Springer protocols feed by Cell Biology 
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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.High-Throughput Fluorometric Assay for Membrane–Protein Interaction
Membrane–protein interaction plays key roles in a wide variety of biological processes. To facilitate rapid and sensitive measurement of membrane binding of soluble proteins, we developed a fluorescence-based quantitative assay that is universally applicable to all proteins. This fluorescence-quenching assay employs fluorescence protein (FP)-tagged proteins whose fluorescence intensity is greatly decreased when they bind vesicles containing synthetic lipid dark quenchers, such as N-dimethylaminoazobenzenesulfonylphosphatidylethanolamine (dabsyl-PE). This simple assay can be performed with either a spectrofluorometer ...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Analyzing Protein–Phosphoinositide Interactions with Liposome Flotation Assays
Liposome flotation assays are a convenient tool to study protein–phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein–lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome li...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Using Surface Plasmon Resonance to Quantitatively Assess Lipid–Protein Interactions
Surface Plasmon Resonance (SPR) is a quantitative, label-free method for determining molecular interactions in real time. The technology involves fixing a ligand onto a senor chip, measuring a baseline resonance angle, and flowing an analyte in bulk solution over the fixed ligand to measure the subsequent change in resonance angle. The mass of analyte bound to fixed ligand is directly proportional to the resonance angle change and the system is sensitive enough to detect as little as picomolar amounts of analyte in the bulk solution. SPR can be used to determine both the specificity of molecular interactions and the kineti...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Detection of Isolated Mitochondria-Associated ER Membranes Using the Sigma-1 Receptor
We describe in detail the preparation and purification of the MAM by using the sigma-1 receptor as the marker and demonstrate the uniqueness of this marker by using a variety of cells, peripheral and neuronal. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Isolation and Analysis of Detergent-Resistant Membrane Fractions
The hypothesis that the Golgi apparatus is capable of sorting proteins and sending them to the plasma membrane through “lipid rafts,” membrane lipid domains highly enriched in glycosphingolipids, sphingomyelin, ceramide, and cholesterol, was formulated by van Meer and Simons in 1988 and came to a turning point when it was suggested that lipid rafts could be isolated thanks to their resistance to solubilization by some detergents, namely Triton X-100. An incredible number of papers have described the composition and properties of detergent-resistant membrane fractions. However, the use of this method has also ra...
Source: Springer protocols feed by Cell Biology - November 12, 2015 Category: Cytology Source Type: news
Single-Molecule RNA In Situ Hybridization (smFISH) and Immunofluorescence (IF) in the Drosophila Egg Chamber
Detection of nucleic acids in whole tissues has become key in our understanding of gene expression during development. In situ hybridization (ISH) has been an invaluable technique in the making of numerous discoveries. Most recently, the technical advance of using short, fluorescently labeled probes has allowed for the detection of single-mRNA molecules. Thus, quantification of RNA levels in single cells or even within subcellular regions is now possible without RNA isolation. In combination with the immunofluorescence (IF) technique, visualization of nucleic acids and associating proteins is achieved with higher resolutio...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Visualization of Actin Cytoskeletal Dynamics in Fixed and Live Drosophila Egg Chambers
Visualization of actin cytoskeletal dynamics is critical for understanding the spatial and temporal regulation of actin remodeling. Drosophila oogenesis provides an excellent model system for visualizing the actin cytoskeleton. Here, we present methods for imaging the actin cytoskeleton in Drosophila egg chambers in both fixed samples by phalloidin staining and in live egg chambers using transgenic actin labeling tools. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Visualizing Microtubule Networks During Drosophila Oogenesis Using Fixed and Live Imaging
The microtubule cytoskeleton is a plastic network of polarized cables. These polymers of tubulin provide orientated routes for the dynamic transport of cytoplasmic molecules and organelles, through which cell polarity is established and maintained. The role of microtubule-mediated transport in the asymmetric localization of axis polarity determinants, in the Drosophila oocyte, has been the subject of extensive studies in the past years. However, imaging the distribution of microtubule fibers in a large cell, where vitellogenesis ensures the uptake of a thick and hazy yolk, presents a series of technical challenges. This ch...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Border Cell Migration: A Model System for Live Imaging and Genetic Analysis of Collective Cell Movement
Border cell migration in the Drosophila ovary has emerged as a genetically tractable model for studying collective cell movement. Over many years border cell migration was exclusively studied in fixed samples due to the inability to culture stage 9 egg chambers in vitro. Although culturing late-stage egg chambers was long feasible, stage 9 egg chambers survived only briefly outside the female body. We identified culture conditions that support stage 9 egg chamber development and sustain complete migration of border cells ex vivo. This protocol enables one to compare the dynamics of egg chamber development in wild-type and ...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Culturing Drosophila Egg Chambers and Investigating Developmental Processes Through Live Imaging
Drosophila oogenesis provides many examples of essential processes in development. A myriad of genetic tools combined with recent advances in culturing egg chambers ex vivo has revealed several surprising mechanisms that govern how this tissue develops, and which could not have been determined in fixed tissues. Here we describe a straightforward protocol for dissecting ovaries, culturing egg chambers, and observing egg development in real time by fluorescent microscopy. This technique is suitable for observation of early- or late-stage egg development, and can be adapted to study a variety of cellular, molecular, or develo...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Genetic Mosaic Analysis of Stem Cell Lineages in the Drosophila Ovary
Genetic mosaic analyses represent an invaluable approach for the study of stem cell lineages in the Drosophila ovary. The generation of readily identifiable, homozygous mutant cells in the context of wild-type ovarian tissues within intact organisms allows the pinpointing of cellular requirements for gene function, which is particularly important for understanding the physiological control of stem cells and their progeny. Here, we provide a step-by-step guide to the generation and analysis of genetically mosaic ovaries using flippase (FLP)/FLP recognition target (FRT)-mediated recombination in adult Drosophila melanogaster...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Mosaic Analysis in the Drosophila melanogaster Ovary
Drosophila melanogaster oogenesis is a versatile model system used to address many important questions of cell and developmental biology such as stem cell regulation, cell determination, cell polarization, cell–cell signaling, cell–cell adhesion, and cell-cycle regulation. The ovary is composed of germline and somatic cells of different origins and functions. Mosaic analysis using the powerful genetic tools available in Drosophila melanogaster allows deciphering the contribution of each cell type in the different processes leading to the formation of a mature egg. Germ cells and follicle cells are produced by a...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Basic Techniques in Drosophila Ovary Preparation
Drosophila melanogaster oogenesis has emerged as an excellent model system to study multiple aspects of eukaryotic cell biology. Ovarian tissue can easily be isolated and analyzed through microscopy or biochemical and molecular biology techniques. Here we describe the isolation of ovarian tissues, techniques to enrich for egg chambers at distinct developmental stages, preparation of protein and nucleic acid extracts, and preparation for microscopic analysis of fixed tissues. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Drosophila melanogaster Oogenesis: An Overview
The Drosophila melanogaster ovary has served as a popular and successful model for understanding a wide range of biological processes: stem cell function, germ cell development, meiosis, cell migration, morphogenesis, cell death, intercellular signaling, mRNA localization, and translational control. This review provides a brief introduction to Drosophila oogenesis, along with a survey of its diverse biological topics and the advanced genetic tools that continue to make this a popular developmental model system. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
Bioinformatics Analysis to Identify RNA–Protein Interactions in Oogenesis
Hundreds of RNA-binding proteins are known, but the biological functions are known for only a few of them. They regulate various aspects of RNA processing or biogenesis such as splicing, polyadenylation, and translation. Here I describe a bioinformatics approach that we developed to identify potential new mRNA target(s) of the Drosophila master sex-switch protein Sex-lethal (SXL) by combining computational analysis with genetic and biochemical investigation. This approach could be used to identify new RNA–protein interactions during oogenesis in the female germline and should be applicable to numerous other posttrans...
Source: Springer protocols feed by Cell Biology - September 5, 2015 Category: Cytology Source Type: news
