Dicty News Electronic Edition Volume 10, number 3 January 24, 1998 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============== Announcement ============== Motion Analysis of Living Cells David R. Soll and Deborah Wessels, Editors a book in the series Techniques in Modern Biomedical Microscopy David M. Shotton, Series Editor, Wiley-Liss, Publisher Most active cellular functions involve motion. From such subtle subcellular actions as the sorting and targeting of vesicles and organelles leading to the release of harmones to the complex rearrangements of a sphere of embryonic cells forming the body plan of a complete organism, motility of cells is exhibited along a wide range of observable behaviors. Despite this phenomenon, however, the views of modern biology historically have been on static images and models constructed from biochemical evidence. Motion Analysis of Living Cells is the first volumeto compile the latest research by prominent specialists in molecular and cell biology, presenting newly developed techniques for studying cellular motions and critical analyses of the information these techniques can yield. Focusing on actin-based motility systems and drawing on the latest advances in the techniques of biochemistry, biophysics, microscopy, computer-assisted motion analysis, and molecular genetics, the works in this collection will prove invaluable to future research in embryogenesis, cancer, and diseases related to the cellular immune system. Among the topics covered: ** Bacterial motility and chemotaxis ** New technologies for characterizing motility-related parameters ** Methods for analyzing the cortical tension of normal cells compared to abnormal cells ** Model for how the cell extends a pseudopod ** Computer-assisted analyses of cytoskeletal mutants of Dictyostelium discoideum ** Emerging methods for studying how cells respond to topographical cues at the substratum ** Polymerization of host actin to facilitate propulsion ** Genetic approaches to the regulatory mechanisms involved in early zebra fish morphogenesis and specification of cell fates ** Specific examples of single cell motility in embryogenesis A groundbreaking treatment of motion analysis of animal cells, Motion Analysis of Living Cells is a thorough review for professional as well as a comprehensive introduction for students and researchers in the field. It is must reading for cell and developmental biologists, microbiologists, immunologists, neuroscientists, cancer researchers, zoologists, and basic researchers in reproductive medicine. Three chapters in the book deal directly with Dictyostelium discoideum motility. The preface of the book, chapters contained, as well as introductions of selected chapters are provided on the W.M. Keck Dynamic Image Analysis Facility home page at http://www.uiowa.edu/~keck/ =========== Abstracts =========== Retrotransposable elements in the Dictyostelium discoideum genome THOMAS WINCKLER Institut fuer Pharmazeutische Biologie, Universitaet Frankfurt/M. (Biozentrum), Marie-Curie-Strasse 9, D-60439 Frankfurt/M, Germany Cell. Mol. Life Sci. (formerly Experientia), in press Abstract: Repetitive DNA is a major component of any living cell. In eukaryotes retrotransposable el ements make up several percent of the genome size, and consequently, retroelements are often identified in experiments aimed to establish physical maps and whole genome sequences. In this review recent progress in the characterization of retrotransposable elements in th e genome of the eukaryotic microorganism Dictyostelium discoideum is summarized with a focus on retroelements which integrate near transfer RNA genes with intriguing position specificity. ------------------------------------------------------------------------- Structural Characterization of a Dynein Motor Domain. Samso, M., Radermacher, M., Frank, J., & Koonce, M.P. Division of Molecular Medicine, Wadsworth Center, Albany, NY 12201-0509 J. Mol. Biol., in press. ABSTRACT. Cytoplasmic dynein is a microtubule-based mechanochemical protein that plays an essential role in cell division, vesicle transport, and cytoplasmic membrane organization. As a molecular motor, dynein utilizes an ATP hydrolysis mechanism to bind and release microtubules and to undergo conformational changes that result in a net displacement towards the microtubule’s minus end. To visualize structural features of this motor protein, we have begun to characterize the dynein head domain by electron microscopy and image processing. Transmission electron microscopy of negatively-stained native dynein from Dictyostelium has been performed and images of the head domain have been aligned and analyzed with the software SPIDER. The resulting 2D averages show an oblong round shape composed of 7-8 globular domains or lobes that encircle a stain-filled area. A recombinant 380 kDa fragment of the dynein heavy chain encodes just the globular head domain; analysis of these particles reveals a high structural similarity with the native head domain. A prominent stalk can be seen in several projections of this fragment, suggesting a structure analogous to the B-link described for some axonemal dyneins. Single tilt pair images were used to compute low resolution 3D reconstructions of the dynein head domain. These show a flattened spheroidal shape of 13.5 nm in length with seven similar domains arranged in a ring. Slices through the reconstructions reveal a large central cavity. This is the first detailed description of the head domain structure for a dynein molecule. The presence of a central cavity and the outer globular features, along with its large size make dynein structurally distinct from either myosin or kinesin. ------------------------------------------------------------------------- Cell-density sensing mediated by a G-protein-coupled receptor activating phospholipase C Derrick T. Brazill, David F. Lindsey, John D. Bishop, and Richard H. Gomer* Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street Houston, TX 77005-1892 J. Biol Chem, in press Abstract When the unicellular eukaryote Dictyostelium discoideum starves, it senses the local density of other starving cells by simultaneously secreting and sensing a glycoprotein called conditioned medium factor (CMF). When the density of starving cells is high, the corresponding high density of CMF permits signal transduction through cAR1, the chemoattractant cAMP receptor. cAR1 activates a heterotrimeric G protein whose alpha subunit is Galpha2. CMF regulates cAMP signal transduction in part by regulating the lifetime of the cAMP-stimulated Galpha2-GTP configuration. We find here that GTPgammaS inhibits the binding of CMF to membranes, suggesting that the putative CMF receptor is coupled to a G protein. Cells lacking Galpha1 (Ga1 null) do not exhibit GTPgammaS inhibition of CMF binding and do not exhibit CMF regulation of cAMP signal transduction, suggesting that the putative CMF receptor interacts with Galpha1. Work by others has suggested that Galpha1 inhibits phospholipase C (PLC), yet when cells lacking either Galpha1 or PLC were starved at high cell densities (and thus in the presence of CMF), they developed normally and had normal cAMP signal transduction. We find that CMF activates PLC. Galpha1 null cells starved in the absence or presence of CMF behave in a manner similar to control cells starved in the presence of CMF in that they extend pseudopods, have an activated PLC, have a low cAMP-stimulated GTPase, permit cAMP signal transduction, and aggregate. Cells lacking Gbeta have a low PLC activity which cannot be stimulated by CMF. Cells lacking PLC exhibit IP3 levels and cAMP-stimulated GTP hydrolysis rates intermediate to what is observed in wild-type cells starved in the absence or in the presence of an optimal amount of CMF. We hypothesize that CMF binds to its receptor, releasing Gbetagamma from Galpha1. This activates PLC, which causes the Galpha2 GTPase to be inhibited, prolonging the lifetime of the cAMP- activated Galpha2-GTP configuration. This, in turn, allows cAR1- mediated cAMP signal transduction to take place. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 3]