Dicty News Electronic Edition Volume 14, number 6 March 4, 2000 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" ================ Announcement ================ NIH has a new model organism. See: http://www.nih.gov/science/models/ Our thanks should go to the effort spearheaded by Peter Devreotes, Rich Kessin, Bill Loomis and Carole Parent and the other members of the "Dictyostelium Advisory Committee". OF course, this just signals the beginning a much hard work to be done. Also, many of you may have noticed a new addition to the Dicty web site, Dicty in the News. When any of you has a paper the receives a "News and Views" type commentary, please let let me know. I'll feature it in a similar way. ============= Abstracts ============= Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host-pathogen interactions Jonathan M. Solomon1, Adam Rupper2, James A. Cardelli2, and Ralph R. Isberg1 1Howard Hughes Medical Institute Dept of Microbiology Tufts University Medical School Boston, MA 02111 2Department of Microbiology and Immunology LSU Health Sciences Center Shreveport, LA 71130 Accepted: Infection and Immunity Abstract Conditions were established in which Legionella pneumophila, an intracellular bacterial pathogen, could replicate within the unicellular organism Dictyostelium discoideum . By several criteria, L. pneumophila grew by the same mechanism within D. discoideum as it does in amoebae and macrophages. Bacteria grew within membrane-bound vesicles associated with rough endoplasmic reticulum, and L. pneumophila dot/icm mutants, blocked for growth in macrophages and amoebae, also did not grow in D. discoideum. Internalized L. pneumophila avoided degradation by D. discoideum and showed evidence of reduced fusion with endocytic compartments. The ability of L. pneumophila to grow within D. discoideum depended on the growth state of the cells. D. discoideum grown as adherent monolayers were susceptible to L. pneumophila infection and to contact-dependent cytotoxicity during high multiplicity infections, whereas D. discoideum grown in suspension were relatively resistant to cytotoxicity and did not support intracellular growth. Some known D. discoideum mutants were examined for their effect on growth of L. pneumophila. The coronin mutant and the double myosin I mutant, myoA/B, were more permissive than wild-type strains for intracellular growth. Growth of L. pneumophila in a G-beta mutant was slightly reduced compared to the parent strain. This work demonstrates the usefulness of the L. pneumophila/D. discoideum system for genetic analysis of host-pathogen interactions. ---------------------------------------------------------------------------- Functional overlap of the Dictyostelium RasG, RasD and RasB proteins. Meenal Khosla1, George B. Spiegelman1,2, Rob Insall3 and Gerald Weeks1,2 1: Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3 2: Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z3 3: Department of Biochemistry, University of Birmingham, Birmingham, UK Journal of Cell Science, in press Abstract Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG- cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects. Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG-) cells, corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG- cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG- cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD. Introduction of the rasB gene under the control of the rasG promoter into rasG- cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG- cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG- cells than they were in the wild type parental, suggesting that RasG also negatively regulates rasB expression in vegetative cells. Overexpression of rasB in the rasG- cells also reduced the level of RasD protein. In view of the fact that alternate Ras proteins correct some, but not all, of the defects exhibited by the rasG- cells, we propose that RasG interacts with more than one downstream effector. In addition, it is clear that the levels of the various Ras proteins are tightly regulated in vegetative cells and that overexpression can be deleterious. ---------------------------------------------------------------------------- Light affects cAMP signaling and cell movement activity in Dictyostelium discoideum Kota Miura and Florian Siegert* Zoologisches Institut, Universität München, Luisenstr. 14, 80333 München, Germany PNAS 2000 97:2111-2116 Abstract The multicellular, slug stage of the slime mould Dictyostelium discoideum lacks specific sensory cells and organs but can never-theless respond in a very sensitive manner to external stimuli such as temperature and light. Within the migrating slug, the behavior of up to 100,000 individual amoebae is coordinated by cAMP mediated cell? cell signaling and chemotaxis. We report here the striking result that light directly modulates the cAMP cell? cell signaling system. Light-induced secretion of cAMP from the slug tips decreased the period length of optical density waves and speeded up cell movement. A local effect of light on cAMP release within the slug tip could modulate cell movement within the slug and thus control its phototactic turning and orientation toward a light source. ---------------------------------------------------------------------------- Dictyostelium DdCP224 is a microtubule-associated protein and a permanent centrosomal resident involved in centrosome duplication Ralph Gräf*, Christine Daunderer and Manfred Schliwa Adolf-Butenandt-Institut / Zellbiologie, Schillerstr. 42, D-80336 München / Germany J. Cell Sci., in press A cDNA encoding a 224-kD Dictyostelium discoideum centrosomal protein (DdCP224) was isolated by immunoscreening. DdCP224 was detected at the centrosome and, more weakly, along microtubules throughout the entire cell cycle. Centrosomal localization does not require microtubules, suggesting that DdCP224 is a genuine centrosomal component. DdCP224 exhibits sequence identity to a weakly conserved class of microtubule-associated proteins including human TOGp and yeast Stu2p. Stu2p has a size of only ~100 kD and corresponds to the N-terminal half of DdCP224. The functions of the N- and C-terminal halves of DdCP224 were investigated in the corresponding GFP-fusion mutants. Surprisingly, the N-terminal construct showed only cytosolic localization, whereas the C-terminal construct localized exclusively to the centrosome. This is unexpected because Stu2p is localized at the spindle pole body. Full-length DdCP224-GFP was present both at centrosomes and along microtubules. Furthermore, it bound to microtubules in vitro, unlike the two truncated mutants. Thus centrosome binding is determined by the C-terminal half and microtubule binding may require the interaction of the N- and C-terminal halves. Interestingly, cells expressing full-length DdCP224-GFP exhibit supernumerary centrosomes and show a cytokinesis defect, suggesting that DdCP224 plays an important role in centrosome duplication. These features are unique among the known centrosomal proteins. ---------------------------------------------------------------------------- Purification and Cloning of Phosphatidylinositol Transfer Proteins from Dictyostelium discoideum: Homologues of Both Mammalian PITPs and S. cerevisiae Sec14p are Found in the Same Cell Philip Swigart, Robert Insall, Andrew Wilkins and Shamshad Cockcroft In press, Biochemical Journal Soluble phosphatidylinositol transfer proteins (PITPs) play important roles in lipid-mediated signalling as well as in membrane traffic. Two PITPs (PITPa and b) have been cloned from mammalian cells, which are unrelated in sequence to yeast PITP (the product of the SEC14 gene). However, all three PITPs can perform interchangeably to reconstitute function in mammalian cells. We have now purified the major PITP from the cytoplasm of Dictyostelium discoideum and cloned the gene. This protein, DdPITP1, is homologous to mammalian PITPs, a and b. We have also cloned a second gene (DdPITP2) which is related in sequence to DdPITP1. In addition, an independently cloned cDNA encodes a relative of the SEC14 family of yeast PITPs. DdPITP1, DdPITP2 and DdSec14 proteins were all able to mediate the transfer of PI from one membrane compartment to another, and thus exhibited the hallmark of PITPs. Secondly, all three PITPs were able to rescue phospholipase C-mediated phosphoinositide hydrolysis in PITP-depleted HL60 cells, indicating that all three PITPs were capable of stimulating phosphoinositide synthesis. The identification of PITPs related to both mammalian PITPs and yeast Sec14p in a single organism will provide a unique opportunity to examine the functions of this class of proteins using genetic approaches. ---------------------------------------------------------------------------- gdt1, a new signal transduction component for negative regulation of the growth-differentiation-transition in Dictyostelium discoideum. Changjiang Zeng1, Christophe Anjard1, Karsten Riemann1, Angelika Konzok2 and Wolfgang Nellen1* Mol. Biol. of the Cell, in press. Abstract Discoidin I expression was used as a marker to screen for mutants affected in the growth-differentiation-transition (GDT) of Dictyostelium. By REMI mutagenesis we have isolated mutant 2-9, an overexpressor of discoidin I. It displays normal morphogenesis but shows premature entry into the developmental cycle. The disrupted gene was denominated gdt1. The mutant phenotype was reconstructed by disruptions in different parts of the gene, suggesting that all had a complete loss of function. gdt1 was expressed in growing cells, the levels of protein and mRNA appear to increase with cell density and rapidly decrease with the onset of development. gdt1 encodes a 175 kDa protein with 4 putative transmembrane domains. In the C-terminus, the derived amino acid sequence displays some similarity to the catalytic domain of protein kinases. Mixing experiments demonstrate that the gdt1- phenotype is cell autonomous. The prestarvation factor PSF is secreted at wild type levels. The response to folate, a negative regulator of discoidin expression, was not impaired in gdt1 mutants. Cells which lack the G protein a2, display a loss of discoidin expression and do not aggregate. gdt1-/Ga2- double mutants show no aggregation but strong discoidin expression. This suggests that gdt1 is a negative regulator of the GDT downstream of, or in a parallel pathway to Ga2. ---------------------------------------------------------------------------- The Biology of Cytokinesis Review as well as original papers will be published in a topical issue of Microscope Research and Technique (MRT) entitled "The Biology of Cytokinesis", Volume 49, Number 2. This issue is scheduled to be published as the cover issue dated April 15, 2000, and will include 3 papers from Dictyostelium labs. Focus of this issue is on the dynamics of calcium, cytoskeletal and signal molecules during cytokinesis in S. pombe, S. saccharomyces, Dictyostelium, Tetrahymena, zebrafish, and tissue culture cells. Below is a tetative Table of Contents of this issue. Table of Contents 1. Yoshio Fukui (Guest Editor: Introductory Review) "Signal to forces: Central themes in cytokinesis" 2. Donald C. Chang, and Pin Lu "Multiple types of calcium signals are associated with cell cleavage in zebrafish embryo" 3. Pascal Madaule, Tomoyuki Furusawa, Masatoshi Eda, Haruhiko Bito, Toshimasa Ishizaki, and Shu Narumiya "Citron: a Rho target that affects contractility during cytokinesis" 4. Osamu Numata, Kohsuke Gonda, Atsushi Watanabe, and Yasuhiro Kurasawa "Cytokinesis in Tetrahymena: Determination of division plane and organization of contractile ring" 5. Taro Uyeda and Shigehiko Yumura "Molecular biological approaches to study myosin functions in cytokinesis of Dictyostelium" 6. Denis Larochelle, Noel Gerald, and Arturo De Lozanne "Molecular analysis of racE function in Dictyostelium" 7. Dan P. Mulvihill, Thein Z. Win, Thomas P. Pack, and Jeremy S. Hyams. "Cytokinesis in fission yeast: a myosin pas de deux" 8. Fred Chang "Microtubule and actin-dependent movement of the formin cdc12p in fission yeast". 9. John Lippincott and Rong Li "The involvement of PCH family proteins in cytokinesis and actin distribution" 10. Hidemasa Goto, Hidetaka Kosako, and Masaki Inagaki "Regulation of intermediate filament organization during cytokinesis: Possible roles of rho-associated kinase" 11. Yoshio Fukui "Real-time high-resolution optical sectioning suggests biphasic cytokinetic mechanism in Dictyostelium discoideum" 12. Jean M. Sanger and Joseph W. Sanger "Assembly of cytoskeletal proteins into cleavage furrows of tissue culture cells" 13. Maurizio Gatti, Maria G. Giansanti, and Silvia Bonaccorsi "The relationships between the central spindle and the contractile ring during cytokinesis in animal cells". ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 6]