Dicty News Electronic Edition Volume 12, number 4 January 30, 1999 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" ======================== Important Announcement ======================== NIH Workshop on Non-Mammalian Model Organisms. February 16 and 17, 1999 Harold Varmus has invited about 100 top scientists from around the country to a meeting at NIH in February to consider the strengths, weaknesses and needs of a variety of non-mammalian model organisms. While S. cerevisiae, Drosophila, C. elegans, zebrafish and Xenopus are established model organisms, other organisms may be added to the list in the future. I have been invited to present a 10 minute pitch for Dictyostelium. I will be joined by Rich Kessin and Carole Parent at this workshop. Other organisms under consideration include Tetrahymena, Neurosporra, S. pombe, Chlamydemonas, Aplysia, Sea urchins, Fugu and chickens. They specifically asked that we discuss the advantages of the organism and outline the needs of the field. They encouraged us to prioritize the specific requests. Each speaker is limited to 3 overheads and so space is at a premium. With this in mind, I have prepared drafts of the overheads presenting the advantages of Dictyostelium and the resources that will advance functional genomics in the field. Since decisions made following this meeting will affect us all, I encourage everyone to consider the Advantages and Resources for the Future of Dictyostelium and let me know their views. We will then be able to present a consensus view of the field. Bill Loomis ADVANTAGES OF DICTYOSTELIUM AS A MODEL SYSTEM 1) Microbial and molecular genetics permit rapid construction of complex genotypes 2) Detailed maps of the 6 chromosomes ( resolution ± 50 kb) 3) 34 Mb genome in the process of being sequenced (near completion in 2001) 4) Efficient homologous recombination permits gene replacement 5) Diverse phenotypes including: a) phagocytosis b) cytokinesis c) cell motility d) chemotaxis e) other cell-cell signaling networks 6) Reliable, inexpensive cell growth and strain storage 7) Ready recovery of each cell type in biochemical quantities throughout the 24 hour developmental cycle. RESOURCES FOR THE FUTURE 1) Completion of the sequence of the genome (finishing steps). 2) Focused funtional analyses of conserved genes in diverse contexts. 3) Arrayed DNA on chips to permit expression studies of every ORF throughout the 24 hour developmental cycle. 4) High resolution computer-assisted microscopy of pertinent mutant strains. 5) Proteome studies - patterns of protein accumulation in wild type and mutant strains throughout the 24 hour developmental cycle. 6) User-friendly databases of genomic, mutational, and analytic information. 7) Centralized stock storage and distribution center. [Editor's note: I would encourage everyone who has thoughts or suggestions to share them with the Dictyostelium community via the listserv (dicty@listserv.acns.nwu.edu). It would be great to have an active discussion of these issues with everyone participating.] ============== Abstracts ============== Multiple developmental roles for CRAC, a cytosolic regulator of adenylyl cyclase. Bin Wang1, Gad Shaulsky2 and Adam Kuspa1 1. Verna and Marrs McLean Department of Biochemistry, 2. Department of Molecular and Human Genetics Baylor College of Medicine, Houston, Texas, USA 77030 Devel. Biol., in press SUMMARY Receptor-mediated activation of adenylyl cyclase (ACA) in Dictyostelium requires CRAC protein. Upon translocation to the membrane, this pleckstrin homology (PH) domain protein stimulates ACA thereby mediating developmental aggregation. CRAC may also have roles later in development since CRAC-null cells can respond to chemotactic signals and participate in developmental aggregation when admixed with wild-type cells, but they do not complete development within such chimeras. To test whether the role of CRAC in post-aggregative development is related to the activation of ACA, chemotactic aggregation was bypassed in CRAC-null cells by activating the cAMP-dependent protein kinase (PKA). While such strains formed mounds, they did not complete fruiting body morphogenesis or form spores. Expression of CRAC in the prespore cells of these strains rescued sporulation and fruiting body formation. This later function of CRAC does not appear to require its PH domain since the C-terminal portion of the protein (CRAC-DPH) can substitute for full-length CRAC in promoting spore cell formation and morphogenesis. No detectable ACA activation was observed in any of the CRAC-null strains rescued by PKA activation and expression of CRAC-DPH. Finally, we found that the development of CRAC- null ACA-null double mutants could be rescued by the activation of PKA together with the expression of CRAC-DPH. Thus, there appears to be a required function for CRAC in post-aggregative development that is independent of its previously described function in the ACA activation pathway. ---------------------------------------------------------------------------- Identification of a suppressor of the Dictyostelium profilin-minus phenotype as a CD36/LIMPII homologue Iakowos Karakesisoglou*‡, Klaus-Peter Janssen*, Ludwig Eichinger, Angelika A. Noegel§, and Michael Schleicher *These authors contributed equally to this work. A.-Butenandt-Institut für Zellbiologie, Ludwig-Maximilians-Universität, 80336 München, FRG. §Institut für Biochemie I, Universität zu Köln, 50931 Köln, FRG. ‡ present address: Howard Hughes Medical Institute, Department of Molecular Genetics, Cell Biology, Biochemistry, and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA. J. Cell Biol., in press Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme- mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2-terminus and the other at the COOH-terminus. It is homologous to mammalian CD36/LIMPII and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ring-like structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is yet unclear how far upstream in a regulatory cascade DdLIMP might be positioned. The PIP2 binding of DdLIMP however points towards a funcion via the phosphatidylinositol pathway, a major regulator of profilin. ---------------------------------------------------------------------------- In vivo observations of myosin II dynamics support a role in rear retraction. Patricia A. Clow and James G. McNally Department of Biology and Institute for Biomedical Computing, Washington University, Box 1229, St. Louis, Missouri 63130 Molecular Biology of the Cell, in press. Summary. To investigate myosin II function in cell movement within a cell mass, we imaged GFP-myosin heavy chain (GFP-MHC) cells moving within the tight mound of Dictyostelium discoideum. In the posterior cortex of cells undergoing rotational motion around the center of the mound, GFP-MHC cyclically formed a "C" which converted to a spot as the cell retracted its rear. Consistent with an important role for myosin in rotation, cells failed to rotate when they lacked the myosin II heavy chain (MHC-) or when they contained predominantly monomeric myosin II (3xAsp). In cells lacking the myosin II regulatory light chain (RLC-), rotation was impaired and eventually ceased. These rotational defects reflect a mechanical problem in the 3xAsp and RLC- cells, since these mutants exhibited proper rotational guidance cues. MHC- cells exhibited disorganized and erratic rotational guidance cues, suggesting a requirement for the MHC in organizing these signals. However, the MHC- cells also exhibited mechanical defects in rotation, since they still moved aberrantly when seeded into wild-type mounds with proper rotational guidance cues. The mechanical defects in rotation may be mediated by the "C"-to-spot, since RLC- cells exhibited a defective "C"-to-spot, including a slower "C"-to-spot transition, consistent with this mutant’s slower rotational velocity. ---------------------------------------------------------------------------- Binding and catalytic properties of the Cdc2 and Crp proteins of Dictyostelium Shiv K. Sharma, Christine Michaelis, Ki-Young Lee, Jerry H. Wang and Gerald Weeks Department of Microbiology and Immunology1, and Department of Medical Genetics5, University of British Columbia, Vancouver, B. C. V6T 1Z3 Canada Exp. Cell Res., in press ABSTRACT Dictyostelium expresses at least two proteins of the Cdk family of protein kinases, Cdc2 and Crp. Cdc2 levels remain relatively constant during differentiation, whereas the levels of Crp increase dramatically as differentiation progresses. Crp is highly related to the mammalian Cdk5 and p25, a truncated form of p35, the activating subunit of Cdk5 from mammalian brain, stimulates the histone H1 kinase activity of GST-Crp by several fold. In contrast, p25 does not stimulate the histone H1 kinase activity of GST-Cdc2 or the Cdc2 activity present in cell extracts from vegetative Dictyostelium cells. GST-Cdc2, in vitro translated Cdc2 and Cdc2 from all stages of differentiation bind to p13suc1. In contrast, GST-Crp, in vitro translated Crp and the Crp protein present in cell extracts do not bind to p13suc1. We have confirmed a previous report by Arakane and Maeda (J. Plant Res. 1997 110, 81-85.) that there is a peak of p13suc1 bound histone H1 kinase activity during late development, but we found that there was no corresponding peak of p13suc1 bound Cdc2 protein that corresponds to this activity. Taken together, these data suggest that neither Cdc2 nor Crp is responsible for the late developmental peak of histone H1 kinase activity that binds to p13suc1. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 4]