dictyNews
Electronic Edition
Volume 39, number 20
July 12, 2013
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Abstracts
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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 Gräf (5), and Jan Faix (1)
(1) Institute for Biophysical Chemistry, Hannover Medical School,
Carl-Neuberg Straße 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 ß-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 [[log in to unmask]]
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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 5–20 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 1–3 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 [[log in to unmask]]
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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. Förstner5, 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 Göttingen, 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 für 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 [[log in to unmask]]
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Chromatin organisation of transgenes in Dictyostelium
Indra Windhof*, Manu J. Dubin*1 and Wolfgang Nellen2
Abt. Genetik, FB 10, Universität 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 [[log in to unmask]]
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WASH is required for lysosomal recycling and efficient autophagic and
phagocytic digestion
Jason S. King1, Aurélie 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-Genève-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 [[log in to unmask]]
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Aeromonas salmonicida Ati2 is an effector protein of the type three secretion
system.
Dallaire-Dufresne S, Barbeau X, Sarty D, Tanaka KH, Denoncourt AM, Lagüe 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 [[log in to unmask]]
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