dictyNews Electronic Edition Volume 37, number 14 December 2, 2011 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 ========= Actin Crosslinking Proteins, Cortexillin I and II, Are Required for cAMP-signaling during Dictyostelium Chemotaxis and Development Shi Shu,1 Xiong Liu,1 Paul W. Kriebel,2 Mathew P. Daniels,3 and Edward D. Korn1 1Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA. 2Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. 3Electron Microscopy Core Facility, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA. Molecular Biology of the Cell, in press Starvation induces Dictyostelium amoebae to secrete cAMP towards which other amoebae stream forming multi-cellular mounds that differentiate and develop into fruiting bodies containing spores. We find that the double deletion of cortexillin (ctx) I and II alters the actin cytoskeleton and substantially inhibits all molecular responses to extracellular cAMP. Synthesis of cAMP-receptor and adenylyl cyclase A (ACA) is inhibited and activation of ACA, RasC and RasG, phosphorylation of ERK2, activation of TORC2 and stimulation of actin polymerization and myosin assembly are greatly reduced. As a consequence, cell streaming and development are completely blocked. Expression of ACA-YFP in the ctxI/ctxII-null cells significantly rescues the wild-type phenotype indicating that the primary chemotaxis and development defect is the inhibition of ACA synthesis and cAMP production. These results demonstrate the critical importance of a properly organized actin cytoskeleton for cAMP-signaling pathways, chemotaxis and development in Dictyostelium. Submitted by Edward Korn [korned2@nhlbi.nih.gov] -------------------------------------------------------------------------------------- Functional characterization of CP148, a novel key component for centrosome integrity in Dictyostelium Oliver Kuhnert, Otto Baumann, Irene Meyer and Ralph Graef* University of Potsdam, Institute for Biochemistry and Biology, Dept. of Cell Biology, D-14476 Potsdam-Golm, Germany Cell. Mol. Life Sci., in press The Dictyostelium centrosome consists of a layered core structure surrounded by a microtubule-nucleating corona. A tight linkage through the nuclear envelope connects the cytosolic centrosome with the clustered centromeres within the nuclear matrix. At G2/M the corona dissociates, and the core structure duplicates yielding two spindle poles. CP148 is a novel coiled coil protein of the centrosomal corona. GFP-CP148 exhibited cell cycle-dependent presence and absence at the centrosome, which correlates with dissociation of the corona in prophase and its reformation in late telophase. During telophase, GFP-CP148 formed cytosolic foci, which coalesced and joined the centrosome. This explains the hypertrophic appearance of the corona upon strong overexpression of GFP-CP148. Depletion of CP148 by RNAi caused virtual loss of the corona and disorganization of interphase microtubules. Surprisingly, formation of the mitotic spindle and astral microtubules was unaffected. Thus, microtubule nucleation complexes associate with centrosomal core components through different means during interphase and mitosis. Furthermore, CP148 RNAi caused dispersal of centromeres and altered Sun1 distribution at the nuclear envelope, suggesting a role of CP148 in the linkage between centrosomes and centromeres. Taken together, CP148 is an essential factor for the formation of the centrosomal corona, which in turn is required for centrosome/centromere linkage. Submitted by Ralph Graef [rgraef@uni-potsdam.de] -------------------------------------------------------------------------------------- The Skp1 Prolyl 4-hydroxylase of Dictyostelium Mediates Glycosylation-Independent and -Dependent Responses to O2 without Affecting Skp1 Stability Dongmei Zhang (1,2), Hanke van der Wel (1), Jennifer M. Johnson (3), Christopher M. West (1,3) (1) Dept. of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, 975 NE 10th St., Oklahoma City, OK, 73104, (2) Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China, (3) Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 975 NE 10th St., Oklahoma City, OK, 73104 J. Biol. Chem., in press Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O2-sensing in animals via modification of the transcriptional factor subunit HIFalpha, resulting in its poly-ubiquitination by the E3VHLubiquitin (Ub)-ligase and degradation in the 26S-proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFalpha-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O2-sensor: for development. Dictyostelium lacks HIFalpha and P4H1 modifies a different protein, Skp1, an adaptor of the SCF-class of E3-Ub ligases related to the E3VHLUb-ligase that targets animal HIFalpha. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc- transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O2-requirement of P4H1-null cells, therefore revealing both glycosylation-independent and -dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Since the effect of the gnt1-deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Though Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub-ligases. Submitted by Chris West [Cwest2@ouhsc.edu] ============================================================== [End dictyNews, volume 37, number 14]