Springer protocols feed by Cell Biology 
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.
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.Efficient Library Preparation for Next-Generation Sequencing Analysis of Genome-Wide Epigenetic and Transcriptional Landscapes in Embryonic Stem Cells
Gene expression in embryonic stem (ES) cells is regulated in part by a network of transcription factors, epigenetic regulators, and histone modifications that influence the underlying chromatin in a way that is conducive or repressive for transcription. Advances in next-generation sequencing technology have allowed for the genome-wide analysis of chromatin constituents and protein–DNA interactions at high resolution in ES cells and other stem cells. While many studies have surveyed genome-wide profiles of a few factors and expression changes at a fixed time point in undifferentiated ES cells, few have utilized an int...
Source: Springer protocols feed by Cell Biology - April 18, 2014 Category: Cytology Source Type: news
Analysis of Mitotic Protein Dynamics and Function in Drosophila Embryos by Live Cell Imaging and Quantitative Modeling
Mitosis depends upon the mitotic spindle, a dynamic protein machine that uses ensembles of dynamic microtubules (MTs) and MT-based motor proteins to assemble itself, control its own length (pole–pole spacing), and segregate chromosomes during anaphase A (chromosome-to-pole motility) and anaphase B (spindle elongation). In this review, we describe how the molecular and biophysical mechanisms of these processes can be analyzed in the syncytial Drosophila embryo by combining (1) time-lapse imaging and other fluorescence light microscopy techniques to study the dynamics of mitotic proteins such as tubulins, mitotic motor...
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
Rapid Measurement of Mitotic Spindle Orientation in Cultured Mammalian Cells
Factors that influence the orientation of the mitotic spindle are important for the maintenance of stem cell populations and in cancer development. However, screening for these factors requires rapid quantification of alterations of the angle of the mitotic spindle in cultured cell lines. Here we describe a method to image mitotic cells and rapidly score the angle of the mitotic spindle using a simple MATLAB application to analyze a stack of Z-images. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
Automated Segmentation of the First Mitotic Spindle in Differential Interference Contrast Microcopy Images of C. elegans Embryos
Differential interference contrast (DIC) microscopy is a non-fluorescent microscopy technique that is commonly used to visualize the first mitotic spindle in C. elegans embryos. DIC movies are easy to acquire and provide data with high spatial and temporal resolution, allowing detailed investigations of the dynamics of the spindle—which elongates, oscillates, and is positioned asymmetrically. Despite the immense amount of information such movies provide, they are normally only used to draw qualitative conclusion based on manual inspection. We have developed an algorithm to automatically segment the mitotic spindle in...
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
Imaging the Mitotic Spindle by Spinning Disk Microscopy in Tobacco Suspension Cultured Cells
Plants are valuable systems for analyzing the acentriolar mitotic spindle. We have developed methods for imaging the mitotic spindle in living tobacco (Nicotiana tabacum) suspension culture cells expressing GFP-α-tubulin. The methods allow the spindle to be observed in living cells at high spatial and temporal resolution and rely on water immersion objectives, spinning disk optics, and high-sensitivity cameras. Here, we describe these methods and provide step-by-step protocols for certain key steps. We also describe a method for application and removal of inhibitors. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
Image-Based Computational Tracking and Analysis of Spindle Protein Dynamics
The spindle is a highly complex and dynamic molecular machine that is assembled during cell division for accurate segregation of replicated chromosomes. Successful completion of cell division relies on the right spindle proteins to be at the right place at the right time to serve their functions. Quantitative characterization and analysis of spatiotemporal behaviors of spindle proteins are therefore essential to understanding related cell division mechanisms. The main goal of this chapter is to introduce basic concepts and methods for computational tracking and analysis of spindle protein spatiotemporal dynamics that is vi...
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
Measuring Microtubule Growth and Gliding in Caenorhabditis elegans Embryos
Microtubule plus-tip tracking is a powerful method to measure microtubule growth dynamics in vivo. Here we outline an approach that exploits live confocal microscopy of a GFP-tagged EB1-like protein to measure microtubule growth behavior and minus-end-directed microtubule motor activity at the cortex of Caenorhabditis elegans embryos. The EB1 velocity assay (EVA) provides a method to reproducibly monitor motor- and non-motor-assisted microtubule movements. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
The Use of Cultured Drosophila Cells for Studying the Microtubule Cytoskeleton
Cultured Drosophila cell lines have been developed into a powerful tool for studying a wide variety of cellular processes. Their ability to be easily and cheaply cultured as well as their susceptibility to protein knockdown via double-stranded RNA-mediated interference (RNAi) has made them the model system of choice for many researchers in the fields of cell biology and functional genomics. Here we describe basic techniques for gene knockdown, transgene expression, preparation for fluorescence microscopy, and centrosome enrichment using cultured Drosophila cells with an emphasis on studying the microtubule cytoskeleton. (S...
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
An Improved Optical Tweezers Assay for Measuring the Force Generation of Single Kinesin Molecules
Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-G...
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
Xenopus Egg Extracts as a Simplified Model System for Structure–Function Studies of Dynein Regulators
Many proteins act in multiple pathways which complicates phenotypic analysis. Xenopus egg extracts reconstitute complex reactions in vitro, and this can be used to develop assays that isolate a single function of a multifunctional protein. We have applied this system to study regulators of cytoplasmic dynein (dynein), which has numerous roles in the cell including trafficking, nuclear migration, and mitotic spindle formation. Here we describe a functional assay to specifically study the regulation of spindle pole self-organization by dynein and summarize an experimental approach that was used to perform a structure–f...
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
Seeded Microtubule Growth for Cryoelectron Microscopy of End-Binding Proteins
End-binding proteins (EBs) have the ability to autonomously track the ends of growing microtubules, where they recruit several proteins that control various aspects of microtubule cytoskeleton organization and function. The structural nature of the binding site recognized by EBs at growing microtubule ends has been a subject of debate. Recently, a fluorescence microscopy assay used for the study of dynamic end tracking in vitro was adapted for cryoelectron microscopy (cryo-EM). In combination with single-particle reconstruction methods, this modified assay was used to produce the first subnanometer-resolution model of how ...
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
The Segmentation of Microtubules in Electron Tomograms Using Amira
The development of automatic tools for the three-dimensional reconstruction of the microtubule cytoskeleton is crucial for large-scale analysis of mitotic spindles. Recently, we have published a method for the semiautomatic tracing of microtubules based on 3D template matching (Weber et al., J Struct Biol 178:129–138, 2012). Here, we give step-by-step instructions for the automatic tracing of microtubules emanating from centrosomes in the early mitotic Caenorhabditis elegans embryo. This approach, integrated in the visualization and data analysis software Amira, is applicable to tomographic data sets from other model...
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
Covalent Immobilization of Microtubules on Glass Surfaces for Molecular Motor Force Measurements and Other Single-Molecule Assays
We present an MT-surface coupling protocol in which aminosilanized glass is formylated using the cross-linker glutaraldehyde, fluorescence-labeled MTs are covalently attached, and the surface is passivated with highly pure beta-casein. The technique presented here yields rigid MT immobilization while simultaneously blocking the remaining glass surface against nonspecific binding by polystyrene optical trapping microspheres. This surface chemistry is straightforward and relatively cheap and uses a minimum of specialized equipment or hazardous reagents. These methods provide a foundation for a variety of optical tweezers exp...
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
Four-Color FISH for the Detection of Low-Level Aneuploidy in Interphase Cells
FISH (fluorescent in situ hybridization) is a molecular cytogenetic technique established in the early 1980s that allows for the detection of DNA copy number changes (gains and losses) mapping to genomic regions of interest (Langer-Safer et al. Proc Natl Acad Sci USA 79:4381–4385, 1982). This technology has been extensively applied to research-based investigations and is routinely used in prenatal diagnosis and oncology. Here we describe a modification of the standard FISH protocol adapted for the detection of low-frequency mosaic aneuploidy in interphase cells. This approach represents a straightforward method for t...
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
Manipulating Cell Shape by Placing Cells into Micro-fabricated Chambers
We describe here a method of physically manipulating sea urchin cells into specified shapes by inserting them into micro-fabricated chambers of different shapes. This method allows for generation of large systematic and quantitative data sets and may be adaptable for different cell types and contexts. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - March 27, 2014 Category: Cytology Source Type: news
