dictyNews Electronic Edition Volume 31, number 15 November 14, 2008 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. ========= Abstracts ========= Differentiation inducing factor-1 (DIF-1) induces gene and protein expression  of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin Danton H. O’Day1,2,4, Yekaterina Poloz2, Michael A. Myre3 1Department of Biology, University of Toronto at Mississauga, Mississauga,  Ontario L5L 1C6, CANADA 2Department of Cell & Systems Biology, University of Toronto, Toronto,  Ontario M5S 3G5, CANADA 3Center for Human Genetic Research, Massachusetts General Hospital,  Harvard Medical School, 185 Cambridge St. , Boston, MA 02114, USA  4danton.oday@utoronto.ca Cellular Signalling, accepted, http://dx.doi.org/10.1016/j.cellsig.2008.10.019 The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain,  calmodulin binding protein that regulates nuclear number. To gain insight into  the regulation of numA, we assessed the effects of the stalk cell differentiation  inducing factor-1 (DIF-1), an extracellular signalling molecule, on the  expression of numA1 RNA and protein. For comparison, the extracellular  signalling molecules cAMP (mediates chemotaxis, prestalk and prespore  differentiation) and ammonia (NH3/NH4+; antagonizes DIF) were also  studied. Starvation, which is a signal for multicellular development, results  in a greater than 80% decrease in numA1 mRNA expression within 4 hours.  Treatment with ammonium chloride led to a greater than 90% inhibition of  numA1 RNA expression within 2 hours. In contrast, the addition of DIF-1  completely blocked the decrease in numA1 gene expression caused by  starvation. Treatment of vegetative cells with cAMP led to decreases in  numA1 RNA expression that were equivalent to those seen with starvation.  Western blotting after various morphogen treatments showed that the  maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells  was reflected in significantly increased numA1 protein levels. Treatment  with cAMP and/or ammonia led to decreased protein expression and each  of these morphogens suppressed the stimulatory effects of DIF-1. Protein  expression levels of CBP4a, a calcium-dependent binding partner of  numA1, were regulated in the same manner as numA1 suggesting this  potential co-regulation may be related to their functional relationship.  NumA1 is the first calmodulin binding protein shown to be regulated by  developmental morphogens in Dictyostelium being upregulated by  DIF-1 and down-regulated by cAMP and ammonia. Submitted by: Danton O'Day [danton.oday@utoronto.ca] -------------------------------------------------------------------------------- An extrachromosomal, inducible expression system for  Dictyostelium discoideum. Douwe M. Veltman*, Ineke Keizer-Gunnink and Peter J.M. Van Haastert * corresponding author Plasmid, in press Inducible expression systems are essential for the expression of toxic proteins  and are very convenient for proteins that induce strong side effects such as  retardation of growth or development. Currently available systems for use in  Dictyostelium either do not have a very tight control over expression levels  or use a combination of an integrating and an extrachromosomal vector. We  designed a new vector in which all components of the available 2-plasmid  tetracycline-inducible system were combined onto a single extrachromosomal  vector. Two types of inducible plasmids are presented, in which transcription  is induced by adding or removing doxycycline respectively. The location and  orientation of the components was optimized in order to obtain a low  background expression combined with high inducibility. The resulting vectors  have a very low expression in the uninduced state (>1,000-fold lower  expression compared to that resulting from the act15 promoter), show a  10,000-fold induction of gene expression in a doxycycline  concentration-dependent manner and are comparatively small (8.5 kb).  With these new vectors, inducible gene expression is as easy as  constitutive gene expression. Submitted by: Douwe M. Veltman [d.veltman@beatson.gla.ac.uk] -------------------------------------------------------------------------------- Efficient cell lysis method for isolation of total RNA from slime mold Dictyostelium:  Applicability in preparation of cDNA Bhavesh Vats and Harish Padh Department of Cell and Molecular Biology B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre,  Thaltej- Gandhinagar Highway, Thaltej, Ahmedabad – 380054, INDIA. Telephone Number: +91-79-27439375, Fax number: +91-79-27450449. Email: perd@perdcentre.com International Journal of Biotechnology and Biochemistry, in press Dictyostelium is a model organism for understanding of many cellular processes,  evolutionary studies, behavioral analysis, gene expression and now even drug  metabolism. RNA analysis is vital to most studies at molecular level. Extracting  RNA is no easy task due to the omnipresence of robust RNases. Attaining full  length mRNAs is the chief task in isolation of pure RNA. Stable ribonucleases  are the major obstacle in isolating RNA of any kind. Many methods and kits are  available for different organisms with differing efficiency, utility and expense.  For isolation of RNA from Dictyostelium, Blumberg and Lodish devised a simple,  in-expensive yet effective method, though for identification of mRNA use of  DNase and poly-dT columns were required. We modified the cell  homogenization process and isolated total RNA with the method. The  modification enables us to detect specific mRNA from the total RNA pool  without purifying through poly-dT columns or processing for DNA removal. The  integrity of RNA isolated by the modified method was similar to that of RNA  extracted with Qiagen RNAasy Mini kit indicative of the quality of the total RNA  extracted. With this modification, we have been able to identify three mRNA  transcripts and specifically amplify the cDNA using sequence specific primers. Submitted by: Harish Padh [hpadh@yahoo.com] -------------------------------------------------------------------------------- Multiple Mechanisms for Accumulation of Myosin II Filaments at the Equator  During Cytokinesis Shigehiko Yumura, Masahiro Ueda, Yasushi Sako, Toshiko  Kitanishi-Yumura,  Toshio Yanagida Traffic, in press Total internal reflection fluorescence microscopy revealed how individual bipolar  myosin II filaments accumulate at the equatorial region in dividing Dictyostelium  cells. Direct observation of individual filaments in live cells provided us with  much convincing information. Myosin II filaments accumulated at the equatorial  region by at least two independent mechanisms: (i) cortical flow, which is driven  by myosin II motor activities and (ii) de novo association to the equatorial cortex.  These two mechanisms were mutually redundant. At the same time, myosin II  filaments underwent rapid turnover, repeating their association and dissociation  with the actin cortex. Examination of the lifetime of mutant myosin filaments in  the cortex revealed that the turnover mainly depended on heavy chain  phosphorylation and that myosin motor activity accelerated the turnover. Double  mutant myosin II deficient in both motor and phosphorylation still accumulated  at the equatorial region, although they displayed no cortical flow and  considerably slow turnover. Under this condition, the filaments stayed for a  significantly longer time at the equatorial region than at the polar regions,  indicating that there are still other mechanisms for myosin II accumulation  such as binding partners or stabilizing activity of filaments in the equatorial  cortex.  Submitted by: Shigehiko Yumura [yumura@yamaguchi-u.ac.jp] -------------------------------------------------------------------------------- Identification of a target for CudA the transcription factor that directs formation  of the Dictyostelium tip organiser Hong Yu Wang and Jeffrey G. Williams  University of Dundee College of Life Sciences University of Dundee  Dow St. Dundee DD1 5EH U. K. Int J Dev Biol, in press The tip of the Dictyostelium slug functions much like an embryonic organiser; when  grafted onto the flank of a recipient slug it recruits a mass of prespore cells and leads them away as part of a secondary slug. CudA is a nuclear protein that is expressed in prespore cells where it acts as a specific transcription factor. CudA is also expressed in an anteriorly located group of cells, the tip-organiser, that is believed to constitute the functional tip. We identify an expansin-like gene, expl7, that is expressed within the tip-organiser region and that is not expressed in a cudA null strain. The expl7promoter contains a region that binds to CudA in vitro and this region is necessary for expression in the tip-organiser. These results provide an end-point for a previously defined signal transduction pathway; wherein regionalised expression of the ACA adenylyl cyclase within the tip-organiser leads to localised cAMP-induced activation of STATa and consequent binding of STATa to the cudA promoter. STATa then induces expression of cudA and  cudA directs the transcription of target genes such as expl7. Submitted by: Jeff Williams [j.g.williams@dundee.ac.uk] -------------------------------------------------------------------------------- Dictyostelium Dock180-related RacGEFs Regulate the Actin Cytoskeleton  during Cell Motility Alessia Para*, Miriam Krischke*, Sylvain Merlot, Zhouxin Shen, Michael Oberholzer,  Susan Lee, Steven Briggs, and Richard A. Firtel * These two authors contributed equally to the work Section of Cell and Developmental Biology Division of Biological Sciences University of California, San Diego 9500 Gilman Drive La Jolla, CA 92093-0380 Molecular Biology of the Cell, in press Cell motility of amoeboid cells is mediated by localized F-actin polymerization  that drives the extension of membrane protrusions to promote forward movements.  We show that deletion of either of two members of the Dictyostelium Dock180  family of RacGEFs, DockA and DockD, causes decreased speed of chemotaxing  cells. The phenotype is enhanced in the double mutant and expression of DockA  or DockD complements the reduced speed of randomly moving DockD null cells’  phenotype, suggesting that DockA and DockD are likely to act redundantly and  to have similar functions in regulating cell movement. In this regard, we find that  overexpressing DockD causes increased cell speed by enhancing F-actin  polymerization at the sites of pseudopod extension. DockD localizes to the cell  cortex upon chemoattractant stimulation and at the leading edge of migrating  cells and that this localization is dependent on PI3K activity, suggesting that  DockD might be part of the pathway that links PtdIns(3,4,5)P3 production to  F-actin polymerization. Using a proteomic approach, we found that DdELMO1  is associated with DockD and that Rac1A and RacC are possible in vivo DockD  substrates. In conclusion, our work provides a further understanding of how  cell motility is controlled and provides evidence that the molecular mechanism  underlying Dock180-related protein function is evolutionarily conserved. Submitted by: Rick Firtel [rafirtel@ucsd.edu] -------------------------------------------------------------------------------- Autophagy contributes to degradation of Hirano bodies Kim, D. H., Davis R. C., Furukawa R. , Fechheimer M. Department of Cellular Biology, University of Georgia, Athens, Georgia,  USA Autophagy, in press. Hirano bodies are actin-rich inclusions reported most frequently in the  hippocampus in association with a variety of conditions including  neurodegenerative diseases and aging. We have developed a model  system for formation of Hirano bodies in Dictyostelium and cultured  mammalian cells to permit detailed studies of the dynamics of these  structures in living cells. Model Hirano bodies are frequently observed  in membrane-enclosed vesicles in mammalian cells consistent with a  role of autophagy in the degradation of these structures. Clearance of  Hirano bodies by an exocytotic process is supported by images from  electron microscopy showing extracellular release of Hirano bodies,  and observation of Hirano bodies in the culture medium of Dictyostelium  and mammalian cells. An autophagosome marker protein Atg8-GFP was  colocalized with model Hirano bodies in wild-type Dictyostelium cells, but  not in atg5(-) or atg1-1 autophagy mutant strains. Induction of model Hirano  bodies in Dictyostelium with a high-level expression of 34 kDa DeltaEF1  from the inducible discoidin promoter resulted in larger Hirano bodies  and a cessation of cell doubling. The degradation of model Hirano bodies  still occurred rapidly in autophagy mutant (atg5(-)) Dictyostelium,  suggesting that other mechanisms such as the ubiquitin-mediated  proteasome pathway could contribute to the degradation of Hirano bodies.  Chemical inhibition of the proteasome pathway with lactacystin significantly  decreased the turnover of Hirano bodies in Dictyostelium, providing direct  evidence that autophagy and the proteasome can both contribute to  degradation of Hirano bodies. Short-term treatment of mammalian cells  with either lactacystin or 3-methyl adenine results in higher levels of  Hirano bodies and a lower level of viable cells in the cultures, supporting  the conclusion that both autophagy and the proteasome contribute to  degradation of Hirano bodies. Submitted by: Chandra Jack [chanj@rice.edu] ============================================================== [End dictyNews, volume 31, number 15]