Dicty News Electronic Edition Volume 11, number 2 August 1, 1998 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" ============== Announcement ============== Jeff Williams has moved. His new address is: The Department of Anatomy and Physiology University of Dundee MSI/WTB Complex Dow Street DUNDEE DD1 5EH tel 01382 345828 e-mail: jgwilliams@bad.dundee.ac.uk =========== Abstracts =========== THE HISTIDINE KINASE dhkC REGULATES THE CHOICE BETWEEN MIGRATING SLUGS AND TERMINAL DIFFERENTIATION IN DICTYOSTELIUM DISCOIDEUM Charles K. Singleton1, Michael J. Zinda, Brook Mykytka, and Ping Yang Department of Molecular Biology, Box 1820 Station B, Vanderbilt University, Nashville TN 37235 Develop. Biol., in press. Abstract An early decision that a newly formed aggregate of Dictyostelium cells must make is whether to form a migrating slug or to proceed through culmination, the process of forming the mature fruiting body. The choice between these alternative morphological pathways is influenced by external and internal cues. dhkC was identified as a potential hybrid sensor kinase possessing domains homologous to the histidine kinase and receiver motifs of two- component signaling systems. Null strains of dhkC show a rapidly developing phenotype for aggregation through finger formation, and culmination commences immediately thereafter and proceeds at a normal rate to generate typical fruiting bodies. Ammonia, an endogenous regulator of the slug versus culmination choice, results in a prolonged slug stage for dhkC + strains while the dhkC - strain bypasses the slug stage in the presence or absence of ammonia. Conversely, expression in wild type cells of a modified DHKC protein composed of only the histidine kinase domain results in normal timing through early aggregation, but subsequent development is significantly delayed. The resulting fingers, once formed, readily convert to slugs that do not undergo culmination but instead migrate until their energy sources are depleted. The slugger phenotype is dependent on the presence of a functional response regulator REGA, and it is rescued by exogenously supplied cAMP. Together, the results indicate that DHKC contributes to the integration of environmental and cellular signals so that the appropriate choice is made between slug formation and culmination. We suggest that DHKC may function as a sensor for ammonia, and that it is the initial component of a phosphorelay signaling system that may modulate the activity of cAMP-dependent protein kinase to either inhibit or promote culmination. Additionally, DhkC - spores were found to be defective in germination, indicating a role for the DHKC signaling pathway activating spore germination. ------------------------------------------------------------------------- Cell-cell signaling during Dictyostelium development Fredrik Söderbom and William F. Loomis Center for Molecular Genetics, Department of Biology University of California San Diego, La Jolla CA 92423 Trends in Microbiology (in press) Summary Specific proteins and peptides, as well as cAMP, are used as intercellular signals in Dictyostelium. Our understanding of the signal transduction pathways activated by these signals has been expanded by inclusion of newly characterized proteins. cAMP-dependent protein kinase (PKA) and its associated phosphodiesterase, RegA, play multiple roles in these pathways. ------------------------------------------------------------------------- A Serum Response Factor Homolog is Required for Spore Differentiation in Dictyostelium Ricardo Escalante* and Leandro Sastre Instituto de Investigaciones Biomédicas del Consejo Superior de Investigaciones Científicas , C/Arturo Duperier,4. 28029 Madrid. Spain. *Author for correspondence: e-mail: rescalante@iib.uam.es Development, in press. Summary A homolog of the Serum Response Factor (SRF) has been isolated from Dictyostelium discoideum and its function studied by analyzing the consequences of its gene disruption. The MADS-box region of Dictyostelium SRF (DdSRF) is highly conserved with those of the human, Drosophila and yeast homologs. srfA is a developmentally regulated gene expressed in prespore and spore cells. This gene plays an essential role in sporulation as its disruption leads to abnormal spore morphology and loss of viability. The mutant spores were round and cellulose deposition seemed to be partially affected. Initial prestalk and prespore cell differentiation did not seem to be compromised in the mutant since the expression of several cell-type specific markers were found to be unaffected. However, the mRNA level of the spore marker spiA was greatly reduced. Activation of the cAMP dependent protein kinase (PKA) by 8-Br-cAMP was not able to fully bypass the morphological defects of srfA- mutant spores, although this treatment induced spiA mRNA expression. Our results suggest that DdSRF is required for full maturation of spores and participates in the regulation of the expression of the spore-coat marker spiA and probably other maturation genes necessary for proper spore cell differentiation. ------------------------------------------------------------------------- High levels of actin tyrosine phosphorylation: correlation with the dormant state of Dictyostelium spores Yoshiro Kishi1,3, Chris Clements2, Dana C. Mahadeo2, David A. Cotter2 and Masazumi Sameshima3 1Department of Physics, Rikkyo (St. Paul's) University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo, 171-0021, Japan. 2Department of Biological Sciences, University of Windsor, Windsor, Ontario, N9B 3P4, Canada. 3Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, 113-8613, Japan J. Cell Sci., in press Abstract: Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores which survive starvation stress. In this study, we demonstrate that half of the actin molecules in the spores are tyrosine-phosphorylated. The phosphorylated actin is distributed around immobile crenate mitochondria and vesicles, as well as, in the cytoplasm of the spores. The actin isolated from spore lysates contains phosphorylated and unphosphorylated forms at the same molar ratio as that of the original whole spore lysate. Under actin polymerizing conditions they form actin filaments and then they are completely depolymerized under actin depolymerizing conditions, indicating that tyrosine phosphorylation of actin may not prohibit actin polymerization nor stimulate depolymerization. The phosphorylation levels increase at the end of the culmination stage when spores have matured morphologically and physiologically, and reach maximum levels after an additional 12 hours of development. The levels are stable for 20 days following spore maturation, and decline to undetectable levels within the next 10 days. Spores having high levels of phosphorylation show high viability, and vice versa. The Dictyostelium dual-specificity kinase, splA, may be a candidate kinase that phosphorylates actin tyrosine during the very late stages in development. In spores of the splA gene null mutant, phosphorylation of actin is not detected by immunoblots with anti- phosphotyrosine antibody. Following activation of spores with nutrient medium containing spore germination promoters, the phosphorylation levels quickly decrease with a half-life of about 5 minutes. After 20 minutes spores begin to swell. At this later time, most of the phosphorylated actin already has been dephosphorylated. Also, in heat-activated spores actin dephosphorylation occurs prior to spore swelling. However, addition of phosphatase inhibitors following heat-activation, prevented spore swelling and dephosphorylation of actin. Our data indicate that the high levels of actin tyrosine phosphorylation, specific to the spore stage, may be required for maintaining dormancy to withstand starvation stress. The rapid dephosphorylation of actin leads to a reactivated dynamic actin system which participates in spore swelling, vesicle movement, and mitochondrial shape changes during the spore germination process. ------------------------------------------------------------------------- A deubiquitinating enzyme that disassembles free polyubiquitin chains is required for development but not growth in Dictyostelium David F. Lindsey1, Alexander Amerik2, William J. Deery1, John D. Bishop1, Mark Hochstrasser2, and Richard H. Gomer1 1Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892 2Department of Biochemistry and Molecular Biology, the University of Chicago, Chicago, IL 60637 Journal of Biological Chemistry, in press Abstract Although cell differentiation usually involves synthesis of new proteins, little is known about the role of protein degradation. In eukaryotes, conjugation to ubiquitin polymers often targets a protein for destruction. This process is regulated by deubiquitinating enzymes (DUBs), which can disassemble ubiquitin polymerases or ubiquitin-substrate conjugates. We find that a DUB, UbpA, is required for Dictyostelium development. ubpA cells have normal protein profiles on gels, grow normally and show normal responses to starvation such as differentiation and secretion of CMF. However, ubpA cells have defective aggregation, chemotaxis, cAMP relay, and cell adhesion. These defects result from low expression of cAMP pulse-induced genes such as those encoding the cAR1 cAMP receptor, phosphodiesterase, and the gp80 adhesion protein. Treatment of ubpA cells with pulses of exogenous cAMP allows them to aggregate and express these genes like wild-type cells, but they still fail to develop fruiting bodies. Unlike wild type, ubpA cells accumulates ubiquitin- containing species that comigrate with ubiquitin polymers, suggesting a defect in polyubiquitin metabolism. UbpA has sequence similarity with yeast Ubp14, which disassembles free ubiquitin chains. Yeast ubp14 cells have a defect in proteolysis, due to excess ubiquitin chains competing for substrate binding to proteasomes. Cross-species complementation and enzyme specificity assays indicate that UbpA and Ubp14 are functional homologs. We suggest that specific developmental transitions in Dictyostelium require the degradation of specific proteins, and that this process in turn requires the disassembly of polyubiquitin chains by UbpA. ------------------------------------------------------------------------- [End Dicty News, volume 11, number 2]