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.Measuring APC/C-Dependent Ubiquitylation In Vitro
The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtai...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Tracking the Cyclin B1-GFP Sensor to Profile the Pattern of Mitosis Versus Mitotic Bypass
This chapter provides a method for quantitative single cell analysis to track the transition of single cells from G2, indicated by high cyclin B1 levels, to G1 polyploidy phase (G1(p)), indicated by low cyclin B1 levels, in a 4n population. The cell tracking methodology described provides a fluorescence fingerprint suitable for deriving G2/M or G2/G1p transitions. Notably, during late G2 the absolute cyclin B1-eGFP reporter levels obtained were high and the switch-off point identifiable, with destruction rates of a similar order across all cell cycle routing avenues. The three principle parameters extracted were defined as...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Detection of Protein–Protein Interactions in Tobacco BY-2 Cells Using Bimolecular Fluorescence Complementation
Knowledge of protein–protein interactions in the plant cell is invaluable for furthering our understanding of the functions of these proteins. Many of the methods available for the study of these interactions, such as yeast two-hybrid and co-immunoprecipitation assays, rely on in vitro techniques. Here we describe the use of bimolecular fluorescence complementation for the study of protein–protein interactions in vivo, using simple techniques and accessible materials. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Cell Cycle Synchronization Using a Microfluidic Synchronizer for Fission Yeast Cells
To produce synchronized cell colonies, many cell cycle synchronization technologies have been developed, among which the baby machine may be considered the most artifact-free. Baby machines incubate “mother cells” under normal conditions and collects their “babies,” producing cell cultures that are similar not only in cell cycle phase but also in age. Several macroscale and microfluidic baby machines have been applied to synchronized cell research. However, for rod-shaped cells like fission yeast (Schizosaccharomyces pombe), it is still a challenge to immobilize only the mother cells in a microfluid...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Combining the Optimized Yeast Cytosine Deaminase Protein Fragment Complementation Assay and an In Vitro Cdk1 Targeting Assay to Study the Regulation of the γ-Tubulin Complex
Cdk1 is the essential cyclin-dependent kinase in the budding yeast Saccharomyces cerevisiae. Cdk1 orchestrates cell cycle control by phosphorylating target proteins with extraordinary temporal and spatial specificity by complexing with one of the nine cyclin regulatory subunits. The identification of the cyclin required for targeting Cdk1 to a substrate can help to place the regulation of that protein at a specific time point during the cell cycle and reveal information needed to elucidate the biological significance of the regulation. Here, we describe a combination of strategies to identify interaction partners of Cdk1, ...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Measuring Activity and Specificity of Protein Phosphatases
Reversible protein phosphorylation plays essential roles in coordinating cell division and many other biological processes. Cell cycle regulation by opposing kinase and protein phosphatase activities is often complex and major challenges exist in identifying the direct substrates of these enzymes and the specific sites at which they act. While cell cycle kinases are known to exhibit strict substrate specificities important for coordinating the complex events of cell division, phosphatases have only recently been recognized to exert similarly precise regulatory control over cell cycle events through timely dephosphorylation...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
A Computational Method for Identifying Yeast Cell Cycle Transcription Factors
The eukaryotic cell cycle is a complex process and is precisely regulated at many levels. Many genes specific to the cell cycle are regulated transcriptionally and are expressed just before they are needed. To understand the cell cycle process, it is important to identify the cell cycle transcription factors (TFs) that regulate the expression of cell cycle-regulated genes. Here, we describe a computational method to identify cell cycle TFs in yeast by integrating current ChIP-chip, mutant, transcription factor-binding site (TFBS), and cell cycle gene expression data. For each identified cell cycle TF, our method also assig...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
The Use of SNAP Labeling to Study Cell Cycle Oscillatory Proteins
This reporter system can be adapted for other cell cycle oscillatory proteins. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Building a Synthetic Transcriptional Oscillator
Reaction circuits mimicking genetic oscillators can be realized with synthetic, switchable DNA genes (so-called genelets), and two enzymes only, an RNA polymerase and a ribonuclease. The oscillatory behavior of the genelets is driven by the periodic production and degradation of RNA effector molecules. Here, we describe the preparation, assembly, and testing of a synthetic, transcriptional two-node negative-feedback oscillator, whose dynamics can be followed in real-time by fluorescence read-out. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
E2F Transcription Factors Control the Roller Coaster Ride of Cell Cycle Gene Expression
Initially, the E2F transcription factor was discovered as a factor able to bind the adenovirus E2 promoter and activate viral genes. Afterwards it was shown that E2F also binds to promoters of nonviral genes such as C-MYC and DHFR, which were already known at that time to be important for cell growth and DNA metabolism, respectively. These findings provided the first clues that the E2F transcription factor might be an important regulator of the cell cycle. Since this initial discovery in 1987, several additional E2F family members have been identified, and more than 100 targets genes have been shown to be directly regulate...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Role of Computational Modeling in Understanding Cell Cycle Oscillators
The periodic oscillations in the activity of the cell cycle regulatory program, drives the timely activation of key cell cycle events. Interesting dynamical systems, such as oscillators, have been investigated by various theoretical and computational modeling methods. Thanks to the insights achieved by these modeling efforts we have gained considerable insights about the underlying molecular regulatory networks that can drive cell cycle oscillations. Here we review the basic features and characteristics of biological oscillators, discussing from a computational modeling point of view their specific architectures and the cu...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Model Organisms for Studying the Cell Cycle
Regulation of the cell-division cycle is fundamental for the growth, development, and reproduction of all species of life. In the past several decades, a conserved theme of cell cycle regulation has emerged from research in diverse model organisms. A comparison of distinct features of several diverse model organisms commonly used in cell cycle studies highlights their suitability for various experimental approaches, and recaptures their contributions to our current understanding of the eukaryotic cell cycle. A historic perspective presents a recollection of the breakthrough upon unfolding the universal principles of cell c...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
Cell Cycle Control: A System of Interlinking Oscillators
The cell cycle is the sequence of events through which a cell duplicates its genome, grows, and divides. Key cell cycle transitions are driven by oscillators comprising cyclin-dependent kinases and other kinases. Different cell cycle oscillators are inextricably linked to ensure orderly activation of oscillators. A recurring theme in their regulation is the abundance of auto-amplifying loops that ensure switch-like and unidirectional cell cycle transitions. The periodicity of many cell cycle oscillators is choreographed by inherent mechanisms that promote automatic inactivation, often involving dephosphorylation and ubiqui...
Source: Springer protocols feed by Cell Biology - August 10, 2015 Category: Cytology Source Type: news
A Novel Extraction Protocol to Probe the Role of Cholesterol in Synaptic Vesicle Recycling
Cholesterol helps to stabilize membrane fluidity and many membrane proteins interact with cholesterol and are functionally clustered in cholesterol rich “rafts.” Synaptic vesicle (SV) membranes are enriched in cholesterol in comparison to other organelles. Attempts to study the function of this high cholesterol content have been hampered by the inability to extract cholesterol from SVs in live presynaptic terminals. Here, we describe a method to extract vesicular cholesterol using a temperature-sensitive Drosophila dynamin mutant, shibire-ts1 (shi), to trap SVs on the plasma membrane. Trapped SVs are more acces...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Visualization of Clathrin-Mediated Endocytosis in Live Drosophila Egg Chambers
In oviparous animals, clathrin-dependent endocytosis is often critical to stockpile a necessary supply of yolk within the maturing oocyte, which enables subsequent embryonic development. In the physically linked chains of maturing egg chambers within the Drosophila melanogaster ovary, a distinct, morphologically discernable subset undergoes a massive burst clathrin-mediated endocytosis to accumulate yolk in a process termed vitellogenesis. Here, we describe how to prepare isolated ovaries to follow endocytosis, and detail approaches to follow live uptake of soluble reporters into vitellogenic Drosophila egg chambers. (Sour...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
