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.Behavioral Methods for the Study of the Ras–ERK Pathway in Memory Formation and Consolidation: Passive Avoidance and Novel Object Recognition Tests
Memory is a high-level brain function that enables organisms to adapt their behavioral responses to the environment, hence increasing their probability of survival. The Ras–ERK pathway is a key molecular intracellular signalling cascade for memory consolidation. In this chapter we will describe two main one-trial behavioral tests commonly used in the field of memory research in order to assess the role of Ras–ERK signalling in long-term memory: passive avoidance and object recognition. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Screening for MAPK Modulators Using an In-Cell Western Assay
Several essential cellular functions are critically influenced by the Ras-Raf-MEK-ERK cascade, and pathological problems have been implicated with loss as well as gain of its activity. Therefore, there is a great demand for chemical probes that act as modulators of the cascade in order to correct medical problems associated with its malfunctions. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Use of the Yeast Two-Hybrid Technology to Isolate Molecular Interactions of Ras GTPases
Since its original description, the yeast two-hybrid system has been extensively used to identify protein–protein interactions from many different organisms, thus providing a convenient mean to both screen for proteins that interact with a protein of interest and to characterize the known interaction between two proteins. In these years the technique has improved to overcome the limitations of the original assay, and many efforts have been made to scale up the technique and to adapt it to large-scale studies. In addition, variations have been introduced to enlarge the range of proteins and interactors that can be ass...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases
Cysteine is one of the most reactive amino acids and is modified by a number of oxidants. The reactivity of cysteines is dependent on the thiol pK
a; however, measuring cysteine pK
a values is nontrivial. Ras family GTPases have been shown to contain a free cysteine that is sensitive to oxidation, and free radical-mediated oxidation of this cysteine has been shown to be activating. Here, we present a new technique that allows for measuring cysteine pK
a values using a fluorescent detection system with the molecule 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F). In addition, we also d...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Ras GTPases Are Both Regulators and Effectors of Redox Agents
Redox agents have been historically considered pathological agents which can react with and damage many biological macromolecules including DNA, proteins, and lipids. However, a growing number of reports have suggested that mammalian cells can rapidly respond to ligand stimulation with a change in intracellular ROS thus indicating that the production of intracellular redox agents is tightly regulated and that they serve as intracellular signaling molecules being involved in a variety of cell signaling pathways. Numerous observations have suggested that some members of the Ras GTPase superfamily appear to regulate the produ...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Ubiquitination of Rac1 by Inhibitors of Apoptosis (IAPs)
Ubiquitination of proteins has emerged as a vital posttranslational modification at the crux of numerous signalling pathways, regulating them in various ways. Most members of the small GTPase family including Ras and Rho proteins are regulated by GEFs, GAPs, and RhoGDIs that modulate their cycling between the active and inactive states. Ubiquitination has added another layer to the regulation of small GTPases. Recently, we have uncovered that inhibitors of apoptosis (IAPs) function as direct E3 ubiquitin ligases for Rho GTPase Rac1 and target it for proteasomal degradation. Here, we describe in vitro and in vivo ubiquitina...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Metabolic Labeling of Ras with Tritiated Palmitate to Monitor Palmitoylation and Depalmitoylation
Metabolic labeling with tritiated palmitate is a direct method for monitoring posttranslational modification of Ras proteins with this fatty acid. Advances in intensifying screens have allowed for the easy visualization of tritium without the need for extended exposure times. While more energetic radioisotopes are easier to visualize, the lack of commercial source and need for shielding make them more difficult to work with. Since radiolabeled palmitate is directly incorporated into Ras, its loss can be monitored by traditional pulse-chase experiments that cannot be accomplished with the method of acyl-exchange chemistry. ...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
A Novel Method for the Production of Fully Modified K-Ras 4B
Post-translational modifications in proteins play a major functional role. Post-translational modifications affect the way proteins interact with each other, bind nucleotides, and localize in cellular compartments. Given the importance of post-translational modifications in protein biology, development of methods to produce post-translationally modified proteins for biochemical and biophysical studies is timely and significant. At the same time, obtaining post-translationally modified proteins in bacterial expression systems is often problematic. Here, we describe a novel recombinant approach to prepare human K-Ras 4B, a p...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Ras Proteins Signaling in the Early Metazoan Dictyostelium discoideum
Since the discovery of Ras, Ras-mediated transforming activity has been the major investigative area of interest. Soon thereafter it has emerged that Ras family members regulate different biological processes, other than cell growth, like development and fine-tune the balance between cell death and survival. The lower metazoan Dictyostelium discoideum is a powerful and genetically accessible model organism that has been used to elucidate the roles played by different Ras members in some biological processes, such as cell motility and development. In the following chapter we describe some very basic techniques aiming to ide...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Methods to Study the Ras2 Protein Activation State and the Subcellular Localization of Ras-GTP in Saccharomyces cerevisiae
Ras proteins were highly conserved during evolution. They function as a point of convergence for different signalling pathways in eukaryotes and are involved in a wide range of cellular responses (shift from gluconeogenic to fermentative growth, breakdown of storage carbohydrates, stress resistance, growth control and determination of life span, morphogenesis and development, and others). These proteins are members of the small GTPase superfamily, which are active in the GTP-bound form and inactive in the GDP-bound form. Given the importance of studies on the Ras protein activation state to understand the detailed mechanis...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Yeast as a Model for Ras Signalling
For centuries yeast species have been popular hosts for classical biotechnology processes, such as baking, brewing, and wine making, and more recently for recombinant proteins production, thanks to the advantages of unicellular organisms (i.e., ease of genetic manipulation and rapid growth) together with the ability to perform eukaryotic posttranslational modifications. Moreover, yeast cells have been used for few decades as a tool for identifying the genes and pathways involved in basic cellular processes such as the cell cycle, aging, and stress response. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Analysis of Rho GTPase-Induced Localization of Nanoscale Adhesions Using Fluorescence Nanoscopy
Rho GTPases are important regulators of the formation of focal adhesions and focal complexes, and thereby they are key regulators of cell adhesion and migration. Here, we describe a method to study the relocalization of proteins within cell–matrix adhesions at a nanoscale level, through the use of super-resolution stimulated emission depletion microscopy imaging. We furthermore describe computational tools for image processing and data analysis. Thus, the method presented in this chapter provides an unbiased approach to the quantitative evaluation of the spatial distribution of the nanoscale protein assemblies by whi...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Analyzing the Roles of Rho GTPases in Cancer Cell Migration with a Live Cell Imaging 3D-Morphology-Based Assay
Rho GTPases are master regulators of cytoskeleton dynamics and therefore regulate cell motility. Rho GTPases, as well as their regulators and effectors, are often deregulated in cancers and thus contribute to tumor progression to metastasis. Cancer progression involves multiple steps, including invasion of the surrounding tissues. Several methods to investigate the invasion of tumors cells in 3D matrices in vitro have been developed. In this chapter we describe a 3D-based morphology assay that can be used for medium-throughput microscopy-based screening to identify regulators of cancer cell invasion. We use this method cou...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Nanoclustering and Heterogeneous Membrane Diffusion of Ras Studied by FRAP and RICS Analysis
Fluorescence Recovery After Photobleaching (FRAP) and Raster Image Correlation Spectroscopy (RICS) are two powerful techniques to study the diffusion dynamics of fluorescently labeled proteins. FRAP and RICS can be easily applied on any commercial confocal microscope. In this chapter, we describe the principles of these methods and provide the reader with a detailed guide on how to apply these methods in the study of Ras nanoclustering and diffusion in the plasma membrane of live cells. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
The Ras Superfamily of Small GTPases: The Unlocked Secrets
The Ras superfamily of small GTPases is composed of more than 150 members, which share a conserved structure and biochemical properties, acting as binary molecular switches turned on by binding GTP and off by hydrolyzing GTP to GDP. However, despite considerable structural and biochemical similarities, these proteins play multiple and divergent roles, being versatile and key regulators of virtually all fundamental cellular processes. Conversely, their dysfunction plays a crucial role in the pathogenesis of serious human diseases, including cancer and developmental syndromes. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
