生物化学技术

The regulation of exocytosis by Rho GTPases is conserved in eukaryotic kingdoms. Given the spatiotemporal dynamics of Rho GTPase activity, a method for visualizing exocytosis with high space and time resolution will be an important tool for the functional analysis of Rho GTPases. During t ...

The model organism D. discoideum is well suited to investigate basic questions of molecular and cell biology, particularly those related to the structure, regulation, and dynamics of the cytoskeleton, signal transduction, cell–cell adhesion, and development. D. discoideum makes use ...

Rho proteins act as molecular switches to control multiple cellular processes. The switch mechanism involves cycling between active and inactive states based on GTP loading and hydrolysis. Assays that quantitatively analyze the GTP loading of Rho proteins have become important mol ...

The cellular branch of the Drosophila larval innate immune system consists of three immunosurveillance (haemocyte) cell types: plasmatocytes, crystal cells, and lamellocytes. In order to examine haemocyte cytoskeletal dynamics or migration, most researchers use embryos or in v ...

We provide information and protocols for the analysis of Rho GTPase function in axon pathfinding in Caenorhabditis elegans. The powerful molecular, genetic, imaging, and transgenic tools available in C. elegans make it an excellent system in which to study the in vivo roles of Rho GTPases. Meth ...

Rho small GTPases play pivotal roles in a variety of dynamic cellular processes including cytoskeleton rearrangement, cell migration, cell proliferation, cell survival, and gene regulation. However, their functions in vivo are much less understood. Recently, the zebrafish, Danio ...

In contrast to typical Rho GTPases the regulation of atypical Rho GTPases, such as the members of the RhoBTB subfamily, rarely depends on GEFs and/or GAPs. Instead, they are regulated at the level of their expression, by post-translational modifications, by their rate of degradation as well as thr ...

We describe a phage display approach to select active Rho-specific scFv sensors. This in vitro technique allows preserving the antigen conformation stability all along the selection process. We used the GTP locked RhoBQ63L mutant as antigen against the Griffin.1 library composed of a hum ...

Extensive knowledge about protein–protein interactions is fundamental to fully understand signaling pathways and for the development of new drugs. Rho GTPases are key molecules in cellular signaling processes and their deregulation is implicated in the development of a variety of ...

Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions, their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this ...

Fluorescence resonance energy transfer (FRET) imaging can measure the spatial and temporal distributions of activated Rho GTPases within living cells. This information is essential for understanding how signaling networks influence Rho-GTPase switching and for elucidat ...

Here, we provide procedures for imaging the Rho GTPase biosensors in both single and multiplex acquisition modes. The multiplex approach enables the direct visualization of two biosensor readouts from a single living cell. Here, we take as an example a combination of the RhoA biosensor based on a ...

Small Rho GTPases are major regulators of actin cytoskeleton dynamics and influence cell shape and migration. The expression of several Rho GTPases is often up-regulated in tumors and this frequently correlates with a poor prognosis for patients. Migration of cancer cells through endo ...

Rac, a member of the Rho family small GTPases, plays a crucial role in activation of Nox family NADPH oxidases in animals, enzymes dedicated to production of reactive oxygen species such as superoxide. The phagocyte oxidase Nox2, crucial for microbicidal activity during phagocytosis, is act ...

Rho GTPases are key regulators of the cytoskeleton during the process of neurite outgrowth. Based on overexpression of dominant-positive and negative Rho GTPase constructs, the classic view is that Rac1 and Cdc42 are important for neurite elongation whereas RhoA regulates neurite re ...

Historically, in vitro culturing of primary osteoclasts involved co-culturing of mononuclear monocytes with bone marrow stromal cells, thereby providing the cytokines required for osteoclast formation and multinucleation. Since the identification and cloning of recept ...

Primary keratinocytes are an important tool to investigate the molecular mechanism underlying the skin phenotype of mice with null mutations in Rho GTPase genes. If the RhoA gene deletion is conditional, the knockout can be induced in vitro by transfection with cre-IRES-GFP and sorting for G ...

Epithelial junction formation and maintenance are multistep processes that rely on the clustering of macromolecular complexes. These events are highly regulated by signalling pathways that involve Rho small GTPases. Usually, when analysing the contribution of different com ...

Tight regulation of Rho GTPase-signaling functions requires the proper localization of proteins to the membrane and cytosolic compartments, which can themselves undergo reconfiguration in response to signaling events. The importance of lipid-mediated membrane signal tra ...

RhoGDI1 is one of the three major regulators of the Rho switch along with RhoGEFs and RhoGAPs. RhoGDI1 extracts prenylated Rho proteins from lipid membranes, sequesters them in the cytosol, and prevents nucleotide exchange or hydrolysis. In addition, RhoGDI1 protects prenylated Rho pro ...