dictyNews Electronic Edition Volume 34, number 12 April 9, 2010 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 ========= SunB, a novel SUN domain-containing protein required for development of Dictyostelium discoideum Nao Shimada 1, 2, Kei Inouye 3, Satoshi Sawai 2 and Takefumi Kawata 1, * 1Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan, 2Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan 3Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan *Author to whom all correspondence should be addressed. Devleop. Growrh & Differ., In press. A gene, sunB, encoding a novel class of Sad1 and UNC-84 (SUN) domain, was isolated from a cDNA screen for suppressors of a mutation in Dd-STATa – a Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription). The SunB protein localized in the area around the nucleus in growing cells, but in the multicellular stages it was predominantly found in prespore vacuoles (PSVs). A disruptant of sunB was multinucleated in the vegetative phase; during development it formed mounds with multiple tips and failed to culminate. The mutation was cell autonomous, and showed reduced expression of the prespore marker gene pspA and elevated expression of marker genes for prestalk AB cells. Interestingly, the level of SunB was abnormally high in the prestalk cells of Dd-STATa mutants, which are defective in culmination. We conclude that SunB is essential for accurate prestalk/prespore differentiation during Dictyostelium development and that its cell-type dependent localization is regulated by a Dd-STATa-mediated signaling pathway. Submitted by Takefumi Kawata [tkawata@bio.sci.toho-u.ac.jp] -------------------------------------------------------------------------------- An unconventional myosin required for cell polarization and chemotaxis Laura M. Breshears, Deborah Wessels, David R. Soll, and Margaret A. Titus Proc.Natl.Acad. Sci., in press MyTH/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) myosins have roles in cellular adhesion, extension of actin-filled projections such as filopiodia and stereocilia, and directional migration. The amoeba Dictyostelium discoideum expresses a simple complement of MyTH/FERM myosins, a class VII (M7) myosin required for cell-substrate adhesion and a unique myosin named MyoG. Mutants lacking MyoG exhibit a wide range of normal actin-based behaviors, including chemotaxis to folic acid, but have a striking defect in polarization and chemotaxis to cAMP. Although the myoG mutants respond to cAMP stimulation by increasing persistence and weakly increasing levels of cortical F-actin, they do not polarize; instead, they maintain a round shape and move slowly and randomly when exposed to a chemotactic gradient. The mutants also fail to activate and localize PI3K to the membrane closest to the source of chemoattractant. These data reveal a role for a MyTH/FERM myosin in mediating early chemotactic signaling and suggest that MyTH/FERM proteins have conserved roles in signaling and the generation of cell polarity. Submitted by Deborah Wessels [deborah-wessels@uiowa.edu] -------------------------------------------------------------------------------- Involvement of the Cytoskeleton in Controlling Leading Edge Function during Chemotaxis Susan Lee, Zhouxin Shen, Douglas N. Robinson, Steven Briggs, and Richard A. Firtel Mol. Biol. Cell., in press In response to directional stimulation by a chemoattractant, cells rapidly activate a series of signaling pathways at the site closest to the chemoattractant source that leads to F-actin polymerization, pseudopod formation, and directional movement up the gradient. Ras proteins are major regulators of chemotaxis in Dictyostelium; they are activated at the leading edge, are required for chemoattractant-mediated activation of PI3K and TORC2, and are one of the most rapid responders with activity peaking at ~3 sec after stimulation. We demonstrate that in myosin II (MyoII) null cells, Ras activation is highly extended and is not restricted to the site closest to the chemoattractant source. This causes elevated, extended, and spatially misregulated activation of PI3K and TORC2 and their effectors Akt/PKB and PKBR1, and elevated F-actin polymerization. We further demonstrate that disruption of specific IQGAP/cortexillin complexes, which also regulate cortical mechanics, causes extended activation of PI3K and Akt/PKB but not Ras activation. Our findings suggest that MyoII and IQGAP/cortexillin play key roles in spatially and temporally regulating leading edge activity and, through this, the ability of cells to restrict the site of pseudopod formation.xation of the MTBD into the stable low affinity state. Submitted by Rick Firtel [rafirtel@ucsd.edu] -------------------------------------------------------------------------------- SCAR/WAVE is activated at mitosis and drives myosin-independent cytokinesis Jason S. King1,3, Douwe M. Veltman1, Marios Georgiou2, Buzz Baum2 and Robert H. Insall1 1Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, UK. G61 1BD. 2MRC-LMCB, University College London, Gower Street, London, UK. WC1E 6BT J. Cell Sci., in press Cell division requires the tight coordination of multiple cytoskeletal pathways. The best understood of these involves myosin II-dependent constriction around the cell equator but both Dictyostelium and mammalian cells also use a parallel, adhesion-dependent mechanism to generate furrows. We show that the actin nucleation factor SCAR/WAVE is strongly activated during Dictyostelium cytokinesis. This activation localises to large polar protrusions, driving separation of the daughter cells. This continues for 10 minutes after division before the daughter cells revert to normal random motility, indicating that this is a tightly regulated process. We demonstrate that SCAR activity is essential to drive myosin II-independent cytokinesis, and stabilises the furrow, ensuring symmetrical division. SCAR is also responsible for the generation of MiDASes, mitosis-specific actin-rich adhesions. Loss of SCAR in both Dictyostelium and Drosophila leads to a similar mitotic phenotype, with severe mitotic blebbing, indicating conserved functionality. We also find that the microtubule end-binding protein EB1 is required to restrict SCAR localisation and direct migration. EB1-null cells also exhibit decreased adhesion during mitosis. Our data reveal a spindle-directed signalling pathway that regulates SCAR activity, migration and adhesion at mitosis. Submitted by Jason King [J.king@beatson.gla.ac.uk] -------------------------------------------------------------------------------- Autophagic cell death in Dictyostelium requires the receptor histidine kinase DhkM Corinne Giusti, Marie-Françoise Luciani, Sarina Ravens, Alexandre Gillet and Pierre Golstein Molecular Biology of the Cell, in press Dictyostelium constitutes a genetically tractable model for the analysis of autophagic cell death (ACD). During ACD Dictyostelium cells first transform into paddle cells, then become round, synthesize cellulose, vacuolize and die. Through random insertional mutagenesis we identified the receptor histidine kinase DhkM as being essential for ACD. Surprisingly, different DhkM mutants showed distinct non-vacuolizing ACD phenotypes. One class of mutants arrested ACD at the paddle cell stage, perhaps through a dominant negative effect. Other mutants, however, progressed further in the ACD programme. They underwent rounding and cellulose synthesis but stopped before vacuolization. Moreover, they underwent clonogenic but not morphological cell death. Exogenous 8-bromo-cAMP restored vacuolization and death. A role for a membrane receptor at a late stage of the ACD pathway is puzzling, raising questions as to which ligand it is a receptor for and which moieties it phosphorylates. Altogether, DhkM is the most downstream known molecule required for this model ACD, and its distinct mutants genetically separate previously undissociated late cell death events. Submitted by Pierre Golstein [golstein@ciml.univ-mrs.fr] ============================================================== [End dictyNews, volume 34, number 12]