Dictyostelium discoideum Protocols presents a comprehensive collection of cutting-edge molecular biological protocols for studying D. discoideum, with emphasis on its utility in the study of fundamental cellular processes including signal transduction, chemotaxis, cell motility, cytokinesis, phagocytosis, and aspects of development such as cell sorting, pattern formation, and cell type differentiation. Written by experts in the field, this important guide provides easy access to current methods and techniques for the study of D. discoideum. For the newcomer to the field, it offers a complete and inclusive introduction to this important model organism. The book is divided into four main parts, which include an introduction to the organism that provides integral community resources and genome-wide approaches; basic methods and available molecular genetic techniques for study of the organism; imaging and localization methods; and a discussion of D. discoideum, with emphasis on its unique advantages as a model system. This in-depth guide provides a complete introduction to and thorough handbook for the study of D. discoideum, including a discussion of the implications of the recently-completed Dictyostelium genome project in strengthening the position of D. discoideum as a model organism for studying fundamental cell processes and aspects of development. Researchers at all levels of experience will benefit from the presentations throughout of the most current, useful and innovative techniques for the study of D. discoideum from leading Dictyostelium scientists.
Part I. The Organism, Community Resources, and Genome-wide Studies; The Secret Lives of Dictyostelium; Richard H. Kessin; The Genome of Dictyostelium discoideum; Adam Kuspa and William F. Loomis; The cDNA Sequencing Project; Hideko Urushihara, Takahiro Morio, and Yoshimasa Tanaka; dictyBase and the Dicty Stock Center; Petra Fey, Pascale Gaudet, Karen E. Pilcher, Jakob Franke, and Rex L. Chisholm; Analysis of Gene Expression Using cDNA Microarrays; Marcel Kaul and Ludwig Eichinger; Proteomic Analysis of Dictyostelium discoideum; Udo Roth, Stefan Muller, and Franz-Georg Hanisch; Part II. Basic Methods and Molecular Genetic Techniques; Cultivation, Spore Production, and Mating; Hideko Urushihara; Parasexual Genetics Using Axenic Cells; Jason King and Robert Insall; Slug Phototaxis, Thermotaxis, and Spontaneous Turning Behavior; Paul R. Fisher and Sarah J. Annesley; Purification Techniques of Subcellular Compartments for Analytical and Preparative Purposes; Laurence Aubry and Gerard Klein; Generation of Multiple Knockout Mutants Using the Cre-loxP System; Alan R. Kimmel and Jan Faix; Restriction Enzyme-Mediated Integration (REMI) Mutagenesis; Adam Kuspa; RNA Interference and Antisense-Mediated Gene Silencing in Dictyostelium; Markus Kuhlmann, Blagovesta Popova, and Wolfgang Nellen; Part III. Imaging and Localization Methods; Application of Fluorescent Protein Tags as Reporters in Live-Cell Imaging Studies; Annette Muller-Taubenberger; Investigating Gene Expression: In Situ Hybridization and Reporter Genes; Ricardo Escalante and Leandro Sastre; Application of 2D and 3D DIAS to Motion Analysis of Live Cells in Transmission and Confocal Microscopy Imaging; Deborah Wessels, Spencer Kuhl, and David R. Soll; Using Quantitative Fluorescence Microscopy and FRET Imaging to Measure Spatiotemporal Signaling Events in Single Living Cells; Xuehua Xu, Joseph A. Brzostowski, and Tian Jin; Visualizing Signaling and Cell Movement During the Multicellular Stages of Dictyostelium Development; Dirk Dormann and Cornelis J. Weijer; Under-Agarose Chemotaxis of Dictyostelium discoideum; David Woznica and David A. Knecht; Optimized Fixation and Immunofluorescence Staining Methods for Dictyostelium Cells; Monica Hagedorn, Eva M. Neuhaus, and Thierry Soldati; Cryofixation Methods for Ultrastructural Studies of Dictyostelium discoideum; Mark J. Grimson and Richard L. Blanton; Part IV. Dictyostelium as Model Organism; Analysis of Signal Transduction: Formation of cAMP, cGMP, and Ins(1,4,5)P3 In Vivo and In Vitro; Peter J. M. Van Haastert; Assaying Chemotaxis of Dictyostelium Cells; Michelle C. Mendoza and Richard A. Firtel; Characterization of Cross-Linked Actin Filament Gels and Bundles Using Birefringence and Polarized Light Scattering; Ruth Furukawa and Marcus Fechheimer; Quantitative and Microscopic Methods for Studying the Endocytic Pathway; Francisco Rivero and Markus Maniak; Preparation of Intact, Highly Purified Phagosomes from Dictyostelium; Daniel Gotthardt, Regis Dieckmann, Vincent Blancheteau, Claudia Kistler, Frank Reichardt, and Thierry Soldati; Assaying Cell-Cell Adhesion; Salvatore Bozzaro; Periodic Activation of ERK2 and Partial Involvement of G Protein in ERK2 Activation by cAMP in Dictyostelium Cells; Mineko Maeda; An Improved Method for Dictyostelium Centrosome Isolation; Irene Schulz, Yvonne Reinders, Albert Sickmann, and Ralph Graf; Epigenetics in Dictyostelium; Markus Kaller, Wolfgang Nellen, and Jonathan R. Chubb; Dictyostelium discoideum as a Model to Study Host-Pathogen Interactions; Can Aunal and Michael Steinert; Pharmacogenetics: Defining the Genetic Basis of Drug Action and Inositol Trisphosphate Analysis; Kathryn E. Adley, Melanie Keim, and Robin S. B.Williams; How to Assess and Study Cell Death in Dictyostelium discoideum; Artemis Kosta, Catherine Laporte, David Lam, Emilie Tresse, Marie-Francoise Luciani, and Pierre Golstein; Index