Dicty News Electronic Edition Volume 23, number 14 October 22, 2004 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 Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Dictyostelium discoideum strains lacking the RtoA protein are defective for maturation of the Legionella pneumophila replication vacuole Zhiru Li2, Jonathan M. Solomon2,ş, and Ralph R. Isberg1,2* 1 Howard Hughes Medical Institute and 2 Department of Molecular Biology and Microbiology, Tufts University School of Medicine 150 Harrison Ave., Boston, MA 02111 Cellular Microbiology, in press To identify host proteins involved in Legionella pneumophila intracellular replication, the soil amoeba Dictyostelium discoideum was analyzed. The absence of the amoebal RtoA protein is demonstrated here to depress L. pneumophila intracellular growth. Uptake of L. pneumophila into a D. discoideum rtoA- strain was marginally defective, but this effect was not sufficient to account for the defective intracellular growth of L. pneumophila. The rtoA mutant was also more resistant to high-multiplicity killing by the bacterium. A targeting assay testing the co-localization of L. pneumophila-containing vacuole with an endoplasmic reticulum/pre-Golgi intermediate compartment marker protein, GFP-HDEL, was used to analyze these defects. In parental D. discoideum, the L. pneumophila vacuole showed recruitment of GFP-HDEL within 40 min after introduction of bacteria to the amoebae. By 6 hours after infection it was clear that the rtoA mutant acquired and retained the GFP-HDEL less efficiently than the parental strain, and that the mutant was defective for promoting the physical expansion of the membranous compartment surrounding the bacteria. Depressed intracellular growth of L. pneumophila in a D. discoideum rtoA- mutant, therefore, appeared to result from a lowered efficiency of vesicle trafficking events that are essential for the modification and expansion of the L. pneumophila-containing compartment. Submitted by: Zhiru Li [li.zhiru@tufts.edu] ----------------------------------------------------------------------------- Dictyostelium PAKc is required for proper chemotaxis Susan Lee1, Francisco Rivero2, Kyung Chan Park1, Emerald Huang1, Satoru Funamoto1, and Richard A. Firtel1 1Section of Cell and Developmental Biology Division of Biological Sciences and Center for Molecular Genetics University of California, San Diego 9500 Gilman Drive La Jolla CA 92093-0634 USA 2Zentrum fźr Biochemie I der Medizinischen FakultŠt UniversitŠt zu Kšln Joseph-Stelzmann-Strasse 52 50931 Kšln Germany Mol. Biol. Cell, in press. We have identified a new Dictyostelium p21-activated protein kinase, PAKc, that we demonstrate to be required for proper chemotaxis. PAKc contains a Rac-GTPase binding (CRIB) and autoinhibitory domain, a PAK-related kinase domain, an N-terminal phosphatidylinositol binding domain, and a C-terminal extension related to the G?? binding domain of S. cerevisiae Ste20, the latter two domains being required for PAKc transient localization to the plasma membrane. In response to chemoattractant stimulation, PAKc kinase activity is rapidly and transiently activated, with activity levels peaking at ~10 sec. pakc null cells exhibit a loss of polarity and produce multiple lateral pseudopodia when placed in a chemoattractant gradient. PAKc preferentially binds the Dictyostelium Rac protein RacB, and point mutations in the conserved CRIB that abrogate this binding result in mis-regulated kinase activation and chemotaxis defects. We also demonstrate that a null mutation lacking the PAK family member myosin I heavy chain kinase (MIHCK) shows mild chemotaxis defects, including the formation of lateral pseudopodia. A null strain lacking both PAKc and the PAK family member MIHCK exhibits severe loss of cell movement, suggesting that PAKc and MIHCK may cooperate to regulate a common chemotaxis pathway. Submitted by: Rick Firtel [rafirtel@ucsd.edu] ----------------------------------------------------------------------------- Involvement of the TRAP-1 homologue, Dd-TRAP1, in spore differentiation during Dictyostelium development Tsuyoshi Morita1, Hitomi Yamaguchi2, Aiko Amagai, and Yasuo Maeda* Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan Present address: 1 Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita City, Osaka 565-0871, Japan; 2 Department of Cell Genetics, National Institute of Genetics, Mishima, Shizuoka-ken 411-8540, Japan. Exp. Cell Res., in press TRAP1 (tumor necrosis factor receptor-associated protein 1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. We have previously demonstrated that Dd-TRAP1 (Dictyostelium discoideum TRAP1) synthesized at the vegetative growth phase is retained during the whole course of D. discoideum development, and that at the multicellular slug stage it is located in prespore-specific vacuoles (PSVs) of prespore cells as well as in the cell membrane and mitochondria. Thereupon, we examined the function of Dd-TRAP1 in prepore and spore differentiation, using Dd-TRAP1-knockdown cells (TRAP1-RNAi cells) produced by the RNA interference method. As was expected, Dd-TRAP1 contained in the PSV was found to be exocytosed during sporulation to constitute the outer-most layer of the spore cell wall. In the TRAP1-RNAi cells, PSV formation and therefore prespore differentiation were significantly impaired, particularly under a heat stress condition. Although the TRAP1-RNAi cells formed apparently normal-shaped spores with a cellulosic wall, the spores were less resistant to heat and detergent treatments, as compared with those of parental MB35 cells derived from Ax-2 cells. These findings strongly suggest that Dd-TRAP1 may be closely involved in late development including spore differentiation, as well as in early development as realized by its induction of prestarvation response. Submitted by: Yasuo Maeda [ymaeda@mail.tains.tohoku.ac.jp] ----------------------------------------------------------------------------- Sphingosine kinase regulates the sensitivity of Dictyostelium discoideum cells to the anticancer drug cisplatin Junxia Min1, David Traynor2, Andrew L. Stegner1, Lei Zhang3, Marie H. Hanigan3, Hannah Alexander1 and Stephen Alexander1# 1Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400; 2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK; 3Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190. Eukaryotic Cell, In press. The drug cisplatin is widely used to treat a number of tumor types. However, resistance to the drug, which remains poorly understood, limits its usefulness. Previous work using D. discoideum as a model for studying drug resistance, showed that mutants lacking sphingosine-1-phosphate (S-1-P) lyase, the enzyme which degrades S-1-P, had increased resistance to cisplatin, while mutants overexpressing the enzyme were more sensitive to the drug. S-1-P is synthesized from sphingosine and ATP by the enzyme sphingosine kinase. We have identified two sphingosine kinase genes in D. discoideum - sgkA and sgkB - which are homologous to those of other species. The biochemical properties of the SgkA and SgkB enzymes suggest that they are the equivalent of the human Sphk1 and Sphk2 enzymes, respectively. Disruption of the kinases by homologous recombination (both single and double mutants) or overexpression of the sgkA gene resulted in altered growth rates, and altered response to cisplatin. The null mutants showed increased sensitivity to cisplatin, while mutants overexpressing the sphingosine kinase resulted in increased resistance compared to the parental cells. The results indicate that both the SgkA and SgkB enzymes function in regulating cisplatin sensitivity. The increase in sensitivity of the sphingosine kinase null mutants was reversed by addition of S-1-P, and the increased resistance of the sphingosine kinase overexpressor mutant was reversed by the inhibitor N, N-dimethylsphingosine. Parallel changes in sensitivity of the null mutants are seen with the platinum based drug carboplatin, but not with doxorubicin, 5-flurouracil and etoposide. This pattern of specificity is similar to that observed with the S-1-P lyase mutants, and should be useful in designing therapeutic schemes involving more than one drug. This work identifies the sphingosine kinases as new drug targets for modulating the sensitivity to platinum based drugs. Submitted by: Hannah Alexander [AlexanderH@missouri.edu] ----------------------------------------------------------------------------- Temperature adaptation in Dictyostelium: role of delta 5 fatty acid desaturase Tamao SAITO1,3, Atsushi KATO1, Hiroshi OCHIAI1 and Naoki MORITA2 1 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810 Japan, 2 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Toyohira-ku, Sapporo 062-8517 Japan. Microbiology, in press Membrane fluidity is critical for proper membrane function and is regulated in part by the proportion of unsaturated fatty acids present in the membrane lipids. The proportion of these lipids in turn varies with temperature and may contribute to temperature adaptation in poikilothermic organisms. The fundamental question in this study is whether the unsaturation of fatty acids contributes to the ability of adaptation to the temperature stress in Dictyostelium. We firstly analyzed fatty acid composition and detected that the relative proportions of dienoic acids changed with temperature. In order to investigate the role of dienoic fatty acids in temperature adaptation, we have created null mutants in the two known delta 5 fatty acid desaturases (FadA and FadB) that are responsible for the production of the dienoic fatty acids. The fadB null mutant showed no significant alteration in fatty acid composition or in phenotype. However, the disruption of fadA resulted in a large drop in dienoic fatty acid content from 51.2% to 4.1 % and a possibly compensatory increase in monoenoic fatty acids (40.9% to 92.4%). We detected no difference for the temperature adaptation with that of wild type cells in the growth phase. However, surprisingly mutant cells develop more efficiently than the wild-type at elevated temperatures. Our results show that fatty acid composition of Dictyostelium changes with temperature and suggest that the regulation of dienoic fatty acid synthesis is involved in the development of Dictyostelium at elevated temperature but not in the growth. Submitted by: Tamao Saito [tasaito@sci.hokudai.ac.jp] ============================================================================== [End Dicty News, volume 23, number 14]