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 describes some of the methods currently being used to examine Rho family GTPase regulation at the biochemical and cellular level.

Key Features

*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. *Over 150 international contributors.


Biochemists, biophysicists, cell biologists, molecular biologists, geneticists, developmental biologists

Table of Contents

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; Ubi


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© 2006
Academic Press
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@from:Praise for the Series @qu:"Incomparably useful." @source:--ANALYTICAL BIOCHEMISTRY @qu:"It is a true 'methods' series, including almost every detail from basic theory to sources of equipment and reagents, with timely documentation provided on each page." @source:--BIO/TECHNOLOGY @qu:"The series has been following the growing, changing and creation of new areas of science. It should be on the shelves of all libraries in the world as a whole collection." @source:--CHEMISTRY IN INDUSTRY