CSM News Electronic Edition Volume 6, number 7 March 23, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Cloning and characterization of a Dictyostelium discoideum cDNA encoding a protein related to the medium chain subunit of clathrin-associated adaptor complexes Lesly A. Temesvari a,b,*, David J. Seastonea,* and James A. Cardelli a,b aDepartment of Microbiology and Immunology and bCenter for Excellence in Cancer Research, Louisiana State University Medical Center,Shreveport, LA 71130, USA *These authors have contributed equally to this study and should be considered co-first authors Gene, in press Abstract We describe the cloning and characterization of a cDNA, DdApm1, encoding a putative medium chain subunit of a clathrin-associated protein (adaptor or assembly protein [AP]) complex in Dictyostelium discoideum. The DdApm1 clone is predicted to encode a polypeptide of 439 amino acids (aa) with a molecular mass of 49.9 kDa. The predicted translation product (DdApm1p) shares at least 51.7% identity and 76.3% similarity with the medium chain subunits of plasma membrane (mb)-associated clathrin AP complexes from rat and Caenorhabditis elegans. The deduced aa sequence also demonstrates significant but lesser homology to a number of medium chain subunits of Golgi-associated clathrin AP complexes. Since DdApm1p demonstrates significantly greater homology to plasma mb-associated clathrin AP complex medium chains than to their Golgi-associated counterparts, we suggest that DdApm1p may be a medium chain subunit of an AP complex involved in clathrin function at the plasma mb of D. discoideum. Southern blot analysis indicated that DdApm1 gene defines a single copy gene in the D. discoideum genome. Northern blot analysis of RNA purified at different times during growth and development demonstrated that the DdApm1 gene is expressed at relatively constant levels throughout the life cycle of the organism. DdApm1 is the first reported full-length cDNA encoding a subunit of an AP complex in D. discoideum, and thus provides the first evidence for the existence of AP complexes in this organism. ---------------------------------------------------------------------- Dictyostelium discoideum myoJ: a member of a broadly defined myosin V class or a class XI unconventional myosin? Michelle D. Peterson, Alexander S. Urioste & Margaret A. Titus Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 J. Musc. Res. Cell Mot., in press. Summary The simple eukaryote Dictyostelium discoideum contains at least twelve unconventional myosin genes. Here we report the characterization of one of these, myoJ, a gene initially identified through a physical mapping screen (Titus et al., Proc. Natl. Acad. Sci. 91:9446-9450). The myoJ gene encodes a high molecular weight myosin, and analysis of the available deduced amino acid sequence reveals that it possesses six IQ motifs and sequences typical of alpha helical coiled coils in the tail region. Therefore, myoJ is predicted to exist as a dimer with up to twelve associated light chains (six per heavy chain). The 7.8 kb myoJ mRNA is expressed all throughout the life cycle of D. discoideum. The myoJ gene has been disrupted and a phenotypic analysis of the mutant cells initiated. Finally, phylogenetic analysis of the head region reveals that myoJ is most similar to two plant myosin genes, Arabidopsis MYA1 and MYA2, that have been alternatively suggested to be either members of the myosin V class or founding members of the myosin XI class. --------------------------------------------------------------------- cAMP dependent protein kinase is required for the expression of a gene specifically expressed in Dictyostelium prestalk cells Natasha Zhukovskaya, Anne Early, Takefumi Kawata, Tomoaki Abe and Jeffrey Williams+ MRC Laboratory of Molecular Cell Biology and Dept of Biology, University College London, Gower St, London WC1E 6BT Dev. Biol., in press. ABSTRACT In the Dictyostelium slug there are two types of prestalk cells, pstA cells and pstO cells, that differ in their ability to utilise the distal and proximal parts of the promoter of ecmA, a gene that is specifically expressed in prestalk cells. When Rm, a dominant inhibitory form of the regulatory subunit of cAMP dependent protein kinase (PKA), is expressed under the control of the complete promoter of the ecmA gene (in a construct termed ecmAO:Rm) development procedes to the slug stage. Although able to form small but outwardly normal slugs, ecmAO:Rm cells are defective in prestalk cell differentiation. In ecmAO:Rm cells, the induction of pstA and pstO-specific gene expression by the stalk cell inducer DIF is greatly inhibited. Paradoxically, a very large fraction of the cells in an ecmAO:Rm slug show evidence of once having expressed the ecmA and ecmO prestalk markers. However, we present evidence that this is due to abortive prestalk cell differentiation that terminates when sufficient Rm protein has accumulated to block PKA activity. This results in regulative transdifferentiation of prespore cells to form prestalk cells. During their transitory period as prestalk cells the ecmAO:Rm cells co-express both the ecmA and ecmO markers, indicating a possible link between PKA activity and divergence of the two prestalk cell sub-types. Finally, we show that the level of the DNA binding activity that is believed to lie at the end of the DIF signal transduction pathway is reduced in ecmAO:Rm slugs. -------------------------------------------------------------------- Myristoylated and non-myristoylated forms of the pH-sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein Frank Hanakam(1), Richard Albrecht, Christoph Eckerskorn, Monika Matzner, and Guenther Gerisch Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany EMBO J., in press. Abstract Hisactophilins are myristoylated proteins that are rich in histidine residues and known to exist in Dictyostelium cells in a plasma-membrane bound and a soluble cytoplasmic state. Intracellular translocation of these proteins in response to pH-changes was monitored using hisactophilin fusions with green fluorescent protein (GFP) and confocal laser scanning microscopy. Both the normal and a mutated non-myristoylated fusion protein shuttled within the cells in a pH-dependent manner. After lowering the pH, proteins translocated within minutes between the cytoplasm, the plasma membrane and the nucleus. The role of histidine clusters on the surface of hisactophilin molecules in binding of the protein to the plasma membrane and in its transfer to the nucleus is discussed on the basis of a pH-switch mechanism. --------------------------------------------------------------------- Oriented binding of a lipid-anchored cell adhesion protein onto a biosensor surface using hydrophobic immobilization and photoactive crosslinking. Thomas Stein(1) and Guenther Gerisch (2) (1) National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Peoria, Illinois, 61604, USA (2) Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany Analytical Biochemistry, in press. Abstract The carboxymethyl-dextran surface of a biosensor instrument was modified to couple, in an active state, the lipid-anchored contact site A (csA) glycoprotein, a homophilic adhesion molecule of aggregating cells of Dictyostelium discoideum. The carboxy groups were modified by heptyl residues for hydrophobic binding of the molecule with its lipid anchor to the dextran matrix. Alternatively, the protein was fixed in a similar orientation by covalent linkage through a perfluorophenylazide-derived hydrophobic crosslinker. Titration of the bound csA molecules with antibodies that recognize either the native or the denatured glycoprotein verified that the csA molecules were coupled in a native state to the sensor surface. Interaction of the immobilized csA protein with csA in solution established that the bound molecules are capable of taking part in homophilic interactions. --------------------------------------------------------------------- Overexpression of Cytoplasmic Dynein's Globular Head Causes a Collapse of the Interphase Microtubule Network in Dictyostelium. Michael P. Koonce and Montserrat Samso Wadsworth Center, Albany, NY 12201-0509 Molecular Biology of the Cell, in press Abstract Cytoplasmic dynein is a minus-end directed microtubule-based motor. Using a molecular genetic approach, we have begun to dissect structure-function relationships of dynein in the cellular slime mold Dictyostelium. Expression of a carboxy-terminal 380-kDa fragment of the heavy chain produces a protein that approximates the size and shape of the globular, mechanochemical head of dynein. This polypeptide cosediments with microtubules in an ATP-sensitive fashion and undergoes a UV-vanadate cleavage reaction. The deleted amino-terminal region appears to participate in dimerization of the native protein and in binding the intermediate and light chains. Overexpression of the 380 kDa carboxy-terminal construct in Dictyostelium produces a distinct phenotype where the interphase radial microtubule array appears collapsed. In many cells, the microtubules form loose bundles that are whorled around the nucleus. Similar expression of a central 107 kDa fragment of the heavy chain does not produce this result. The data presented here suggest that dynein may participate in maintaining the spatial pattern of the interphase microtubule network. ---------------------------------------------------------------------- [End CSM News, volume 6, number 7]