CSM News Electronic Edition Volume 6, number 15 June 8, 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" ======================== Dicty Proteome Project ======================== Keith Williams writes: As some will know, we have been developing a complementary approach to DNA sequencing based on mass protein screening which is becoming known as Proteome Analysis. The Australian Government has awarded us 7 million to establish a national facility for mass protein screening based on 2D gel separation and post separation analysis of separated proteins using a range of techniques (Edman sequencing, amino acid analysis, mass spectrometry). To conduct individual projects in the facility, one is required to supply staff and consumables. Because of our long experience in Dictyostelium, we will be using Dictyostelium to sort our some of the technical problems involved with making proteome studies routine eg. sample extraction, analysis of post-translational modifications etc. The intention to sequence the Dictyostelium genome is a wonderful opportunity to couple this with a complete proteome study on Dictyostelium. This would make Dictyostelium the first eukaryotic organism where such a complete survey is attempted although similar studies are planned for yeast currently in Europe. The developmental side of Dictystelium makes it a more challenging project than yeast. I suggest that we should look to coordinating the genome and proteome work in Dictyostelium and look forward to your suggestions as to how this can be conducted. Funding is obviously an issue that needs to be addressed. Comments please. Both the Proteomic and Genomic programs will presumably form a section at the Dictyostelium meeting in Japan but we may need to initiate some things before then. The proteome stuff has become possible using immobilised pH gradients and high loading (mgs) of protein on the 2D gels as pioneered by a number of European groups, notably Hochstrasser in Switzerland. Best regards Keith Williams Professor Keith Williams School of Biological Sciences Macquarie University email keith.williams@mq.edu.au =========== Abstracts =========== A Dictyostelium mutant with defective aggregate size determination Debra A. Brock1, Greg Buczynski2, Timothy P. Spann1*, Salli A. Wood1, James Cardelli2, and Richard H. Gomer1 1Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892 and 2Department of Microbiology and Immunology, LSU Medical Center, Shreveport, LA 71130 Development, in press. Abstract Starved Dictyostelium cells aggregate into groups of roughly 10e5 cells. We have identified a gene which, when repressed by antisense transformation or homologous recombination, causes starved cells to form large numbers of small aggregates. We call the gene smlA for small aggregates. A roughly 1.0 kb smlA mRNA is expressed in vegetative and early developing cells, and the mRNA level then decreases at about 10 hours of development. The sequence of the cDNA and the derived amino acid sequence of the SmlA protein show no significant similarity to any known sequence. There are no obvious motifs in the protein or large regions of hydrophobicity or charge. Immunofluorescence and staining of Western blots of cell fractions indicates that SmlA is a 35 x 103 Mr cytosolic protein present in all vegetative and developing cells and is absent from smlA cells. The absence of SmlA does not affect the growth rate, cell cycle, motility, differentiation, or developmental speed of cells. Synergy experiments indicate that mixing 5% smlA cells with wild type cells will cause the wild type cells to form smaller fruiting bodies and aggregates. Although there is no detectable SmlA protein secreted from cells, starvation medium conditioned by smlA cells will cause wild-type cells to form large numbers of small aggregates. The component in the smlA CM which affects aggregate size is a molecule with a molecular mass greater than 100 x 103 Mr that is not CMF, phosphodiesterase or the phosphodiesterase inhibitor. The data thus suggest that the cytosolic protein SmlA regulates the secretion or processing of a secreted factor which regulates aggregate size. ----------------------------------------------------------------------- Chemoattractant-Mediated Increases in cGMP Induce Changes in Dictyostelium Myosin II Heavy Chain Specific Protein Kinase C Activities Adi Dembinsky, Hila Rubin, and Shoshana Ravid* Department of Biochemistry, Hadassah Medical School, The Hebrew University Jerusalem 91120, Israel J. Cell Biol., in press. Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP [Abu-Elneel et al (1996) J. Biol. Chem. 271, 977-984]. Recent studies have indicated that cAMP-induced cGMP accumulation plays a role in the regulation of myosin II phosphorylation and localization [Liu, G., and Newell, P. (1991) J. Cell Sci. 98, 483-490]. This report describes the roles of cAMP and cGMP in the regulation of MHC-PKC membrane-association, phosphorylation, and activity (hereafter termed MHC-PKC activities). cAMP stimulation of Dictyostelium cells resulted in translocation of MHC-PKC from the cytosol to the membrane fraction, as well as increasing in MHC-PKC phosphorylation and in its kinase activity. We present evidence that MHC is phosphorylated by MHC-PKC in the cell cortex which leads to myosin II dissociation from the cytoskeleton. Use of Dictyostelium mutants that exhibit aberrant cAMP-induced increases in cGMP accumulation revealed that MHC-PKC activities are regulated by cGMP. Dictyostelium streamer F mutant (stmF), which produces a prolonged peak of cGMP accumulation upon cAMP stimulation, exhibits prolonged increases in MHC-PKC activities. In contrast, Dictyostelium KI-10 mutant that lacks the normal cAMP-induced cGMP response, or KI-4 mutant that shows normal cAMP-induced cGMP response but has low cGMP binding activity, show no changes in MHC-PKC activities. We provide evidence that cGMP may affect MHC-PKC activities via the activation of cGMP-dependent protein kinase which, in turn, phosphorylates MHC-PKC. The results presented here indicate that cAMP-induced cGMP accumulation regulates myosin II phosphorylation and localization via the regulation of MHC-PKC. ---------------------------------------------------------------------- Dictyostelium Protein Kinase C-delta-like Protein is Localized in the Cell Nucleus Yingcai Wang, Hila Rubin and Shoshana Ravid* Department of Biochemistry, Hadassah Medical School, The Hebrew University Jerusalem 91120, Israel Biol Cell, in press The molecular mechanism whereby protein kinase C (PKC) molecules transduce signals into the cell nucleus are unknown. In this study, we provide an evidence that Dictyostelium discoideum contains PKCd-like protein that is localized in the nucleus. The Dictyostelium PKCd-like protein has an apparent molecular mass of 76 kDa. This protein is already highly expressed in vegetative Dictyostelium cells. The expression level remained constant up to 12 h of development, and sharply decreased after 16 h. The PKCd-like protein is phosphorylated in vivo in response to cAMP and phorbol ester stimulation. Immunofluorescent studies, as well as subcellular fractionation experiments, have indicated that Dictyostelium PKCd-like protein is permanently located in the nucleus. Our results may indicate that PKCd-like protein in Dictyostelium functions as a link between cAMP and the tumor-promoting phorbol esters, and events that take place in the nucleus. ---------------------------------------------------------------------- Inhibitory effects of tunicamycin on cell-cell adhesion and cell reaggregation of the cellular slime mold, Polysphondylium pallidum Nobuyuki Nakata and Hiroshi Ochiai Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido, 060 Japan Journal of Plant Research, in press Abstract To assess the role in cell-cell adhesion of gp64, a putative cell-cell adhesion molecule of Polysphondylium pallidum, we treated the cells with tunicamycin (TM), a known inhibitor of the synthesis of the N-linked oligosaccharide precursor, and examined TM's effect on cell-cell adhesion. The vegetative growth of Polysphondylium cells was inhibited with TM in a dose-dependent manner. When cells were treated with TM (2.0 mg/ml) during only the first 4 hr of starvation and further starved for 8 hr without TM, the cells dissociated considerably, although even the growth phase cells of Polysphondylium normally show EDTA-resistant (Ca2+-independent) cell adhesions. In parallel with the above effects, the amounts of intact gp64 decreased considerably in time with the lengths of incubation (0 hr>4 hr>8 hr). When TM-treated cells were washed free of TM, and shaken for a further 12 hr, the cells began to aggregate again, accompanied by an increase of gp64. In conclusion, TM affected cell-cell adhesion of Polysphondylium cells, but we were not able to distinguish whether the inhibition of cell aggregation was due to defects in glycosylation on glycoproteins and/or due to reduced levels of glycoproteins themselves. --------------------------------------------------------------------- [End CSM-News, volume 6, number 15]