dictyNews Volume 39 Number 20

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dictyNews 
Electronic Edition 
Volume 39, number 20 
July 12, 2013 

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========= 
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 GrŠf (5), and Jan Faix (1) 

(1) Institute for Biophysical Chemistry, Hannover Medical School,  
Carl-Neuberg Stra§e 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 5Ð20 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 1Ð3 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. Fšrstner5, 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 Gšttingen, 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 fŸr 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, UniversitŠt 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, AurŽlie 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-Genve-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, LagŸe 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] 
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[End dictyNews, volume 39, number 20]