Dicty News Electronic Edition Volume 10, number 11 April 18, 1998 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/dicty.html" =========== Abstracts =========== SKIPPER, AN LTR RETROTRANSPOSON OF DICTYOSTELIUM Ping Leng**, David H. Klatte**, Gerald Schumann**, Jef D. Boeke, and Theodore L. Steck ** These authors contributed equally and are considered joint first authors Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637 and Dept. of Molecular Biology & Genetics, Johns Hopkins Univ. School of Medicine, Baltimore, MD 21205 Nucleic Acids Research, in press Abstract The complete sequence of a retrotransposon from Dictyostelium discoideum, named skipper, was obtained from cDNA and genomic clones. The sequence of a nearly full-length skipper cDNA was similar to that of three other partially sequenced cDNAs. The corresponding retro- transposon is represented in about 15-20 copies and is abundantly transcribed. Skipper contains three open reading frames (ORFs) with an unusual sequence organization, aspects of which resemble certain mammalian retroviruses. ORFs 1 and 3 correspond to gag and pol genes; the second ORF, pro, corresponding to protease, was separated from gag by a single stop codon followed shortly thereafter by a potential pseudoknot. ORF3 (pol) was separated from pro by a +1 frameshift. ORFs 2 and 3 overlapped by 32 bp. The computed amino acid sequences of the skipper ORFs contain regions resembling retrotransposon polyprotein domains, including a nucleic acid binding protein, aspartyl protease, reverse transcriptase and integrase. Skipper is the first example of a retrotransposon with a separate pro gene. Skipper is also novel in that it appears to use stop codon suppression rather than frameshifting to modulate pro expression. Finally, skipper and its components may provide useful tools for the genetic characterization of Dictyostelium. ------------------------------------------------------------------------- A novel Myb homologue initiates Dictyostelium development by induction of adenylyl cyclase expression Hideshi Otsuka and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Genes Dev., in press ABSTRACT Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation and differentiation in neighbouring cells. Using insertional mutagenesis we have isolated a mutant that does not aggregate upon starvation but is rescued by adding extracellular cAMP. Sequencing of the mutated locus revealed a new gene, DdMYB2, whose product contains three Myb repeats, the DNA-binding motif of Myb-related transcription factors. Ddmyb2-null cells show undetectable levels of ACA transcript and no cAMP production. Ectopic expression of ACA from a constitutive promotor rescues differentiation and morphogenesis of Ddmyb2-null mutant. The results suggest that development in Dictyostelium starts by starvation-mediated DdMyb2 activation, which induces adenylyl cyclase activity producing the differentiation-inducing signal cAMP. ------------------------------------------------------------------------- In Vitro Microtubule-based Organelle Transport in Wild-Type Dictyostelium and Cells Overexpressing a Truncated Dynein Heavy Chain Nira Pollock(1), Michael P. Koonce(3), Eugenio L. de Hostos(4), and Ronald D. Vale(2,1) (1)Dept. of Pharmacology and (2)Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94143; (3)Division of Molecular Medicine, Wadsworth Center, Empire State Plaza, Albany, New York 12201-0509; and (4)Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005-1892. Cell Motil. Cytoskel., in press Abstract The transport of vesicular organelles along microtubules has been well documented in a variety of systems, but the molecular mechanisms underlying this process are not well understood. We have developed a method for preparing extracts from Dictyostelium discoideum which support high levels of bidirectional, microtubule-based vesicle transport in vitro. This organelle transport assay was also adapted to observe specifically the motility of vesicles in the endocytic pathway. Vesicle transport can be reconstituted by recombining a high-speed supernatant with KI-washed organelles, which do not move in the absence of supernatant. Furthermore, a microtubule affinity -purified motor fraction supports robust bidirectional movement of the salt-washed organelles. The plus and minus end-directed transport activities can be separated by exploiting differences in their affinities for microtubules in the presence of 0.3 M KCl. We also used our assay to examine organelle transport in a strain of Dictyostelium overexpressing a 380-kDa carboxy-terminal fragment of the cytoplasmic dynein heavy chain, which displays an altered microtubule pattern (380-kDa cells; Koonce and Samso, Mol. Biol. Cell, 7: 935-948, 1996). We have found that the frequency and velocity of minus end-directed membrane organelle movements were significantly reduced in 380-kDa cells relative to wild- type cells, while the frequency and velocity of plus end-directed movements were equivalent in the two cell types. The 380-kDa carboxy-terminal fragment cosedimented with membrane organelles, although its affinity was significantly lower than that of native dynein. An impaired membrane-microtubule interaction may be responsible for the altered microtubule patterns in the 380-kDa cells. ------------------------------------------------------------------------- Conditional Loss of Myosin-II Function Mutants Reveal a Position in the Tail that is Critical for Filament Nucleation Sheri L. Moores and James A. Spudich Department of Biochemistry, Stanford University School of Medicine Stanford, CA 94305 Molecular Cell, in press Summary Myosin-II must be assembled into filaments to perform its cellular functions. Two conditional loss of myosin-II function mutants were recovered from a genetic screen (Patterson and Spudich, 1995) with defects that were mapped to the coiled coil tail region of Dictyostelium myosin-II. Strikingly, both tail mutations affected the same arginine residue at position 1880. A single amino acid substitution, R1880P, disrupted both the dimerization and tetramerization steps of filament nucleation. Even a single charge reversal at this position, R1880D, was sufficient to inhibit filament assembly, while other single charge reversals in the region of antiparallel contact suppressed these filament assembly mutants. The considerable impact of small electrostatic forces on nucleation suggests that these steps are delicately balanced. ------------------------------------------------------------------------- EXPRESSION OF STARVATION-INDUCED GENES IN ZYGOTES OF Dictyostelium discoideum MIHO IIJIMA, KAZUHIRO AIBA*, YOSHIMASA TANAKA and HIDEKO URUSHIHARA INSTITUTE OF BIOLOGICAL SCIENCES UNIVERSITY OF TSUKUBA TSUKUBA-SHI, IBARAKI 305 JAPAN * Present address: Doi Bioassymmetry Project, ERATO, JST, Tsukuba Research Consortium, Tsukuba-shi, Ibaraki 300-26, Japan Journal of Plant Research, in press. ABSTRACT Two cDNA fragments induced in developing zygotes of Dictyostelium discoideum were isolated by mRNA differential display. The relevant genes were also found to be expressed during asexual development, suggesting that sexual and asexual development share common molecular mechanisms in D. discoideum. -------------------------------------------------------------------------- A Dictyostelium discoideum homologue to Tcp-1 is essential for growth and development Miho Iijima, Hajime Shimizua, Yoshimasa Tanaka and Hideko Urushihara* Institute of Biological Sciences, University of Tsukuba Tsukuba-shi, 305-8572, JAPAN *Address for correspondence: Institute of Biological Sciences, 305-8572 Japan; TEL. +81-298-53-4664; FAX. +81-298-53-6614; e-mail: d402hu@sakura.cc.tsukuba.ac.jp GENE, in press. Abstract Tcp-1 (t-complex polypeptide 1 gene) was first identified in the mouse as relevant for tail-less and embryonic lethal phenotypes. Since then, its homologous sequences have been isolated in several other species, and the yeast Tcp-1 has been shown to encode a molecular chaperon for actin and tubulin. In a random sample of genes expressed in the gamete of Dictyostelium discoideum (Dd), we encountered a sequence containing the TCP1 motifs. The complete ORF of the gene (DdTcp-1) showed more than 60% similarity to TCP-1 of several organisms including human. DdTcp-1 was found to be expressed in both sexually mature and immature cells at the growth phase. Although the sexual process itself was not affected, antisense interference of this gene resulted in severe retardation of cell growth, leading to the complete cessation of division. In addition, the antisense transformants stopped asexual development at the finger stage. These results suggest an important function of DdTcp-1 in growth and development of this organism. ------------------------------------------------------------------------- Microtubule mediated centrosome motility and the positioning of cleavage furrows in multinucleate myosin II-null cells Ralph Neujahr, Richard Albrecht, Jana Köhler, Monika Matzner, Jean-Marc Schwartz, Monika Westphal, and Günther Gerisch Max-Planck-Institut für Biochemie D-82152 Martinsried, Germany J. Cell Science, in press. Summary To study centrosome motility and the interaction of microtubules with the cell cortex in mitotic, post-mitotic and interphase cells, a-tubulin was tagged in Dictyostelium discoideum with GFP. Multinucleate cells formed by myosin II-null mutants proved to be especially suited for the analysis of the control of cleavage furrow formation by the microtubule system. After docking of the mitotic apparatus onto the cell cortex during anaphase, the cell surface is activated to form ruffles on top of the asters of microtubules that emanate from the centrosomes. Cleavage furrows are initiated at spaces between the asters independent of the positions of spindles. Once initiated, the furrows expand as deep folds without a continued connection to the microtubule system. Occurrence of unilateral furrows indicates that a closed contractile ring is dispensable for cytokinesis in Dictyostelium. The progression of cytokinesis in the multinucleate cells underlines the importance of proteins other than myosin II in specifying a cleavage furrow. The analysis of centrosome motility suggests a major role for a minus-end directed motor protein, probably cytoplasmic dynein, in applying traction forces on guiding microtubules that connect the centrosome with the cell cortex. ------------------------------------------------------------------------- Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342 I. Tatischeff(a)*, M. Bomsel(b), C. de Paillerets(c), H. Durand(b), B. Geny(b), D. Segretain(d), E. Turpin(b) and A. Alfsen(c) (a) : Laboratoire de Physicochimie Biomoléculaire et Cellulaire, CNRS URA 2056, Université Pierre et Marie Curie, 4, Place Jussieu, Case 138,F-75252 Paris Cedex 05, France. e-mail: tati@lpbc.jussieu.fr (b) : Signalisation, Inflammation et Transformation Cellulaire, U. 332, Institut Cochin de Génétique Moléculaire, 22, rue Méchain,F-75014 Paris, France © : Etats Liés Moléculaires, Université René Descartes, 45, rue des Saints-Pères,F-75270 Paris Cedex 06, France (d) : Laboratoire d'Histologie et Embryologie, CHU Paris-Ouest, Faculté de Médecine, 45, rue des Saints-Pères,F-75270 Paris Cedex 06, France Cellular and Molecular Life Sciences, in press. Abstract Dictyostelium discoideum cells are highly resistant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170 kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but non-functional in Dictyostelium cells. We show here that Dictyostelium discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size >21 kb. Shedding of microvesicles during cell growth is not concomitant to programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 11]