Dicty News Electronic Edition Volume 16, number 7 April 7, 2001 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ================================================== Post-doctoral position in proteomics research ================================================== We are looking to recruit an experienced post-doctoral researcher in order to use proteomics to study signalling pathways in Dictyostelium and identify novel components of signalling networks. 2D gel analysis will be used to establish proteome maps covering Dictyostelium development and changes in strains lacking known components of signalling pathways will be characterised. Changes in post-translational modification will be identified as will unknown proteins that co-immunoprecipitate with components of a signalling network. The pathways to be investigated involve STAT, GSK3 and ERK2 proteins. The analysis will exploit a sophisticated proteome facility in Oxford that will permit rapid-throughput computer-assisted 2-D gel analysis of complex protein mixtures. Proteins of interest will be identified from the very rapidly expanding Dictyostelium sequence databases by MALDI-TOF fingerprinting of in-gel tryptic digests and ESI sequencing. The project, based in Oxford, is a collaboration between three UK Dictyostelium groups (Cathy Pears, Jeff Williams and Adrian Harwood) and the Oxford proteomics unit. The successful candidate should have proven research skills in protein characterisation and experience with Dictyostelium would be a major advantage. Experience in running 2D gels, mass spectroscopy, HPLC or database analysis would also be preferable. The ability to manage the running of the research project, to work as part of a team and good communication skills are essential. Applicants should have a sound publication record. The post is funded by The Wellcome Trust for three years in the first instance. Salary will be in the range £16,775 - £25,213, the scale position dependent on age and experience. Informal enquiries by email to pears@bioch.ox.ac.uk. Applications, including two copies of a detailed CV, the names and addresses of two referees (one of which must be your current employer) and two copies of a letter setting out, with examples, how you meet the above selection criteria, should be sent to: The Deputy Administrator Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU Please quote reference number: BR/965/W ============== Abstracts ============== Cell type proportioning in Dictyostelium slugs: lack of regulation within a 2.5-fold tolerance range Ismael Rafols1, Aiko Amagai2, Yasuo Maeda2, Harry K. MacWilliams3 and Yasuji Sawada1 1Research Institute of Electrical Communication, Tohoku Univ., Japan 2Biological Institute, Graduate School of Science, Tohoku Univ., Japan 3Zoologisches Institut, Ludwig-Maximilians-Univ., München, Germany Corresponding Author: rafols@sawada.riec.tohoku.ac.jp Differentiation, in press. Abstract The proportion of prestalk and prespore cells in Dictyostelium discoideum slugs is often cited as an example of "almost perfect" regulation. The pattern is similar over a very wide range of cell number; furthermore, removal of either of the cell types leads to compensatory transdifferentiation. Several studies of Dictyostelium fruiting bodies, however, have suggested that proportioning in Dictyostelium differs systematically from true constancy. We have confirmed this in the slug stage using a short-lived beta-galactosidase as a reporter of the prestalk specific ecmA gene expression: the prestalk proportion decreases from 24% in slugs of 10^3 cells to 10% when 10^5 cells are present. Regeneration experiments suggest that this difference is not due to a modulation of the proportioning set-point by size, as one might have expected; instead there appears to be a regulatory "tolerance zone" at all sizes. After amputation of the whole posterior region, transdifferentiation stops after the fraction of prestalk has been reduced from 100% to 28%, well above the initial value of 10%, while after anterior removal the transdifferentiation endpoint is about 10%. Most strikingly, we find no regulation at all after partial amputations of the prespore region. It seems that any prestalk proportion is stable between a 10% lower threshold and a 30% upper threshold. To explain this we propose a regulation mechanism based on a negative feedback plus cell type bistability. In both intact and regenerating slugs we find that the slug morphology is regulated so that the length-to-width ratio of the anterior region is constant. ----------------------------------------------------------------------------- Unconventional mRNA processing in the expression of two calcineurin B isoforms in Dictyostelium Annette Aichem and Rupert Mutzel1 Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany 1Present address: Institut für Biologie - Mikrobiologie, Freie Universität Berlin, Königin-Luise-Strasse 12 - 16, 14195 Berlin, Germany J. Mol. Biol. in press Summary The genome of Dictyostelium discoideum contains a single gene (cnbA) for the regulatory (B) subunit of the Ca2+/calmodulin dependent protein phosphatase, calcineurin. Two mRNA species and two protein products differing in size were found. The apparent molecular masses of the protein isoforms corresponded to translation products starting from the first and second AUG codons of the primary transcript, respectively. The smaller mRNA and protein isoforms accumulated during early differentiation of the cells. Whereas the amount of the higher Mr protein isoform remained constant throughout development, the larger mRNA disappeared to virtually undetectable levels during aggregation. 5´RACE amplification of the smaller transcript yielded cDNAs lacking the 5´ nontranslated region and the first ATG initiator codon. Expression of truncated cDNAs and various chimeric genes encoding CNB-green fluorescent protein fusions in Dictyostelium indicate that the mature cnbA transcript is processed by an unconventional mechanism that leads to truncation of the 5´ untranslated region and at least the first AUG initiator codon and to utilization of the second AUG codon for translation initiation of the small CNB isoform. Determinants for this processing mechanism reside within the coding region of the cnbA gene. ----------------------------------------------------------------------------- Cytoskeletal Alterations in Dictyostelium Induced by Expression of Human Cdc42 Eunkyung Lee1 and David A. Knecht2 Department of Molecular and Cell Biology, University of Connecticut Storrs, CT 06269 Eur. J. Cell Biol., in press. Abstract The rho family of small G proteins has been shown to be involved in controlling actin filament dynamics in cells. To evaluate the functional overlap between human and Dictyostelium G proteins, we conditionally expressed constitutively active human cdc42 (V12-cdc42) in Dictyostelium cells. Upon induction, cells adopted a unique morphology: a flattened shape with wrinkles running from the cell edge toward the center. The appearance of these wrinkles is highly dynamic so that the cells cycle between the wrinkled and relatively normal morphologies. Phalloidin staining indicate that the stellate wrinkles contain dense actin structures and also that numerous filopods project vertically from the center of these cells. Consistent with the hypothesis that cdc42 induces actin polymerization in vivo, cells expressing V12-cdc42 have an increase in the amount of F-actin associated with the cytoskeleton. This is accompanied by an increase in the association of the actin binding proteins; 34 kDa bundler, ABP-120 and a-actinin with the cytoskeleton. In conclusion, human cdc42 has various effects on the Dictyostelium actin cytoskeleton consistent with a conserved role of small GTPases in control of the cytoskeleton. ----------------------------------------------------------------------------- Lysophosphatidic Acid- and Gb-Dependent Activation of Dictyostelium MAP Kinase ERK2 Paul W. Schenk, Stefan J.P. Epskamp, Menno L.W. Knetsch, Veronique Harten, Ellen L. Lagendijk, Bert van Duijn, and B. Ewa Snaar-Jagalska BBRC, in press Exogenous lysophosphatidic acid (LPA) has been shown to evoke a chemotactic response in aggregative cells of the social amoeba Dictyostelium discoideum. In this paper, we demonstrate that extracellular LPA is also able to induce activation of mitogen-activated protein (MAP) kinase DdERK2 (extracellular signal regulated kinase 2) in these cells. This activation is independent of cyclic AMP receptors, yet fully dependent on the single Gb subunit, hinting to the presence of functional heptahelical LPA receptors in a primitive eukaryote. We did not observe LPA-dependent cyclic GMP accumulation, which suggests that the pathways for LPA-induced and "classical" chemotaxis of D. discoideum cells are substantially different. ----------------------------------------------------------------------------- Role of PI3 kinase and a downstream PH domain-containing protein in controlling chemotaxis in Dictyostelium Satoru Funamoto, Kristina Milan, Ruedi Meili, and Richard A. Firtel1 J. Cell Biology, in press. ABSTRACT We show that cells lacking two Dictyostelium Class I phosphatidylinositol 3' kinases (PI3K; pi3k1/2 null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated F-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid binding sites for localization of PH-domain-containing proteins. We demonstrate that, in response to chemoattractants, three PH-domain-containing proteins do not localize to the leading edge in pi3k1/2 null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge, and have chemotaxis phenotypes that are distinct from those previously described for PKB (pkbA) null cells. Phenotypes of PhdA dominant-interfering mutations suggests that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis. ----------------------------------------------------------------------------- [End Dicty News, volume 16, number 7]