dictyNews Electronic Edition Volume 30, number 3 January 25, 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 ========= Rho GTPases of the RhoBTB subfamily and tumorigenesis Jessica Berthold(1), Kristina Schenkova(1) and Francisco RIVERO(1,2) (1)Center for Biochemistry and Center for Molecular Medicine, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany (2)The Hull York Medical School, University of Hull, Hull HU6 7RX, United Kingdom. Acta Pharmacol. Sin., review in press RhoBTB proteins constitute a subfamily of atypical members within the Rho family of small GTPases. Their most salient feature is their domain architecture: a GTPase domain (in most cases non-functional) is followed by a proline-rich region, a tandem of two BTB domains and a conserved C-terminal region. In human the RhoBTB subfamily consists of three isoforms: RhoBTB1, RhoBTB2 and RhoBTB3. Orthologs are present in several other eukaryotes, like Drosophila and Dictyostelium, but have been lost in plants and fungi. Interest in RhoBTB arose when RHOBTB2 was identified as the gene homozygously deleted in breast cancer samples and was proposed as a candidate tumor suppressor gene, a property that has been extended to RHOBTB1. The functions of RhoBTB proteins have not been defined yet, but may be related to the roles of BTB domains in the recruitment of cullin3, a component of a family of ubiquitin ligases. A model emerges in which RhoBTB proteins are required to maintain constant levels of putative substrates involved in cell cycle regulation or vesicle transport t hrough targeting for degradation in the 26S proteasome. RhoBTB proteins are engrossing the list of Rho GTPases involved in tumorigenesis. Unlike typical Rho GTPases (usually overexpressed or hyperactive), RhoBTB proteins appear to play a part in the carcinogenic process through a mechanism that involves decreased or abolished expression of the corresponding genes or, more rarely, mutations that result in impaired functioning of the protein, presumably leading to accumulation of RhoBTB substrates and alterations of the cellular homeostasis. Submitted by: Francisco Rivero [francisco.rivero@uni-koeln.de] -------------------------------------------------------------------------------- Evidence that DIF-1 and hyper-osmotic stress activate a Dictyostelium STAT by inhibiting a specific protein tyrosine phosphatase Tsuyoshi Araki1*, Judith Langenick1*, Marianne Gamper2,3, Richard A. Firtel2 and Jeffrey G. Williams1+ 1 University of Dundee, College of Life Sciences, Dow Street, Dundee DD1 5EH, UK 2 University of California, San Diego, Natural Sciences Building Room 6111, 9500 Gilman Drive, La Jolla, CA 92093-0380, USA 3 Biomedical Research Foundation (SBF), Lauchefeld 31, CH-9548 Matzingen, Switzerland * Contributed equally + author for correspondence Development, in press STATc becomes tyrosine phosphorylated and accumulates in the nucleus when Dictyostelium cells are exposed to the prestalk cell inducer, Differentiation Inducing Factor-1 (DIF-1), or are subjected to hyper-osmotic stress. We show that the protein tyrosine phosphatase PTP3 interacts directly with STATc and that STATc is refractory to activation in PTP3 over-expressing cells. Conversely, over-expression of a dominant inhibitor of PTP3 leads to constitutive tyrosine phosphorylation and ectopic nuclear localisation of STATc. Treatment of cells with DIF-1 or exposure to hyper-osmotic stress induce a decrease in biochemically assayable PTP3 activity and both agents also induce serine-threonine phosphorylation of PTP3. These observations suggest a novel mode of STAT activation; whereby serine-threonine phosphorylation of a cognate protein tyrosine phosphatase results in the inhibition of its activity, shifting the phosphorylation-dephosphorylation equilibrium in favour of phosphorylation. Submitted by: Tsuyoshi Araki [t.araki@dundee.ac.uk] -------------------------------------------------------------------------------- Molecular dynamics and forces of a motile cell simultaneously visualized by TIRF and force microscopies Yoshiaki IWADATE and Shigehiko YUMURA Department of Functional Molecular Biology, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8512, Japan Biotechniques, in press Cells must exert traction forces onto the substratum for continuous migration. Molecular dynamics such as actin polymerization at the front of the cell and myosin II accumulation at the rear should play important roles in the exertion of forces required for migration. Therefore, it is important to reveal the relationship between the traction forces and molecular dynamics. Traction forces can be calculated from the deformation of the elastic substratum under a migrating cell. A transparent and colorless elastic substratum with a high reflective index (1.40) and a low Young's Modulus (1.0 kPa) were made from a pair of platinum-catalyzed silicones. We used this substratum to develop a new method for simultaneous recording of molecular dynamics and traction forces under a migrating cell in which total internal reflection fluorescence (TIRF) and force microscopy were combined. This new method allows the detection of the spatiotemporal distribution of traction forces produced by individual filopodia in migrating Dictyostelium cells, as well as simultaneous visualization of these traction forces and the dynamics of filamentous myosin II. Submitted by: Yoshiaki Iwadate [iwadate@yamaguchi-u.ac.jp] -------------------------------------------------------------------------------- The P450 oxidoreductase, RedA, controls development beyond the mound stage in Dictyostelium discoideum Daniela C. Gonzalez-Kristeller,Layla Farage, Leonardo C. Fiorini, William F. Loomis, and Aline M. da Silva BMC Development (in press) Background: NADPH-cytochrome-P450 oxidoreductase (CPR) is a ubiquitous enzyme that belongs to a family of diflavin oxidoreductases and is required for activity of the microsomal cytochrome-P450 monooxygenase system. CPR gene-disruption experiments have demonstrated that absence of this enzyme causes developmental defects both in mouse and insect. Results: Annotation of the sequenced genome of D. discoideum revealed the presence of three genes (redA, redB and redC) that encode putative members of the diflavin oxidoreductase protein family. redA transcripts are present during growth and early development but then decline, reaching undetectable levels after the mound stage. redB transcripts are present during growth and in all stages of development while redC expression was detected only in vegetative growing cells. We isolated a mutant strain of Dictyostelium discoideum following restriction enzyme-mediated integration (REMI) mutagenesis in which redA was disrupted. This mutant develops only to the mound stage and accumulates a bright yellow pigment. The mound-arrest phenotype is cell-autonomous suggesting that the defect occurs within the cells rather than in intercellular signaling. Conclusion: The developmental arrest due to disruption of redA implicates CPR in the metabolism of compounds that control cell differentiation. Submitted by: Bill Loomis [wloomis@ucsd.edu] ============================================================== [End dictyNews, volume 30, number 3]