CSM News Electronic Edition Volume 8, number 15 June 21, 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" =========================== Dicty Researcher Database =========================== Also, please if you have not completed your entry in the Dicty Researcher Database on the Dicty web page or by following this link "http://apps.basic.northwestern.edu/dicty/dictyqbe.html" . =========== Abstracts =========== Efficient induction by DIF-1 and 8-bromo cyclic AMP of prespore-to-stalk conversion in Dictyostelium discoideum Yuzuru Kubohara and Mineko Maeda Department of Molecular Physiology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371, Japan, and Department of Biology, Faculty of Science, Osaka University, Osaka 560, Japan. Comp. Biochem. Physiol. In press. We have examined the in vitro effects of DIF-1 (differentiation-inducing factor-1), DIF-2, cAMP, and/or 8-bromo cAMP (Br-cAMP) on prespore cells isolated from normally formed slugs of the cellular slime mold, Dictyostelium discoideum (strain NC4). Without these factors, the isolated cells remained amoeboid at 24 h of incubation (at 5 x 103 cells/cm2). DIF-1 (100 nM) poorly induced the prespore cells to differentiate into stalk cells (<15%), and DIF-2 (100 nM) was a less effective inducer of stalk cells (<1%). Cyclic AMP (200 micro M) itself induced neither stalk- nor spore-formation but was inhibitory to the DIF-1-induced stalk cell formation. Br-cAMP (10 mM) alone induced prespore-to-spore maturation. It was found that prespore cells were converted to stalk cells efficiently (>85%) in the presence of both Br-cAMP and DIF-1. --------------------------------------------------------------------- Dictyostelium Development in the Absence of cAMP Bin Wang and Adam Kuspa* Verna and Mars McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA. Science, in press. Summary During the formation of multicellular aggregates, Dictyostelium cells use extracellular cAMP as a chemoattractant. Later in development they use cAMP, acting through cAMP-dependent protein kinase (PKA), as an intracellular signal for cell differentiation. Although there are two genes encoding adenylyl cyclase in the Dictyostelium genome, only one is expressed during development and its product, ACA, accounts for the synthesis of all of the measurable cAMP prior to germination. While acaA-null cells are incapable of aggregation, we have found that moderate over-expression of the catalytic subunit of PKA (PKA-C) in these cells leads to almost normal development under certain conditions. When deposited at 1.4 to 5.5 million cells per square-cm, acaA- cells carrying an act15::pkaC expression construct aggregate and proceed through morphogenesis to form properly proportioned fruiting bodies. They are unable to aggregate at lower cell densities. When they have aggregated, prestalk cells sort to the apex and can be seen later in the anterior of finger-like structures. Under appropriate conditions, these fingers fall over and migrate phototactically like wild-type slugs. All of these developmental events are carried out without any measurable adenylyl cyclase activity or accumulation of cAMP. Thus, as long as PKA is active, cell-type divergence and sorting, slug migration, morphogenesis and terminal differentiation can proceed in the absence of cAMP. It appears that most of the cAMP regulation required for development is effected through activation of PKA, and that precise spatial and temporal control of PKA activity may not be required for development following aggregation. These results also suggest that signals other than extracellular cAMP coordinate morphogenesis in Dictyostelium. --------------------------------------------------------------------- Talin - null cells of Dictyostelium are strongly defective in adhesion to particle and substrate surfaces and slightly impaired in cytokinesis Jens Niewöhner, Igor Weber, Markus Maniak, Annette Müller-Taubenberger, and Günther Gerisch J. Cell. Biol., in press. Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous crosstalk between cell surface and the underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell-surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast to the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin-dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells. --------------------------------------------------------------------- Identification of a Microtubule-Binding Domain in a Cytoplasmic Dynein Heavy Chain. Michael P. Koonce. J. Biol. Chem. in press SUMMARY As a molecular motor, dynein must coordinate ATP-hydrolysis with conformational changes that lead to processive interactions with a microtubule and generate force. To understand how these processes occur, we have begun to map functional domains of a dynein heavy chain from Dictyostelium. The carboxy-terminal 10 kb region of the heavy chain encodes a 380 kDa polypeptide that approximates the globular head domain. Attempts to further truncate this region fail to produce polypeptides that either bind microtubules or UV-vanadate cleave, indicating that the entire 10 kb fragment is necessary to produce a properly folded functional dynein head. We have further identified a region just downstream from the fourth P-loop that appears to constitute at least part of the microtubule-binding domain (aa 3182 - 3818). When deleted, the resulting head domain polypeptide no longer binds microtubules; when the excised region is expressed in vitro, it cosediments with added tubulin polymer. This microtubule-binding domain falls within an area of the molecule predicted to form extended a-helices. At least four discrete sites appear to coordinate activities required to bind the tubulin polymer, indicating that dynein's interaction with microtubules is complex. --------------------------------------------------------------------- Twisted scroll waves organize Dictyostelium mucoroides slugs Dirk Dormann, Cornelis Weijer and Florian Siegert Zoologisches Institut, Universität München, Luisenstr. 14, 80333 München, Germany J.Cell Science, in press ABSTRACT Cellular slime moulds (Dictyosteloids) are characterised by at least two different modes of slug migration. Most species, e.g. Dictyostelium mucoroides, produce a stalk continuously during slug migration, while a few species, e.g. Dictyostelium discoideum are characterised by stalk-less slug migration and only produce a stalk upon culmination. Experiments on D. discoideum and theoretical model calculations have shown that D. discoideum slugs are organized by a cAMP scroll wave in the tip which produces planar waves in the back. These waves guide cell movement in slugs: spiralling in the tip and forward movement parallel to the slug axis in the back. Simple changes in model parameters can lead to the formation of a twisted scroll wave which extends throughout the slug (Bretschneider et al., (1995). Proc. Natl. Acad. Sci. USA 92, 4387). In order to investigate whether such twisted scroll waves occure naturally we have analysed the movement of fluorescently labelled single cells in migrating D. mucoroides slugs. The results show that cells in the prespore zone of D. mucoroides slugs move in a spiral path. Although the velocity of single cells in D. mucoroides is faster than in D. discoideum, the net forward component of their movement is less due to their spiral trajectories. As a result D. mucoroides slugs move slower than D. discoideum slugs. The entire D. mucoroides slug also describes a spiralling path leaving corkscrew shaped stalks behind. Based on these observations we propose that cell movement in D. mucoroides slugs is controlled by a propagating twisted scroll wave of cAMP which extends throughout the length of the slug. --------------------------------------------------------------------- Mechanism of cAMP-induced Ca2+-influx in Dictyostelium: the role of phospholipase A2 Ralph Schaloske and Dieter Malchow University of Konstanz, Faculty of Biology, 78457 Konstanz, FRG Biochem. J. (in press) Abstract cAMP-induced Ca2+-influx in Dictyostelium follows two pathways: a G-protein dependent one where influx is reduced by 50 - 70 % in G alpha 2 and G beta-negative strains. The remaining influx is heterotrimeric G-protein independent. Using a pharmacological approach we found that phospholipase A2 is the target of both pathways. The products of phospholipase A2-activity arachidonic acid (AA) and palmitic acid induced Ca2+-influx to a similar extent as cAMP. Half maximal activation occurred at 3 µM AA, saturation at 10 µM AA. The response to AA was quantitatively similar throughout early differentiation independent of cAMP receptor concentration. Synergy experiments revealed that cAMP and AA acted through identical pathways. The phospholipase A2 activating peptide (PLAP), a peptide with sequence similarity to the G-protein beta-subunit, activated Ca2+-influx. The G-protein-independent pathway was sensitive to genistein but not to blockers of protein kinase C (PKC) and other kinases, suggesting that tyrosine kinase may directly or indirectly activate phospholipase A2 in this case. --------------------------------------------------------------------- [End CSM News, volume 8, number 15]