2013 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 ========= Abstracts ========= ForC lacks canonical formin activity but bundles actin filaments and is required and multicellular development of Dictyostelium cells Alexander Junemann (1), Moritz Winterhoff (1), Benjamin Nordholz (1), Klemens Rottner (2,3), Ludwig Eichinger (4), Ralph Grf (5), and Jan Faix (1) (1) Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg Strae 1, 30625 Hannover, Germany (2) Institute of Genetics, University of Bonn, Karlrobert-Kreiten-Strasse 13, 53115 Bonn, Germany (3) Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany (4) Centre for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Cologne, Germany (5) Department of Cell Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany EJCB, in press Diaphanous-related formins (DRFs) drive the nucleation and elongation of linear actin filaments downstream of Rho GTPase signaling pathways. Dictyostelium formin C (ForC) resembles a DRF, except that it lacks a genuine formin homology domain 1 (FH1), raising the questions whether or not ForC can nucleate and elongate actin filaments. We found that a recombinant ForC-FH2 fragment does not nucleate actin polymerization, but moderately decreases the rate of spontaneous actin assembly and disassembly, although the barbed-end elongation rate in the presence of the formin was not markedly changed. However, the protein bound and crosslinked actin filaments into loose bundles of mixed polarity. Furthermore, ForC is an important regulator of morphogenesis since ForC-null cells are severely impaired in development resulting in the formation of aberrant fruiting bodies. Immunoblotting revealed that ForC is absent during growth, but becomes detectable at the onset of early aggregation when cells chemotactically stream together to form a multicellular organism, and peaks around the culmination stage. Fluorescence microscopy of cells ectopically expressing a GFP-tagged, N-terminal ForC fragment showed its prominent accumulation in the leading edge, suggesting that ForC may play a role in cell migration. In agreement with its expression profile, no defects were observed in random migration of vegetative mutant cells. Notably, chemotaxis of starved cells towards a source of cAMP was severely impaired as opposed to control. This was, however, largely due to a marked developmental delay of the mutant, as evidenced by the expression profile of the early developmental marker csA. In line with this, chemotaxis was almost restored to wild type levels after prolonged starvation. Finally, we observed a complete failure of phototaxis due to abolished slug formation and a massive reduction of spores consistent with forC promoter-driven expression of -galactosidase in presporecells. Together, these findings demonstrate ForC to be critically involved in regulation of the cytoskeleton during various stages of development. Submitted by Jan Faix [faix.jan@mh-hannover.de] --------------------------------------------------------------------------- Mitochondria are the target organelle of differentiation-inducing factor-3, an anti-tumor agent isolated from Dictyostelium discoideum Yuzuru Kubohara 1*, Haruhisa Kikuchi 2, Yusuke Matsuo 2, Yoshiteru Oshima 2 and Yoshimi Homma 3 1 Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan 2 Laboratory of Natural Product Chemistry, Tohoku University Graduate School of Pharmaceutical Sciences, Aoba-yama, Aoba-ku, Sendai 980-8578, Japan 3 Department of Biomolecular Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan PLoS ONE, in press. Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives such as butoxy-DIF-3 (Bu-DIF-3) are potent anti-tumor agents. However, the precise mechanisms underlying the actions of DIF-3 remain to be elucidated. In this study, we synthesized a green fluorescent derivative of DIF-3, BODIPY-DIF-3, and a control fluorescent compound, Bu-BODIPY (butyl-BODIPY), and investigated how DIF-like molecules behave in human cervical cancer HeLa cells by using both fluorescence and electron microscopy. BODIPY-DIF-3 at 520 microM suppressed cell growth in a dose-dependent manner, whereas Bu-BODIPY had minimal effect on cell growth. When cells were incubated with BODIPY- DIF-3 at 20 microM, it penetrated cell membranes within 0.5 h and localized mainly in mitochondria, while Bu-BODIPY did not stain the cells. Exposure of cells for 13 days to DIF-3, Bu-DIF-3, BODIPY-DIF-3, or CCCP (a mitochondrial uncoupler) induced substantial mitochondrial swelling, suppressing cell growth. When added to isolated mitochondria, DIF-3, Bu-DIF-3, and BOIDPY-DIF-3, like CCCP, dose-dependently promoted the rate of oxygen consumption, but Bu-BODIPY did not. Our results suggest that these bioactive DIF-like molecules suppress cell growth, at least in part, by disturbing mitochondrial activity. This is the first report showing the cellular localization and behavior of DIF-like molecules in mammalian tumor cells. Submitted by Yuzuru Kubohara [kubohara@showa.gunma-u.ac.jp] --------------------------------------------------------------------------- Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA) Sara Mueller1,2, Indra M. Windhof1, Vladimir Maximov1,3, Tomasz Jurkowski4, Albert Jeltsch4, Konrad U. Frstner5, Cynthia M. Sharma5, Ralph Graef6, Wolfgang Nellen1* 1 Department of Genetics, University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany 2 current address: Institute for Biochemistry and Molecular Cell Biology, Georg-August-University, Humboldtallee 23, 37073 Gttingen, Germany 3 current address: Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, PO-Box 7080, SE-75007 Uppsala, Sweden 4 Institute of Biochemistry, University Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany 5 Research Center for Infectious Diseases (ZINF), University of Wuerzburg, Josef-Schneider-Str. 2/Bau D15, 97080 Wuerzburg 6 Universitaet Potsdam, Institut fr Biochemie und Biologie, Abt. Zellbiologie, Karl-Liebknecht-Strasse 24-25,, 14476 Potsdam - Golm Nucleic Acids Research, in press Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on tRNA than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNAAsp(GUC) in vitro and in vivo. By a UV-crosslinking and immunoprecipitation (CLIP) approach we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNAGlu(CUC/UUC) and tRNAGly(GCC) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. Since expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation. Submitted by Wolfgang Nellen [nellen@uni-kassel.de] --------------------------------------------------------------------------- Chromatin organisation of transgenes in Dictyostelium Indra Windhof*, Manu J. Dubin*1 and Wolfgang Nellen2 Abt. Genetik, FB 10, Universitt Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany * these authors equally contributed to this paper 1 present address: Gregor Mendel Institute of Molecular Plant Biology, Doktor-Bohr-Gasse 3, 1030 Vienna, Austria 2 corresponding author Die Pharmazie 7/2013, page 595-600. The introduction of transgenes in Dictyostelium discoideum typically results in the integration of the transformation vector into the genome at one or a few insertion sites as tandem arrays of approx. 100 copies. Exceptions are extrachromosomal vectors, which do not integrate into chromosomes, and vectors containing resistance markers such as Blasticidin, which integrate as single copies at one or a few sites. Here we report that low copy number vector inserts display typical euchromatic features while high copy number insertions are enriched for modifications associate with heterochromatin. Interestingly, high copy number insertions also colocalise with heterochromatin, are enriched for the centromeric histone CenH3 and display centromere-like behaviour during mitosis. We also found that the chromatin organisation on extrachromosmal transgenes is different from those integrated into the chromosomes. Submitted by Wolfgang Nellen [nellen@uni-kassel.de] --------------------------------------------------------------------------- WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion Jason S. King1, Aurlie Gueho2, Monica Hagedorn3, Navin Gopaldass,2 Florence Leuba2, Thierry Soldati2 and Robert H. Insall1. 1 Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow, UK. G61 1BD 2 Department of Biochemistry, University of Geneva, 30 quai Ernest Ansermet, Sciences II, CH-1211-Genve-4, Switzerland. 3 Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht Strasse 74, 20359 Hamburg, Germany. Molecular Biology of the Cell, In press WASH is an important regulator of vesicle trafficking. By generating actin on the surface of intracellular vesicles, WASH is able to directly regulate endosomal sorting and maturation. Here we report that in Dictyostelium, WASH is also required for the lysosomal digestion of both phagocytic and autophagic cargo. Consequently, Dictyostelium cells lacking WASH are unable to grow on many bacteria, or digest their own cytoplasm to survive starvation. WASH is required for efficient phagosomal proteolysis, and proteomic analysis demonstrates that this is due to reduced delivery of lysosomal hydrolases. Both protease and lipase delivery are disrupted, and lipid catabolism is also perturbed. Starvation-induced autophagy therefore leads to phospholipid accumulation within WASH null lysosomes. This causes the formation of multilamellar bodies typical of many lysosomal storage diseases. Mechanistically, we show that in cells lacking WASH, cathepsin D becomes trapped in a late endosomal compartment, unable to be recycled to nascent phagosomes and autophagosomes. WASH is therefore required for the maturation of lysosomes to a stage where hydrolases can be retrieved and reused. Submitted by Jason King [j.king@beatson.gla.ac.uk] --------------------------------------------------------------------------- Aeromonas salmonicida Ati2 is an effector protein of the type three secretion system. Dallaire-Dufresne S, Barbeau X, Sarty D, Tanaka KH, Denoncourt AM, Lage P, Reith ME, Charette SJ. Microbiology. 2013 Jul 7. [Epub ahead of print] The bacterium Aeromonas salmonicida, a fish pathogen, uses the type three secretion system (TTSS) to inject effector proteins into host cells to promote the infection. The study of the genome of A. salmonicida has revealed the existence of Ati2, a potential TTSS effector protein. In the present study, a structure-function analysis of Ati2 has been done to determine its role in the virulence of A. salmonicida. Biochemical assays revealed that Ati2 is secreted into the medium in a TTSS-dependent manner. Protein sequence analyses, molecular modeling and biochemical assays demonstrated that Ati2 is an inositol polyphosphate 5-phosphatase which hydrolyzes PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in a way similar to VPA0450, a protein from Vibrio parahaemolyticus having high sequence similarity with Ati2. Mutants of Ati2 with altered amino acids at two different locations in the catalytic site displayed no phosphatase activity. Wild-type and mutant forms of Ati2 were cloned into expression systems for Dictyostelium discoideum, a soil amoeba used as alternative host to study A. salmonicida virulence. Expression tests allowed us to demonstrate that Ati2 is toxic for the host cell in a catalytic-dependent manner. Finally, this study has demonstrated the existence of a new TTSS effector protein in A. salmonicida. Submitted by Steve Charette [steve.charette@bcm.ulaval.ca] ============================================================== [End dictyNews, volume 39, number 20]