Dicty News Electronic Edition Volume 16, number 1 January 20, 2001 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 the Dictyostelium Web Page "http://dicty.cmb.northwestern.edu/dicty" ============== Abstracts ============== The Dictyostelium discoideum family of Rho-related proteins Francisco Rivero(1), Heidrun Dislich(1), Gernot Glöckner(2) and Angelika A. Noegel(1) (1) Institut für Biochemie I, Medizinische Fakultät, Universität zu Köln. Joseph-Stelzmann-Strasse 52, D-50931 Köln, Germany (2) Institut für Molekulare Biotechnologie, Beutenbergstrasse 11, D-07745 Jena, Germany Nucleic Acids Research, accepted ABSTRACT Taking advantage of the ongoing genome sequencing project, we have assembled more than 73 kb genomic DNA in 15 contigs harboring 15 genes and one pseudogene of Rho-related proteins. Comparison with EST sequences revealed that every gene is interrupted by at least one and up to four introns. For racC extensive alternative splicing was identified. Northern blot analysis showed that mRNAs for racA, racE, racG, racH and racI were present at all stages of development, whereas racJ and racL were expressed only at late stages. Amino acid sequences have been analyzed in the context of Rho-related proteins of other organisms. Rac1a/1b/1c, RacF1/F2 and to a lesser extent RacB and the GTPase domain of RacA can be grouped in the Rac subfamily. None of the additional Dictyostelium Rho-related proteins belongs to any of the well-defined subfamilies, like Rac, Cdc42 or Rho. RacD and RacA are unique in that they lack the prenylation motif characteristic of Rho proteins. RacD possesses a 50 residues C-terminal extension and RacA a 400 residues C-terminal extension that contains a proline-rich region, two BTB domains and a novel C-terminal domain. We have also identified homologues for RacA in Drosophila and mammals, thus defining a new subfamily of Rho proteins, RhoBTB. ----------------------------------------------------------------------------- Inducible nuclear translocation of a STAT protein in Dictyostelium prespore cells: implications for morphogenesis and cell-type regulation Dirk Dormann*, Tomoaki Abe*, Cornelis J Weijer and Jeffrey Williams School of Life Sciences University of Dundee Wellcome Trust Biocentre Dow Street DUNDEE, DD1 5EH UK * These two authors contributed equally to this work Development, in press. SUMMARY Dd-STATa, the Dictyostelium STAT (Signal Transducer and Activator of Transcription) protein, is selectively localised in the nuclei of a small subset of prestalk cells located in the slug tip. Injection of cAMP into the extracellular spaces in the rear of the slug induces rapid nuclear translocation of a Dd-GFP:STATa fusion protein in prespore cells surrounding the site of injection. This suggests that cAMP signals emanating from the tip direct the localised nuclear accumulation of Dd-STATa. It also shows that prespore cells are competent to respond to cAMP, by Dd-STATa activation, and it implies that cAMP signalling is in some way limiting in the rear of the slug. Co-injection of a specific inhibitor of the cAR1 serpentine cAMP receptor almost completely prevents the cAMP-induced nuclear translocation, showing that most or all of the cAMP signal is transduced by cAR1. Dd-GFP:STATa also rapidly translocates into the nuclei of cells adjoining the front and back cut edges when a slug is bissected. Less severe mechanical disturbances, such as pricking the rear of a slug with an unfilled micro-pipette, also cause a more limited nuclear translocation of Dd-GFP:STATa. We propose that these signalling events form part of a repair mechanism that is activated when the migrating slug suffers mechanical damage. ----------------------------------------------------------------------------- Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate Zahara M. Jaffer1, Meenal Khosla1, George B. Spiegelman1,2, and Gerald Weeks1,2 Departments of Microbiology and Immunology1, and Medical Genetics2, University of British Columbia, 300 - 6174 University Boulevard Vancouver, B.C., V6T 1Z3, Canada e-mail: gweeks@interchange.ubc.ca Development, in press. ABSTRACT There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the over-expression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. In order to determine if the defects were due to the presence of activated RasG in prestalk or prespore cells, rasG was expressed under the control of either the ecmAO or the psA promoters. When activated rasG was specifically expressed in prestalk cells in ecmAO::rasG(G12T) transformants, a single tipped aggregate was formed and cell type specific gene expression was not appreciably altered. In contrast, when activated rasG was specifically expressed in prespore cells in psA::rasG(G12T) transformants, multi-tipped aggregates were formed and the expression of the prespore specific gene cotC was dramatically reduced while the expression of the prestalk cell specific gene ecmA gene was markedly enhanced. Thus, it is the expression of activated ras in prespore cells that causes the deregulation of cell type specific gene expression and tip formation. However, the psA::rasG(G12T) transformants continued to develop beyond the multi-tipped aggregate stage, suggesting that the block in development at the multi-tipped aggregate stage in the rasD::rasG(G12T) transformant is due to the presence of RasG(G12T) in the prestalk cell population. The psA::rasG(G12T) transformants produced mounds that supported multiple stalk like structures, some with small apical sori. Spore formation was inhibited and this was not corrected by the presence of wild type cells in chimeric mixtures, indicating that the defect in spore formation was cell autonomous. Prespore cells were initially formed in the mound but they then transdifferentiated into prestalk cells, which eventually formed stalk cells. The single tipped aggregates of the ecmAO::rasG(G12T) transformants produced slugs that appeared normal, but these went on to form abnormal terminal structures with very few spores and large numbers of stalk cells. Although the slugs appeared to be normal, there was considerable prestalk cell mislocalization. PstA cells were found in the rear rather than at the tip and PstO cells were greatly reduced in number and also found in the rear. In addition, there was an increase in the number of PstB cells in both the anterior and posterior regions of the slug, but the anterior PstB cells were not specifically localized as a cone behind the tip. The slugs also exhibited impaired motility and phototaxis and there was no formation of stalk tubes at the onset of culmination. The defect in spore formation was corrected by developing the transformant in chimeric mixtures with wild type cells, indicating that the defect is not cell autonomous. Examination of cell localization in chimeras revealed that wild type cells occupied the anterior tip of the slug, presumably providing the signals that allowed the prespore cells of the transformant to form spores. These results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations. ----------------------------------------------------------------------------- [End Dicty News, volume 16, number 1]