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.HaloTag as a Tool to Investigate Peroxisome Dynamics in Cultured Mammalian Cells
Peroxisomes are multifunctional organelles that can rapidly modulate their morphology, number, and function in response to changing environmental stimuli. Defects in any of these processes can lead to organelle dysfunction and have been associated with various inherited and age-related disorders. Progress in this field continues to be driven by advances in live-cell imaging techniques. This chapter provides detailed protocols for the use of HaloTag to fluorescently pulse-label peroxisomes in cultured mammalian cells. In contrast to the use of classical fluorescent proteins, this technology allows researchers to optically d...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Combining Pulsed SILAC Labeling and Click-Chemistry for Quantitative Secretome Analysis
Secreted proteins, such as cytokines, chemokines, and hormones, exhibit central functions in intercellular communication, which is crucial to maintain homeostasis in every multicellular organism. A common approach to identify secreted proteins is by proteomic analysis of culture media after conditioning with a cell type of interest. This is preferably done in serum-free conditions to enable the detection of low-abundance secretory factors that would otherwise be masked by serum proteins. However, serum starvation introduces the risk of bringing cells in a stressed or perturbed state. A superior approach employs the enrichm...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Fractionation of Subcellular Membrane Vesicles of Epithelial and Non-epithelial Cells by OptiPrep™ Density Gradient Ultracentrifugation
Density gradient ultracentrifugation (DGUC) is widely used for physical isolation (enrichment rather than purification) of subcellular membrane vesicles. It has been a valuable tool to study specific subcellular localization and dynamic trafficking of proteins. While sucrose has been the main component of density gradients, several years ago, synthetic OptiPrep™ (iodixanol) began being used for separation of organelles due to its iso-osmotic property. Here, we describe a detailed protocol for density gradient fractionation of various mammalian subcellular vesicles, including endoplasmic reticulum (ER), Golgi apparatu...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Secretion of Circular Proteins Using Sortase
Circular proteins occur naturally and have been found in microorganisms, plants, and eukaryotes where they are commonly involved in host defense. Properties of circular proteins include enhanced resistance to exoproteases, increased thermostability, longer life spans, and increased activity. Using an enzymatic approach based on the bacterial sortase A (SrtA) transpeptidase, N- and C-termini of conventional linear proteins can be linked resulting in a circular protein. Circularization of bioengineered linear substrate proteins can indeed confer the desirable properties associated with circular proteins. Here, we describe ho...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Profiling Lysine Ubiquitination by Selective Enrichment of Ubiquitin Remnant-Containing Peptides
Protein ubiquitination plays critical roles in many biological processes. However, functional studies of protein ubiquitination in eukaryotic cells are limited by the ability to identify protein ubiquitination sites. Unbiased high-throughput screening methods are necessary to discover novel ubiquitination sites that play important roles in cellular regulation. Here, we describe an immunopurification approach that enriches ubiquitin remnant-containing peptides to facilitate downstream mass spectrometry (MS) identification of lysine ubiquitination sites. This approach can be utilized to identify ubiquitination sites from pro...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Real-Time Detection of SNARE Complex Assembly with FRET Using the Tetracysteine System
Small tetracysteine insertions are more suitable for fluorescence resonance energy transfer (FRET) studies of protein folding and small complex assembly than bulky GFP-based fluorophores. Here, we describe a procedure for expression, purification, and fluorescent labeling of a FRET-based probe, called CSNAC that can track the conformational changes undergone by SNAP-25 as it folds in the exocytic complex. The fluorescent protein Cerulean was attached to the N-terminus and served as a FRET donor. The biarsenical dye FlAsH, served as a FRET acceptor, was bound to a short tetracysteine motif positioned in the linker domain of...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Systematic Analysis of Endocytosis by Cellular Perturbations
Endocytosis is an essential process of eukaryotic cells that facilitates numerous cellular and organismal functions. The formation of vesicles from the plasma membrane serves the internalization of ligands and receptors and leads to their degradation or recycling. A number of distinct mechanisms have been described over the years, several of which are only partially characterized in terms of mechanism and function. These are often referred to as novel endocytic pathways. The pathways differ in their mode of uptake and in their intracellular destination. Here, an overview of the set of cellular proteins that facilitate the ...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Pharmacological Inhibitors of Exocytosis and Endocytosis: Novel Bullets for Old Targets
Pharmacological inhibitors of vesicle trafficking possess great promise as valuable analytical tools for the study of a variety of biological processes and as potential therapeutic agents to fight microbial infections and cancer. However, many commonly used trafficking inhibitors are characterized by poor selectivity that diminishes their use in solving basic problems of cell biology or drug development. Recent high-throughput chemical screens intensified the search for novel modulators of vesicle trafficking, and successfully identified a number of small molecules that inhibit exocytosis and endocytosis in different types...
Source: Springer protocols feed by Cell Biology - June 20, 2014 Category: Cytology Source Type: news
Analyzing G1–S Transcriptional Control
G1–S transcriptional control involves the coordination of the expression of a large set of co-regulated genes as a function of cell cycle progression (Bertoli et al., Nat Rev Mol Cell Biol 14:518–528, 2013). Confining transcription to the G1 phase of the cell cycle requires the regulation of specific transcription factor activity through either co-factors or regulation of promoter DNA binding. Therefore, the analysis of G1–S transcriptional control involves cell cycle synchronization and monitoring cell cycle synchrony, in order to establish DNA binding of G1–S transcription factors to G1–S pr...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Analyzing RB and E2F During the G1–S Transition
The G1/S-phase restriction point is an important landmark in the mammalian cell division cycle. The key regulator of the G1/S transition is the retinoblastoma gene product (pRB). It prevents the transcription of genes required for S-phase progression by repressing E2F transcription factors. An increase in Cdk phosphorylation of pRB causes the release of E2F transcription factors and advancement into S phase. Here we describe two simple techniques used to assess pRB phosphorylation and E2F transcription during G1/S progression. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Detecting Senescence: Methods and Approaches
The detection of senescent cells has become an important area of research in the aging field. Due to the complexity of the senescence program and the lack of a unique signature for senescence, the detection of these cells remains problematic. This is especially true for in vivo detection in aged or diseased tissue samples. This chapter outlines approaches for the detection of senescent cells based upon methods established for mesenchymal cells in culture. A stepwise approach to the detection of senescent cells using multiple techniques is provided. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Telomere Regulation During the Cell Cycle in Fission Yeast
The fission yeast Schizosaccharomyces pombe has emerged as a useful model organism to study telomere maintenance mechanisms. In this chapter, we provide detailed protocols for quantitative ChIP and BrdU incorporation analyses to investigate how fission yeast telomeres are regulated during the cell cycle by utilizing cdc25-22 synchronized cell cultures. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Analyzing Ras-Associated Cell Proliferation Signaling
Ras-dependent signaling is an important regulator of cell cycle progression, proliferation, senescence, and apoptosis. Several of the downstream effectors of Ras play dual roles in each of these processes. Under one set of conditions, they promote cell cycle progression and proliferation; yet, in a different paradigm, they drive cell cycle arrest and apoptosis. Furthermore, there is cross talk between certain downstream effectors of Ras including the PI3K–AKT and Raf–MEK–ERK pathways. Here we describe a series of experiments used to dissect the effect of different Ras-dependent signaling pathways on cell ...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
Analyzing Cdc2/Cdk1 Activation During Stress Response in Schizosaccharomyces pombe
Stress leads to multiple changes in the physiology of the cell. One of the most important is the adaptation of the cell cycle to the changing conditions of the environment. Cellular responses after stress can be followed by cellular synchronization previous to the insult. In this chapter, we use centrifugal elutriation to synchronize Schizosaccharomyces pombe cells and outline methods to investigate the hallmarks of cell cycle progression upon stress. These include analyses of cyclin-dependent kinase phosphorylation and cell size change. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
PCNA-Dependent Ubiquitination of Cdt1 and p21 in Mammalian Cells
PCNA is a DNA clamp, acting on chromatin as a platform for various proteins involved in many aspects of DNA replication-linked processes. Most of these proteins have the PCNA-interaction protein motif (PIP box) that associates with PCNA. Recent works show that PCNA plays an important role as a matchmaker, connecting PCNA-interacting proteins to the ubiquitin ligase CRL4Cdt2 for their degradation. Proteins degraded by CRL4Cdt2 include Cdt1, p21, and Set8 in mammalian cells. These CRL4Cdt2 substrates have a PIP degron that consists of the canonical PIP-box sequence and additional conserved amino acids required for ubiquitina...
Source: Springer protocols feed by Cell Biology - June 10, 2014 Category: Cytology Source Type: news
