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.Imaging Analysis of Cell Cycle-Dependent Degradation of Cdt1 in Mammalian Cells
Numerous cell cycle-regulating proteins are controlled by protein degradation. Recent work shows that ubiquitination-dependent proteolysis plays an important role in once-per-cell cycle control of DNA replication. Cdt1 is a licensing factor essential for assembling the pre-replicative complex on replication origins. Cdt1 is present in G1 phase, but after S phase ubiquitin-mediated proteolysis maintains Cdt1 at low levels. This is important to prevent the re-replication of chromosomal DNA. The cell cycle-dependent degradation of Cdt1 can be monitored by dual staining of the cell nuclei with antibodies against Cdt1- and S/G2...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Analyzing Cell Cycle-Dependent Degradation and Ubiquitination in Budding Yeast
Cell cycle progression is tightly regulated to prevent uncontrolled growth and division. Specific cell cycle factors are responsible for driving the cell from one cell cycle stage to the next. Many of these proteins are targeted for degradation by the ubiquitin proteasome system when their function is no longer required or may disrupt cell cycle progression. Here we describe a series of experiments used to study the ubiquitin-mediated degradation of cell cycle proteins. This collection of assays may be used to determine the requirement for individual proteins in the degradation and ubiquitination of cell cycle proteins in ...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Analyzing DNA Replication Checkpoint in Budding Yeast
Checkpoints are conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. Cells also possess a surveillance mechanism called the DNA replication checkpoint, which consists of a conserved kinase cascade that is provoked by insults that block or slow down replication fork progression. In the budding yeast Saccharomyces cerevisiae, the DNA replication checkpoint controls the timing of S-phase events such as origin firing and spindle elongation. This checkpoint also upregulates dNTP pools and maintains the replication fork structure in o...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Analyzing Cell Cycle Checkpoints in Response to Ionizing Radiation in Mammalian Cells
Exposure of cells to DNA-damaging agents, such as ionizing radiation (IR), results in perturbation of cell cycle progression. IR activates cell cycle checkpoints that arrest the cell cycle at the G1/S, S, and G2/M phases. The DNA damage-signaling network involves a number of important DNA damage response factors that are required for maintaining genome stability and prevention of cancer. These factors are involved in the regulation of cell cycle checkpoints and include ATM, NBS1, BRCA1, Chk2, and p53. Here we describe a series of assays that are often used to analyze cell cycle checkpoints after IR. These assays include a ...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Analyzing Transcription Dynamics During the Budding Yeast Cell Cycle
Assaying global cell cycle-regulated transcription in budding yeast involves extracting RNA from a synchronous population and proper normalization of detected transcript levels. Here, we describe synchronization of Saccharomyces cerevisiae cell populations by centrifugal elutriation, followed by the isolation of RNA for microarray analysis. Further, we outline the computational methods required to directly compare RNA abundance from individual time points within an experiment and to compare independent experiments. Together, these methods describe the complete workflow necessary to observe RNA abundance during the cell cyc...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Cell Cycle Synchronization and Flow Cytometry Analysis of Mammalian Cells
Analysis of cellular DNA content and measurement of pulse-labeled newly replicated DNA by flow cytometry are useful techniques for cell cycle studies. In this chapter, we describe the protocols for cell cycle synchronization of mammalian cells, including time course designs and consideration of cell types to achieve successful experiments, along with the methods for detection of DNA. Some selected applications dealing with siRNA-mediated knockdown are also presented. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Oncogenic Ras Pushes (and Pulls) Cell Cycle Progression Through ERK Activation
The Ras–Raf–MEK–ERK signaling cascade is capable of channeling a wide variety of extracellular signals into control of cell proliferation, differentiation, senescence, and death. Because aberrant regulation at all steps of this signaling axis is observed in cancer, it remains an area of great interest in the field of tumor biology. Here we present evidence of the intricate and delicate levels of control of this pathway as it pertains to cell cycle regulation and illustrate how this control is not simply a rheostat. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
The Senescence Arrest Program and the Cell Cycle
All living organisms are subject to progressive loss of function and damage to their tissues, a process known as aging. At the cellular level, the accumulation of damage to DNA, proteins, and organelles induces cellular senescence, a stress-response pathway that likely influences the aging process. Although the senescence arrest program was initially described in vitro, accumulating evidence suggests that this damage response program occurs in a variety of pathologic settings. This review discusses aspects of the senescence program, their interrelationships with damage arrest pathways, the cell cycle, and the impact of sen...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Cell Cycle Regulation by the Nutrient-Sensing Mammalian Target of Rapamycin (mTOR) Pathway
Cell division involves a series of ordered and controlled events that lead to cell proliferation. Cell cycle progression implies not only demanding amounts of cell mass, protein, lipid, and nucleic acid content but also a favorable energy state. The mammalian target of rapamycin (mTOR), in response to the energy state, nutrient status, and growth factor stimulation of cells, plays a pivotal role in the coordination of cell growth and the cell cycle. Here, we review how the nutrient-sensing mTOR-signaling cascade molecularly integrates nutritional and mitogenic/anti-apoptotic cues to accurately coordinate cell growth and ce...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
The Greatwall–PP2A Axis in Cell Cycle Control
Cell cycle progression is largely controlled by reversible protein phosphorylation mediated by cyclically activated kinases and phosphatases. It has long been known that cyclin B–Cdk1 activation triggers mitotic entry, and the enzymatic network controlling its activation and inactivation has been well characterized. Much more recently protein phosphatase 2A (PP2A) together with its B55 regulatory subunit has been recognized as the major activity dephosphorylating Cdk1 targets. Moreover, PP2A-B55 activity is high in late M phase and interphase, but low at mitotic entry. A series of discoveries in the fly and frog mode...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Linking Chromosome Duplication and Segregation via Sister Chromatid Cohesion
DNA replication during S phase generates two identical copies of each chromosome. Each chromosome is destined for a daughter cell, but each daughter must receive one and only one copy of each chromosome. To ensure accurate chromosome segregation, eukaryotic cells are equipped with a mechanism to pair the chromosomes during chromosome duplication and hold the pairs until a bi-oriented mitotic spindle is formed and the pairs are pulled apart. This mechanism is known as sister chromatid cohesion, and its actions span the entire cell cycle. During G1, before DNA is copied during S phase, proteins termed cohesins are loaded ont...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Cell Cycle Regulation by Protein Degradation
Cell division is controlled by a highly regulated program to accurately duplicate and segregate chromosomes. An important feature of the cell cycle regulatory program is that key cell cycle proteins are present and active during specific cell cycle stages but are later removed or inhibited to maintain appropriate timing. The ubiquitin–proteasome system has emerged as an important mechanism to target cell cycle proteins for degradation at critical junctures during cell division. Two key E3 ubiquitin ligase complexes that target key cell cycle proteins are the Skp1–Cul1–F-box protein complex and the anaphas...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Interplay Between the Cell Cycle and Double-Strand Break Response in Mammalian Cells
The cell cycle is intimately associated with the ability of cells to sense and respond to and repair DNA damage. Understanding how cell cycle progression, particularly DNA replication and cell division, are regulated and how DNA damage can affect these processes has been the subject of intense research. Recent evidence suggests that the repair of DNA damage is regulated by the cell cycle, and that cell cycle factors are closely associated with repair factors and participate in cellular decisions regarding how to respond to and repair damage. Precise regulation of cell cycle progression in the presence of DNA damage is esse...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Cell Cycle Regulation by Checkpoints
Cell cycle checkpoints are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events of the cell cycle. These include growth to the appropriate cell size, the replication and integrity of the chromosomes, and their accurate segregation at mitosis. Many of these mechanisms are ancient in origin and highly conserved, and hence have been heavily informed by studies in simple organisms such as the yeasts. Others have evolved in higher organisms, and control alternative cell fates with significant impact on tumor suppression. Here, we consider these different checkpoint pathways and the consequ...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Cell Cycle-Regulated Transcription: Effectively Using a Genomics Toolbox
The cell cycle comprises a series of temporally ordered events that occur sequentially, including DNA replication, centrosome duplication, mitosis, and cytokinesis. What are the regulatory mechanisms that ensure proper timing and coordination of events during the cell cycle? Biochemical and genetic screens have identified a number of cell-cycle regulators, and it was recognized early on that many of the genes encoding cell-cycle regulators, including cyclins, were transcribed only in distinct phases of the cell cycle. Thus, “just in time” expression is likely an important part of the mechanism that maintains th...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
