CSM News Electronic Edition Volume 7, number 16 December 28, 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" We wish everyone a happy and productive new year! =========== Abstracts =========== Spatial patterning of the distribution of Ca2+ in Dictyostelium discoideum as assayed in fine glass capillaries. M. Azhar and Vidyanand Nanjundiah Developmental Biology and Genetics Laboratory, Indian Institute of Science, Bangalore 560012, India Pre- and post-aggregative amoebae of D. discoideum were labelled with the Ca2+-specific fluorescent dyes chlortetracycline (CTC) or indo-1 and their fluorescence monitored after being drawn into a fine glass capillary using the technique of Bonner et al. [Proc. Natl. Acad. Sci. USA 92, 8249-8253, 1995]. The cell mass rapidly forms two zones of Ca2+-CTC or Ca2+-indo-1 fluorescence. The anterior ('air side') zone, which we identify as a prestalk zone, shows a high level of fluorescence demarcated by an especially bright band towards its posterior margin. The prestalk zone constitutes a fraction of about 18% of the entire length of the cell mass; the width of the bright band is about 19% of that of the prestalk zone. The level of fluorescence drops in the middle portion of the capillary and rises again to a lesser extent in the posteriormost region ('oil side'). When bounded by air on both sides, the cells display high fluorescence at both ends. When oil is present at both ends of the capillary, there is little fluorescence except for small regions at the ends. These patterns appear within a couple of minutes and the regions displaying strong fluorescence have a tendency to move away from the anterior end. In the 'double air' configuration, two bands of high Ca2+ are seen, one at each end. Amoebae from prestalk or prespore regions of slugs displayed a similar pattern of fluorescence as freshly starved amoebae. With neutral red (NR)-stained pre- or post-aggregative amoebae in a 'double-air' setup, both ends of the capillary showed dark staining within 5 minutes. These NR-stained regions moved inwards and stabilized their positions in 20 minutes. Cell movement was not responsible for any of the patterns seen. Thus, freshly starved amoebae, which were previously shown to fall into two classes on the basis of their Ca2+ levels and in the absence of any spatial cues [Azhar et al., Exp. Cell Res. 227, 344-351, 1996], are now seen to organize themselves into the Ca2+ classes but on a purely spatial basis. --------------------------------------------------------------------- The cysteine proteinase gene cprG in Dictyostelium discoideum has a serine-rich domain that contains GlcNAc-1-P Tonis Ord*, Celine Adessi#, Liying Wang* and Hudson H. Freeze* *The Burnham Institute, La Jolla, CA, #CENG/DBMS, Chimie des Proteines, Grenoble, France Arch. Biochem. Biophys., in press. A lysosomal cysteine proteinase called proteinase-1 is the major proteolytic enzyme in vegetative cells of Dictyostelium discoideum. This phosphoglycosylated protein contains multiple residues of GlcNAc-1-P linked to peptidyl serines. Here we report the cloning, structure and expression of its cDNA (cprG). Another cDNA (cprF) closely related to cprG was also cloned and characterized. mRNAs of both genes are present during the vegetative phase and decrease in developing cells. However, the level of cprG mRNA is about 100-fold higher than that of cprF. The predicted protein products of both genes contain a unique serine-rich domain that was previously found only in two Dictyostelium proteinases (CP4 and CP5) that also carry a GlcNAc-1-P-Ser modification. The cprG product, renamed CP7, was tagged with the FLAG-epitope (FLAG-CP7) and shown to bind to cystatin, a highly specific inhibitor of cysteine proteinases. The FLAG-CP7 product also contained both N-linked oligosaccharides and GlcNAc-1-P. Deletion of the serine-rich domain from FLAG-CP7 yields a product that still binds to cystatin, but no longer carries GlcNAc-1-P. This finding supports the idea that the GlcNAc-1-P residues are normally added to the serine-rich domain, found only in vegetative Dictyostelium cysteine protienases. --------------------------------------------------------------------- Cloning, sequencing and developmental expression of phosphofructokinase from Dictyostelium discoideum Departamento de Bioquimica UAM and Instituto de Investigaciones Biomedicas CSIC, Facultad de Medicina de la Universidad Autonoma de Madrid, Madrid, Spain Antonio M. Estevez, Oscar H. Martenez-Costa, Valentina Sanchez and Juan J. Aragon Eur. J. Biochem., in press Phosphofructokinase (PFK) from Dictyostelium discoideum is a non-allosteric enzyme that lacks any of the characteristic regulatory mechanisms of PFK from other cells. We have determined the DNA sequence and analyzed the amino acid sequence of D. discoideum PFK, as an initial step toward understanding the peculiar properties of this enzyme. Three overlapping fragments, two of cDNA and one of genomic DNA, were isolated, which together could encode the complete sequence of D. discoideum PFK. The constructed full-length cDNA coded for a protein of 834 amino acids, with a calculated molecular mass of 92.4 kDa, that was homologous to other eukaryotic and prokaryotic PFKs. Alignments of the amino acid sequence with other isozymes revealed that many of the amino acid residues assigned to binding sites of substrates and allosteric effectors are conserved in this enzyme, but changes were also found that may contribute to the absence of allosteric mechanisms. A phylogenetic tree for the eukaryotic PFK family was constructed and showed that the N-terminal domain clustered with those of yeast subunits, whereas the C-terminal domain was more related to PFK from metazoa. Southern blotting indicated that this PFK is encoded by a single gene. The enzyme is present throughout the life-cycle of D. discoideum with a gradual decrease of its expression during development. --------------------------------------------------------------------- A voltage- and K+-dependent K+ channel from a membrane fraction enriched in contractile vacuole of Dictyostelium discoideum Kunito Yoshida *, Toru Ide, Kei Inouye, Koichi Mizuno, Takahisa Taguchi, Michiki Kasai Biophys. Biochim. Acta, in press We obtained a membrane fraction enriched in the contractile vacuole by aqueous-polymer two-phase partitioning and its channel activities were analysed by incorporating it into artificial planar lipid bilayers. In asymmetrical KCl solutions (cis, 300 mM/100 mM, trans), we observed single-channel currents of a highly K+-selective channel with slope conductance of 102 pS and reversal potential of -20.4 mV, which corresponded to PK+/PCl-=7. They showed bursts separated by infrequent quiescent periods. At 0 mV the mean open time was 2.0 ms. Among monovalent cations, Na+ and Li+ were impermeant, whereas Rb+ showed permeability equivalent to that of K+, although the unitary conductance was apparently reduced when the current flowed from the Rb+ containing side, suggesting that Rb+ is a permeant blocking ion. The open probability within bursts remained constant at approx. 0.6 as long as the holding potential was positive on the cis side with respect to the trans side, but it decreased to 0 at negative potential. This channel was blocked by submillimolar concentrations of quinine and 30 mM TEA+. The open probability-voltage relationship showed a striking dependency on the KCl concentration on either side. This channel may play a role in water transport in this organelle. ---------------------------------------------------------------- Myosin II-independent processes in mitotic cells of Dictyostelium discoideum: redistribution of the nuclei, re-arrangement of the actin system, and formation of the cleavage furrow Ralph Neujahr, Christina Heizer, and Guenther Gerisch Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany J. Cell Science, in press. Mitosis was studied in multinucleated and mononucleated mutant cells of Dictyostelium discoideum that lack myosin II (Manstein et al., EMBO J. 8, 923-932, 1989). Multinucleated cells were produced by culture in suspension, mononucleated cells were enriched by growth on a solid surface (DeLozanne and Spudich, Science 236, 1086-1091,1987). The multinucleated cells disclosed interactions of mitotic complexes with the cell cortex that were not apparent in normal, mononucleated cells. During the anaphase stage, entire mitotic complexes consisting of spindle, microtubule asters, and separated sets of chromosomes were translocated to the periphery of the cells. These complexes were appended at a distance of about 3 mm from the cell surface, in a way that the spindle became orientated in parallel to the surface. Subsequently, lobes of the cell surface were formed around the asters of microtubules. These lobes were covered with tapered protrusions rich in coronin, an actin associated protein that typically accumulates in dynamic cell-surface projections (DeHostos et al., EMBO J. 10, 4097-4104, 1991). During their growth on a solid surface, mononucleated myosin II-null cells passed through the mitotic cleavage stages with a speed comparable to wild-type cells. Cytokinesis as linked to mitosis is distinguishable by several parameters from traction mediated cytofission, which results in the pinching off of pieces of a multinucleated cell in the interphase. The possibility is discussed that cells can cleave during mitosis without forming a contractile ring at the site of the cleavage furrow. --------------------------------------------------------------------- Diphospho-myo-inositol phosphates from Dictyostelium identified as D-6-PP-InsP5 and D-5,6-bis-PP-InsP4 Tim Laussmann, Komandla Malla Reddy, K. Kishta Reddy, J.R. Falck and Gunter Vogel Fachbereich 9 - Chemie, Bergische Universitat GHS Wuppertal, Gausstrasse 20, D-42097 Wuppertal, FRG; Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9038 Biochem. J., in press Two diphospho-myo-inositolphosphates from Dictyostelium were recently investigated by two- dimensional 1H-31P NMR analysis and assigned to be either D-4-PP-InsP5 and D-4,5-bis-PP-InsP4 or their corresponding enantiomers D-6-PP-InsP5 and D-5,6-bis-PP-InsP4. In the present study the naturally occuring enantiomers were identyfied by using defined synthetic PP-InsP5 isomers as substrates for a partially purified PP-InsP5 5-kinase from Dictyostelium. This enzyme specifically phosphorylates the naturally occuring PP-InsP5 and the synthetic D-6-PP-InsP5 leading to 5,6-bis-PP-InsP4. In contrast, neither D-4-PP-InsP5 nor D-1-PP-InsP5 or D-3-PP-InsP5 are converted by the enzyme. --------------------------------------------------------------------- The wacA Gene of Dictyostelium discoideum is a Developmentally Regulated Member of the MIP Family Karin M. Flick, Gad Shaulsky and William F. Loomis Department of Biology, University of California, San Diego, La Jolla, CA 92093-0322, USA Gene, in press We isolated a cDNA from Dictyostelium discoideum that encodes a 30 kd protein with significant similarity to members of the MIP (major intrinsic protein) family of membrane transporters. The most closely related protein in the public data bases is an aquaporin from Cicadella viridis which shows 34% identity. The cDNA was used to isolate and characterize genomic fragments carrying the Dictyostelium gene which we named wacA. Genomic probes were used to recognize wacA mRNA isolated at various stages of development. The results showed that the gene is developmentally regulated such the mRNA first appears at 12h of development and is retained throughout the remainder of development. In situ hybridization of whole mounts prepared at fifteen hours of development showed that wacA mRNA accumulates exclusively in prespore cells and is absent from prestalk cells. Although wacA expression is prespore specific, disruption of the gene by homologous recombination did not result in observable alterations in the formation of spores or their resistance to osmotic challenges. --------------------------------------------------------------------- [End CSM News, volume 7, number 16]