Dicty News Electronic Edition Volume 14, number 7 April 1, 2000 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============= Abstracts ============= Dictyostelium, A Model Organism For Microtubule-Based Transport. Michael Koonce Division of Molecular Medicine, Wadsworth Center, Albany, NY 12201-0509. in press: Protist. 151 (June 2000) No abstract, but here is a paragraph from the introduction. Although investigated at a lower level than in many other cell types or organisms, the number of Dictyostelium microtubule-associated proteins characterized in the past decade has steadily increased (e.g. Kimble et al. 1992, Sellitto et al. 1992, Kalt and Schliwa, 1996, Gräf et al. 1999). Recent cloning of the single copy a-, b-, and g-tubulin genes has also opened up several molecular avenues that increase microtubule visibility and manipulation (Triviños-Lagos et al. 1993a, Euteneuer et al. 1998). The development of an in vitro motility assay now allows detailed analyses of microtubule-based motor protein functions, especially when coupled with molecular genetic disruptions or modifications (Pollock et al. 1998, 1999). It seems reasonable to propose that a critical threshold of reagents has been assembled that will greatly facilitate investigation of microtubule-based movement in this organism. This mini-review focuses on the dynein and kinesin-like mechanoenzymes of Dictyostelium and their roles here in microtubule-based intracellular transport. A few recent experiments are also highlighted, to stress the utility of Dictyostelium as a model organism for microtubule-based motility. ---------------------------------------------------------------------------- Optimizing heterologous expression in Dictyostelium: importance of 5' codon adaptation Elisa B. Vervoort, Arno van Ravestein, Noël N.M.E. van Peij, Judith C. Heikoop, Peter J.M. van Haastert, Gijs F.Verheijden, Maarten H.K. Linskens Cell Engineering Facility GBB, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Nucleic Acids Research, in press ABSTRACT Expression of heterologous proteins in Dictyostelium discoideum presents unique research opportunities, such as the functional analysis of complex human glycoproteins after random mutagenesis. In one study, human chorionic gonadotropin (hCG) and human follicle stimulating hormone were expressed in Dictyostelium. During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start codon. The Dictyostelium genome has a higher AT content than the human, resulting in a different codon preference. The hCG-beta gene contains three clusters with infrequently used codons that were changed to codons that are preferred by Dictyostelium. The results reported here show that optimizing the first 5-17 codons of the hCG gene contributes to 4 to 5- fold increased expression levels, but that further optimization has no significant effect. These observations suggest that optimal codon usage contributes to ribosome stabilization, but does not play an important role during the elongation phase of translation. Furthermore, adapting the 5' sequence of the hCG gene to the Dictyostelium 'Kozak'- like sequence increased expression levels about 1.5 fold. Thus, using both codon optimization and 'Kozak' adaptation, a 6 to 8 fold increase of expression levels could be obtained for hCG. ---------------------------------------------------------------------------- Inactivation of lmpA, Encoding a LIMPII-Related Endosomal Protein, Suppresses the Internalization and Endosomal Trafficking Defects in Profilin-Null Mutants Lesly Temesvari1,*, Linyi Zhang2, Brent Fodera1, Klaus-Peter Janssen3, Michael Schleicher3 and James A. Cardelli1,2 1Feist-Weiller Cancer Center and 2Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, LA 71130 and 3A.-Butenandt-Institut fuer Zellbiologie, Ludwig-Maximilians-Universtaet, 80336 Muenchen, FRG. Mol. Biol. Cell, in press. ABSTRACT Profilin is a key phosphoinositide and actin binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. We demonstrate using biochemical assays and microscopic approaches that profilin-null cells are defective in macropinocytosis, fluid phase efflux and secretion of lysosomal enzymes, but are unexpectedly more efficient in phagocytosis compared to wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging to the CD36/LIMPII family suppressed, to different degrees, most of the profilin minus defects, including the increase in F-actin, but did not rescue the secretion defect. Immunofluorescence microscopy indicated that DdLIMP, also capable of binding phosphoinositides, was associated with macropinosomes, but not detected in the plasma membrane. Also, inactivation of the lmpA gene in wild-type strains resulted in defects in macropinocytosis and fluid phase efflux, but not in phagocytosis. These results suggest an important role for profilin in regulating the internalization of fluid and particles, and movement of material along the endosomal pathway, and demonstrate a functional interaction between profilin and DdLIMP that may connect phosphoinositide-based signaling through the actin cytoskeleton with endo-lysosomal membrane trafficking events. ---------------------------------------------------------------------------- Analysis of the promoter of the cudA gene reveals novel mechanisms of Dictyostelium cell type differentiation Masashi Fukuzawa and Jeffrey G. Williams* Department of Anatomy and Physiology, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH Development, in press. SUMMARY The cudA gene encodes a nuclear protein that is essential for normal multicellular development. At the slug stage cudA is expressed in the prespore cells and in a sub-region of the prestalk zone. We show that cap site distal promoter sequences direct cudA expression in prespore cells, while proximal sequences direct expression in the prestalk sub-region. The promoter domain that directs prespore-specific transcription consists of a positively acting region, that has the potential to direct expression in all cells within the slug, and a negatively acting region that prevents expression in the prestalk cells. Dd-STATa is the STAT protein that regulates commitment to stalk cell gene expression, where it is known to function as a transcriptional repressor. We show that Dd-STATa binds in vitro to the positively acting part of the prespore domain of the cudA promoter. However, Dd-STATa cannot be utilised for this purpose in vivo, because analysis of a Dd-STATa null mutant strain shows that Dd-STATa is not necessary for cudA transcription in prespore cells. In contrast, the part of the cudA promoter that directs prestalk-specific expression contains a binding site for Dd-STATa that is essential for its biological activity. Dd-STATa appears therefore to serve as a direct activator of cudA transcription in prestalk cells, while a protein with a DNA binding specificity highly related to that of Dd-STATa is utilised to activate cudA transcription in prespore cells. ---------------------------------------------------------------------------- Developmental changes in the spatial expression of genes involved in myosin function in Dictyostelium Mineko Maeda1*, Hidekazu Kuwayama1, Masako Yokoyama1, Keiko Nishio1, Takahiro Morio2, Hideko Urushihara2, Mariko Katoh2 Yoshimasa Tanaka2, Tamao Saito3, Hiroshi Ochiai3, Keiko Takemoto4, Hiroo Yasukawa5, and Ikuo Takeuchi6 1Department of Biology, Graduate School of Science, Osaka Univ., Machikaneyama 1-16, Toyonaka, Osaka 560-0043, Japan, 2Institute of Biological Sciences, Univ., Tsukuba, Tsukuba, Ibaraki 305-8572, Japan, 3Division of Biological Science, Graduate School of Science, Hokkaido Univ., Sapporo, Hokkaido 060-0810, Japan, 4Institute for Virus Research, Kyoto Univ., Kyoto 606-8507, Japan, 5Faculty of Engineering, Toyama Univ., Toyama, Toyama 930-8555, Japan, 6Novartis Foundation for the Promotion of Science, Takarazuka, Hyogo 665-0042, Japan Develop. Biol. in press. Abstract We analyzed the spatial expression patterns of the genes involved in myosin function by in situ hybridization at the tipped aggregate and early culmination stages of Dictyostelium. Myosin heavy chain II (MHC-II) mRNA was enriched in the anterior prestalk region of the tipped aggregates, whereas it disappeared from there and began to appear in both upper and lower cups of the early culminants. Similarly, mRNAs for essential light chain, regulatory light chain, myosin light chain kinase A and myosin heavy chain kinase C were enriched in the prestalk region of the tipped aggregates. However, expression of these genes was distinctively regulated in the early culminants. These findings suggest the existence of mechanisms responsible for the expression of particular genes. ---------------------------------------------------------------------------- Differential developmental expression and cell type specificity of Dictyostelium catalases and their response to oxidative stress and UV-light Ma. Xenia U. Garcia1, Christopher Foote1, Saskia van Es2, Peter N. Devreotes2, Stephen Alexander1 and Hannah Alexander1 1Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400 2 Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 In press, Biochimica et Biophysica Acta ABSTRACT Cells of Dictyostelium discoideum are highly resistant to DNA damaging agents such as UV-light, gamma-radiation and chemicals. The genes encoding nucleotide excision repair (NER) and base excision repair (BER) enzymes are rapidly upregulated in response to UV-irradiation and DNA-damaging chemicals, suggesting that this is at least partially responsible for the resistance of this organism to these agents. Although Dictyostelium is also unusually resistant to high concentrations of H2O2, little is known about the response of this organism to oxidative stress. To determine if transcriptional upregulation is a common mechanism for responding to DNA damaging agents, we have studied the Dictyostelium catalase and Cu/Zn superoxide dismutase antioxidant enzymes. We show that there are two catalase genes, and that each is differentially regulated both temporally and spatially during multicellular development. The catA gene is expressed throughout growth and development and its corresponding enzyme is maintained at a steady level. In contrast, the catB gene encodes a larger protein and is only expressed during the final stages of morphogenesis. Cell type fractionation showed that the CatB enzyme is exclusively localized to the prespore cells, and the CatA enzyme is found exclusively in the prestalk cells. Each enzyme has a different subcellular localization. The unique developmental timing and cell type distribution suggest that the role for catB in cell differentiation is to protect the dormant spores from oxidative damage. We found that exposure to H2O2 does not result in the induction of the catalase, superoxide dismutase, NER or BER mRNAs. A mutant with greatly reduced levels of catA mRNA and enzyme has greatly increased sensitivity to H2O2 but normal sensitivity to UV. These results indicate that the natural resistance to oxidative stress is not due to an ability to rapidly raise the level of antioxidant or DNA repair enzymes, and that the response to UV-light is independent from the response to reactive oxygen compounds. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 7]