Dicty News Electronic Edition Volume 12, number 7 March 20, 1999 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" ============================== Dicty Research Lab Web Pages ============================== If your lab has a web page describing your work using Dictyostelium please be sure that it is listed on the Dicty web site at http://dicty.cmb.nwu.edu/dicty/dicty.html. If your site is not listed send the address of your web site to dicty@nwu.edu. ============== Abstracts ============== Cisplatin inhibits folicacid chemotaxis and phagocytotic functions in Dictyostelium discoideum. T.B.K.Reddy* and S.Chatterjee School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067. INDIA. *corresponding author&present address. Center for Molecular Genetics, University of California, San Diego 9500 Gilman Drive, La Jolla, CA-92093-0634 USA. Cell Biology International., in press Summary. Here we report the action of cisplatin (a widely used anticancer drug) on some basic cellular functions of this eucaryotic model system. Administration of 100 and 200 ug/ml of cisplatin [cis-diammine dichloro platinum (II)] for 1hr to growing Dictyostelium discoideum cells severely affects folic acid chemotaxis and phagocytotic functions in this organism. Following cisplatin treatment cells show a much lower uptake of FITC labelled bacteria and a reduced plaque forming ability when plated on E.coli seeded normal agar. Folic acid chemotaxis and folate deaminase activity are greatly inhibited in cisplatin treated Dictyostelium cells. SDS-PAGE analysis show a greater association of actin and myosin with cell cortex of the treated cells. These results have beeen discussed in relation to cisplatin’s known function of rising the levels of cytosolic calcium. ---------------------------------------------------------------------------- Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium Ruedi Meili, Charlene Ellsworth, Susan Lee, T. B. K. Reddy, Hui Ma, and Richard A. Firtel EMBO J., in press. SUMMARY Chemotaxis-competent cells respond to a variety of ligands by activating second messenger pathways leading to changes in the actin/myosin cytoskeleton and directed cell movement. We demonstrate that Dictyostelium Akt/PKB, a homologue of mammalian Akt/PKB, is very rapidly and transiently activated by the chemoattractant cAMP. This activation takes place through G protein- coupled chemoattractant receptors via a pathway that requires homologues of mammalian p110 phosphoinositide-3 kinase. pkbA null cells exhibit aggregation- stage defects that include aberrant chemotaxis, a failure to properly polarize in a chemoattractant gradient and aggregate at low densities. Mechanistically, we demonstrate that the PH domain of Akt/PKB fused to GFP transiently translocates to the plasma membrane in response to cAMP with kinetics similar to those of Akt/PKB kinase activation and is localized to the leading edge of chemotaxing cells in vivo. Our results indicate Akt/PKB is part of the regulatory network required for sensing and responding to the chemoattractant gradient that mediates chemotaxis and aggregation. ---------------------------------------------------------------------------- The tip Genes Act in Parallel Pathways of Early Dictyostelium Development Justin T. Stege, Michael T. Laub and William F. Loomis Center for Molecular Genetics, Department of Biology, University of California San Diego, La Jolla California 92093 Developmemtal Genetics, in press ABSTRACT Analysis of Dictyostelium strains carrying null mutations in tipA revealed a primary defect in cell sorting and the formation of tips on the developing mound. To further study the process affected in tipA- mutants, other mutants with a similar phenotype were isolated and characterized. These studies revealed three new Dictyostelium genes, tipB, tipC, and tipD. All of the tip mutants aggregate into larger than average mounds which split up and form many tips on their surfaces. Furthermore, each mutant exhibits reduced or aberrant cell sorting behavior, never makes migrating slugs, and has severely reduced fruiting body and spore production. The mRNA of each tip gene is present in vegetative cells and does not vary significantly with development. Prespore and prestalk gene expression is reduced or delayed in the tip mutants indicating cell type differentiation is dependent on the function of these genes. Developing mutant cells in chimeric mixtures with wild type cells revealed that the defects in each tip mutant behave cell autonomously. The overexpression of TipA in a tipB-background and the overexpression of TipB in a tipA- background significantly improved the morphogenesis of these mutants. These were the only situations where the expression of one tip gene could compensate for the lack of a different tip gene. Except for the tipA-/tipB- strain, double mutations in the tip genes have additive effects, causing a more severe mutant phenotype with defects earlier in development than single mutants. The tipA-/tipB- double mutant does not show additive effects and is very similar to the tipA- single mutant. Analysis of the effects of double mutations and overexpression indicates that members of this class of genes appear to act through parallel pathways of differentiation and tip formation in early Dictyostelium development. Furthermore, TipA and TipB appear to have some overlapping functions or are involvement in the same pathway. The multitipped phenotype observed in all the mutants may be a general result of perturbing early developmental events such as cell type differentiation and cell type proportioning. ---------------------------------------------------------------------------- Architectural dynamics of F-actin in eupodia suggests their role for invasive locomotion in Dictyostelium. Fukui, Y., E. de Hostos, S. Yumura, T. Kitanishi-Yumura, and S. Inoué Exp. Cell Res., in press Summary Eupodia are F-actin-containing cortical structures similar to vertebrate podosomes or invadopodia found in metastatic cells. Eupodia are rich alpha-actinin and myosin IB/D, but not a Dictyostelium homologue of talin [1]. In the present study, we localized other actin-binding proteins ABP120, cofilin, coronin and fimbrin in the eupodia, and examined the three-dimensional organization of their F-actin system by confocal microscopy and transmission electron microscopy. To examine their function, we analyzed the assembly and disassembly dynamics of the F-actin system in eupodia and its relation to lamellipodial protrusion. Actin dynamics was examined by monitoring S65T-GFP- coronin and rhodamine-actin using a real-time confocal unit and a digital microscope system. Fluorescence morphometric analysis demonstrates the presence of a precise spatiotemporal coupling between F-actin assembly in eupodia and lamellipodial protrusion. When a lamellipodium advances to invade a tight space, additional rows of eupodia are sequentially formed at the base of that lamellipodium. These results indicate that mechanical stress at the leading edge modulates the structural integrity of actin and its binding proteins, such that eupodia are formed when anchorage is needed to boost for invasive protrusion of the leading edge. ---------------------------------------------------------------------------- A unique talin homologue with a villin headpiece-like domain is required for multicellular morphogenesis in Dictyostelium M. Tsujioka, L. M. Machesky*, S. L. Cole**, K. Yahata, and K. Inouye Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. *MRC Laboratory for Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. (Present address : Department of Biochemistry, University of Birmingham, Birmingham, B15-2TT, UK) **MRC Laboratory for Molecular Cell Biology, University College London, Gower St, London WC1E 6BT, UK Current Biology, in press Abstract One important aspect of multicellular morphogenesis is the mechanical one involving the intracellular and extracellular architecture on which forces are exerted to cause changes of tissue shape. Molecules involved in the interaction between the extracellular matrix, cell membrane and cytoskeleton are therefore of central importance in morphogenesis. Talin is a large cytoskeletal protein with a modular structure consisting of an N-terminal membrane-interacting domain with sequence similarities to members of the band 4.1 family and a C-terminal region containing F-actin and vinculin-binding domains [1, 2]. It also interacts with the cytoplasmic tail of beta integrins which on the external face of the membrane bind to extracellular matrix proteins [3]. However, the possible roles of talin in multicellular morphogenesis in development remain largely unexplored. In Dictyostelium, a eukaryotic microorganism capable of multicellular morphogenesis, a talin homologue has been identified and shown to play an important role in cell-to-substrate adhesion and maintenance of normal elastic properties of the cell [4, 5, 6]. Here we describe a second talin homologue which is required for multicellular morphogenesis in the development of Dictyostelium. Unlike any other talin known so far, it contains an additional C-terminal domain homologous to the villin headpiece. ---------------------------------------------------------------------------- Interaction Mapping of a Dynein Heavy Chain: Identification of Dimerization and Intermediate-Chain Binding Domains. Andrea Habura, Irina Tikhonenko, Rex L. Chisholm, and Michael P. Koonce. Division of Molecular Medicine, Wadsworth Center, Empire State Plaza, Albany, NY 12201-0509. Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL, 60611-3072. J. Biol. Chem, in press SUMMARY. Cytoplasmic dynein is a multisubunit microtubule-based motor protein that is involved in several eukaryotic cell motilities. Two dynein heavy chains each form a motor domain that connects to a common cargo-binding tail. While this tail domain is composed of multiple polypeptides, subunit organization within this region is poorly understood. Here we present an in vitro dissection of the tail-forming region of the dynein heavy chain from Dictyostelium. Our work identifies a sequence important for dimerization and for binding the dynein intermediate chain. The core of this motif localizes within a ~ 150 aa region that is strongly conserved among other cytoplasmic dyneins. This level of conservation does not extend to the axonemal dynein heavy chains, suggesting functional differences between the two. Dimerization appears to occur through a different mechanism than the heavy chain-intermediate chain interaction. We corroborate the in vitro interactions with in vivo expression of heavy chain fragments in Dictyostelium. Fragments lacking the interaction domain express well, without an obvious phenotype. On the other hand, the region crucial for both interactions appears to be lethal when overexpressed. ---------------------------------------------------------------------------- Kinetic Analysis of Dictyostelium discoideum Myosin Motor Domains with Glycine-to-Alanine Mutations in the Reactive Thiol Region Renu Batra, Michael A. Geeves§, and Dietmar J. Manstein Max-Planck-Institut für Medizinische Forschung, Jahnstr. 29, D-69120 Heidelberg, Germany and Max-Planck-Institut für Molekulare Physiologie, Postfach 102664, D-44026 Dortmund, Germany § Current address: Department of Biosciences, University of Kent, Canterbury, Kent, UK. Biochemistry, in press ABSTRACT Three conserved glycine residues in the reactive thiol region of Dictyostelium discoideum myosin II were replaced by alanine residues. The resulting mutants G680A, G684A and G691A were expressed in the soluble myosin head fragment M761-2R [Anson, M., Geeves, M.A., Kurzawa, S.E. and Manstein, D.J. (1996) EMBO J. 15, 6069-6074] and characterized using transient kinetic methods. Mutant G691A shows no major alterations except for a marked increase in basal Mg2+-ATPase activity. Phosphate release seems to be facilitated by this mutation and the addition of actin to G691A stimulates ATP turnover not more than 3-fold. In comparison to M761-2R, mutant constructs G691A and G684A show a 4-fold reduction in the rate of the ATP cleavage step. Most other changes in the kinetic properties of G684A are small (~2-fold). In contrast, substitution of G680 by an alanine residue leads to large changes in nucleotide binding. Compared to M761-2R, rates of nucleotide binding are 20-30-fold slower and the affinity for mantADP is approximately 10-fold increased due to a 200- fold reduction in dissociation rate constant of mantADP. The ATP-induced dissociation of actin from the acto.G680A complex is normal but coupling between binding of actin and ADP appears disrupted as G680A and acto.G680A show the same affinity for ADP. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 7]