Dicty News Electronic Edition Volume 18, number 11 June 15, 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 ====================== Research Assistant 1A, Department of Biological Sciences, Imperial College of Science, Technology and Medicine Salary range 19,681 - 21,503 plus 2,134 London Allowance per annum Our Cell Dynamics group is located in the Sir Alexander Fleming Building, South Kensington campus which offers excellent research facilities in modern laboratories, including state-of the-art instrumentation. Applications are invited for the post of Research Assistant 1A (postdoctoral level) in our young and dynamic group to study the molecular and cellular functions of a myosin I in actin polymerisation, cell cortex dynamics and motility. Our aim is to understand the cellular and molecular mechanims of cell motility, and particularly the function of actin- dependent molecular motors. We previously demonstrated that MyoK, a class I myosin from Dictyostelium, plays an important role in the maintenance of cell cortex tension, motility and phagocytosis. As the components of the complex machineries involved are evolutionarily conserved, their molecular and cellular dissection in Dictyostelium is directly relevant to unravel their functional importance in higher organisms. A description of this and other projects of the group is available at: http://www.bio.ic.ac.uk/research/tps/ The candidate should be motivated and enthusiastic about this area of research. Proficiency in a variety of techniques, including cell culture, single cell assays, genetic screenings, recombinant protein expression, biophysical and enzymatic assays, and in a range of standard molecular biology and biochemistry methods will be a determining asset for the successful candidate. For further details, please contact Dr. Thierry Soldati, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Exhibition Road, London, SW7 2AZ. E-mail t.soldati@ic.ac.uk. To apply, please, send a full CV, a description of current research and interests, and the name of two referees at the same address. Closing date July 15. The work is supported by a BBSRC Research Grant for 3 years. The position is available immediately or upon other agreement. The College is committed to equality of opportunity. ============= Abstracts ============= Visualising PI3 kinase mediated cell-cell signalling during Dictyostelium development Dirk Dormann, Gerti Weijer, Carole A. Parent, Peter N. Devreotes and Cornelis J. Weijer Current Biology, in press Background. Starving amoebae of Dictyostelium discoideum communicate by relaying extracellular cAMP signals, which direct chemotactic movement, resulting in the aggregation of thousands of cells into multicellular aggregates. Both cAMP relay and chemotaxis require the activation of PI3 kinase signalling. The spatio-temporal dynamics of PI3 kinase signalling can be followed in individual cells via the cAMP induced membrane recruitment of a GFP tagged PH-domain containing protein, CRAC, which is required for the activation of adenylylcyclase. Results. We show that polarised periodic CRAC-GFP translocation occurs during the aggregation and mound stages of development in response to periodic cAMP signals. The duration of CRAC translocation to the membrane is determined by the duration of the rising phase of the cAMP signal. The system shows fast adaptation and responds to the rate of change of the extracellular cAMP concentration. When the cells are in close contact it takes 10 seconds for the signal to propagate from one cell to the next. In slugs all cells show a permanent polarised PI3 kinase signalling in their leading edge, which is dependent on cell-cell contact. Conclusions. Measuring the redistribution of GFP tagged CRAC has enabled us to study the dynamics of PI3 kinase mediated cell-cell communication at the individual cell level in the multicellular stages of Dictyostelium development. This approach should also be useful to study the interactions between cell-cell signalling, cell polarisation and movement in the development other organisms. ----------------------------------------------------------------------------- Dictyostelium mobile elements: strategies to amplify in a compact genome Thomas Winckler(1), Theodor Dingermann(1) and Gernot Glckner(2) (1) Institut fuer Pharmazeutische Biologie, Universitaett Frankfurt/M. (Biozentrum), Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany and (2) IMB Jena, Department of Genome Analysis, Beutenbergstrasse 11, D-07745 Jena, Germany Review, Cell. Mol. Life Sci., in press Dictyostelium discoideum is a eukaryotic microorganism that is attractive for the study of fundamental biological phenomena such as cell-cell communication, formation of multicellularity, cell differentiation, and morphogenesis. Large-scale sequencing of the D. discoideum genome has provided new insights into evolutionary strategies evolved by transposable elements (TEs) to settle in compact microbial genomes and to maintain active populations over evolutionary time. The high gene density (about 1 gene/2.6 kb) of the D. discoideum genome leaves limited space for selfish molecular invaders to move and amplify without causing deleterious mutations that eradicate their host. Targeting of tRNA gene loci appears to be a generally successful strategy for TEs residing in compact genomes to insert away from coding regions. In D. discoideum tRNA gene-targeted retrotransposition has evolved independently at least three times by both non-long terminal repeat (LTR) retrotransposons and retrovirus-like LTR retrotransposons. Unlike the nonspecifically inserting D. discoideum TEs, which have a strong tendency to insert into preexisting TE copies and form large and complex clusters near the ends of chromosomes, the tRNA gene-targeted retrotransposons have managed to occupy 75% of the tRNA gene loci spread on chromosome 2 and represent 80% of the TEs recognized on the assembled central 6.5 Mb part of chromosome 2. In this review we update the available information about D. discoideum TEs which emerges both from previous work and current large-scale genome sequencing with special emphasis on the fact that tRNA genes are principle determinants of retrotransposon insertions into the D. discoideum genome. ----------------------------------------------------------------------------- Ga-Mediated Inhibition of Developmental Signal-Response Joseph A. Brzostowski, Cynthia Johnson + and Alan R. Kimmel Laboratory of Cellular and Developmental Biology, NIDDK (Bldg. 50/3351) National Institutes of Health, Bethesda, MD 20892-8028. +The University of Texas Southwestern Medical Center Dallas, TX 75390. ark@helix.nih.gov Current Biology, in press. SUMMARY Background: Seven-transmembrane receptor (7-TMR)-G protein networks are molecular sensors of extracellular signals in all eukarya. These pathways cycle through activated (sensitized) and inhibited (de-sensitized) states, and, while many of the molecular components for signal activation have been described, inhibitory mechanisms are not well characterized. In Dictyostelium, 7-TM cAMP receptors direct chemotaxis and development, but also regulate the periodic synthesis of their own ligand, the chemoattractant/morphogen cAMP. We now demonstrate through loss-of-function, gain-of -function studies that the novel heterotrimeric Ga9 protein subunit regulates an inhibitory pathway during early Dictyostelium development for cAMP signal-response. Results: ga9-null cells form more cAMP signaling centers, are more resistant to compounds that inhibit cAMP signaling, and complete aggregation sooner and at lower cell densities than wild-type. These phentoypes are consistent with the loss of an inhibitory signaling pathway during development of ga9-null cells. Cells expressing constitutively activated Ga9 are defective in cAMP signal center formation and development at low cell density and display an increased sensitivity to cAMP signal inhibition that is characteristic of enhanced suppression of cAMP signal-response. Finally, we demonstrate that ga9-null cells, which have been co-developed with a majority of wild-type cells, primarily establish cAMP signaling centers and are able to non- autonomously direct wild-type cells to adopt a ga9-null-like phenotype. Conclusions: We suggest that Ga9 functions in an inhibitory-feedback pathway that regulates cAMP signal center formation and propagation. Ga9 may be part of the mechanism that regulates lateral signal inhibition or that modulates receptor de-sensitization. ----------------------------------------------------------------------------- Cell polarity and locomotion, as well as endocytosis, depend on NSF. Chris R.L. Thompson and Mark S. Bretscher MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England. Development, in press NEM-sensitive factor (NSF) is an essential protein required during membrane transport. We replaced part of the endogenous D. discoideum NSF gene (nsfA) by a pcr-mutagenised library and isolated 11 mutants temperature-sensitive (ts) for growth. Two of these have been studied in detail. As expected, both are ts for FITC-dextran uptake by macropinocytosis, for internalising their surface membrane (monitored with FM1-43) and for phagocytosis. However, after 10-20 minutes at 28C, they round up and cease to chemotax, move or cap ConA receptors. They fully recover when returned to 22C. These cells carry out a normal "cringe" reaction in response to cAMP, indicating that the actin cytoskeleton and this signal transduction pathway are still functional at 28C . The behaviour of these mutants shows that NSF-catalysed processes are required not only for the different endocytic cycles but also for the maintenance of cell polarity. As cell locomotion depends on a cell having a polarity, the mutants stop moving at high temperature. A tentative model is proposed to explain the surprising link between membrane recycling and cell polarity revealed here. ----------------------------------------------------------------------------- [End Dicty News, volume 18, number 11]