CSM News Electronic Edition Volume 9, number 1 July 5, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== cAMP-induced changes in the cytosolic free Ca2+ concentration in Dictyostelium discoideum are light sensitive Jürgen Sonnemann, Gerd Knoll and Christina Schlatterer Fakultät für Biologie, Universität Konstanz, D-78434 Konstanz Cell Calcium, accepted Summary The cytosolic free calcium concentration ([Ca2+]i) of the social amoeba Dictyostelium discoideum was analyzed after challenge with the chemoattractant cAMP. [Ca2+]i was measured by digital imaging in single cells loaded with the Ca2+-indicator fura2-dextran. Global stimulation with low concentrations of cAMP (0.1-1 mM) led to a global transient [Ca2+]i-increase. This increase was abolished when cells were illuminated with high doses of light. However, after a short recovery period of several minutes the cells again displayed the normal response. Inhibition of the [Ca2+]i-elevation depended on the wavelength of illumination light. We compared the required recovery period of cells irradiated with either 340, 380, 405, 450 or 490 nm at defined intensities. Light of 405 nm had a pronounced effect; 340 nm alone or in combination with 380 nm was also effective, but to a lesser extent, whereas neither 450 nm nor 490 nm inhibited the [Ca2+]i-increase, even at very high irradiance. The wavelength dependence matched the absorption spectrum of amoebae grown in darkness that contain a photopigment which seems to be responsible for phototaxis of single cells. Cells grown in darkness exhibited an increased sensitivity of the cAMP-induced [Ca2+]i-transient towards light compared to light-grown cells. From these data we conclude that phototactic signaling could interfere with chemotactic signaling at the level of [Ca2+]i-changes. --------------------------------------------------------------------- DIF-1, putative morphogen of D. discoideum, suppresses cell growth and promotes retinoic acid-induced cell differentiation in HL-60 Yuzuru Kubohara Department of Molecular Physiology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371, Japan Biochem. Biophys. Res. Commun. In press. Abstract Differentiation-inducing factor-1 (DIF-1) is a putative morphogen that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. In this study, we have examined the effects of DIF-1 on the human leukemia HL-60 cells. DIF-1 at 10-40 micro M suppressed cell growth in a dose-dependent manner, and approximately 50% growth inhibition was attained with 15-20 micro M DIF-1. FACS analysis of cell-cycle phase distribution using propidium iodide revealed that many cells were accumulated in the G1 phase after treatment with 15-20 micro M DIF-1. These concentrations of DIF-1 also raised [Ca2+]i in a dose-dependent manner irrespective of the presence of extracellular Ca2+, indicating that DIF-1 elicited Ca2+-release from some intracellular Ca2+ store(s). Most importantly, relatively low concentrations of DIF-1 (1-5 micro M) were found to promote retinoic acid-induced cell differentiation. The present results indicate that DIF-1 may be a useful tool for the analysis of myeloid cell differentiation and have therapeutic potential in the treatment of human myeloid leukemia. --------------------------------------------------------------------- SDF, a new spore differentiation factor secreted by Dictyostelium cells is phosphorylated by the cAMP dependent protein kinase Anjard, C., van Bemmelen, M., Véron, M. and Reymond, C. D. 1 Unité de régulation enzymatique des activités cellulaires, CNRS-UMR 321, Institut Pasteur, 25 rue du D. Roux, 75724 Paris Cedex 15, France. 2 Institut de biologie cellulaire et de morphologie, Université de Lausanne, rue du Bugnon 9, CH-1005 Lausanne, Switzerland. Differentiation, in press Abstract Upon starvation, Dictyostelium discoideum unicellular amoebae form a multicellular organism leading to the development of a fruiting body containing spores. Single cells of sporogenous mutants, unlike wild type cells, are able to differentiate into spores under specific conditions. We show in this report that overexpression of the catalytic subunit of the cAMP dependent protein kinase (PKA), not only renders the cells sporogenous, but is also accompanied by the production/release of a diffusible spore differentiation factor (SDF). SDF is a small, thermostable phospho-polypeptide. In vitro dephosphorylation reduces SDF spore differentiation capacity, which can be re-gained in vitro by PKA phosphorylation. These results indicate that SDF is a PKA substrate and might be activated in vivo by this protein kinase. Since spore differentiation requires PKA catalytic subunit activation, we conclude that the response of prespore cells to SDF involves an intracellular pathway dependent on PKA. --------------------------------------------------------------------- OSMOTIC HOMEOSTASIS IN DICTYOSTELIUM DISCOIDEUM: EXCRETION OF AMINO ACIDS AND INGESTED SOLUTES Theodore L. Steck, Lucius Chiaraviglio and Stephen Meredith J. Euk. Micro. 44:503-510 (Sept. issue) The response to osmotic stress in axenically cultured Dictyostelium discoideum was examined. Hypo-osmotic buffers elicited two changes in the large (~50 mM) cytosolic pool of amino acids: a) the total size of the pool diminished, while b) about half of the initial pool was excreted. Hyperosmotic stress had the opposite effect. Among the predominant amino acids in the pool were glycine, alanine and proline. Putrescine, the major diamine, was neither excreted nor modulated. Recently ingested radioactive amino acids were excreted in preference to those in the cytoplasm, suggesting that the endocytic pathway might be involved in water excretion. Furthermore, hypo-osmotic stress stimulated the selective excretion of small, membrane-impermeable fluorescent dyes which had been ingested into endocytic vacuoles. Caffeine inhibited the excretion of the fluorophores but not the amino acids. We conclude that the response of Dictyostelium to osmotic stress is complex and includes both modulation of the cytoplasmic amino acid pool and the excretion of amino acids and other small solutes from the endocytic pathway. --------------------------------------------------------------------- [End CSM News, volume 9, number 1]