Regulators and Effectors of Small GTPases: Rho Family (eBook)

W. E. Balch, Channing J. Der, Alan Hall (Herausgeber)

eBook Download: PDF

2006 | 1. Auflage
848 Seiten
Elsevier Science (Verlag)
978-0-08-047939-2 (ISBN)

Infos

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 - Over 150 international contributors

Inhalt

Cover 1
Copyright Page 5
Table of Contents 6
Contributors to Volume 406 12
Preface 20
Volumes in Series 22
Chapter 1: Purification and Biochemical Properties of Rac1, 2, 3 and the Splice Variant Rac1b 45
Chapter 2: Biochemical Analyses of the Wrch Atypical Rho Family GTPases 55
Chapter 3: Purification of P-Rex1 from Neutrophils and Nucleotide Exchange Assay 70
Chapter 4: In Vitro Guanine Nucleotide Exchange Activity of DHR-2/DOCKER/CZH2 Domains 85
Chapter 5: Biochemical Characterization of the Cool (Cloned-Out-of-Library)/Pix (Pak-Interactive Exchange Factor) Proteins 102
Chapter 6: GEF and Glucosylation Assays on Liposome-Bound Rac 114
Chapter 7: Phosphorylation of RhoGDI by p21-Activated Kinase 1 124
Chapter 8: Purification of ARAP3 and Characterization of GAP Activities 135
Chapter 9: Regulation of RhoGAP Specificity by Phospholipids and Prenylation 148
Chapter 10: Purification and Activity of the Rho ADP-Ribosylating Binary C2/C3 Toxin 161
Chapter 11: Purification of TAT-C3 Exoenzyme 172
Chapter 12: Imaging and Photobleach Correction of Mero-CBD, Sensor of Endogenous Cdc42 Activation 184
Chapter 13: Cdc42 and PI(4,5)P2-Induced Actin Assembly in Xenopus Egg Extracts 200
Chapter 14: In Vitro Reconstitution of Cdc42-Mediated Actin Assembly Using Purified Components 218
Chapter 15: Biochemical Analysis of Mammalian Formin Effects on Actin Dynamics 234
Chapter 16: Formin Proteins: Purification and Measurement of Effects on Actin Assembly 259
Chapter 17: Purification and Kinase Assay of PKN 278
Chapter 18: Purification and Enzyme Activity of ACK1 294
Chapter 19: Direct Activation of Purified Phospholipase C Epsilon by RhoA Studied in Reconstituted Phospholipid Vesicles 304
Chapter 20: Regulation of PLCbeta Isoforms by Rac 316
Chapter 21: Biochemical Properties and Inhibitors of (N-)WASP 325
Chapter 22: The Use of GFP to Localize Rho GTPases in Living Cells 340
Chapter 23: Analysis of the Spatiotemporal Activation of Rho GTPases Using Raichu Probes 359
Chapter 24: Measurement of Activity of Rho GTPases During Mitosis 376
Chapter 25: Inhibition of Rho GTPases by RNA Interference 389
Chapter 26: RNA Interference Techniques to Study Epithelial.Cell Adhesion and Polarity 406
Chapter 27: Nucleofection of Primary Neurons 418
Chapter 28: Dock180-ELMO Cooperation in Rac Activation 432
Chapter 29: Rho GTPase Activation by Cell-Cell Adhesion 446
Chapter 30: Activation of Rap1, Cdc42, and Rac by Nectin Adhesion System 459
Chapter 31: Analysis of Activated GAPs and GEFs in Cell Lysates 469
Chapter 32: Degradation of RhoA by Smurf1 Ubiquitin Ligase 481
Chapter 33: Ubiquitin-Mediated Proteasomal Degradation of Rho Proteins by the CNF1 Toxin 491
Chapter 34: Regulation of Superoxide-Producing NADPH Oxidases in Nonphagocytic Cells 500
Chapter 35: Activation of MEKK1 by Rho GTPases 512
Chapter 36: Activation of the Apoptotic JNK Pathway Through the Rac1-Binding Scaffold Protein POSH 523
Chapter 37: Quantification of Isozyme-Specific Activation of Phospholipase C-beta2 by Rac GTPases and Phospholipase C-epsiv by Rho GTPases in an Intact Cell Assay System 533
Chapter 38: Activation of Rho and Rac by Wnt/Frizzled Signaling 544
Chapter 39: Fluorescent Assay of Cell-Permeable C3 Transferase Activity 556
Chapter 40: Use of TIRF Microscopy to Visualize Actin and Microtubules in Migrating Cells 564
Chapter 41: Inhibition of ROCK by RhoE 577
Chapter 42: Conditional Regulation of a ROCK-Estrogen Receptor Fusion Protein 585
Chapter 43: Rational Design and Applications of a Rac GTPase-Specific Small Molecule Inhibitor 598
Chapter 44: In Vitro Assay of Primary Astrocyte Migration as a Tool to Study Rho GTPase Function in Cell Polarization 609
Chapter 45: Real-Time Centrosome Reorientation During Fibroblast Migration 623
Chapter 46: Lentiviral Delivery of RNAi in Hippocampal Neurons 637
Chapter 47: Methods for Studying Neutrophil Chemotaxis 649
Chapter 48: Measurement of Epidermal Growth Factor Receptor Turnover and Effects of Cdc42 658
Chapter 49: Tumor Cell Migration in Three Dimensions 669
Chapter 50: An In Vitro Model to Study the Role of Endothelial Rho GTPases During Leukocyte Transendothelial Migration 687
Chapter 51: Analysis of a Mitotic Role of Cdc42 700
Chapter 52: Plexin-Induced Collapse Assay in COS Cells 709
Chapter 53: Morphological and Biochemical Analysis of Rac1 in Three-Dimensional Epithelial Cell Cultures 720
Chapter 54: Using Three-Dimensional Acinar Structures for Molecular and Cell Biological Assays 736
Chapter 55: TC10 and Insulin-Stimulated Glucose Transport 745
Chapter 56: GTPases and the Control of Neuronal Polarity 759
Chapter 57: In Vitro Assembly of Filopodia-Like Bundles 771
Author Index 785
Subject Index 821

Produkt-Details

• Erscheint lt. Verlag: 21.2.2006
• Sprache: englisch
• Themenwelt: Sachbuch/Ratgeber
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• Themenwelt: Technik
• ISBN-10: 0-08-047939-1 / 0080479391
• ISBN-13: 978-0-08-047939-2 / 9780080479392