dictyNews Electronic Edition Volume 33, number 4 July 31, 2009 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. ========= Abstracts ========= A Dictyostelium chalone requires G proteins to regulate proliferation Deenadayalan Bakthavatsalam, Jonathan M. Choe, Nana E. Hanson, and Richard H. Gomer Department of Biochemistry and Cell Biology, Rice University, Houston, TX BMC Biology, in press Several studies have shown that organ size, and the proliferation of tumor  metastases, may be regulated by negative feedback loops in which  autocrine secreted factors called chalones inhibit proliferation.  However,  very little is known about chalones, and how cells sense them.  We  previously identified two secreted proteins, AprA and CfaD, which act as  chalones in Dictyostelium.  Cells lacking AprA or CfaD proliferate faster  than wild type cells, and adding recombinant AprA or CfaD to cells slows  their proliferation. We show here that cells lacking the G protein components Galpha8,  Galpha9, and Gbeta proliferate faster than wild-type cells despite  secreting normal or high levels of AprA and CfaD.  Compared to wild-type  cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only  weakly inhibited by recombinant AprA (rAprA).  Like AprA and CfaD,  Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate  of increase in mass and protein per nucleus), whereas Galpha9 inhibits  both proliferation and growth.  galpha8- cells show normal cell-surface  binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface  rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the  synthesis or processing of the AprA receptor.  Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced  [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require  Galpha8 and Gbeta but not Galpha9.  Like aprA- cells, galpha8- cells have  reduced spore viability.   This study shows that Galpha8 and Gbeta are part of the signal  transduction pathway used by AprA to inhibit proliferation but not growth  in Dictyostelium, whereas Galpha9 is part of a different pathway that  regulates both proliferation and growth, and that a chalone signal  transduction pathway uses G proteins. Submitted by Richard Gomer [richard@rice.edu] -------------------------------------------------------------------------------- Dictyostelium discoideum CenB Is a Bona Fide Centrin Essential for Nuclear Architecture and Centrosome Stability Sebastian Mana-Capelli,1 Ralph Gräf,2 and Denis A. Larochelle1* Department of Biology, Clark University, 950 Main Street, Worcester, Massachusetts 01610,1 Institut für Biochemie und Biologie, Zellbiologie, Universität Potsdam, Karl-Liebknecht-Strasse 24-25, Haus 26, 14476 Potsdam-Golm, Germany2 Eukaryotic Cell, in press Centrins are a family of proteins within the calcium-binding EF-hand superfamily. In addition to their archetypical role at the MTOC, centrins have acquired multiple functionalities throughout the course of evolution. For example, centrins have been linked to different nuclear activities, including mRNA export and DNA repair. Dictyostelium discoideum centrin B is a divergent member of the centrin family. At the amino acid level, DdCenB shows 51% identity with its closest relative and only paralog, DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a well-supported monophyletic and divergent group within the centrin family of proteins. Interestingly, fluorescently tagged versions of DdCenB were not found at the centrosome (in whole cells or in isolated centrosomes). Instead, DdCenB localized to the nuclei of interphase cells. This localization disappeared as the cells entered mitosis, although Dictyostelium cells undergo a closed mitosis in which the nuclear envelope (NE) does not break down. DdCenB knockout cells exhibited aberrant nuclear architecture, characterized by enlarged and deformed nuclei and loss of proper centrosome-nucleus anchoring (observed as NE protrusions). At the centrosome, loss of DdCenB resulted in defects in the organization and morphology of the MTOC and supernumerary centrosomes and centrosome-related bodies. The multiple defects that the loss of DdCenB generated at the centrosome can be explained by its atypical division cycle, transitioning into the NE as it divides at mitosis. On the basis of these findings, we propose that DdCenB is required at interphase to maintain proper nuclear architecture, and before delocalizing from the nucleus, DdCenB is part of the centrosome duplication machinery. Submitted by Denis Larochelle [dlarochelle@clarku.edu] -------------------------------------------------------------------------------- Dynamic localization of G proteins in Dictyostelium discoideum Carrie A. Elzie, Jennifer Colby, Morgan A. Sammons, and Chris Janetopoulos Journal of Cell Science,in press   Extracellular stimuli exert their effects on eukaryotic cells via serpentine G-protein-coupled receptors and mediate a vast number of physiological responses. Activated receptors stimulate heterotrimeric G-proteins, consisting of three subunits, alpha, beta and gamma. In Dictyostelium discoideum, cAMP binds to the cAMP receptor cAR1, which is coupled to the heterotrimer containing the G{alpha}2 subunit. These studies provide in vivo evidence as to how receptors influence the localization of the G-protein complex prior to and after ligand binding. Previous work has shown that the state of the heterotrimer could be monitored by changes in fluorescence (or Förster) resonance energy transfer (FRET) between the alpha2- and beta-subunits of D. discoideum. We now report the kinetics of G-protein activation as a loss of FRET prior to and after cAMP addition by using total internal reflection fluorescence microscopy (TIRFM). We also performed photobleaching experiments to measure G-protein recovery times. Our data show that inactive and active G-proteins cycle between the cytosol and plasma membrane. These data suggest that cAR1 activation slows the membrane dissociation ('off') rate of the alpha2 subunit, while simultaneously promoting beta/gamma-subunit dissociation. Submitted by: Chris Janetopoulos [c.janetopoulos@Vanderbilt.Edu] ============================================================== [End dictyNews, volume 33, number 4]