Dicty News Electronic Edition Volume 19, number 3 August 3, 2002 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. ====================== Position Available ====================== Postdoctoral position in the Wellcome Trust Biocentre at the University of Dundee A postdoctoral position is available in Dr. Inke Nthke s laboratory to join a team studying the function of the Adenomatous Polyposis Coli tumour suppressor protein (APC). The project will involve using Dictyostelium Discoideum to investigate the role of APC in cytoskeletal dynamics and organisation. The general aim of work in the laboratory is to determine how the diverse functions of APC relate to its role in cancer and how these functions are co-ordinated and regulated. Experimental approaches include general cell- and molecular biology techniques combined with high-resolution fluorescence microscopy and use of Dictyostelium as a model organism. The candidate will join a small, dynamic, highly interactive research team with active collaborations worldwide. A proven research ability and motivation are required and experience in working with Dictyostelium is requisite. Experience in general cell biology and molecular biology is essential. The position is available immediately for 2 years in the first instance. For informal enquiries, please contact Dr. Inke S. Nthke (+ 44 1382 345821; e-mail: i.s.nathke@dundee.ac.uk). Applications should be directed to: Janette Cordiner, School Administrator, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH; j.m.cordiner@dundee.ac.uk. Please cite reference SC/763/1 on the application. The Wellcome Trust Biocentre is part of the internationally renowned WTB/MSI complex at the University of Dundee. The complex provides an extremely interactive research environment and houses nearly 400 research staff. Further information about research in the WTB/MSI complex can be obtained at http://www.dundee.ac.uk/biocentre/. ============= Abstracts ============= Differential localization in cells of myosin II heavy chain kinases during cytokinesis and polarized migration. Wenchuan Liang, Lucila Licate, Hans Warrick, James Spudich, and Thomas Egelhoff BMC Cell Biology 2002 3: 19 Abstract Background Cortical myosin-II filaments in Dictyostelium discoideum display enrichment in the posterior of the cell during cell migration, and in the cleavage furrow during cytokinesis. Filament assembly in turn is regulated by phosphorylation in the tail region of the myosin heavy chain (MHC). Early studies have revealed one enzyme, MHCK-A, which participates in filament assembly control, and two other structurally related enzymes, MHCK-B and -C. In this report we evaluate the biochemical properties of MHCK-C, and using fluorescence microscopy in living cells we examine the localization of GFP-labeled MHCK-A, -B, and -C in relation to GFP-myosin-II localization. Results Biochemical analysis indicates that MHCK-C can phosphorylate MHC with concomitant disassembly of myosin II filaments. In living cells, GFP-MHCK-A displayed frequent enrichment in the anterior of polarized migrating cells, and in the polar region but not the furrow during cytokinesis. GFP-MHCK-B generally displayed a homogeneous distribution. In migrating cells GFP-MHCK-C displayed posterior enrichment similar to that of myosin II, but did not localize with myosin II to the furrow during the early stage of cytokinesis. At the late stage of cytokinesis, GFP-MHCK-C became strongly enriched in the cleavage furrow, remaining there through completion of division. Conclusion MHCK-A, -B, and -C display distinct cellular localization patterns suggesting different cellular functions and regulation for each MHCK isoform. The strong localization of MHCK-C to the cleavage furrow in the late stages of cell division may reflect a mechanism by which the cell regulates the progressive removal of myosin II as furrowing progresses. ----------------------------------------------------------------------------- cAMP dependent protein kinase regulates Polysphondylium pallidum development Satoru Funamoto1,3, Christophe Anjard2,4, Wolfgang Nellen2 and Hiroshi Ochiai1,5 1Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan; 2Department of Genetics, University of Kassel, Heinrich-Plett-Str. 40 D-34132 Kassel, Germany; 3 Present address: Department of Neuropathology, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan; 4Present address: Center for Molecular Genetics, Department of Biology University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0634 Author for correspondence. E-mail: hochiai@sci.hokudai.ac.jp Differentiation, in press. Abstract In eukaryotic cells the universal second messenger cAMP regulates various aspects of development and differentiation. The primary target for cAMP is the regulatory subunit of cAMP-dependent protein kinase A (PKA), which, upon cAMP binding, dissociates from the catalytic subunit and thus activates it. In the soil amoeba Dictyostelium discoideum the function of PKA in growth, development and cell differentiation has been thoroughly investigated and substantial information is available. To obtain a more general view we investigated the influence of PKA on development of the related species Polysphondylium pallidum. Cells were transformed to overexpress either a dominant negative mutant of the regulatory subunit (Rm) from Dictyostelium that cannot bind cAMP, or the catalytic subunit (PKA-C) from Dictyostelium. Cells overexpressing Rm rarely aggregated and the few multicellular structures developed slowly into very small fruiting bodies without branching of secondary sorogens, the prominent feature of Polysphondylium. Few round spores with reduced viability were formed. When mixed with wild type cells and allowed to develop, the Rm cells were randomly distributed in aggregation streams, but were later found in the posterior region of the culminating slug or were left behind on the surface of the substratum. The PKA-C overexpressing cells exhibited precocious development and formed more aggregates of smaller size. Moreover, expression of PKA-C under the control of the prestalk specific ecmB promoter of Dictyostelium, lead to protrusions from aggregation streams. We conclude that Dictyostelium PKA subunits introduced into Polysphondylium cells are functional as signal components, indicating that a biochemically similar PKA mechanism works in Polysphondylium. ----------------------------------------------------------------------------- Element analysis of the Polysphondylium pallidum gp64 promoter Naohisa Takaokaa, Masashi Fukuzawab, Atsushi Katoa, Tamao Saitoa and Hiroshi Ochiaia, c a. Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810 Japan; b. Wellcome Trust Building, Department of Anatomy and Physiology, University of Dundee, Dow Street, Dundee, DD1 4HN, UK Author for corresponding. E-mail: hochiai@sci.hokudai.ac.jp BBA, in press. Abstract gp64 mRNA in Polysphondylium pallidum is expressed extensively during vegetative growth, and begins to rapidly decrease at the onset of development. To examine this unique regulation, 5 deletion analysis of the gp64 promoter was undertaken, and two growth-phase activated elements have been found: a food-dependent, upstream regulatory region (FUR, -222 to 170) and a vegetatively activated, downstream region (VAD, -110 to 63). Here we concentrate our analysis on an A1 and A2 sequences in the FUR region: A1 consists of a GATTTTTTTA sequence called a corresponding sequence and A2 consists of the direct repeat TTTGTTGTG. The cells carrying a combined construct of A1 and A2 acted synergistically in a reporter activity. A point mutation analysis in A1 indicates that a G residue is required for the activation of A1. From analyses of promoter regulation in a liquid or a solid medium, the promoter activity of the cells fed on bacteria in A-medium (axenic medium for Polysphondylium) or grown in A-medium alone was only one-fourth of that of the cells fed on bacteria. ----------------------------------------------------------------------------- A novel Dictyostelium Cdk8 is required for aggregation, but dispensable for growth Kosuke Takeda, Tamao Saito, Hiroshi Ochiai* Division of Biological Sciences, Graduate School of Science, Hokkaido University Sapporo, Hokkaido 060-0810, Japan Author for correspondence. E-mail: hochiai@sci.hokudai.ac.jp Develop. Growth Differ., in press Abstract When Dictyostelium cells starve, they express genes necessary for aggregation. Using insertional mutagenesis, we have isolated a mutant that does not aggregate upon starvation and that forms small plaques on bacterial lawns, thus indicating slow growth. The sequencing of the mutated locus showed a strong similarity to the catalytic domain of cdc2-related kinase genes. Phylogenetic analysis further indicated that the amino acid sequence was more close to cyclin-dependent kinase 8 than to those of other cyclin- dependent kinases. Thus we designated this gene as Ddcdk8. The Ddcdk8-null cells do not aggregate and grow somewhat more slowly than parental cells when being shaken in axenic medium or laid on bacterial plates. To confirm whether these defective phenotypes were caused by disruption of this gene, the Ddcdk8-null cells were complemented with DdCdk8 protein expressed from an endogenous promoter but not an actin promoter, and when the complemented cells were then allowed to grow on a bacterial lawn, they began to aggregate as the food supply was depleted, and finally became fruiting bodies. The results suggest that properly regulated DdCdk8 activity is essential for aggregation. Since when starved, Ddcdk8-null cells do not express at all the acaA transcripts required for aggregation, we deduce that Ddcdk8 is epistatic for acaA expression, indicating that the Ddcdk8 products may regulate expression of acaA and/or other genes. ----------------------------------------------------------------------------- Cell adhesion during the migratory slug stage of Dictyostelium discoideum. *Vivienne M. Bowers-Morrow, *Sinan O. Ali, and +Keith L. Williams *Department of Biological Sciences, Macquarie University, Sydney, NSW, 2109 AUSTRALIA +Proteome Systems Ltd, North Ryde, Sydney, NSW, 2113 AUSTRALIA All correspondence to Vivienne Bowers-Morrow, email: vmorrow@rna.bio.mq.edu.au CELL BIOLOGY INTERNATIONAL, in press ABSTRACT Prespore-specific Antigen (PsA) is selectively expressed on the surface of prespore cells at the multicellular migratory slug stage of Dictyostelium discoideum development. It is a developmentally regulated glycoprotein which is anchored to the cell membrane through a glycosyl phosphatidylinositol (GPI) anchor. We present the results of an in vitro immunological investigation of the hypothesis that PsA functions as a cell adhesion molecule (CAM), and of a ligand binding assay indicating that PsA has cell membrane binding partner(s). This is the first evidence to implicate a direct role for a putative CAM in cell-cell adhesion during the multicellular migratory slug stage of D. discoideum development. Cell-cell adhesion assays were carried out in the presence or absence of the monoclonal antibody (mAb) MUD1 which has a single antigenic determinant: a peptide epitope on PsA. These assays showed specific inhibition of cell-cell adhesion by MUD1. Further, it was found that a purified recombinant form of PsA (rPsA), can neutralize the inhibitory effect of MUD1; the inhibitory effect on cell-cell adhesion is primarily due to the blocking of PsA by the mAb. The resistance of aggregates to dissociation in the presence of 10mM EDTA (ethylenediamintetraacetic acid) indicates that PsA mediates EDTA-stable cell-cell contacts, and that PsA-mediated cell adhesion is likely to be independent of divalent cations such as Ca 2+ or Mg 2+. ----------------------------------------------------------------------------- The three-dimensional model of Dictyostelium discoideum racE based on the human rhoA-GDP crystal structure Madhavi Agarwala, Donald J. Nelsonb and Denis A. Larochellea a Department of Biology, Clark University, 950 Main Street, Worcester, MA 01610, USA; b Department of Chemistry, Clark University, 950 Main Street, Worcester, MA 01610, USA Corresponding author. Tel.: +1-508-793-7664; fax: +1-508-793-8861; email: magarwal@clarku.edu Journal of Molecular Graphics and Modelling 21, 3-18 Abstract The three-dimensional structure of racE was modeled using several homologous small G proteins, and the best model obtained using the human rhoA as modeling template is reported. The three-dimensional fold of the racE model is remarkably similar to the cellular form of human ras p21 crystal structure. Its secondary structure consists of six -helices, six -strands and three 310 helices. The model retains its secondary structure after a 300 K, 300 ps molecular dynamics (MD) simulation. Important domains of the protein include its effector loop (residues 3446), the insertion domain (residues 121136), and the polybasic motif (between 210 and 220) not modeled in the current structure. The effector loop is inherently flexible and the structure docked with GDP exhibits the effector loop moving significantly closer to the nucleotide binding pocket, forming a tighter complex with the bound GDP. The mobility of the effector loop is conferred by a single residue `hinge' point at residue 34Asp, also allowing the Switch I region, immediately preceding the effector loop, to be equally mobile. In comparison, the Switch II region shows average mobility. The insertion domain is highly flexible, with the insertion taking the form of a helical domain, with several charged residues forming a complex charged interface over the entire insertion region. While the GDP moiety is loosely held in the active site, the metal cation is extensively co-ordinated. The critical residue 38Thr exhibits high mobility, and is seen interacting directly with the metal ion at a distance of 2.64 , and indirectly via an intervening water molecule. 64Gln, a key residue involved in GTP hydrolysis in ras, is seen facing the -phosphate group and the metal ion. Certain residues (i.e. 51Asn, 38Thr and 65Glu) exhibit unique characteristics and these residues, together with 158Val, may play important roles in the maintenance of the protein's integrity and function. There is strong consensus of secondary structural elements between models generated using various templates, such as h-rac1, h-rhoA and h-cdc42 bound to RhoGDI, all sharing only 5055% sequence identity with racE, which suggests that this model is in all probability an accurate prediction of the true tertiary structure of racE. ----------------------------------------------------------------------------- A novel cGMP-signaling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium Leonard Bosgraaf#, Henk Russcher#, Janet L. Smith^, Deborah Wessels*, David R. Soll* and Peter J.M. Van Haastert# # Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands. ^ Boston Biomedical Research Institute, 64 Grove Street, Watertown, Massachusetts 02472-2829, U.S.A. * W.M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, U.S.A. EMBO J, in press Chemotactic stimulation of Dictyostelium cells results in a transient increase in cGMP levels, and transient phosphorylation of myosin II heavy and regulatory light chains. In Dictyostelium, two guanylyl cyclases and four candidate cGMP-binding proteins (GbpA-D) are implicated in cGMP signaling. GbpA and GbpB are homologous proteins with a Zn2+-hydrolase domain. A double gbpA/gbpB gene disruption leads to a reduction of cGMP- phosphodiesterase activity and a ten-fold increase of basal and stimulated cGMP levels. Chemotaxis in gbpA-B- cells is associated with increased myosin II phosphorylation compared to wild type cells; formation of lateral pseudopodia is suppressed resulting in enhanced chemotaxis. GbpC is homologous to GbpD, and contains Ras, MAPKKK and Ras-GEF domains. Inactivation of the gbp genes indicates that only GbpC harbours high affinity cGMP-binding activity. Myosin phosphorylation, assembly of myosin in the cytoskeleton as well as chemotaxis are severely impaired in mutants lacking GbpC and D, or mutants lacking both guanylyl cyclases. Thus a novel cGMP-signaling cascade is critical for chemotaxis in Dictyostelium, and plays a major role in myosin II regulation during this process. ----------------------------------------------------------------------------- An observation of the initial stage towards a symbiotic relationship Masahiko Todoriki 1, Shinya Oki 1, Shin-Ichi Matsuyama1, Elizabeth P. Ko-Mitamura1, Itaru Urabe 1 and Tetsuya Yomo 1,2,3* 1Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita City, 2PRESTO, JST, 2-1 Yamadaoka, Suita City, Osaka 565-0871, Japan and 3Department of Pure and Applied Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan. *Corresponding author. Mailing address: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita City, Osaka 565-0871, Japan. E-mail: yomo@bio.eng.osaka-u.ac.jp; Tel. :+81-6-6879-7427; Fax: +81-6-6879-7428 BioSystems 65 (2002) 105?112 Abstract Two well-characterized and phylogenetically different species, Escherichia coli and Dictyostelium discoideum, were used as the model organisms. When the two species were mixed and allowed to grow on minimal agar plates at 22?C, the two species remarkably achieved a state of coexistence at about two weeks. In addition, the emerged colonies housing the coexisting species have a mucoidal nature that was not observed from its origin. Moreover, the state of coexistence was confirmed to be stable, and so does the mucoidal nature of the emerged colonies. Comparing with the pure E.coli origin, mucoidal colony showed a significant increase in higher molecular weight extracellular components, with polysaccharide as the major constituent. Qualitative analysis of the monosaccharide contents in the extracellular components of the mucoidal colony revealed not only a significant increase in the glucose content, but also significant amount of additional xylose and galactose. The system permits the initial stages of the development of relationship between two species be captured within a short period of time. This feature, together with being simple and reproducible on laboratory conditions, hence provides a new model system for the study of symbiosis, especially when initial stages are concerned. ----------------------------------------------------------------------------- [End Dicty News, volume 19, number 3]