Dicty News Electronic Edition Volume 18, number 9 May 25, 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. ============= Abstracts ============= Identification of four candidate cGMP targets in Dictyostelium. Goldberg JM, Bosgraaf L, Van Haastert PJ, Smith JL. Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472-2829; and Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. Proc Natl Acad Sci U S A 2002 May 14;99(10):6749-54 In Dictyostelium, a transient increase in intracellular cGMP is important for cytoskeletal rearrangements during chemotaxis. There must be cGMP- binding proteins in Dictyostelium that regulate key cytoskeletal components after treatment with chemoattractants, but to date, no such proteins have been identified. Using a bioinformatics approach, we have found four candidate cGMP-binding proteins (GbpA-D). GbpA and -B have two tandem cGMP-binding sites downstream of a metallo beta-lactamase domain, a superfamily that includes cAMP phosphodiesterases. GbpC contains the following nine domains (in order): leucine-rich repeats, Ras, MEK kinase, Ras guanine nucleotide exchange factor N-terminal (RasGEF-N), DEP, RasGEF, cGMP-binding, GRAM, and a second cGMP-binding domain. GbpD is related to GbpC, but is much shorter; it begins with the RasGEF-N domain, and lacks the DEP domain. Disruption of the gbpC gene results in loss of all high- affinity cGMP-binding activity present in the soluble cellular fraction. GbpC mRNA levels increase dramatically 8 h after starvation is initiated. GbpA, -B, and -D mRNA levels show less dramatic changes, with gbpA mRNA levels highest 4 h into starvation, gbpB mRNA levels highest in vegetative cells, and gbpD levels highest at 8 h. The identification of these genes is the first step in a molecular approach to studying downstream effects of cGMP signaling in Dictyostelium. ----------------------------------------------------------------------------- A Comparative Analysis of Spore Coat Formation, Structure and Function in Dictyostelium Christopher M. West Deptartment of Anatomy & Cell Biology, University of Florida College of Medicine, 1600 SW Archer Road, Gainesville, FL 32610-0235 USA Telephone: 352-392-3329, Fax: 352-392-3305, Email: westcm@ufl.edu Int. Rev. Cyt., in press Dictyostelium produces spores at the end of its developmental cycle to propagate the lineage. The spore coat is an essential feature of spore biology contributing a semipermeable chemical and physical barrier to protect the enclosed amoeaba. The coat is assembled from proteins and a branched polysaccharide that are secreted and from cellulose produced at the cell surface. They are organized into a polarized molecular sandwich with proteins forming layers surrounding the microfibrillar cellulose core. Genetic and biochemical studies are beginning to provide insight into how the delivery of protein and cellulose to the cell surface are coordinated, and how cysteine-rich domains of the proteins interact to form the layers. A multi-domain inner layer protein, SP85/PsB, has a central role in regulating coat assembly and contributing to a core structural module that bridges proteins to cellulose. Coat formation and structure have many parallels in walls from plant, algal, yeast, protist and animal cells. ----------------------------------------------------------------------------- The different components of a multisubunit cell number-counting factor have both unique and overlapping functions Debra A. Brock, R. Diane Hatton, Dan-Victor Giurgiutiu, Brenton Scott, Robin Ammann, and Richard H. Gomer Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892 Development, in press Summary Dictyostelium aggregation streams break up into groups of 103 to 2 x 104 cells. The cells sense the number of cells in a stream or group by the level of a secreted counting factor (CF). CF is a complex of at least 5 polypeptides. When the gene encoding countin (one of the CF polypeptides) was disrupted, the cells could not sense each other's presence, resulting in non-breaking streams that coalesced into abnormally large groups. To understand the function of the components of CF, we have isolated cDNA sequences encoding a second component of CF, CF50. CF50 is 30% identical to lysozyme (but has very little lysozyme activity) and contains distinctive serine-glycine motifs. Transformants with a disrupted cf50 gene, like countin cells, form abnormally large groups. Addition of recombinant CF50 protein to developing cf50 cells rescues their phenotype by decreasing group size. Abnormalities seen in aggregating countin cells (such as high cell-cell adhesion and low motility) are also observed in the cf50 cells. Western blot analysis of conditioned medium sieve column fractions showed that the CF50 protein is present in the same fraction as the 450 KD CF complex. In the absence of CF50, secreted countin is degraded, suggesting that one function of CF50 may be to protect countin from degradation. However, unlike countin cells, cf50 cells differentiate into an abnormally high percentage of prespore cells, and this difference can be rescued by exposing cells to recombinant CF50. These observations indicate that unlike other known multisubunit factors, CF contains subunits with both overlapping and unique properties. ----------------------------------------------------------------------------- [End Dicty News, volume 18, number 9]