Dicty News Electronic Edition Volume 25, number 10 October 28, 2005 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 Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Effects of chemoattractant pteridines upon speed of D. discoideum vegetative amoebae. Jared L. Rifkin1 & Robert R. Goldberg2 1Biology Department and 2Computer Science Department, Queens College of CUNY, Flushing, NY 11367. Cell Motility and the Cytoskeleton, in press Movements of D. discoideum vegetative amoebae responding to pteridine chemoattractants, folate acid and pterin, were recorded. A vector analysis of these images was performed to partition the speed and orientation components of these motility patterns. This study demonstrates that in addition to orientation (chemotaxis), stimulated speed (chemokinesis) is an important component of the directed migration of these amoebae. Furthermore, the primary difference in their response to folate vs. pterin is in speed rather than orientation. The data support a model of directed migration of these cells in which there are (1) separate signal translation pathways consequent from folate vs. pterin reception and (2) specific pathways leading to increase in orientation vs. speed. Submitted by: Jared Rifkin [jared_rifkin@qc.edu] ----------------------------------------------------------------------------- DNA-PKcs-Dependent Signaling of DNA Damage in Dictyostelium discoideum Jessica J.R. Hudson1, Duen-Wei Hsu1, Kunde Guo1, Natasha Zhukovskaya2, Po- Hsien Liu1, Jeffrey G. Williams2, Catherine J. Pears1 and Nicholas D. Lakin1, 1Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom 2School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom Current Biology Volume 15, Issue 20, 25 October 2005, Pages 1880-1885 DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ) [1]. In vertebrates, the first step in NHEJ is recruitment of the DNA-dependent protein kinase (DNA-PK) to DNA termini [2]. DNA-PK consists of a catalytic subunit (DNA- PKcs) that is recruited to DNA ends by the Ku70/Ku80 heterodimer [3]. Although Ku has been identified in a wide variety of organisms, to date DNA- PKcs has only been identified experimentally in vertebrates. Here, we report the identification of DNA-PK in the nonvertebrate Dictyostelium. Dictyostelium Ku80 contains a conserved domain previously implicated in recruiting DNA-PKcs to DNA [4] and consistent with this observation, we have identified DNA-PKcs in the Dictyostelium genome. Disruption of the gene encoding Dictyostelium DNA-PKcs results in sensitivity to DNA DSBs and defective H2AX phosphorylation in response to this form of DNA damage. However, these phenotypes are only apparent when DNA damage is administered in G1 phase of the cell cycle. These data illustrate a cell cycle-dependent requirement for Dictyostelium DNA-PK in signaling and combating DNA DSBs and represent the first experimental verification of DNA-PKcs in a nonvertebrate organism. Submitted by: Jessica J.R. Hudson [jessica.hudson@bioch.ox.ac.uk] ----------------------------------------------------------------------------- Developmentally Regulated DNA Methylation in Dictyostelium Mariko Katoh, Tomaz Curk, Qikai Xu, Blaz Zupan, Adam Kuspa and Gad Shaulsky Department of Molecular and Human Genetics, Graduate Program in Structural Computational Biology and Molecular Biophysics, Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia Eukaryotic Cell, in press Methylation of cytosine residues in DNA plays a critical role in the silencing of gene expression, organization of chromatin structure and cellular differentiation of eukaryotes. Previous studies failed to detect 5- methylcytosine in Dictyostelium genomic DNA, but the recent sequencing of the Dictyostelium genome revealed a candidate DNA methyltransferase gene (dnmA). The genome sequence also uncovered an unusual distribution of potential methylation sites, CpG islands, throughout the genome. DnmA belongs to the Dnmt2 subfamily and contains all the catalytic motifs necessary for cytosine methyltransferases. Dnmt2 activity is typically weak in Drosophila, mouse and human cells and the gene function in these systems is unknown. We have investigated the methylation status of Dictyostelium genomic DNA with antibodies raised against 5-methylcytosine and detected low levels of the modified nucleotide. We also found that DNA methylation increased during development. We searched the genome for potential methylation sites and found them in retrotransposable elements and in several other genes. Using Southern blot analysis with methylation sensitive and insensitive restriction endonucleases we found that the DIRS retrotransposon and the guaB gene were indeed methylated. We then mutated the dnmA gene and found that DNA methylation was reduced to about 50% of the wild-type level. The mutant cells exhibited morphological defects in late development, indicating that DNA methylation has a regulatory role in Dictyostelium development. Our findings establish a role for a Dnmt2 methyltransferase in eukaryotic development. Submitted by: Gad Shaulsky [gadi@bcm.tmc.edu] ----------------------------------------------------------------------------- The making of filopodia Jan Faix(1) and Klemens Rottner(2) (1)Institute of Biophysical Chemistry, Hannover Medical School, Carl-Neuberg- Str. 1, D-30623 Hannover, Germany. (2)Cytoskeleton Dynamics Group, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany. Current Opinion in Cell Biology, in press Filopodia are rod-like cell surface projections filled with bundles of parallel actin filaments. They are found on a variety of cell types and have been ascribed sensory or exploratory functions. Filopodia formation is frequently associated with protrusion of sheet-like actin filament arrays called lamellipodia or membrane ruffles, but in comparison to these structures, the molecular details underpinning the initiation and maintenance of filopodia are only just beginning to emerge. Recent advances have improved our understanding of the molecular requirements for filopodium protrusion and have yielded insights into the interrelationships between lamellipodia and filopodia, the two ‘sub-compartments’ of the protrusive actin cytoskeleton. Submitted by: Jan Faix [faix@bpc.mh-hannover.de] ----------------------------------------------------------------------------- The Shwachman-Diamond Syndrome Gene Encodes an RNA-Binding Protein That Localizes to the Pseudopod of Dictyostelium Amoebae During Chemotaxis Deborah Wessels, Thyagarajan Srikantha, Song Yi, Spencer Kuhl, L. Aravind and David R. Soll Journal Cell Science, in press The Shwachman-Diamond Syndrome (SDS) is an autosomal disorder with multisystem defects. The SBDS gene, which contains mutations in a majority of SDS patients, encodes a protein of unknown function, although it has been strongly implicated in RNA metabolism. There is also some evidence that it interacts with molecules that regulate cytoskeletal organization. Recently, it was demonstrated by computer-assisted methods that the single behavioral defect of polymorphonuclear leukocytes (PMNs) of SDS patients is the incapacity to orient correctly in a spatial gradient of chemoattractant. We considered the social amoeba Dictyostelium discoideum, a model for PMN chemotaxis, an excellent system for elucidating the function of the SDS protein. We first identified the homolog of SBDS in D. discoideum and found that the amino acids that are altered in human disease were conserved. Given that several proteins involved in chemotactic orientation localize to the pseudopods of cells undergoing chemotaxis, we tested whether the SBDS gene product did the same. We produced an SBDS-GFP chimeric in-frame fusion gene, and generated transformants either with multiple ectopic insertions of the fusion gene or multiple copies of a non-integrated plasmid carrying the fusion gene. In both cases, the SBDS-GFP protein was dispersed equally through the cytoplasm and pseudopods of cells migrating in buffer. However, we observed differential enrichment of SBDS in the pseudopods of cells treated with the chemoattractant cAMP, suggesting that the SBDS protein may play a role in chemotaxis. In light of these results, we discuss how SBDS may function during chemotaxis. Submitted by: Deborah Wessels [deborah-wessels@uiowa.edu] ============================================================================== [End Dicty News, volume 25, number 10]