Springer protocols feed by Microbiology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Fluorescent Reporters and Methods to Analyze Fluorescent Signals
The use of fluorescent reporters and the development of new imaging technologies have revolutionized studies in cell biology. During recent years the number of fluorescent proteins offering the ability to visualize the distribution of proteins, organelles, and cells has increased tremendously. In parallel, the imaging tools available were refined rapidly enabling now the use of a huge spectrum of specialized methods to explore the cellular and subcellular localization and dynamics of fluorescently tagged markers. This chapter presents an overview of fluorescent reporters and methods available, and describes a selection of ...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
One Stop Shop for Everything Dictyostelium: dictyBase and the Dicty Stock Center in 212
dictyBase (http://dictybase.org), the model organism database for Dictyostelium discoideum, includes the complete genome sequence and expression data for this organism. Relevant literature is integrated into the database, and gene models and functional annotation are manually curated from experimental results and comparative multigenome analyses. dictyBase has recently expanded to include the genome sequences of three additional Dictyostelids and has added new software tools to facilitate multigenome comparisons. The Dicty Stock Center, a strain and plasmid repository for Dictyostelium research, has relocated to Northweste...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Comparative Genomics of the Dictyostelids
The complete genomes of Dictyostelium discoideum, Dictyostelium purpureum, Polysphondylium pallidum and Dictyostelium fasciculatum have been sequenced. The proteins predicted to be encoded by the genes in each species have been compared to each other as well as to the complete compilation of nonredundant proteins from bacteria, plants, fungi, and animals. Likely functions have been assigned to about half of the proteins on the basis of sequence similarity to proteins with experimentally defined functions or properties. Even when the sequence similarity is not sufficiently high to have much confidence in the predicted funct...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
The Model Organism Dictyostelium discoideum
Much of our knowledge of molecular cellular functions is based on studies with a few number of model organisms that were established during the last 50 years. The social amoeba Dictyostelium discoideum is one such model, and has been particularly useful for the study of cell motility, chemotaxis, phagocytosis, endocytic vesicle traffic, cell adhesion, pattern formation, caspase-independent cell death, and, more recently, autophagy and social evolution. As nonmammalian model of human diseases D. discoideum is a newcomer, yet it has proven to be a powerful genetic and cellular model for investigating host–pathogen inte...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Monitoring Autophagy in Dictyostelium
We describe here the use of confocal microscopy to detect the pattern of different autophagic markers and the differences expected in strains deficient in autophagy. Autophagy dysfunction might also lead to the formation of intracellular ubiquitinated protein aggregates that can be easily detected by immunofluorescence. In addition, we describe two different techniques that allow the assessment of the so-called autophagic flux, the progression of autophagosomes until their fusion with lysosomes. The first one is a proteolytic cleavage assay of cytosolic markers such as GFP-PgkA and the second the visualization of the RFP-G...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Secretory Lysosomes in Dictyostelium: Visualization, Characterization, and Dynamics
Dictyostelium discoideum is a unicellular eukaryotic model largely used for the study of the endocytic pathway, mainly due to its high resemblance to the mammalian pathway. Over the last years, Dictyostelium has been increasingly employed for investigating the biogenesis and secretion of secretory lysosomes, given its advantage over mammalian cells. Here we describe protocols for determination of ionic concentration and rates of maturation and exocytosis of these compartments. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Immunomagnetic Purification of Fluorescent Legionella-Containing Vacuoles
Protozoa are natural reservoirs of the environmental bacterium Legionella pneumophila. Upon inhalation of Legionella-laden aerosols, the amoeba-resistant bacteria replicate within human alveolar macrophages causing the severe pneumonia “Legionnaires’ disease.” Within host cells, including Dictyostelium discoideum, L. pneumophila establishes a custom-tailored compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system and involves a plethora of “effector” proteins, some of which specifically decorate the LCV membrane. This unique f...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Isolation of Pathogen-Containing Vacuoles
Dictyostelium discoideum cells are “professional phagocytes,” as they ingest a large variety of bacteria, yeast, and inert particles. Several bacterial pathogens are able to survive intracellularly within specialized vacuoles of D. discoideum by interfering with host signaling pathways. To better understand the molecular mechanisms underlying these evolutionary conserved processes we have established a method for the isolation of pathogen-containing vacuoles (PCVs). The isolation protocol describes the infection of D. discoideum cells with the intracellular pathogen Legionella pneumophila, loading of the lysoso...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Setting Up and Monitoring an Infection of Dictyostelium discoideum with Mycobacteria
We present here a “starter kit” of detailed methods that allows to establish an infection of Dictyostelium with M. marinum and to monitor quantitatively the intracellular bacterial growth. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Quantitative Analysis of Phagocytosis and Phagosome Maturation
Phagocytosis and phagosome maturation lead to killing and digestion of bacteria by protozoans and innate immune phagocytes. Phagocytosis of particles expressing or coupled to various fluorescent reporters and sensors can be used to monitor quantitatively various parameters of this central biological process. In this chapter we detail different labeling techniques of bacteria and latex beads used to measure adhesion and uptake by FACS analysis. We also describe methods to use fluorescent reporter dyes (FITC or DQgreen) coupled to silica beads to measure the kinetics of acidification and proteolysis. Measurements can be perf...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Micropipette Aspiration for Studying Cellular Mechanosensory Responses and Mechanics
Micropipette aspiration (MPA) is a widely applied method for studying cortical tension and deformability. Based on simple hydrostatic principles, this assay allows the application of a specific magnitude of mechanical stress on cells. This powerful method has revealed insights about cell mechanics and mechanosensing, not only in Dictyostelium discoideum but also in other cell types. In this chapter, we present how to set up a micropipette aspiration system and the experimental procedures for determining cortical tension and mechanosensory responses. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
The Amoebozoa
The model organism Dictyostelium discoideum is a member of the Amoebozoa, one of the six major divisions of eukaryotes. Amoebozoa comprise a wide variety of amoeboid and flagellate organisms with single cells measuring from 5 μm to several meters across. They have adopted many different life styles and sexual behaviors and can live in all but the most extreme environments. This chapter provides an overview of Amoebozoan diversity and compares roads towards multicellularity within the Amoebozoa with inventions of multicellularity in other protist divisions. The chapter closes with a scenario for the evolution of Dic...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Mitochondrial Respiratory Complex Function and the Phenotypic Consequences of Dysfunction
Dictyostelium provides a well established model system for the study of mitochondrial biology and disease. Mitochondrial dysfunction in Dictyostelium has been generated by knockout of nonessential nuclear genes encoding mitochondrial proteins, by knockout of targeted mitochondrial genes in a subset of the mitochondria, and by knock down of essential nuclear-encoded mitochondrial proteins. The resulting effects on mitochondrial electron transport and membrane potential can be studied by directly measuring the activities, composition, and assembly or stability of individual mitochondrial respiratory complexes and by using fl...
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Analysis of Mitochondrial Gene Expression
Dictyostelium provides a well-established model system for the study of mitochondrial biology and disease. A complete mitochondrial transcription and RNA-processing map has been generated, while the start site for transcription and the responsible RNA polymerase have also been identified, as have the major cotranscriptional cleavage sites that generate the mature mitochondrial RNA molecules. Here we describe the methods deployed to study mitochondrial gene transcription and RNA processing in Dictyostelium. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
Transcript Localization in Dictyostelium discoideum Cells by RNA FISH
We describe methods for the generation of suitable fluorescently labeled probes and the fixation of cells, by which we have localized different precursor and mature rRNA molecules to the nucleolus or the cytoplasm, respectively. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - March 6, 2013 Category: Microbiology Source Type: news
