Dicty News Electronic Edition Volume 23, number 18 December 10, 2004 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Activation of soluble guanylyl cyclase at the leading edge during Dictyostelium chemotaxis Douwe M. Veltman, Jeroen Roelofs, Ruchira Engel, Antonie J.W.G. Visser and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Molecular Biology of the Cell, in press Dictyostelium contains two guanylyl cyclases, GCA, a twelve-transmembrane enzyme, and sGC, a homolog of mammalian soluble adenylyl cyclase. sGC provides nearly all chemoattractant-stimulated cGMP formation and is essential for efficient chemotaxis towards cAMP. We show that in resting cells the major fraction of the sGC-GFP fusion protein localizes to the cytosol and a small fraction is associated to the cell cortex. With the artificial substrate Mn2+/GTP, sGC activity and protein exhibit a similar distribution between soluble and particulate fraction of cell lysates. However, with the physiological substrate Mg2+/GTP, sGC in the cytosol is nearly inactive, while the particulate enzyme shows high enzyme activity. Reconstitution experiments reveal that inactive cytosolic sGC acquires catalytic activity with Mg2+/GTP upon association to the membrane. Stimulation of cells with cAMP results in a 2-fold increase of membrane-localized sGC-GFP, which is accompanied by an increase of the membrane-associated guanylyl cyclase activity. In a cAMP gradient, sGC-GFP localizes to the anterior cell cortex, suggesting that in chemotacting cells, sGC is activated at the leading edge of the cell. Submitted by: Peter van Haastert [P.J.M.van.Haastert@chem.rug.nl] ----------------------------------------------------------------------------- Reverse genetic analyses of gamete-enriched genes revealed a novel regulator of the cAMP signaling pathway in Dictyostelium discoideum Tetsuya Muramoto, Shugaku Takedaa, Yoko Furuya, and Hideko Urushihara Mechanisms of Development, in press. Sexual development in Dictyostelium discoideum is initiated by the fusion of oppo-site mating type cells to form zygote giant cells, which subsequently gather and phagocytose surrounding cells for nutrition to form macrocysts. Here we performed the targeting of 24 highly gamete-enriched genes we previously isolated, and successfully generated knockout mutants for 16 genes and RNAi mutants for 20 genes including 6 genes without disruptants. In the knockout mutants of two genes, cell aggregation to-ward the giant cells was much less extensive and many cells remained around poorly formed macrocysts. We named these genes tmcB and tmcC. Although macrocyst formation of wild type cells was suppressed by the addition of exogenous cAMP, that of knockout mutants of tmcB was much less sensitive. The mRNA level of phosphodi-esterase (pde) was higher and that of its inhibitor (pdi) was lower in the latter cells compared to the parental strains during sexual development. Thus, tmcB appeared to be a novel regulator of the cAMP signaling pathway specific to sexual development. Knockout mutants of tmcC were indistinguishable from the wild type cells with respect to the cAMP response, suggesting that this gene is relevant to other processes. Submitted by: Hideko Urushihara [hideko@biol.tsukuba.ac.jp] ----------------------------------------------------------------------------- Regulation of growth and differentiation in Dictyostelium Yasuo Maeda Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan International Review of Cytology, in press In general, growth and differentiation are mutually exclusive, but cooperatively regulated during the course of development. Thus the process of cellŐs transition from growth to differentiation is of general importance not only for the development of organisms but also the initiation of malignant transformation, in which this process is reversed. The cellular slime mold Dictyostelium, a wonderful model organism, grows and multiplies as long as nutrients are supplied, and its differentiation is triggered by starvation. A strict checkpoint (GDT-point), from which cells start differentiating in response to differentiation, has been specified in the cell cycle of D. discoideum Ax-2 cells. Accordingly, integration of GDT-point specific events with starvation-induced events is needed to understand the mechanism regulating growth/differentiation transition (GDT). A variety of inter- and intra-cellular signals are involved positively or negatively in the initiation of differentiation, making a series of cross-talks. As was expected from the presence of GDT-point, the cellŐs positioning in cell masses and subsequent cell-type choice occur depending on the cellŐs phase in the cell cycle at the onset of starvation. Since novel and somewhat unexpected multiple functions of mitochondria in cell movement, differentiation and pattern formation have been realized well in Dictyostelium cells, they are reviewed in this article. Submitted by: Yasuo Maeda [ymaeda@mail.tains.tohoku.ac.jp] ============================================================================== [End Dicty News, volume 23, number 18]