Dictyostelium Discoideum Protocols

ISBN-13: 9781617376771 / Angielski / Miękka / 2010 / 564 str.

Ludwig Eichinger

Opis

Dictyostelium discoideum is a simple but fascinating eukaryotic microorg- ism, whose natural habitat is deciduous forest soil and decaying leaves, where the amoebae feed on bacteria and grow as independent single cells. Exhaustion of the bacterial food source triggers a developmental program, in which up to 100,000 cells aggregate by chemotaxis towards cAMP. Morphogenesis and cell different- tion then culminate in the production of spores enabling the organism to survive unfavorable conditions. Dictyostelium offers unique advantages for studying f- damental cellular processes with the aid of powerful molecular genetic, bioche- cal, and cell biological tools. These processes include signal transduction, chemotaxis, cell motility, cytokinesis, phagocytosis, and aspects of development such as cell sorting, pattern formation and cell type differentiation. Recently, D- tyostelium was also described as a suitable host for pathogenic bacteria in which one can conveniently study the process of infection. In addition, Dictyostelium has many of the experimental conveniences of Saccharomyces cerevisiae and is pr- ably the best experimentally manipulatable protozoan, providing insight into this diverse group of organisms, which includes some of the most dangerous human parasites. The recent completion of the Dictyostelium genome sequencing project strengthens the position of D. discoideum as a model organism. The completed genome sequence and other valuable community resources constitute the source for basic biological and biomedical research and for genome-wide analyses.

Spis treści

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 Müller, 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 Gérard 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 Müller-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 UsingQuantitative 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, Régis 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 Gräf Epigenetics in Dictyostelium Markus Kaller, Wolfgang Nellen, and Jonathan R. Chubb Dictyostelium discoideum as a Model to Study

Blurb

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.