dictyNews Electronic Edition Volume 39, number 3 January 25, 2013 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 dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Stress and development in Dictyostelium discoideum: the involvement of the catalytic calcineurin A subunit. Sascha Thewes, Sebastian K. Schubert, Kyuhyeon Park, and Rupert Mutzel Journal of Basic Microbiology, in press Calcium signaling is one of the most important signaling-pathways in all eukaryotes. One important target activated by an increased intracellular calcium concentration via calmodulin is the protein phosphatase calcineurin, which is composed of a catalytic subunit (calcineurin A) and a regulatory subunit (calcineurin B). The importance of calcium and calcineurin for the differentiation and development of the social amoeba Dictyostelium discoideum has already been shown by pharmacological approaches. However, so far only a RNAi-silenced calcineurin B mutant has been investigated on a molecular level. Here we describe the construction and phenotypic investigation of a RNAi-silenced calcineurin A mutant. Phenotypic aberrations during development resemble those produced by silencing of calcineurin B with ectopic tip formation of the fruiting bodies. Additionally we tested the response of the mutants under various stress conditions in liquid culture as well as during development. Both, calcineurin A and B RNAi-mutants, are hypersensitive during development towards cation stress. Besides its role in development calcineurin is thus also involved in the stress response in D. discoideum. Further, our data imply that many functions of calcineurin are conserved among the eukaryotes. Submitted by Sascha Thewes [sascha.thewes@fu-berlin.de] --------------------------------------------------------------------------- Conserved gene-regulatory function of the carboxy-terminal domain of dictyostelid C-module-binding factor Anika Schmith, Marco Groth, Josephine Ratka, Sara Gatz, Thomas Spaller, Oliver Siol, Gernot Glšckner and Thomas Winckler Eukaryotic Cell, in press C-module-binding factor (CbfA) is a jumonji-type transcription regulator that is important for maintaining the expression and mobility of the retrotransposable element TRE5-A in the social amoeba Dictyostelium discoideum. CbfA-deficient cells have lost TRE5-A retrotransposition, are impaired in the ability to feed on bacteria, and do not enter multicellular development due to a block in cell aggregation. In this study, we performed Illumina RNA sequencing on growing CbfA mutant cells to obtain a list of CbfA-regulated genes. We demonstrate that the carboxy-terminal domain of CbfA alone is sufficient to mediate the majority of CbfA-dependent gene expression. The carboxy-terminal domain of CbfA from the distantly related social amoeba Polysphondylium pallidum restored the expression of CbfA-dependent genes in the D. discoideum CbfA mutant, indicating a deep conservation in the gene regulatory function of this domain in the dictyostelid clade. The CbfA-like protein CbfB displays ~25% sequence identity with CbfA in the amino- terminal region, which contains a JmjC domain and two zinc finger regions and is thought to mediate chromatin-remodeling activity. In contrast to CbfA proteins, where the carboxy-terminal domains are strictly conserved in all dictyostelids, CbfB proteins have completely unrelated carboxy- terminal domains. Outside the dictyostelid clade, CbfA-like proteins with the CbfA-archetypical JmjC/zinc finger arrangement and individual carboxy- terminal domains are prominent in filamentous fungi but are not found in yeasts, plants, and metazoans. Our data suggest that two functional regions of the CbfA-like proteins evolved at different rates to allow the occurrence of species-specific adaptation processes during genome evolution. Submitted by Thomas Winckler [t.winckler@uni-jena.de] --------------------------------------------------------------------------- Matricellular Signal Transduction Involving Calmodulin in the Social Amoebozoan Dictyostelium Danton H. OĠDay1,2,* and Robert J. Huber3 1 Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6 2 Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, Canada M5S 3G5 3 Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Richard B. Simches Research Center, 185 Cambridge Street, Boston, MA, USA 02114 Genes, in press Abstract: The social amoebozoan Dictyostelium discoideum undergoes a developmental sequence wherein an extracellular matrix (ECM) sheath surrounds a group of differentiating cells. This sheath is comprised of proteins and carbohydrates, like the ECM of mammalian tissues. One of the characterized ECM proteins is the cysteine-rich, EGF-like (EGFL) repeat-containing, calmodulin (CaM)-binding protein (CaMBP) CyrA. The first EGFL repeat of CyrA increases the rate of random cell motility and cyclic AMP-mediated chemotaxis. Processing of full-length CyrA (~63kDa) releases two major EGFL repeat-containing fragments (~45kDa and ~40kDa) in an event that is developmentally regulated. Evidence for an EGFL repeat receptor also exists and downstream intracellular signaling pathways involving CaM, Ras, protein kinase A and vinculin B phosphorylation have been characterized. In total, these results identify CyrA as a true matricellular protein comparable in function to tenascin C and other matricellular proteins from mammalian cells. Insight into the regulation and processing of CyrA has also been revealed. CyrA is the first identified extracellular CaMBP in this eukaryotic microbe. In keeping with this, extracellular CaM (extCaM) has been shown to be present in the ECM sheath where it binds to CyrA and inhibits its cleavage to release the 45kDa and 40kDa EGFL repeat-containing fragments. The presence of extCaM and its role in regulating a matricellular protein during morphogenesis extends our understanding of CaM-mediated signal transduction in eukaryotes. Submitted by Danton H. OĠDay (danton.oday@utoronto.ca) --------------------------------------------------------------------------- A SAP-domain containing protein shuttles between the nucleus and cell membranes and plays a role in adhesion and migration in D. discoideum. Jessica S. Kelsey and Daphne D. Blumberg Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, Maryland 21250 BiologyOpen, accepted The AmpA protein reduces cell adhesion, thereby influencing cell migration in Dictyostelium. To understand how ampA influences cell migration, second site suppressors of an AmpA overexpressing cell line were created by REMI mutagenesis. Mutant candidates were identified by their ability to suppress the large plaques that the AmpA overexpressing cells form on bacterial lawns as a result of their increased rate of migration. One suppressor gene, sma, encodes an uncharacterized protein which contains a SAP DNA binding domain and a PTEN like domain. Using sma gene knockouts and Sma-mRFP expressing cell lines, a role for sma in influencing cell migration was uncovered. Knockouts of the sma gene in a wildtype background enhanced chemotaxis. An additional role for Sma in influencing cell-cell adhesion was also demonstrated. Sma protein transitions between cytosolic and nuclear localizations as a function of cell density. In growing cells migrating to folic acid it is localized to regions of actin polymerization and absent from the nucleus. A role for Sma in influencing ampA mRNA levels is also demonstrated. Sma additionally appears to be involved in ampA pathways regulating cell size, actin polymerization, and cell substrate adhesion. We present insights to the SAP domain-containing group of proteins in Dictyostelium and provide evidence of a role for a SAP domain-containing protein shuttling from the nucleus to sites of actin polymerization during chemotaxis to folic acid and influencing the efficiency of migration. Submitted by Daphne D. Blumberg [blumberg@umbc.edu] ============================================================== [End dictyNews, volume 39, number 3]