Dicty News Electronic Edition Volume 15, number 7 October 14, 2000 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" =========================== Postdoctoral Position =========================== Two postdoctoral associate positions are available, focusing on the following two projects. We previously found that Dictyostelium cells sense the density of cells of one type using a secreted 80 kD glycoprotein; this is essentially the eukaryotic equivalent of a quorum sensor. The cells sense this factor using a 2-transmembrane receptor. Opportunities are available to characterize the basic biochemistry of this receptor, determine the functional domains of the receptor, and elucidate its signal transduction pathway. When Dictyostelium cells starve, they form aggregates of roughly 20,000 cells. To determine how the cells can sense their number, we isolated mutants which have an abnormal number of cells in the aggregates. We found a mutant which oversecretes a factor the cells use to sense the number of cells in a group. The factor is a 450 kD complex of polypeptides (Brock and Gomer, Genes&Development. 13, 1960-1969. (1999)). Opportunities are available to study both this cell number counting factor and its signal transduction pathway. Applicants must be within two months of obtaining their Ph.D., or have received their Ph.D. within the last two years. Send CV and cover letter by email to richard@bioc.rice.edu, or send by regular mail to Richard Gomer, HHMI, Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892. ============== Abstracts ============== Glucose-induced pathways for actin tyrosine dephosphorylation during Dictyostelium spore germination. Yoshiro Kishi1, Dana Mahadeo2, David N. Cervi2, Chris Clements2, David A. Cotter2 and Masazumi Sameshima1 1Electron Microscopy Center, The Tokyo Metro Inst of Med Sci, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, 113-8613, Japan 2Department of Biological Sciences, University of Windsor, 401 Sunset Avenue, Windsor, Ontario, N9B 3P4, Canada Experimantal Cell Research, in press In the presence of germination signals, dormant spores of Dictyostelium discoideum rapidly germinate to start a new life cycle. Previously we have shown that half of the actin molecules in spores are maintained in a tyrosine- phosphorylated state, and a decline of the actin phosphorylation levels is a prerequisite for spore swelling. In this study, we have established D-glucose as a trigger molecule for the actin dephosphorylation. Present in a nutrient germination medium, D-glucose may act both as a trigger molecule and/or may serve as a substrate within a pathway for actin dephosphorylation depending upon spore age. However, the glucose-induced actin dephosphorylation was insufficient for spores to swell. Other factors in the nutrient medium were required for complete germination of young spores aged 1 to 5 days. In contrast, dispersion in non-nutrient buffer was necessary and sufficient for a decline of actin phosphorylation levels and even the emergence of amoebae in older spores (6 days and beyond). Moreover, the dephosphorylation pathway in the older spores was independent of energy production. We propose that the diversification of the actin dephosphorylation pathway may enable spores to increase their probability of germination upon spore aging. ---------------------------------------------------------------------------- Aberrant folate response and premature development in a mutant of Dictyostelium discoideum Klaus Salger and Birgit W. Wetterauer* Zoologisches Institut, Ludwig-Maximilians-Universität of München Luisenstraße 14, D-80333 München, Germany Differentiation, in press. Summary: Growth and development are mutually exclusive in Dictyostelium discoideum. The transition between the two stages of the life cycle is regulated by the relative abundance of nutrients and proteins secreted by the cells which reflect population density. At the transition from growth to development, the discoidin genes - developmental markers - are induced by the "quorum" protein PSF. The effect of PSF is counteracted by food bacteria and by folate [8]. We show that folate treatment during growth delays morphologic development. Furthermore, we demonstrate that in a mutant of Dictyostelium discoideum (VI88, renamed HBW3), which expresses discoidinI during growth and which develops rapidly [46], discoidinI expression is less sensitive to folate than in wild type cells. Finally, we present evidence that fragments of the discoidinIg promoter which are unresponsive to PSF and CM are sufficient for misregulation in the mutant. The only known regulator of these promoter elements is folate. Changes in the expression of other early developmental genes are shown as well. Taken together these data suggest that the reduced sensitivity to folate might be the cause for the "rapid development" phenotype of the mutant and that folate regulates developmental timing. ---------------------------------------------------------------------------- A Novel Dictyostelium RasGEF is Required for Normal Endocytosis, Cell Motility and Multicellular Development Andrew Wilkins, Jonathan Chubb, and Robert H. Insall Current Biology, in press. Background: Ras proteins are regulators of multiple signalling pathways, which are found in all eukaryotes. Ras pathways are activated by guanine nucleotide exchange factors (GEFs). The social amoeba Dictyostelium possesses an unexpectedly large number of ras genes, which suggests a complex pattern of Ras signalling. Loss of these genes causes a range of phenotypes, including defects in cell polarity, cytokinesis, development, endocytosis and phototaxis. However, very little is known about which GEFs activate these Ras proteins, and which signals are mediated through GEFs. It is also unclear, in Dictyostelium or higher eukaryotes, whether Ras pathways are linear, with each Ras controlled by its own GEF, or networked, with multiple GEFs able to act on multiple Ras proteins. Results: We have identified the gene which encodes RasGEFB, a protein with strong homology to known RasGEFs such as Sos. Like the other known Dictyostelium RasGEF, Aimless, it has no obvious sites for regulation such as PH domains or cyclic nucleotide binding sites. Loss of RasGEFB causes multiple phenotypes. Mutant cells move unusually rapidly, but lose the ability to perform macropinocytosis and therefore to grow in liquid medium. These phenotypes correlate with a change in morphology; crowns, the sites of macropinocytosis, are replaced by polarised lamellipodia. Mutant cells are also profoundly defective in early development, although they eventually form tiny but normally proportioned fruiting bodies. This defect correlates with loss of discoidin Ig mRNA, a starvation-induced gene, although other genes required for development are expressed normally or even precociously. RasGEFB is able to rescue a Saccharomyces CDC25 mutant, which indicates that it is a genuine GEF for Ras proteins. Conclusions: RasGEFB appears to be the principal activator of the RasS protein, which regulates macropinocytosis and cell speed, but also appears to regulate one or more other Ras proteins. Dictyostelium Ras pathways therefore appear not to be simply linear. ---------------------------------------------------------------------------- Folate Reception by Vegetative Dictyostelium Discoideum Amoebae: Distribution of Receptors and Trafficking of Ligand. Jared L. Rifkin. Biology Department, Queens College of CUNY, Flushing NY 11367-1597, USA. Tel.:718-997-3432. Fax: 718-997-3445. E-mail: jared-rifkin@qc.edu. Cell Motil. and Cytoskeleton, in press ABSTRACT We report the first explicit demonstration of post-reception processing of a Dictyostelium chemoattractant. Folic acid stimulates reorganization of the cytoskeleton of vegetative amoebae of D. discoideum. In particular, folic acid is a potent chemoattractant and it causes enlargement of the filopodial array. The distribution of folic acid receptors and the fate of bound folate was investigated by presenting an agonist consisting of the conjugate, folic acid-lactalbumin-FITC (Folate*), to these amoebae. This novel probe was specifically bound to folic acid receptors of these amoebae and it stimulated chemotaxis and enlargement of their filopodial array. Hence, Folate* is a physiologically competent probe. The probe sans-folate moiety was not bound anywhere to living or fixed amoebae. Since Folate* did not bind to amoebae after incubation with equimolar folic acid, this probe is a receptor-specific agonist. We report here the first description, by confocal visualization of a competent agonist, of the distribution of folate receptors of D. discoideum vegetative amoebae and of the fate of this ligand. Examination of fixed amoebae revealed that bound Folate* was distributed generally over their entire surface including their filopodia. However, in living amoebae, Folate* was bound only at the cell body and this bound Folate* was almost completely internalized as concentrated packets into vacuoles. This endocytosis of the probe and the clustering of endocytosed Folate* is consistent with receptor-mediated internalization of a ligand. Possible routes for internalization of the folate probe and the implications of this endocytosis for signal molecule ---------------------------------------------------------------------------- [End Dicty News, volume 15, number 7]