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dictyNews Volume 25 Number 10
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.
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Abstracts
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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]
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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]
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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]
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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]
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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]
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[End Dicty News, volume 25, number 10]
