Dicty News Electronic Edition Volume 10, number 7 February 28, 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 =========== Isolation of nucleation-competent centrosomes from Dictyostelium discoideum Ralph Graef, Ursula Euteneuer, Masahiro Ueda and Manfred Schliwa Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-Universit�t M�nchen, Schillerstr. 42, D-80336 M�nchen, Germany. Eur. J. Cell Biol., in press The centrosome of Dictyostelium discoideum is a box-shaped, layered core structure surrounded by a corona which is made up of dense nodules embedded in amorphous material. It is also known as nucleus-associated body. Because of its tight association with the nucleus the centrosome has resisted so far all attempts for isolation in sufficient purity and quantity for biochemical analysis. Here we report on the large- scale isolation of D. discoideum centrosomes after treatment of nucleus-centrosome complexes with a buffer containing sodium pyrophosphate. Following heparin treatment and a filtration step, centrosomes were further purified by density gradient centrifugation. Immunofluorescence analysis of the isolated centrosomes revealed the presence of the D. discoideum 350-kDa antigen, a centrosomal marker protein, g-tubulin, and the D. discoideum homologues of pericentrin, Spc110p, and Cdc31p. The structural integrity of the isolated centrosomes was demonstrated by confocal laser microscopy and electron microscopy. Microtubule nucleation assays with purified pig brain tubulin showed that the isolation procedure did not only preserve the structure but also the functionality of the isolated centrosomes. D. discoideum centrosomes should now become an attractive new model system in addition to, and for comparison with, centriolar centrosomes and yeast spindle pole bodies. ------------------------------------------------------------------------- Calcium levels correlate with cell cycle phase and affect the level of the cyclin B transcript in Dictyostelium discoideum. M. Azhar(a), M. Krefft(b), S. Saran(c), G. Weeks(d), and Vidyanand Nanjundiah(a) a Developmental Biology and Genetics Laboratory, Indian Institute of Science, Bangalore 560012, India b Fachhochschule Rheinland-Pfalz, Abteilung Bingen, FB- Verfahrenstechnik, Berlinerstrasse 109, Germany c School of Life Sciences, Jawaharlal Nehru University, New Delhi- 11067, India d Department of Microbiology and Immunology and Department of Medical Genetics, University of British Columbia, Vancouver BC V6T, Canada FEMS Microbiology Letters, in press Abstract In pre-aggregation amoebae of D. discoideum, phenotypic differences with respect to cellular Ca2+ and cell cycle phases act coordinately and bias post-aggregative cell-type choice. We have shown that it is possible to enhance (/reduce) the proportion of prestalk cells in the slug and generate a stalky (/spory) phenotype in D. discoideum by artificially increasing (/decreasing) cellular Ca2+ by using the right combination of the calcium ionophore A23187 and Ca2+-EGTA buffers. Also, sequestered and free calcium are both relatively high or relatively low in the same cell. This enables us to make use of chlortetracycline (CTC) fluorescence, which is primarily a correlate of sequestered calcium, indirectly to monitor overall cellular calcium. Based on the temporal pattern of (3-H) thymidine uptake and MUD-9 antibody staining, we infer that Ca2+ levels are highest at the S phase of the cell cycle. Upon increasing the level of Ca2+ with the help of A23187, there is a 2 to 3-fold decrease in the cyclin B (clb1) mRNA level; the cdc2 mRNA level shows a marginal decrease. A decrease in cellular Ca2+ does not appear to influence either the clb1 or the cdc2 mRNA levels. These results suggest that the effect of Ca2+ and the cell cycle on cell fate could be exerted at the level of transcription, or message stability, of specific genes. ------------------------------------------------------------------------- Presteady-state of reaction of Nucleoside Diphosphate Kinase with anti-HIV Nucleotides SCHNEIDER, B. (1) , XU, Y.W., (2) SELLAM, O., (1) SARFATI, R., (1) JANIN, J., (2) VERON, M. (1) & DEVILLE-BONNE, D. (1) (1) Institut Pasteur, Paris and (2) CNRS, Gif-sur-Yvette, France J. Biol. Chem., in press SUMMARY: The presteady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) was studied by quenching of protein fluorescence after manual mixing or by stopped-flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addition according to a monoexponential time course. The pseudo-first order rate constant was determined for different concentrations of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme where fast association of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate constants and dissociation equilibrium constants determined for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymatic assay in the presence of the two substrates. It is shown that ddNTPs are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s-1 and a KS of 1 to 5 mM. The equilibrium dissociation constants for ADP, GDP, ddADP and ddGDP were also determined by fluorescence titration of a mutant F64W NDP kinase where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3'OH in ddNTP causes a ten fold increase in KD. Contrary to � natural � NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in kcat values. ------------------------------------------------------------------------- Fucose-beta-1-P-Ser is a new type of glycosylation: Using antibodies to identify a novel structure in Dictyostelium discoideum and study multiple types of fucosylation during growth and development Geetha Srikrishna, Liying Wang, and Hudson H. Freeze The Burnham Institute, La Jolla Cancer Research Center, La Jolla, California 92037 Three antibodies that recognize distinct fucose epitopes were used to study fucosylation during growth and development of Dictyostelium discoideum. mAb83.5 is known to recognize an undefined "fucose epitope" on several proteins with serine-rich domains, while mAb CAB4, and a component of anti-horse-radish peroxidase, specifically recognize Fuca1,6GlcNAc and Fuca1,3GlcNAc residues respectively in the core of N-linked oligosaccharides. We show that mAb 83.5 defines a new type of O-glycosylation. Serine-containing peptides incubated with GDPb[3H]Fuc and microsomes formed two fucosylated products. A neutral product accounting for 30% of the label did not react with the antibody, while the rest of the label was incorporated into a charged product which contained all the mAb83.5 reactive material. b-elimination of the labeled peptide or endogenous products produced [3H]Fuc-1-P, indicating phosphodiester linkage to serine. Fucb-1-P and GDP-bFuc at 100�M blocked mAb83.5 binding to endogenous and peptide products, but their a-linked anomers did not. Electrospray ionization mass spectra of the neutral and anionic labeled products showed major peaks of mass units corresponding to O-Fuc-Ser peptide and O-Fuc-phospho-Ser peptide respectively. The activity of Fuc-phosphotransferase exactly paralleled the accumulation of reactive glycans during growth and development. The expressions of N-glycan core Fuca1,6GlcNAc and Fuca1,3GlcNAc, and their respective fucosyl transferase activities were also synchronous, but their developmental regulation differed from one another. Fuca1,6GlcNAc was expressed maximally during growth, but declined during development. In contrast core Fuca1,3GlcNAc epitopes were expressed almost exclusively during development. These findings provide direct evidence for a novel type of O-phosphofucosylation, demonstrate the existence of an O-fucosyl transferase, and identify two different types of core fucosylation in the N-glycans of Dictyostelium. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 7]