Dictyostelium Discoideum Protocols

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.

Includes supplementary material: sn.pub/extras

The Organism, Community Resources, and Genome-wide Studies.- The Secret Lives of Dictyostelium.- The Genome of Dictyostelium discoideum.- The cDNA Sequencing Project.- dictyBase and the Dicty Stock Center.- Analysis of Gene Expression Using cDNA Microarrays.- Proteomic Analysis of Dictyostelium discoideum.- Basic Methods and Molecular Genetic Techniques.- Cultivation, Spore Production, and Mating.- Parasexual Genetics Using Axenic Cells.- Slug Phototaxis, Thermotaxis, and Spontaneous Turning Behavior.- Purification Techniques of Subcellular Compartments for Analytical and Preparative Purposes.- Generation of Multiple Knockout Mutants Using the Cre-loxP System.- Restriction Enzyme-Mediated Integration (REMI) Mutagenesis.- RNA Interference and Antisense-Mediated Gene Silencing in Dictyostelium.- Imaging and Localization Methods.- Application of Fluorescent Protein Tags as Reporters in Live-Cell Imaging Studies.- Investigating Gene Expression.- Application of 2D and 3D DIAS to Motion Analysis of Live Cells in Transmission and Confocal Microscopy Imaging.- Using Quantitative Fluorescence Microscopy and FRET Imaging to Measure Spatiotemporal Signaling Events in Single Living Cells.- Visualizing Signaling and Cell Movement During the Multicellular Stages of Dictyostelium Development.- Under-Agarose Chemotaxis of Dictyostelium discoideum.- Optimized Fixation and Immunofluorescence Staining Methods for Dictyostelium Cells.- Cryofixation Methods for Ultrastructural Studies of Dictyostelium discoideum.- Dictyostelium as Model Organism.- Analysis of Signal Transduction.- Assaying Chemotaxis of Dictyostelium Cells.- Characterization of Cross-Linked Actin Filament Gels and Bundles Using Birefringence and Polarized Light Scattering.- Quantitative and Microscopic Methods for Studyingthe Endocytic Pathway.- Preparation of Intact, Highly Purified Phagosomes from Dictyostelium.- Assaying Cell-Cell Adhesion.- Periodic Activation of ERK2 and Partial Involvement of G Protein in ERK2 Activation by cAMP in Dictyostelium Cells.- An Improved Method for Dictyostelium Centrosome Isolation.- Epiggenetics in Dictyostelium.- Dictyostelium discoideum as a Model to Study Host-Pathogen Interactions.- Pharmacogenetics.- How to Assess and Study Cell Death in Dictyostelium discoideum.