dictyNews Electronic Edition Volume 31, number 2 July 11, 2008 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 ========= The GPI-anchored Superoxide Dismutase, SodC, is essential for regulating  basal Ras activity and chemotaxis of Dictyostelium discoideum Sudhakar Veeranki, Bohye Kim, and Leung Kim*   Dept of Biological Sciences, Florida International University, Miami, Fl 33199 USA Journal of Cell Sciences, in press A genetic screen for Dictyostelium mutant displaying high level of constitutive Phosphatidylinositol-3,4,5-triphosphate (PIP3) led to the finding that the   glycosylphosphatidylinositol (GPI) anchored superoxide dismutase, SodC, regulates small GTPase Ras. Cells lacking SodC exhibited constitutively high levels of active Ras, more membrane localization of GFP-PHcrac, and defects in chemoattractant sensing, cell polarization, and motility. These defects of sodC- cells were partially restored by expression of wild type SodC but not with the catalytically inactive mutant SodC (H245R, H247Q). Furthermore, an inhibition of PI3K activity in sodC- cells by LY294002 only partially restored chemoattractant sensing and cell polarization, consistent with the fact that sodC- cells have aberrantly high level of active Ras, which functions upstream of PI3K. Higher level of active GFP-RasG was observed in sodC- cells, which significantly decreased upon incubation of sodC- cells with the superoxide scavenger XTT. Having constitutively high levels of active Ras proteins and more membrane localization of GFP-PHcrac, sodC- cells exhibited severe defects in chemoattractant sensing, cell polarization, and motility.   Submitted by: Kim Leung [kiml@fiu.edu] -------------------------------------------------------------------------------- Dictyostelium kinase DPYK3 negatively regulates STATc signaling in cell fate decision. Nam-Sihk Lee, Marbelys Rodriguez, Bohye Kim, and Leung Kim* Department of Biological Sciences Florida International University Miami, FL 33199 USA Development, Growth and Differentiation, in press DPYK3, a member of the Dictyostelium TKL (Tyrosine Kinase Like) kinase family, was ablated by homologous recombination. dpyk3- cells displayed aberrant pattern formation during development. The prestalk O zone was not properly formed and, instead, the prespore zone was expanded in dpyk3- slugs. During development, the transcription factor STATc was persistently phosphorylated and ecmAO expression level was kept low in dpyk3- cells. Furthermore, in response to DIF-1 in suspension culture, dypk3- cells displayed persistent STATc phosphorylation and re-introduction of DPYK3 in dypk3- cells restored transient STATc phosphorylation similarly to wild type cells. In contrast to the positive STAT regulation by Janus Kinase in metazoans, Dictyostelium DPYK3 negatively regulates STATc during development in response to DIF-1 signaling. Submitted by: Kim Leung [kiml@fiu.edu] -------------------------------------------------------------------------------- Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding Kyuwon Baek*†, Xiong Liu*‡, François Ferron†, Shi Shu‡, Edward D. Korn‡§, Roberto Dominguez†§ †University of Pennsylvania School of Medicine, Department of Physiology, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA. ‡Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. *These authors contributed equally to this work Proc. Natl. Acad. Sci. USA, in press On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin-Dictyostelium-actin is strikingly similar to previously determined structures of profilin-beta-actin and profilin-alpha-actin. By comparing this representative set of profilin-actin structures with other structures of actin we highlight the effects of profilin on the actin conformation. In the profilin-actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7º rotation of the two  major domains of actin relative to each other. As a result, the nucleotide cleft  becomes moderately more open in the profilin-actin complex, probably  explaining the stimulation of nucleotide exchange on actin by profilin. Submitted by: Edward Korn [edk@nih.gov] -------------------------------------------------------------------------------- Actin-Cytoskeleton Dynamics in Non-monotonic Cell Spreading Doris Heinrich1, Simon Youssef1, Britta Schroth-Diez2, Ulrike Engel3, Daniel Aydin4, Jacques Bluemmel4, Joachim Spatz4, and Guenther Gerisch5* 1 Department fuer Physik, Ludwig-Maximilians-University, Geschwister-Scholl-Platz 1, D-80539 Muenchen, Germany. 2 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, D-01307 Dresden, Germany. 3 Nikon Imaging Center at the University of Heidelberg, Bioquant BQ 0004, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany. 4 Max Planck Institute for Metals Research, Dept. of New Materials and Biosystems, Heisenbergstr. 3, D-70569 Stuttgart, Germany, and University of Heidelberg, Dept. of Biophysical Chemistry. 5  Max-Planck-Institut fuer Biochemie, Am Klopferspitz 18, D-82152 Martinsried, Germany.   Cell Adhesion & Migration, in press The spreading of motile cells on a substrate surface is accompanied by  reorganization of their actin network. We show that spreading in the highly  motile cells of Dictyostelium is non-monotonic, and thus differs from the  passage of spreading cells through a regular series of stages. Quantification  of the gain and loss of contact area revealed fluctuating forces of protrusion  and retraction that dominate the interaction of Dictyostelium cells with a  substrate. The molecular basis of these fluctuations is elucidated by  dual-fluorescence labeling of filamentous actin together with proteins that  highlight specific activities in the actin system. Front-to-tail polarity is  established by the sorting out of myosin-II from regions where dense actin  assemblies are accumulating. Myosin-IB identifies protruding front regions,  and the Arp2/3 complex localizes to lamellipodia protruded from these regions.  Coronin is used as a sensitive indicator of actin disassembly to visualize  the delicate balance of polymerization and depolymerization in spreading  cells. Short-lived actin patches that co-localize with clathrin suggest that  membrane internalization occurs even when the substrate-attached cell  surface expands. We conclude that non-monotonic cell spreading is  characterized by spatiotemporal patterns formed by motor proteins together  with regulatory proteins that either promote or terminate actin polymerization  on the scale of seconds. Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de] -------------------------------------------------------------------------------- Regulation of Contractile Vacuole Formation and Activity in Dictyostelium Fei Du, Kimberly Edwards, Zhouxin Shen, Binggang Sun, Arturo De Lozanne, Steven Briggs, and Richard A. Firtel Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0380, USA and Section of Molecular Cell & Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA EMBO J., in press The contractile vacuole (CV) system is the osmoregulatory organelle required for survival for many free-living cells under hypotonic conditions. We identified a new CV regulator, Disgorgin, a TBC domain-containing protein, which translocates to the CV membrane at the late stage of CV charging and regulates CV/plasma membrane fusion and discharging. disgorgin- cells produce large CVs due to impaired CV/plasma membrane fusion. Disgorgin is a specific GAP for Rab8A-GTP, which also localizes to the CV and whose hydrolysis is required for discharging. We demonstrate that Drainin, a previously identified TBC-domain-containing protein, lies upstream from Disgorgin in this pathway. Unlike Disgorgin, Drainin lacks GAP activity but functions as a Rab11A effector. The BEACH family proteins LvsA and LvsD were identified in a suppressor/enhancer screen of the disgorgin- large CV phenotype and demonstrated to have distinct functions in regulating CV formation. Our studies help define the pathways controlling CV function. Submitted by: Rick Firtel [rafirtel@ucsd.edu] -------------------------------------------------------------------------------- Article Addendum Vacuole membrane protein 1, autophagy and much more Javier Calvo-Garrido, Sergio Carilla-Latorre and Ricardo Escalante Instituto de Investigaciones Biomedicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autonoma de Madrid, Calle Arturo Duperier 4, 28029 Madrid, Spain Autophagy, In press Vacuole membrane protein 1 (Vmp1) is a putative transmembrane protein that has been associated with different functions including autophagy, cell adhesion and membrane traffic. Highly similar proteins are present in lower eukaryotes and plants although a homologue is absent in the fungi lineage. We have recently described the first loss-of-function mutation for a Vmp1 homologue in a model system, Dictyostelium discoideum. Our results give a more comprehensive view of the intricate roles played by this new gene. Dictyostelium Vmp1 is an endoplasmic reticulum-resident protein. Cells deficient in Vmp1 display pleiotropic defects in the context of the secretory pathway such as organelle biogenesis, the endocytic pathway and protein secretion.  The biogenesis of the contractile vacuole, an organelle necessary to survive under hypoosmotic conditions, is compromised as well as the structure of the endoplasmic reticulum and the Golgi apparatus. Transmission electron microscopy also shows abnormal accumulation of aberrant double-membrane vesicles, suggesting a defect in autophagosome biogenesis or maturation. The expression of a mammalian Vmp1 in the Dictyostelium mutant complements the phenotype suggesting a functional conservation during evolution. We are taking the first steps in understanding the function of this fascinating protein and recent studies have brought us more questions than answers about its basic function and its role in human pathology. Submitted by: Ricardo Escalante [rescalante@iib.uam.es] -------------------------------------------------------------------------------- Kinesin-5 is Not Essential for Mitotic Spindle Elongation in Dictyostelium Irina Tikhonenko, Dilip K. Nag, Nora Martin, and Michael P. Koonce Wadsworth Center, Albany, NY Cell Motil. Cytoskel, in press The proper assembly and operation of the mitotic spindle is essential to ensure the accurate segregation of chromosomes and to position the cytokinetic furrow during cell division in eukaryotes. Not only are dynamic microtubules required, but also the concerted actions of multiple motor proteins are necessary to effect spindle pole separation, chromosome alignment, chromatid segregation, and spindle elongation. Although a number of motor proteins are known to play a role in mitosis, there remains a limited understanding of their full range of functions and the details by which they interact with other spindle components. The kinesin-5 (BimC/Eg5) family of motors is largely considered essential to drive spindle pole separation during the initial and latter stages of mitosis. We have deleted the gene encoding the kinesin-5 member in Dictyostelium, (kif13), and find that, in sharp contrast with results found in vertebrate, fly, and yeast organisms, kif13- cells continue to grow at rates indistinguishable from wild type. Phenotype analysis reveals a slight increase in spindle elongation rates in the absence of Kif13. More importantly, there is a dramatic, premature separation of spindle halves i n kif13- cells, suggesting a novel role of this motor in maintaining spindle integrity at the terminal stages of division. Submitted by: Michael Koonce [koonce@wadsworth.org] ============================================================== [End dictyNews, volume 31, number 2]