Dicty News Electronic Edition Volume 19, number 14 December 13, 2002 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 ============= Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton in Dictyostelium Ruth Furukawa, Andrew Maselli, Susanne A. M. Thomson, Rita W. L. Lim, John V. Stokes and Marcus Fechheimer Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA Journal of Cell Science In Press The actin cytoskeleton is sensitive to changes in calcium,which affect contractility, actin-severing proteins, actin-crosslinking proteins and calmodulin-regulated enzymes. To dissect the role of calcium control on the activity of individual proteins from effects of calcium on other processes, calcium-insensitive forms of these proteins were prepared and introduced into living cells to replace a calcium-sensitive form of the same protein. Crosslinking and bundling of actin filaments by the Dictyostelium 34 kDa protein is inhibited in the presence of micromolar free calcium. A modified form of the 34 kDa protein with mutations in the calcium binding EF hand (34 kDa DEF2) was prepared using site-directed mutagenesis and expressed in E. coli. Equilibrium dialysis using [ 45 Ca]CaCl2 revealed that the wild-type protein is able to bind one calcium ion with a Kd of 2.4 mM. This calcium binding is absent in the 34 kDa DEF2 protein. The actin-binding activity of the 34 kDa DEF2 protein was equivalent to wildtype but calcium insensitive in vitro. The wild-type and 34 kDa DEF2 proteins were expressed in 34-kDa-null and 34 kDa/a-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo. The 34 kDa DEF2 failed to supply function of the 34 kDa protein important for control of cell size and for normal growth to either of these 34-kDa-null strains. Furthermore, the distribution of the 34 kDa protein and actin were abnormal in cells expressing 34 kDa DEF2. Thus, calcium regulation of the formation and/or dissolution of crosslinked actin structures is required for dynamic behavior of the actin cytoskeleton important for cell structure and growth. submitted by: amaselli@uconnvm.uconn.edu ---------------------------------------------------------------------------- Constitutively Active Protein Kinase A Disrupts Motility and Chemotaxis in Dictyostelium Hui Zhang*, Paul J. Heid*, Deborah Wessels*, Karla J. Daniels*, Tien Pham*, William F. Loomis?, and David R. Soll*? *W.M. Keck Dynamic Image Analysis Facility Department of Biological Sciences University of Iowa Iowa City, IA 52242 ?Department of Biology University of California, San Diego La Jolla, CA 92039 Eukaryotic Cell, in press. ABSTRACT Deletion of the gene for the regulatory subunit of PKA results in constitutively active PKA in the mutant pkaR-. To investigate the role of PKA in the basic motile behavior and chemotaxis of Dictyostelium, pkaR- cells were subjected to computer-assisted 2D and 3D motion analysis. pkaR- cells crawled at only half the speed of wild type cells in buffer, chemotaxed in spatial gradients of cAMP, but with reduced efficiency, were incapable of suppressing lateral pseudopods in the front of temporal waves of cAMP, a requirement for natural chemotaxis, did not exhibit the normal velocity surge in response to the front of a wave, and were incapable of chemotaxing towards an aggregation center in natural waves generated by a majority of wild type cells in mixed cultures. Many of the behavioral defects appeared to be the result of the constitutively ovoid shape of pkaR- cells, which forced the dominant pseudopod off the substratum and to the top of the cell body. The behavioral abnormalities that pkaR- cells shared with regA- cells are discussed in considering the pathway ERK2 | RegA | [cAMP] PKA, which emanates from the front of a wave. The results demonstrate that cells must suppress PKA activity in order to elongate along a substratum, suppress lateral pseudopod formation, and crawl and chemotax efficiently. The results also implicate PKA activation in dismantling cell polarity at the peak and in the back of a natural cAMP wave. submitted by: Hui Zhang [hui-zhang@uiowa.edu] ---------------------------------------------------------------------------- Co-occurrence in nature of different clones of the social amoeba, Dictyostelium discoideum Angelo Fortunato, Joan E. Strassmann, Lorenzo Santorelli, and David C. Queller Department of Ecology and Evolutionary Biology MS170, Rice University, PO Box 1892, Houston, Texas, 77251-1892, USA Molecular Ecology, in press Abstract. The social amoeba, Dictyostelium discoideum, produces a multicellular fruiting body and has become a model system for cell-cell interactions such as signaling, adhesion, and development. However, unlike most multicellular organisms, it forms by aggregation of cells and, in the lab, it forms genetic chimeras where there may be competition among clones. Here we show that chimera formation is also likely in nature, because different clones commonly co-occur on a very small scale. This suggests that D. discoideum will likely have evolved strategies for competing in chimeras, and that the function of some developmental genes will be competitive. Natural chimerism also makes D. discoideum a good model organism for the investigation of issues relating to coexistence and conflict between cells. submitted by: David Queller [queller@pop.ruf.rice.edu] ---------------------------------------------------------------------------- A diverse family of inositol 5-phosphatases playing a role in growth and development in Dictyostelium discoideum Harrit M. Loovers, Kees Veenstra, Helena Snippe, Xavier Pesesse, Christophe Erneux, and Peter J.M. van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands and Institute of Interdisciplinary Research, Free University of Brussels, Campus Erasme, Bldg. C, 808 Route de Lennik, B-1070 Brussels, Belgium. J. Biol. Chem, in press Inositol phosphate containing molecules play an important role in a broad range of cellular processes. Inositol 5-phosphatases participate in the regulation of these signaling molecules. We have identified four inositol 5-phosphatases in Dictyostelium discoideum, Dd5P1-4, showing a high diversity in domain composition. Dd5P1 possesses only a inositol 5-phosphatase catalytic domain. An unique domain composition is present in Dd5P2, containing a RCC1-like domain; RCC1 has a seven-bladed propeller structure and interacts with G-proteins. Dd5P3 and Dd5P4 have a domain composition similar to human Synaptojanin, with a Sac1 domain, and OCRL, with a RhoGAP domain, respectively. We have expressed the catalytic domains and show that these inositol 5-phosphatases have different substrate preferences. Single and double gene inactivation suggest a functional redundancy for Dd5P1, Dd5P2 and Dd5P3. Inactivation of the gene coding for Dd5P4 leads to defects in growth and development. These defects are restored by expression of the complete protein, but not by the 5-phosphatase catalytic domain. submitted by: P.J.M.van.Haastert [P.J.M.van.Haastert@chem.rug.nl] ---------------------------------------------------------------------------- [End Dicty News, volume 19, number 14]