The Ras superfamily (>150 human members) encompasses Ras GTPases involved in cell proliferation, Rho GTPases involved in regulating the cytoskeleton, Rab GTPases involved in membrane targeting/fusion and a group of GTPases including Sar1, Arf, Arl and dynamin involved in vesicle budding/fission. These GTPases act as molecular switches and their activities are controlled by a large number of regulatory molecules that affect either GTP loading (guanine nucleotide exchange factors or GEFs) or GTP hydrolysis (GTPase activating proteins or GAPs). In their active state, they interact with a continually increasing, functionally complex array of downstream effectors. Since the last Methods in Enzymology volume on this topic in 2000, Rho GTPases have continued to receive a huge amount of attention. The human genome sequence has revealed the full extent of the Rho GEF and Rho GAP families (over 80 members for each) and the challenge of identifying the molecular interactions and cellular pathways influenced by each of these regulators is a daunting prospect. This new volume, Regulators and Effectors of Small GTPases: Rho Family, describes some of the methods currently being used to examine Rho family GTPase regulation at the biochemical and cellular level.
Describes the methods currently being used to examine Rho family GTPase regulation at the biochemical and cellular levels
Includes new imaging techniques that revolutionize the ability to visualize GTPase activities
Purification and Biochemical Properties of Rac1,2,3 and the Splice Variant Rac1b; Biochemical Analyses of the Wrch Atypical Rho Family GTPases; Purification of P-Rex1 from neutrophils and nucleotide exchange assay; In vitro guanine nucleotide exchange activity of DHR-2/DOCKER/ CZH2 domains; Biochemical characterization of the Cool (Coned-out-of-Library)/Pix (Pak-interactive exchange factor) proteins; GEF and glucosylation assays on liposome bound Rac; Phosphorylation of RhoGDI by p21-activated kinase 1; Purification of ARAP3 and characterization of GAP activities; Regulation of RhoGAP specificity by phospholipids and prenylation; Purification and activity of the Rho ADP-ribosylating binary C2/C3 toxin; Purification of Tat-C3 exoenzymes; Imaging and photobleach correction of MeroCBD, sensor of endogenous Cdc42 activation ; Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts; In Vitro Reconstitution of Cdc42-Mediated Actin Assembly Using Purified Components; Biochemical Analysis of Mammalian Formin Effects on Actin Dynamics; Formin proteins: purification and measurement of effects on actin assembly; Purification and Kinase Assay of PKN; Purification and Enzyme Activity of ACK1; Direct activation of purified phospholipase C epsilon by RhoA studied in reconstituted phospholipid vesicles; Regulation of PLCâ isoforms by Rac; Biochemical properties and inhibitors of (N-)WASP; The Use of GFP to localize Rho GTPases in Living Cells; Analysis of the Spatio-Temporal Activation of Rho GTPases using Raichu Probes; Measurement of Activity of Rho GTPases during Mitosis; RNAi inhibition of Rho GTPases; RNA interference techniques to study epithelial cell adhesion and polarity; Nucleofection of primary neurons; Dock180-ELMO Cooperation in Rac Activation; Rho GTPase activation by cell-cell adhesion; Activation of Rap1, Cdc42, and Rac by nectin adhesion system; Analysis of activated GAPs and GEFs in cell lysates; Degradation of RhoA by Smurf1 ubiquitin ligase; Ubiquitin-mediated proteasomal degradation of Rho proteins by the CNF1 toxin; Regulation of superoxide-producing NADPH in non-phagocytic cells; Activation of MEKK1 by Rho GTPases; Activation of Apoptotic JNK Pathway through the Rac1-Binding Scaffold Protein POSH; Quantification of isozyme-specific activation of phospholipase C-â2 by Rac GTPases and phospholipase C-å by Rho GTPases in an intact cell assay system; Activation of Rho and Rac by Wnt/Frizzled signalling; Fluorescent assay of cell permeable C3 transferase activity; Use of TIRF microscopy to visualize actin and microtubules in migrating cells; Inhibition of ROCK by RhoE; Conditional regulation of a ROCK-estrogen receptor fusion protein; Rational design and applications of a Rac GTPase-specific small molecule inhibitor; In vitro assay of primary astrocyte migration as a tool to study Rho GTPase function in cell polarization; Real time centrosome reorientation during fibroblast migration; Lentiviral delivery of RNAi in Hippocampal Neurons; Methods for studying neutrophil Chemtoaxis; Measurement of Epidermal Growth Factor Receptor turnover and effects of Cdc42; Tumor cell migration in three dimensions; Reciprocal regulation of cyclin D1 expression by Rac and Rho; Regulation of ERK signal duration and cyclin D1 expression by Rhodependent stress fiber formation; An in vitro model to study the role of endothelial Rho GTPases during leukocyte transendothelial migration; Analysis of a Mitotic role of Cdc42; Plexin-induced collapse assay in COS cells; Morphological and Biochemical Analysis of Rac1 in Three-Dimensional Epithelial Cell Cultures; Using Three Dimensional Acinar Structures for Molecular and Cell Biological Assays; TC10 and insulin-stimulated glucose transport; GTPases and the control of neuronal polarity; In vitro assembly of filopodia-like bundles