Dicty News Electronic Edition Volume 10, number 15 June 20, 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 =========== Spatiotemporal Dynamics of Actin Concentration during Cytokinesis and Locomotion in Dictyostelium Shigehiko Yumura*1) and Yoshio Fukui*2) *1) Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan, *2) Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA. Journal of Cell Science, in press Summary To study the spatial and temporal regulation of the actin cytoskeleton, we have analyzed the actin concentration dynamics in live Dictyostelium. The relative actin concentration was analyzed with respect to cell behavior by fluorescence morphometry. We electroporated rhodamine-actin into Dictyostelium cells and acquired images with 200-300 millisecond temporal and ~250 nm spatial resolutions. To convert fluorescence intensity into actin concen tration, the observation was made on nearly two-dimensional cells, and the actin signal was ratioed over a volume marker (FITC -BSA or GFP). Since the emission of FITC and GFP is pH-dependent, we first measured the cytoplasmic pH in live cells and determined that the pHi in pseudopods is same as that of general cytoplasm. During cytokinesis, the relative concentration of acti n in the cleavage furrow was significantly higher than in the general cytopl asm. In migrating cells, actin was recruited surprisingly rapidly, particul arly in the pseudopod. We found that the region of high actin concentration moves relative to the leading edge when a pseudopod projects or retracts. W hen the pseudopod retracts, the actin density dissipates within 5 seconds. We have also found that actin accumulates in developing pseudopods in an osc illatory manner, and this timing coordinates with advancement of the centroid. This is the first study to reveal the dynamic changes in relative concen tration of actin in live cells and to quantitatively correlated these changes with the locomotive behavior of the amoeba. ------------------------------------------------------------------------- Rapid changes of Nucleotide Excision Repair gene expression following UV-irradiation and cisplatin treatment of Dictyostelium discoideum Sung-Lim Yu1,2, Sung-Keun Lee1,2, Hannah Alexander1 and Stephen Alexander1* Division of Biological Sciences, 422 Tucker Hall, University of Missouri, Columbia, MO 65211-7400. Nucleic Acids Research, in press ABSTRACT Organisms use different mechanisms to detect and repair different types of DNA damage, and different species vary in their sensitivity to DNA damaging agents. The cellular slime mold Dictyostelium discoideum has been long recognized for its unusual resistance to UV and ionizing radiation. We have recently cloned three Nucleotide Excision Repair (NER) genes from Dictyostelium, the repB, D and E genes (the homologs of the human xeroderma pigmentosum group B, D and E genes, respectively). Each of these genes has a unique pattern of expression during the multicellular development of this organism. We have now examined the response of these genes to DNA damage. The repB and D DNA helicase genes are rapidly and transiently induced in a dose dependent manner following exposure to both UV-light and the widely used chemotherapeutic agent cisplatin. Interestingly, the repE mRNA level is repressed by UV but not by cisplatin, implying unique signal transduction pathways for recognizing and repairing different types of damage. Cells from all stages of growth and development display the same pattern of NER gene expression following exposure to UV-light. These results suggest that the response to UV is independent of DNA replication, and that all the factors necessary for rapid transcription of these NER genes are either stable throughout development, or are continuously synthesized. It is significant that the up-regulation of the repB and D genes in response to UV and chemical damage has not been observed to occur in cells from other species. We suggest that this rapid expression of NER genes is at least in part responsible for the unusual resistance of Dictyostelium to DNA damage. ---------------------------------------------------------------------------------- A mutation in repB, the Dictyostelium homolog of the human xeroderma pigmentosum B gene, has increased sensitivity to UV-light but normal morphogenesis Sung-Keun Lee1,2, Sung-Lim Yu1,2, Hannah Alexander1 and Stephen Alexander1* Division of Biological Sciences, 422 Tucker Hall, University of Missouri, Columbia, MO 65211-7400. Biochemica et Biophysica Acta, in press Summary Nucleotide Excision Repair (NER) is an important cellular defense mechanism which protects the integrity of the genome by removing DNA damage caused by UV- light or chemical agents. In humans, defects in the NER pathway result in the disease xeroderma pigmentosum (XP) which is characterized by increased UV- sensitivity, with increased propensity for skin cancer, and an array of developmental abnormalities. Some XP patients exhibit in addition symptoms of Cockayne’s syndrome (CS) and Trichothiodystrophy (TTD), which are characterized by increased UV- sensitivity, without increased cancer incidence, and an array of developmental abnormalities. Some NER genes, including the DNA helicases XPB and XPD, have been shown to function in transcription as well as repair, by virtue of being an integral part of the transcription initiation factor TFIIH. This dual function may account for the above mentioned wide pleiotropy of phenotypes associated with defects in NER genes, and may explain why some XP patients exhibit developmental abnormalities in addition to XP symptoms. To date, only five XPB patients with three different mutations in the XPB gene have been reported. One of these mutations is a C to A transversion at the splice site at the beginning of the last exon, which resulted in a frameshift throughout the last exon. This patient shows combined clinical symptoms of XP and CS. The recent cloning of the repB gene, the Dictyostelium discoideum homolog of XPB, allowed us to generate a similar C-terminal mutation in the Dictyostelium, in order to test whether the defect in this NER gene has an effect on growth or development. To this end we have constructed a C-terminal deletion repB mutant in Dictyostelium. To avoid the possibility that a null mutant would be lethal, we used direct homologous recombination to create a 46 amino acid C-terminal deletion mutant. Indeed, we were unable to obtain mutants with a longer 95 amino acid deletion. The repBDC46 mutants showed an increased sensitivity to UV-light, but a normal pattern of UV-induced expression of repair genes, and no immediately obvious defect in either growth rate or development. The results suggest that the associated developmental defects in the human XPB patients may be due to mutations in another gene. ------------------------------------------------------------------------- A novel Dictyostelium cell surface protein important for both cell adhesion and cell sorting Rebecca S. Ginger, Lucy Drury, Christine Baader, Natasha V. Zhukovskaya and Jeffrey G. Williams MRC Laboratory of Molecular Cell Biology and Department of Biology, University College London, Gower St., London WC1E 6BT DEVELOPMENT, in press SUMMARY A mutant of Dictyostelium that is aberrant in the process of tip formation (dtfA-: defective in tip formation A) has been isolated by gene tagging. The dtfA gene is predicted to encode a protein of 163 Kd. There are no extensive sequence homologies between DTFA and previously identified proteins, but four short N-terminal sequence motifs show partial homology to repeats found in mammalian mucins. Immuno-fluorescence reveals a lattice-like arrangement of DTFA protein at the cell surface. When developing on a bacterial lawn, cells of the mutant strain (dtfA- cells) aggregate to form tight mounds, but development then becomes arrested. When developed in the absence of nutrients, a fraction of dtfA- cells complete development, but there is a long delay at the tight mound stage and the culminants that eventually form are aberrant. In such dtfA- mounds the prestalk cells fail to move to the apex on cue and so tip formation is delayed. DtfA- cells also show a conditional defect in early development, in that they are unable to aggregate when plated at low density. In addition dtfA- cells do not agglomerate efficiently when shaken in suspension. In combination, these results suggest that DTFA may form part of a cell-cell adhesion system that is needed both for optimal aggregation and for efficient cell sorting during multicellular development. The DTFA protein also apears to be important during cell growth, because cytokinesis is defective and the actin cytoskeleton aberrant in growing dtfA- cells. ------------------------------------------------------------------------- Developmentally and spatially regulated activation of a Dictyostelium STAT protein by a serpentine receptor Tsuyoshi Araki1*, Marianne Gamper2*, Anne Early1, Masashi Fukuzawa1, Tomoaki Abe1, Takefumi Kawata1, Eugene Kim2, Richard A. Firtel2 and Jeffrey G. Williams1,3 1MRC Laboratory of Molecular Cell Biology and Department of Biology University of College London, Gower Street London WC1E 6BT United Kingdom 2Department of Biology, Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla CA 92093-0634, USA *Co-first authors, both authors contributed equally. EMBO J., in press SUMMARY Dd-STAT, the protein that in part controls Dictyostelium stalk cell differentiation, is a structural and functional homolog of metazoan STATs. Although present during growth and throughout development, Dd-STAT's tyrosine phosphorylation and nuclear localization are developmentally and spatially regulated. Prior to late aggregation, Dd-STAT is not tyrosine phosphorylated and is not selectively localized in the nucleus. During mound formation, the time at which cell-type-specific gene expression initiates, Dd-STAT becomes tyrosine phosphorylated and translocates into the nuclei of all cells. The tyrosine phosphorylation and nuclear localization of Dd-STAT are very rapidly induced by extracellular cAMP through the serpentine cAMP receptor cAR1, with Dd-STAT tyrosine phosphorylation being detectable within 10 sec of stimulation. This activation is independent of the only known Gb subunit, suggesting that it may be G protein independent. Nuclear enrichment of Dd-STAT is selectively maintained within the sub-population of prestalk cells that form the tip, the organizing center of the slug, but is lost in most of the other cells of the slug. This spatial patterning of Dd-STAT nuclear localization is consistent with its known role as a negative regulator of stalk cell differentiation. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 15]