CSM News Electronic Edition Volume 5, number 8 Sept. 9, 1995 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmsbio.nwu.edu/dicty.html" =========== Abstracts =========== Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain Guyu Ho, Tung-Ling L. Chen, and Rex L. Chisholm* Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611 J. Biol. Chem., in press. SUMMARY Dictyostelium myosin deficient in the essential light chain (ELC) does not function normally either in vivo or in vitro (1). Since normal myosin function requires association of ELC, we investigated the domains of ELC that are necessary for binding to the myosin heavy chain (MHC). Deleting the N-terminal 11 or 28 amino acid residues (DN11 or DN28) or the C-terminal 15 amino acid residues (C15) abolished binding of the ELC to the MHC when the mutants were expressed in wild type (wt) cells. In contrast, the ELC carrying deletion or insertion of four amino acid residues (D4 or I4) in the central linker segment bound the MHC in wt cells, although less efficient competition with wt ELC suggested that the affinity for the MHC is reduced. When these mutants were expressed in ELC-minus (mlcE-) cells, where the binding to the heavy chain is not dependent on efficient competition with the endogenous ELC, DN28 and DN11 bound to the MHC at 15% of wt levels and C15 did not bind to a significant degree. I4 and D4, however, bound with normal stoichiometry. These data indicate that residues at both termini of the ELC are required for association with the MHC, while the central linker domain appears to be less critical for binding. When the mutants were analyzed for their ability to complement the cytokinesis defect displayed by mlcE- cells, a correlation to the level of ELC carried by the MHC was observed, indicating that a stoichiometric ELC-MHC association is necessary for normal myosin function in vivo. ---------------------------------------------------------------------- ISOLATION OF INACTIVE AND G PROTEIN-RESISTANT ADENYLYL CYCLASE MUTANTS USING RANDOM MUTAGENESIS Carole A. Parent and Peter N. Devreotes Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Journal of Biological Chemistry, in press ABSTRACT We used random mutagenesis and phenotypic rescue of adenylyl cyclase-null Dictyostelium cells to isolate loss-of-function mutations in the enzyme. Mutants were (i) catalytically inactive or (ii) resistant to chemoattractant receptor and guanosine 5'-3'-O-(thio)triphosphate stimulation. Both classes of mutants harbored substitutions within the cytoplasmic C1a domain. Mutations that inactivated the enzyme were often at highly conserved positions. Those that blocked activation were grouped in two distinct regions: one close to the plane of the plasma membrane and another halfway within the C1 loop. Missense mutations or deletions within the transmembrane domains resulted in missorting of the protein. Our screen provides a simple and efficient method to separately define the sites of catalysis and regulation of this important class of enzymes. --------------------------------------------------------------------------- Initial cell-type choice in a simple eukaryote: Cell-autonomous or morphogen-gradient dependent? Jennifer L. Clay, Robin R. Ammann and Richard H. Gomer Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892 Developmental Biology, in press Abstract Dictyostelium discoideum is a simple eukaryote that lives as an amoeba until starvation triggers aggregation. The aggregate forms a slug which then develops into a fruiting body with two main cell types, stalk and spore cells. Two mechanisms have been proposed to explain cell-type differentiation. Studies using expression of the ecmA gene as a prestalk cell marker indicated that gradients of morphogens determine cell fate in the slug. However, studies using dyes or the CP2 gene product as a prestalk cell marker indicated that cell autonomous factors such as cell-cycle phase at the time of starvation cause an initial choice of cell fate. To help resolve these differences, we have used transformed cells containing the promoter of the prestalk gene ecmA fused to b-galactosidase (K.A. Jermyn and J. G. Williams, Development 111, 779-787, 1991) to study the differentiation of Dictyostelium cells at low cell density, where cell-to-cell interactions and morphogen gradients are minimal. We find that under all conditions of low cell density where expression of the ecmA fusion gene occurs, it is invariably detected in less than 25% of the cells from a clonal population. This suggests that a cell-autonomous mechanism is involved in ecmA expression. We then used double label immunofluorescence to examine the ontogeny of the CP2-positive and the ecmA-positive cells. In developing aggregates, 9 to 12% of the cells are CP2-positive from 12 until 24 hours of development. The ecmA-positive cells are first detected at 16 hours as a subset of the CP2-positive cells, and then increase in number. At approximately 20 hours, the CP2-positive cells and the ecmA-positive cells are almost completely overlapping sets. By late development, all of the CP2-positive cells are ecmA-positive and an additional 10% of the CP2-negative cells are also ecmA-positive. This indicates that up to 20 hours of development, ecmA is expressed only in CP2-positive cells. The data thus suggest that cell-cycle phase at the time of starvation causes an initial choice of cell type, and that during later development other factors influence cell fate. ------------------------------------------------------------------------- [End CSM News, volume 5, number 8]