dictyNews Electronic Edition Volume 36, number 8 March 11, 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 ========= An Extracellular Matrix, Calmodulin-Binding Protein from Dictyostelium with EGF-Like Repeats that Enhance Cell Motility Andres Suareza, Robert Huberb, Michael Myrec, Danton H. OĠDaya,b,* aDepartment of Biology, University of Toronto at Mississauga, Mississauga, Ontario, Canada bDepartment of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada cMolecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, USA Cellular Signalling, in press CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca2+-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility it likely also represents the first matricellular protein identified in a lower eukaryote. Submitted by Danton OĠDay [danton.oday@utoronto.ca] -------------------------------------------------------------------------------- Functional analysis of Dictyostelium IBARa suggests a conserved role of the I-BAR domain in endocytosis Douwe M. Veltman, Giulio Auciello, Heather J. Spence, Laura M. Machesky, Joshua Z. Rappoport and Robert H. Insall Biochemical Journal, in press I-BAR domain containing proteins such as IRSp53 associate with outwardly curved membranes and connect them to proteins involved in actin dynamics. Research on I-BAR proteins has focussed on possible roles in filopod and lamellipod formation, but their full physiological function remains unclear. The social amoeba Dictyostelium encodes a single I- BAR/SH3 protein, called IBARa, along with homologues of proteins that interact with IRSp53 family proteins in mammalian cells, providing an excellent model to study its cellular function. Disruption of the gene encoding IBARa leads to a mild defect in development, but filopod and pseudopod dynamics are unaffected. Furthermore, ectopically expressed IBARa does not induce filopod formation and does not localize to filopods. Instead, IBARa associates with clathrin puncta immediately before they are endocytosed. This role is conserved- human BAIAP2L2 also tightly colocalizes with clathrin plaques, though its homologues IRSp53 and IRTKS associate with other punctate structures. Our data suggest that I-BAR-containing proteins help generate the membrane curvature required for endocytosis and implies an unexpected role for IRSp53 family proteins in vesicle trafficking. Submitted by Robert Insall [R.Insall@beatson.gla.ac.uk] -------------------------------------------------------------------------------- Actin Polymerization Driven by WASH Causes V-ATPase Retrieval and Vesicle Neutralization Before Exocytosis. Michael Carnell, Tobias Zech, Simon Johnston, Robin May, Thierry Soldati, Laura Machesky and Robert Insall J. Cell Biol., in press WASH is a recently identified and evolutionarily conserved regulator of actin polymerization. Here we show that WASH coats mature Dictyostelium lysosomes and is essential for exocytosis of indigestible material. A related process, expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles and cells expressing dominant negative WASH mutants inefficiently expel Cryptococcus. Dictyostelium WASH causes F-actin patches to form on lysosomes, leading to removal of vacuolar ATPase (V-ATPase) and neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coat are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive separation and recycling of proteins such as the V-ATPase. Submitted by Robert Insall [R.Insall@beatson.gla.ac.uk] -------------------------------------------------------------------------------- Modelling cell movement and chemotaxis using pseudopod-based feedback. M.P. Neilson, J.A. Mackenzie, S.D. Webb and R.H. Insall SIAM journal on Scientific Computation, in press A computational framework is presented for the simulation of eukaryotic cell migration and chemotaxis. An empirical pattern formation model, based on a system of non-linear reaction-diffusion equations, is approximated on an evolving cell boundary using an Ar- bitrary Lagrangian Eulerian surface finite element method (ALE-SFEM). The solution state is used to drive a mechanical model of the protrusive and retractive forces exerted on the cell boundary. Movement of the cell is achieved using a level set method. Results are presented for cell migration with and without chemotaxis. The simulated behaviour is compared with experimental results of migrating Dictyostelium discoideum cells. Submitted by Robert Insall [R.Insall@beatson.gla.ac.uk] -------------------------------------------------------------------------------- A Polarized Epithelium Organized by Beta- and Alpha-Catenin Predates Cadherin and Metazoan Origins Daniel J. Dickinson, W. James Nelson,* William I. Weis* *To whom correspondence should be addressed Science, 11 March 2011, Vol. 331, #6022 A fundamental characteristic of metazoans is the formation of a simple, polarized epithelium. In higher animals, the structural integrity and functional polarization of simple epithelia require a cell-cell adhesion complex containing a classical cadherin, the Wnt-signaling protein beta-catenin and the actin-binding protein alpha-catenin. We show that the non-metazoan Dictyostelium discoideum forms a polarized epithelium that is essential for multicellular development. Although D. discoideum lacks a cadherin homolog, we identify an alpha-catenin ortholog that binds a beta-catenin-related protein. Both proteins are essential for formation of the epithelium, polarized protein secretion and proper multicellular morphogenesis. Thus the organizational principles of metazoan multicellularity may be more ancient than previously recognized, and the role of the catenins in cell polarity predates the evolution of Wnt signaling and classical cadherins. Submitted by Daniel Dickinson [ddickins@stanford.edu] ============================================================== [End dictyNews, volume 36, number 8]