Dicty News Electronic Edition Volume 20, number 8 May 02, 2003 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============= Abstracts ============= Genetic Interactions of the E3 Ubiquitin Ligase Component FbxA with cAMP Metabolism and a Histidine Kinase Signaling Pathway During Dictyostelium discoideum development Turgay Tekinay, Herbert L. Ennis, Mary Y. Wu, Margaret Nelson1, Richard H. Kessin*, and David I. Ratner2,* Accepted: Eukaryotic Cell Abstract Dictyostelium amoebae altered in a gene called fbxA, which is thought to encode a component of an SCF E3 ubiquitin ligase, have defective regulation of cell-type proportionality. In chimeras with wild-type cells, the mutant amoebae form mainly spores, leaving the construction of stalks to wild-type cells. To examine the role of fbxA and regulated proteolysis we have recovered the promoter of fbxA and shown that it is expressed in a pattern resembling that of a prestalk-specific gene until late in development when it is also expressed in developing spore cells. Because fbxA cells are developmentally deficient in pure culture, we were able to select suppressor mutations that promote sporulation of the original mutant. One suppressor mutation resides within the gene regA, which encodes a cAMP phosphodiesterase linked to an activating response regulator domain. In another suppressor, there has been a disruption of dhkA, a gene encoding a two-component histidine kinase known to influence Dictyostelium development. RegA appears precociously and in greater amounts in the fbxA mutant but not in an fbxA/dhkA double mutant, where RegA is restored to wild-type levels. Because the basis of regA suppression might involve alterations in cAMP levels during development, the concentration of cAMP was determined in all strains. The level of cAMP is relatively constant during multi-cellular development in all strains except the dhkA mutant, in which it is reduced at least 6-fold. The level of cAMP in the double mutant dhkA/fbxA is relatively normal. The level of cAMP in the various mutants does not correlate with spore formation, as would be expected on the basis of our present understanding of the signaling pathway leading to the induction of spores. Altered amounts of RegA and cAMP early in the development of the mutants suggest that both fbxA and dhkA genes act earlier than previously thought. Submitted by: Turgay Tekinay [tt420@columbia.edu] ----------------------------------------------------------------------------- Dictyostelium and Acanthamoeba myosin II assembly domains go to the cleavage furrow of Dictyostelium myosin II-null cells Shi Shu*, Xiong Liu* and Edward D. Korn? Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA *These authors contributed equally. ?To whom correspondence should be addressed: Building 50, Room 2517, National Institutes of Health, Bethesda, MD 20892. Tel.: (301) 496-1616. Fax: (301) 402-1519. E-mail: edk@nih.gov Pro. Natl. Acad. Sci. USA in press ABSTRACT How myosin II localizes to the cleavage furrow of dividing cells is largely unknown. We show here that a 283-residue protein, AD1, corresponding to the assembly domain in the tail of Dictyostelium myosin II assembles into bundles of long tubules when expressed in myosin II-null cells and localizes to the cleavage furrow of dividing cells. AD1 mutants that do not polymerize in vitro do not go to the cleavage furrow in vivo. An assembly-competent polypeptide corresponding to the C-terminal 256 residues of Acanthamoeba myosin II also goes to the cleavage furrow of Dictyostelium myosin II-null cells. When over-expressed in wild-type cells, AD1 co-localizes with endogenous myosin II (possibly as a co-polymer) in interphase, motile and dividing cells and under caps of concanavalin A receptors but has no effect on myosin II-dependent functions. These results suggest that neither a specific sequence, other than that required for polymerization, nor interaction with other proteins is required for localization of myosin II to the cleavage furrow. Submitted by: Edward Korn [KornE@NHLBI.NIH.GOV] ----------------------------------------------------------------------------- The Dictyostelium discoideum prespore specific catalase B functions to control late development and to protect spore viability Ma. Xenia U. Garcia+@ Hannah Alexander+, Dana Mahadeo#&, David A. Cotter#, and Stephen Alexander+* +Division of Biological Sciences, University of Missouri, 303 Tucker Hall, Columbia, MO 65211-7400 # Department of Biological Sciences, University of Windsor, Windsor, Ontario, N9B 3P4 Canada @ Current address: Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110 & Current address: Laboratory of Cellular and Molecular Biology, NCI, NIH, 37 Covet Dr., Bethesda, MD 20892-4255 Accepted: Biochimica Biophysica Acta (BBA) - Molecular Cell Research Abstract Changes in the levels of reactive oxygen species (ROS) have been associated previously with cell differentiation and development in several systems. Thus, there is interest in studying the developmental regulation of antioxidant enzymes, whose activities may modulate ROS levels and subsequent oxidant-mediated signal transduction events in specific tissues. Our recent identification in Dictyostelium discoideum of the prespore specific catalase B (CatB) enzyme suggested a) that the CatB enzyme functions to provide protection to the mature spores, and b) that the CatB enzyme may have a regulatory role in cell differentiation and morphogenesis. We have now confirmed both these hypotheses. We specifically disrupted the catB gene by homologous recombination. The resulting catB null strain displays a 4-hour delay in development at the time of normal catB gene expression, followed by slow and asynchronous development of fruiting bodies, taking 10 hours longer than the isogenic parent strain. The expression of both prestalk and prespore specific genes was altered in the mutant both temporally and quantitatively, and the resultant mutant spores had increased sensitivity to H2O2. This study supports the idea that catalase B functions in the development of D. discoideum by regulating the level of ROS, and adds to the growing body of evidence for regulatory roles for ROS. Submitted by: Hannah Alexander [AlexanderH@missouri.edu] ============================================================ [End Dicty News, volume 20, number 8]