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.Real-Time Visualization and Quantification of Native Ras Activation in Single Living Cells
Members of the Ras family of small guanosine triphosphate phosphohydrolases are GDP/GTP-binding proteins that function as pivotal molecular switches in multiple cell biological processes. The prototypical Ras family members K-Ras, N-Ras, and H-Ras, in particular, have been the focus of intense research for the last 30 years owing to their critical function as signalling nodes in the control of cell growth and proliferation and as drivers of oncogenic transformation. One aspect that has attracted much attention in recent times is the spatial control of Ras activity, which is dictated largely by a series of posttranslational...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Effector Recruitment Method to Study Spatially Regulated Activation of Ras and Rho GTPases
We describe an example of a recruitment assay using the GBD of PAK1 to monitor Rac1 activity and explain how the assay can be expanded to determine the subcellular localization of activation of other GTPases. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
High-Resolution Scanning Electron Microscopy for the Imaging of Nuclear Pore Complexes and Ran-Mediated Transport
High-resolution scanning electron microscopy provides three-dimensional surface images of nuclear pore complexes (NPCs) embedded in the nuclear envelope. Here, we describe a method for exposing the nuclear surface in mammalian tissue culture cells for imaging by scanning electron microscopy. Hypotonic treatment is followed by low-speed centrifugation onto polylysine-coated silicon chips, without the use of detergents. This helps to preserve NPCs close to their native morphology, embedded in undamaged nuclear membranes. This method is particularly advantageous for combining high-resolution imaging of NPCs with mammalian gen...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Immunofluorescence Methods in Studies of the GTPase Ran and Its Effectors in Interphase and in Mitotic Cells
The GTPase RAN, the regulators of its nucleotide-bound state, and its effectors represent a specialized network in the RAS GTPase superfamily and regulate the localization of macromolecules (RNAs and proteins) in subcellular compartments in interphase cells and at the mitotic apparatus when cells divide. Essential cell cycle processes, e.g., replication, repair, transcription, export of transcribed RNAs out of the nucleus, assembly of the mitotic apparatus, kinetochore function, chromosome segregation, nuclear reorganization, and rebuilding of the nuclear envelope and nuclear pores at mitotic exit, ultimately depend on RAN...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Analysis of the Rit Subfamily GTPase-Mediated Signaling and Neuronal Differentiation and Survival
The Rit subfamily of GTPases is a founding branch within the Ras family of small G-proteins and preserves unique sequences in the G2 effector loop domain and the C-terminus. Rit proteins regulate a diversity of signal transduction pathways, some of which are similar to and others of which differ from the pathways that are regulated by other Ras family GTPases. Rit proteins have been demonstrated to be essential regulators in neuronal differentiation and survival. Here, we describe the materials and methods utilized to characterize cellular signaling for the Rit subfamily of G-proteins in neuronal differentiation and surviv...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
An In Vitro System to Evaluate the Scaffold Function of the RalA Effector Protein RalBP1
We describe here a method to evaluate the contribution of specific scaffold proteins to kinase reactions using a modified in vitro kinase assay. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Fluorescence Microscopy Study of Rap1 Subcellular Localization
The Ras-related GTPase Rap has been implicated in multiple cellular functions. In particular, Rap1 is a crucial regulator of both inside-out integrin activation and outside-in E-cadherin-mediated signaling. Thus, Rap1 was proposed as a fundamental regulator of the cross talk between cadherins and integrins. We provide microscopic techniques to study subcellular localization of Rap1 protein in the crosstalk between integrins and cadherins. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Combined Pulldown and Time-Lapse Microscopy Studies for Determining the Role of Rap1 in the Crosstalk Between Integrins and Cadherins
The coordinate modulation of the cellular functions of cadherins and integrins plays an essential role in fundamental physiological and pathological processes, including morphogenesis, tissue differentiation and renewal, wound healing, immune surveillance, inflammatory response, tumor progression, and metastasis. Recent findings state the molecular mechanisms underlying the fine-balanced relationship between cadherins and integrins. In particular, some of the novel results recently obtained raise the possibility of a pivotal role for the small GTPase Rap1 in the functional crosstalk between cadherins and integrins. Conside...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Pull-Down Assay for Analysis of Integrin-Mediated Activation of Rap Proteins in Adherent Platelets
Rap1 GTPases operate as molecular switches by cycling between a GDP-bound inactive state and a GTP-bound active state and regulate several cellular pathways in response to different stimuli. Circulating blood platelets express high levels of Rap1 proteins, mainly Rap1b, which plays a critical role in platelet adhesion and activation. Rap1 is a key element in the inside-out signaling pathway leading to the conversion of integrins into the high-affinity state for their ligands. In platelets, Rap1b regulates inside-out activation of both integrin αIIbβ3 and α2β1. In addition, Rap1b is also involved in in...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Functional Phosphoproteomics of Oncogenic KRAS Signaling
Identification of oncogene-mediated phosphorylation events is essential to understanding the molecular determinants responsible for cancer development and progression. Here, we identify KRAS-regulated phosphorylation events using label-free quantitation-based comparative phosphoproteomics analyses of immortalized human bronchial epithelial cells that express oncogenic KRAS as well as cells that do not. Further, we demonstrate integration of the identified phosphorylation events with the Pathway Interaction Database to infer KRAS-regulated pathways, which may have implications in KRAS-associated lung adenocarcinoma developm...
Source: Springer protocols feed by Cell Biology - January 29, 2014 Category: Cytology Source Type: news
Polarization Microscopy of Extended Chromatin Fibers
Analysis of the formation of extended chromatin fibers (ECFs) in response to the action of gravity following lysis by hypertonic and detergent solutions is a useful technical procedure relevant for studies of the positioning of particular DNA signals on chromatin filaments. Additionally, if toluidine blue molecules are allowed to bind electrostatically to available DNA phosphates on ECFs, the birefringence brightness generated in these filaments, as observed by polarization microscopy, facilitates the description of the frequency of ECF formation and extension of the chromatin filaments generated. Thus, different patterns ...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Alkaline Nuclear Dispersion Assays for the Determination of DNA Damage at the Single Cell Level
Over the past three decades the development of methods for visualizing at the cell level the extent of DNA breakage significantly contributed to genotoxicity testing: their availability greatly improved the knowledge in the field of genetic toxicology. These procedures are based on the separation and visualization of DNA fragments resulting from cleavage of nuclear DNA. The separation process can be obtained either electrically (comet assay, linear migration of DNA fragments) or chemically (alkaline dispersion assays, radial diffusion of DNA fragments). Once separated and stained, intact and fragmented DNA can be observed ...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Analysis of DNA Damage and Repair by Comet Fluorescence In Situ Hybridization (Comet-FISH)
A useful tool in the detection of overall and region-specific DNA damage is the Comet-FISH technique. This method combines two well-established methods, the Comet assay (single cell gel electrophoresis), which makes it possible to detect and quantify DNA damage at the single cell level, and FISH (fluorescence in situ hybridization), a technique that allows the specific detection of selected DNA sequences. The influence of specific substances such as water pollutants or food ingredients on individual cells can be measured with the alkaline version of the Comet assay, which involves the embedding of cells in agarose on micro...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Identifying Different Types of Chromatin Using Giemsa Staining
Mixtures of polychrome methylene blue-eosin Y (i.e., Giemsa stain) are widely used in biological staining. They induce a striking purple coloration of chromatin DNA (the Romanowsky-Giemsa effect), which contrasts with the blue-stained RNA-containing cytoplasm and nucleoli. After specific prestaining treatments that induce chromatin disorganization (giving banded or harlequin chromosomes), Giemsa staining produces a differential coloration, with C- and G-bands appearing in purple whereas remaining chromosome regions are blue. Unsubstituted (TT) and bromo-substituted (BT) DNAs also appear purple and blue, respectively. The s...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
Using Microchip Gel Electrophoresis to Probe DNA–Drug Binding Interactions
Binding of small molecules with DNA plays an important role in many biological functions such as DNA replication, repair, and transcription. These interactions also offer enormous potential as targets for diagnostics and therapeutics, leading to intense interest in development of methods to probe the underlying binding events. In this chapter, we present a new approach to investigate the structural changes that accompany binding of DNA and small molecules. Instead of relying on conventional yet delicate single-molecule imaging methods, we show how a single microchip gel electrophoresis experiment incorporating both constan...
Source: Springer protocols feed by Cell Biology - October 30, 2013 Category: Cytology Source Type: news
