Dicty News Electronic Edition Volume 18, number 12 June 22, 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 ====================== Scientific Curator, DictyBase A position is available for a scientific curator to work with DictyBase. This is a great opportunity for someone experienced with Dictyostelium as an experimental system, who is also interested in bioinformatics. The individual occupying this position will become part of the DictyBase team which includes other scientific curators and bioinformatics support. Responsibilities will include curating entries in DictyBase, working closely with the sequencing centers involved in sequencing the Dictyostelium genome as well as the Dictyostelium research community, to integrate the scientific knowledge derived from research using Dictyostelium with genome sequence and the scientific literature. The DictyBase scientific curators will also interact with scientific curators from other model organism databases such as FlyBase, WormBase, SGD (Sacchromyces Genome Database) and MGI (Mouse Genome Informatics). Desired experience includes research using Dictyostelium, familiarity with genome informatics, and the use of computers in research. This position will be located in the Northwestern University Center for Genetic Medicine in Chicago. Appointment will be on the research scientist track at Northwestern University and includes a generous salary and full benefits. Interested candidates should submit a current CV and a brief description of research experience and interest in bioinformatics (preferably by email) to: Rex L. Chisholm (r-chisholm@northwestern.edu) Center for Genetic Medicine Northwestern University 303 E. Chicago Ave. Chicago, IL 60611 ============= Abstracts ============= Regulated expression of the MADS-box transcription factor SrfA mediates activation of gene expression by PKA during Dictyostelium sporulation Ricardo Escalante* and Leandro Sastre Instituto de Investigaciones Biomdicas C.S.I.C/U.A.M. C/Arturo Duperier, 4, 28029 Madrid, Spain. *Author for correspondence: Fax: +34-91-585-4587. E-mail: rescalante@iib.uam.es Mechanisms of Development (in press) Abstract Cell differentiation and morphogenesis are tightly regulated during sporulation in the lower eukaryote Dictyostelium discoideum. The control of the cAMP-dependent protein kinase (PKA) is essential to coordinate these processes. Several signal transduction pathways are being recognized that lead to the regulation of intracellular cAMP levels. However, very little is known about the events lying downstream of PKA that are essential to activate late gene expression and terminal differentiation of the spores. We have studied the relationship between PKA and the MADS-box transcription factor SrfA, essential for spore differentiation. Constitutive activation of PKA was not able to rescue sporulation in a strain that lacks srfA suggesting the possibility that srfA functions downstream of PKA in a signal transduction pathway leading to spore maturation. A distal promoter region regulates the induction of srfA expression in the prespore region during culmination. We found that this promoter can be induced precociously by activating PKA with 8-Br-cAMP suggesting a transcriptional regulation by PKA. Moreover, precocious sporulation and expression of the spore marker spiA in a strain that over-expresses PKA correlates with a precocious induction of srfA expression. The temporal and spatial pattern of expression was also studied in a mutant strain lacking the main adenylyl cyclase that functions during culmination, ACR. This strain is expected to have lower PKA activity and consistently, the level of srfA expression was reduced. Moreover, the temporal induction of srfA in the prespore region was also delayed during culmination. Our results strongly suggest that PKA activation during culmination leads to the induction of the expression of srfA. The correct temporal and spatial pattern of srfA expression appears to be part of a mechanism that ensures the adequate coordination of gene expression and morphogenesis. ----------------------------------------------------------------------------- Cells respond to and bind countin, a component of a multisubunit cell-number counting factor Tong Gao1, Karen Ehrenman1, Lei Tang2, Matthias Leippe3, Debra A. Brock1, and Richard H. Gomer1, 2 1Howard Hughes Medical Institute and 2Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892 and 3Molecular Parasitology Group, Research Center for Infectious Diseases, Rntgenring 11, 97070 Wuerzburg, Germany Journal of Biological Chemistry, in press Summary In Dictyostelium discoideum counting factor (CF), a secreted ~450kDa complex of polypeptides, inhibits group and fruiting body size. When the gene encoding countin (a component of CF) was disrupted, cells formed large groups. We find that recombinant countin causes developing cells to form small groups with an EC50 of ~3 ng/ ml, and affects cAMP signal transduction in the same manner as semipurified CF. Recombinant countin increases cell motility, decreases cell-cell adhesion, and regulates gene expression in a manner similar to the effect of CF. However, countin does not decrease adhesion or group size to the extent that semipurified CF does. A 1 minute exposure of developing cells to countin causes an increase in F-actin polymerization and myosin phosphorylation, and a decrease in myosin polymerization, suggesting that countin activates a rapid signal transduction pathway. 125I-labelled countin has countin bioactivity, and binding experiments suggest that vegetative and developing cells have ~53 cell-surface sites which bind countin with a KD of approximately 1.5 ng/ ml or 60 pM. We hypothesize that countin regulates cell development through the same pathway as CF and that other proteins within the complex may modify the activity of countin and/or have independent size-regulating activities. ----------------------------------------------------------------------------- A single cell-density sensing factor stimulates distinct signal transduction pathways through two different receptors William J. Deery, Tong Gao, Robin Ammann, and Richard H. Gomer Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street Houston, TX 77005-1892 Journal of Biological Chemistry, in press Summary In Dictyostelium discoideum, cell density is monitored by levels of a secreted protein, conditioned-medium factor (CMF). CMFR1 is a putative CMF receptor necessary for CMF-induced G protein-independent accumulation of the SP70 prespore protein, but not for CMF-induced G protein-dependent IP3 production. Using recombinant fragments of CMF, we find that stimulation of the IP3 pathway requires amino acids 170 - 180 whereas SP70 accumulation does not, corroborating a two-receptor model. Cells lacking CMFR1 do not aggregate, due to the lack of expression of several important early developmentally regulated genes, including gp80. Although many aspects of early developmental cAMP-stimulated signal transduction are mediated by CMF, CMFR1 is not essential for cAMP-stimulated cAMP and cGMP production or Ca++ uptake, suggesting the involvement of a second CMF receptor. Exogenous application of antibodies against either the region between a first and second or a second and third possible transmembrane domain of CMFR1 induces SP70 accumulation. Antibody- and CMF-induced gene expression can be inhibited by recombinant CMFR1 corresponding to the region between the first and third potential transmembrane domains, indicating that this region is extracellular and likely contains the CMF binding site. These observations support a model where a one- or two-transmembrane CMFR1 regulates gene expression and a G protein-coupled CMF receptor mediates cAR1 signal transduction. ----------------------------------------------------------------------------- A second UDP glucose pyrophosphorylase is required for differentiation and development in Dictyostelium discoideum John D. Bishop1, Byoung C. Moon2, Faith Harrow2, Richard H. Gomer1, Robert P. Dottin2, and Derrick T. Brazill2 1Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, MS-140, Rice University, Houston, TX 77251-1892 2Hunter College, Department of Biological Sciences, 695 Park Avenue, New York, NY 10021 J. Biological Chemistry, in press. Summary Uridine diphosphoglucose pyrophosphorylase (UDPGP) is a developmentally regulated enzyme in Dictyostelium discoideum, which is involved in trehalose, cellulose and glycogen synthesis. Two independent UDPGP proteins are believed to be responsible for this activity. To determine the relative contributions of each protein, the genes encoding them were disrupted individually. Cells lacking the udpgp1 gene exhibit normal growth and development and make normal levels of cellulose. In agreement with these phenotypes, udpgp1- cells still have UDPGP activity, although at a reduced level. This supports the importance of the second UDPGP gene. This newly identified gene, ugpB, encodes an active UDPGP as determined by complementation in E. coli. When this gene is disrupted, cells undergo aberrant differentiation and development ending with small, gnarled fruiting bodies. These cells also have decreased spore viability and decreased levels of glycogen, whose production requires UDPGP activity. These phenotypes suggest that UgpB constitutes the major UDPGP activity produced during development. Sequence analysis of the two UDPGP genes shows that UgpB has higher homology to other eukaryotic UDPGPs than does UDPGP1. This includes the presence of 5 conserved lysine residues. Udpgp1 only has 1 of these lysines. ----------------------------------------------------------------------------- RabD, a Dictyostelium Rab14-related GTPase, Regulates Phagocytosis and Homotypic Phagosome and Lysosome Fusion Edward Harris and James Cardelli Department of Microbiology and Immunology and Feist-Weiller Cancer Center LSU Health Sciences Center Shreveport, LA J. Cell Science (in press) Abstract RabD, a Dictyostelium Rab14-related GTPase, localizes in the endo-lysosomal pathway and contractile vacuole system of membranes, and cell-lines expressing dominant negative RabD were defective in endocytosis, endosomal membrane flow and homotypic lysosome fusion. In support of support of a role in fusion, cells overexpressing constitutively active RabDQ67L accumulated enlarged hydrolase-rich acidic vesicles ringed with GFP-RabD, consistent with RabD directly regulating lysosome fusion. To determine if RabD also regulated phagocytosis and/or homotypic phagosome fusion (a process stimulated by many intracellular pathogens), cells overexpressing dominant active (RabDQ67L+ cells) or dominant negative (RabN121I+ cells) RabD were analyzed microscopically and biochemically. The rate of phagocytosis was increased 2-fold in RabDQ67L+ cells and reduced by 50% in RabDN121I+ cells as compared to control cells. To examine the role of RabD in the formation of multi-particle phagosomes, we performed a series of pulse-chase experiments using fluorescently labeled bacteria and fluorescent latex beads. The rate of fusion of newly formed phagosomes was 5 times higher in the RabDQ67L+ cells and reduced over 50% in RabDN121I+ cells as compared to control cells. GFP-RabDQ67L+ was found to ring multi-particle spacious phagosomes, supporting a direct role for this protein in regulating fusion. Inhibition of PI 3-kinase activity, known to regulate phagosome fusion in the wild-type cells, reduced the rate of phagosome fusion in RabDQ67L+ cells, indicating that RabD acted upstream of or parallel with PI 3-kinase. We hypothesize that RabD and, possibly, Rab14, a related GTPase that associates with phagosomes in mammalian cells, are important regulators of homotypic phagosome and endo-lysosome fusion ----------------------------------------------------------------------------- Expression Pattern of Alkaline Phosphatase in Dictyostelium Muatasem Ubeidat, Bradley R. Joyce and Charles L. Rutherford* Biology Department, Molecular and Cellular Biology Section, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406, USA Mechanisms of Development, In Press Abstract We used two different methods to study the expression pattern of alkaline phosphatase (alp) in Dictyostelium. In situ staining of the endogenous enzyme activity at different stages of development showed that the enzyme was active early in the aggregation stage and localized to the area where the tip of the first finger was initiated. The activity was localized to the anterior region of developing slugs, then became restricted to the region between the prestalk and prespore cells at the culmination stage. In the complete fruiting body, the activity was confined to the lower and upper cup. A second method to study alp expression utilized a beta- galactosidase reporter gene under the control of the alp promoter. A low level of (-galactosidase activity was observed in vegetative cells, then increased during development. Reporter gene activity was restricted to PstO cells at the slug stage. At the culmination stage, the expression was restricted to prestalk cells at the interface between the prestalk and prespore cells. In the completed fruiting body, the expression was observed in the upper and lower cup. ----------------------------------------------------------------------------- [End Dicty News, volume 18, number 12]