Dicty News Electronic Edition Volume 12, number 13 June 26, 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" =================================== Postdoctoral Position Available =================================== A postdoctoral associate position is available beginning August 1 in the Singleton lab. The project centers around a recently identified phosphorelay signaling system that controls cAMP dependent protein Kinase A activity. DHKC is a sensor histidine kinase that we propose transfers phosphates down the phosphorelay in response to endogenous ammonia production. The result is a decrease in the intracellular cAMP concentration brought about by an activation of the cAMP phosphodiesterase activity of the postulated downstream response regulator REGA. The resulting alteration of PKA activity is hypothesized to contribute to the maintenance of appropriate timing of aggregation through finger formation and to making the appropriate choice between slug formation versus culmination. DHKC signaling, stimulated by ammonia, is also postulated to be required for activating the spore germination pathway. Another sensor kinase, DHKB, is proposed to act as a phosphatase within spores to inhibit the transfer of phosphates to REGA. In doing so in response to discadenine, an autoinhibitor of germination, DHKB signaling prevents premature activation of the germination pathway. More information about the project, including an abstact and specific aims, about the background leading up to the project, and about other activities in the lab can be obtained at the following web site: http://www.biocourses.cas.vanderbilt.edu/singleton/singletonlab/index.html. If you are interested in this position, please send via email your CV (charles.k.singleton@vanderbilt.edu). ============= Abstracts ============= [Editor's note: the following abstract appeared in the last issue with errors: Betas had been inappropriately replaced by alphas. The correct abstract appears in its entirety below.] A Serpentine Receptor-dependent, Gbeta- and Ca2+ Influx-independent Pathway Regulates Mitogen-activated Protein Kinase ERK2 in Dictyostelium Paul W. Schenk, Thomas Nebl, Paul R. Fisher and B. Ewa Snaar-Jagalska Section of Cell Biology, Institute of Molecular Plant Sciences, Leiden University, P.O. Box 9505, 2300 RA Leiden, The Netherlands and School of Microbiology, La Trobe University, Bundoora, VIC 3083, Australia Biochem. Biophys. Res. Commun., in press Abstract Ca2+ influx and mitogen-activated protein (MAP) kinase activation are important phenomena in signal transduction, which are often interconnected. We investigated whether serpentine receptor-dependent, G beta-independent activation of MAP kinase ERK2 by chemoattractant cyclic AMP (cAMP) is mediated by Ca2+ influx in the social amoeba Dictyostelium discoideum. We generated a D. discoideum double mutant, which harbours a temperature- sensitive G beta subunit and expresses the apoaequorin protein. Utilizing this mutant, we demonstrate that cAMP induced Ca2+ influx into intact D. discoideum cells can be blocked completely at both the permissive and the restrictive temperature, by using either gadolinium ions or Ruthenium Red. Under the same experimental conditions, these substances do not abolish cAMP stimulation of ERK2 at either temperature. We conclude that there is a G beta- and Ca2+ influx-independent pathway for the receptor-dependent activation of MAP kinase ERK2 in Dictyostelium discoideum. ---------------------------------------------------------------------------- Co-loss of Profilin I, II and Cofilin with Actin from Maturing Phagosomes in Dictyostelium discoideum Aidong Yuan and Catherine P. Chia School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Neraska 68588-0118, USA Protoplasma, in press ABSTRACT: Although it is known that actin polymerizes rapidly at the plasma membrane during the ingestion phase of phagocytosis, not yet fully understood are the mechanisms by which actin is recruited to form a phagocytic cup and then subsequently dissociated from the phagosome. The aim of this study was to identify actin-binding proteins that mediated actin filament dynamics during phagosome formation and processing. We report that profilins I and II, which promote filament assembly, and cofilin, which stimulates filament disassembly, were constituents of phagosomes isolated from Dictyostelium discoideum fed latex beads, and associated with actin. Biochemical analyses detected one isoform only of cofilin, which bound actin in unstimulated cells as well as in cells engaged in phagocytosis, subjected to various stress treatments, and through development. At membranes of young phagosomes, profilins I and II co-localized with monomeric actin labeled with fluorescent DNase, and cofilin co-localized with filamentous actin labeled with rhodamine phalloidin. Both immunocytochemical and quantitative immunoblotting data indicated that the kinetic loss of profilins I, II and cofilin of maturing phagosomes closely followed the falling levels of actin associated with the vesicles. As evidence of vesicle processing, D. discoideum crystal protein (an esterase) was recruited rapidly to phagosomes and its levels increased while those of actin, profilins I, II and cofilin jointly decreased. The localization data and concurrent losses of profilins and cofilin with actin from phagosomes are consistent with the roles of these actin-binding proteins in filament dynamics, and indicated that they were involved in regulating the assembly and disassembly of the actin coat of phagosomes. ---------------------------------------------------------------------------- Three actin cross-linking proteins, the 34 kDa actin-bundling protein, a-actinin, and ABP-120, have both unique and redundant roles in the growth and development of Dictyostelium Francisco Rivero(1), Ruth Furukawa(2), Marcus Fechheimer(2) and Angelika A. Noegel(1) 1 Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany 2 Department of Cellular Biology, University of Georgia, Athens, Georgia 30602 Journal of Cell Science, in press. Abstract The contribution of three actin cross-linking proteins, a-actinin (aA), ABP-120, and the 34 kDa actin-bundling protein to cellular functions has been studied in three single mutant (aA-, 120-, and 34-) and three double mutant (aA-/120-, 34-/aA-, 34-/120-) strains of Dictyostelium generated by homologous recombination. Strains aA-/120- and 34-/aA- exhibited a reduced rate of pinocytosis, grew to lower saturation densities, and produced small cells in shaking cultures. All strains grew normally in bacterial suspensions and on agar plates with a bacterial lawn. Slow growth under conditions of reduced temperature and increased osmolarity was observed in single mutants 34- and aA-, respectively, as well as in some of the double mutant strains. Motility, chemotaxis, and development were largely unaltered in 34-/aA- and 34-/120- cells. However, 34-/aA- cells showed enhanced aggregation when starved in suspension. Moreover, morphogenesis was impaired in both double mutant strains and fruiting bodies of aberrant morphology were observed. These defects were reverted by re-expression of one of the lacking cross-linking proteins. The additive and synthetic phenotypes of these mutations indicate that actin cross-linking proteins serve both unique and overlapping functions in the actin cytoskeleton. ---------------------------------------------------------------------------- The Dictyostelium genome project: an invitation to species hopping Robert R. Kay1 & Jeffrey G. Williams2 1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK CB2 2QH. 2 Wellcome Trust Building, University of Dundee, Dundee, UK DD1 5EH. TIGS, in press Summary Dictyostelium allows a number of general problems of eukaryotic biology to be addressed using molecular genetic tools more normally associated with yeast. The genome project, now underway, marks an important increase in the attractiveness of Dictyostelium as an experimental organism and will invite increased ‘species hopping’ by experimenters. We provide a brief guide to the problems that are being addressed in Dictyostelium, to the genome project itself and to the molecular genetic tools available for its exploitation. ---------------------------------------------------------------------------- Regulation of the ribonucleotide reductase small subunit gene by DNA- damaging agents in Dictyostelium discoideum GAUDET, P. (1,3) and TSANG, A. (1,2,3) (1) Department of Chemistry and Biochemistry, (2) Department of Biology, and (3) Centre for Structural and Functional Genomics, Concordia University, 1455 de Maisonneuve Blvd. W., Montreal, Quebec, H3G 1M8, Canada Nucleic Acids Research, in press ABSTRACT In E. coli, yeast, and mammalian cells, the genes encoding ribonucleotide reductase, an essential enzyme for de novo DNA synthesis, are up-regulated in response to DNA damaging agents. We have examined the response of the rnrB gene, encoding the small subunit of ribonucleotide reductase in Dictyostelium discoideum, to DNA damaging agents. We show here that the accumulation of rnrB transcript is increased in response to methyl methane sulfonate, 4-nitroquinoline-1-oxide and irradiation with UV-light, but not to the ribonucleotide reductase inhibitor hydroxyurea. This response is rapid, transient, and independent of protein synthesis. Moreover, cells from different developmental stages are able to respond to the drug in a similar fashion, regardless of the basal level of expression of the rnrB gene. We have defined the cis-acting elements of the rnrB promoter required for the response to methyl methane sulfonate and 4-nitroquinoline-1-oxide by deletion analysis. Our results indicate that there is one element, named box C, that can confer response to both drugs. Two other boxes, box A and box D, specifically conferred response to methyl methane sulfonate and 4-nitroquinoline-1-oxide, respectively. ---------------------------------------------------------------------------- Hyperosmotic Stress-induced Reorganization of Actin Bundles in Dictyostelium Cells Overexpressing Cofilin. Hiroyuki Aizawa1, Maiko Katadae1,$, Mikako Maruya1, Masazumi Sameshima1, Kimiko Murakami-Murofushi$, and Ichiro Yahara1,*. 1Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113, Japan, and $Department of Biology, Faculty of Science, Ochanomizu University, Bunkyo-ku, Tokyo 112, Japan. Genes to Cells, in press Abstract Background: Cofilin is a low-molecular weight actin-modulating protein, which binds to, severs, and depolymerizes actin filaments in vitro. Aip1, an actin-interacting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature-sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae. Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum. Roles of cofilin and Aip1 in the resistance are not known. Results: In response to hyperosmotic stress, D. discoideum cells round up. This stress-induced morphological change involves redistribution of cofilin together with actin filaments into cortical contractile portions of the cells, followed by their contraction. Overexpression of cofilin increases and thickens cortical actin bundles in cells. The bundles became tight and reorganized into a ring-shaped structure in response to hyperosmotic stress. The ring structure of actin bundles had two characteristic bands across them; bright and dark bands which were heavily stained and were not stained with phalloidin. In the bundles, straight filaments with a diameter of 5.3-nm were aligned parallel by cross-bridge structures. In cells lacking myosin-II heavy chain, the bundles, which were induced by overexpression of cofilin, shortened and became straight upon hyperosmotic stress, forming a polygonal structure. D. discoideum Aip1/Wrp2 enhanced severing actin filaments by cofilin in vitro and colocalized with cofilin in cells including those overexpressing cofilin before and after exposure to hyperosmotic stress. Conclusions: Cofilin plays a pivotal role together with Aip1/Wrp2 in reorganization of actin architectures into bundles that contract in a myosin-II-independent manner, in response to hyperosmotic stress. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 13]