细胞技术

Gene silencing techniques have brought new insights into mammalian oocyte and embryo development. More specifically, the use of Morpholino oligonucleotides which sterically inhibit translation from target mRNAs thereby compromising gene function, allowed the identifi ...

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 ...

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 cor ...

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 ...

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 ra ...

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 stimulat ...

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 the ...

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. Wh ...

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 modific ...

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 betw ...

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 carbohydrate ...

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 ...

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 ...

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 s ...

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 c ...

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 ...

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 foc ...

Ras and Rho family GTPases control a wide variety of cellular processes, and the signaling downstream of these GTPases is influenced by their subcellular localization when activated. Since only a minority of total cellular GTPases is active, observation of the total subcellular distri ...

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 elec ...

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 appar ...