dictyNews Electronic Edition Volume 35, number 20 Dec 30, 2010 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. Follow dictyBase on twitter: http://twitter.com/dictybase HAPPY NEW YEAR! ========= Abstracts ========= The histone methyltransferase Dot1 is required for DNA damage repair and proper development in Dictyostelium Annette MŸller-Taubenberger, Clemens Bšnisch, Marlis FŸrbringer, Finni Wittek, Sandra B. Hake BBRC, in press Posttranslational histone modifications play an important role in modulating gene expression and chromatin structure. Here we report the identification of histone H3K79 dimethylation in the simple eukaryote Dictyostelium discoideum. We have deleted the D. discoideum Dot1/KMT4 homologue and demonstrate that it is the sole enzyme responsible for histone H3K79me2. Cells lacking Dot1 are reduced in growth and delayed in development, but do not show apparent changes in cell cycle regulation. Furthermore, our results indicate that Dot1 contributes to UV damage resistance and DNA repair in D. discoideum. In summary, the data support the view that the machinery controlling the setting of histone marks is evolutionary highly conserved and provide evidence that D. discoideum is a suitable model system to analyze these modifications and their functions during development and differentiation. Submitted by Annette MŸller-Taubenberger [amueller@lrz.uni-muenchen.de] -------------------------------------------------------------------------------- EGF-like peptide-enhanced cell motility in Dictyostelium functions independently of the cAMP-mediated pathway and requires active Ca2+/calmodulin signaling Robert Huber (1) and Danton H. OĠDay (1,2) (1) Department of Cell & Systems Biology, 25 Harbord Street, University of Toronto, Toronto, ON, Canada M5S 3G5 (2) Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, ON, Canada L5L 1C6 Cellular Signalling, in press Current knowledge suggests that cell movement in the eukaryotic slime mold Dictyostelium discoideum is mediated by different signaling pathways involving a number of redundant components. Our previous research has identified a specific motility-enhancing function for Epidermal Growth Factor-Like (EGFL) repeats in Dictyostelium, specifically for the EGFL repeats of cyrA, a matricellular, calmodulin (CaM)-binding protein in Dictyostelium. Using mutants of cAMP signaling (carA-, carC-, gpaB-, gpbA-), the endogenous calcium (Ca2+) release inhibitor TMB-8, the CaM antagonist W-7, and a radial motility bioassay, we show that DdEGFL1, a synthetic peptide whose sequence is obtained from the first EGFL repeat of cyrA, functions independently of the cAMP-mediated signaling pathways to enhance cell motility through a mechanism involving Ca2+ signaling, CaM, and RasG. We show that DdEGFL1 increases the amounts of polymeric myosin II heavy chain and actin in the cytoskeleton by 24.1 ħ 10.7% and 25.9 ħ 2.1% respectively and demonstrate a link between Ca2+/CaM signaling and cytoskeletal dynamics. Finally, our findings suggest that carA and carC mediate a brake mechanism during chemotaxis since DdEGFL1 enhanced the movement of carA-/carC- cells by 844 ħ 136% compared to only 106 ħ 6% for parental DH1 cells. Based on our data, this signaling pathway also appears to involve the G-protein beta subunit, RasC, RasGEFA, and protein kinase B. Together, our research provides insight into the functionality of EGFL repeats in Dictyostelium and the signaling pathways regulating cell movement in this model organism. It also identifies several mechanistic components of DdEGFL1-enhanced cell movement, which may ultimately provide a model system for understanding EGFL repeat function in higher organisms. Submitted by: Danton H. OĠDay [danton.oday@utoronto.ca] -------------------------------------------------------------------------------- Tyrosine Phosphorylation of Actin during Microcyst Formation and Germination in Polysphondylium pallidum Aldona Budniak (1) and Danton H. OĠDay (1,2) (1) Department of Cell & Systems Biology, 25 Harbord Street, University of Toronto, Toronto, ON, Canada M5S 3G5 (2) Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, ON, Canada L5L 1C6 Protist, in press High osmolarity causes amoebae of the cellular slime mould Polysphondylium pallidum to individually encyst, forming microcysts. During microcyst differentiation, actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent of encystment conditions and occurs during the final stages of microcyst formation. During microcyst germination, actin undergoes dephosphorylation prior to amoebal emergence. Renewed phosphorylation of actin in germinating microcysts can be triggered by increasing the osmolarity of the medium which inhibits emergence. Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in dormant microcysts. Following the onset of germination, actin is observed around vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin localizes to patches near the cell surface. Increasing osmolarity disrupts this l ocalization and causes actin to redistribute throughout the cytoplasm, a situation similar to that observed in dormant microcysts. The tyrosine phosphorylation state of actin does not appear to influence the long-term viability of dormant microcysts. Together, these results indicate an association between actin tyrosine phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy. Submitted by: Danton H. OĠDay [danton.oday@utoronto.ca] -------------------------------------------------------------------------------- Expression, identification and purification of Dictyostelium acetoacetyl-CoA thiolase expressed in Escherichia coli Ó Tanaka T, Shima Y, Ogawa N, Nagayama K , Yoshida T, Ohmachi T* Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, 036-8561, Japan   Present address: Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK Int. J. Biol. Sci., in press Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH2)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn- Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase. Submitted by: Tetsuo Ohmachi [tohmachi@cc.hirosaki-u.ac.jp] -------------------------------------------------------------------------------- Artificial compounds differentially control Dictyostelium chemotaxis and cell differentiation. Hidekazu Kuwayama 1, Haruhisa Kikuchi 2, Yoshiteru Oshima 2, Yuzuru Kubohara 3 1 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan 2 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan 3 Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512, Japan Correspondence: Yuzuru Kubohara, Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512, Japan. Cell Structure and Function, in press Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2) are small lipophilic signal molecules that control both cell differentiation and chemotaxis in the cellular slime mold Dictyostelium discoideum. In this study, we examined the effects of four amide derivatives of DIF-1 on stalk cell differentiation and chemotaxis. The DIF derivatives differentially affected cell differentiation and chemotaxis, suggesting the possible existence of at least three receptors for DIFs: one receptor responsible for stalk cell induction, and two receptors responsible for chemotaxis modulation. Furthermore, our results indicate that DIF derivatives can be utilized to analyze the DIF-signaling pathways. Submitted by: Yuzuru Kubohara [kubohara@showa.gunma-u.ac.jp] ============================================================== [End dictyNews, volume 35, number 20]