Rho GTPases (Methods in Molecular Biology, v827)

✍ Scribed by Francisco Rivero

• Publisher: Springer
• Year: 2011
• Tongue: English
• Leaves: 422
• Edition: 2012
• Category: Library

✦ Synopsis

Although initially described as major regulators of cytoskeletal remodeling, Rho GTPases have been implicated in the establishment of polarity, endocytosis, vesicle trafficking, morphogenesis, cytokinesis, transcriptional activation, cell cycle progression, and apoptosis, to mention a few. In addition, Rho GTPases have acquired medical relevance because of their participation in tumorigenesis and metastasis, in cardiovascular conditions, and as targets of infectious agents. The field has broadened even more with the contribution of studies in model organisms (plants, amoebas, fungi, invertebrates), each adding a particular view to the complexity of the family and vastly enriching our perception of these important signaling components. Divided into five convenient sections, Rho GTPase: Methods and Protocols provides an historical overview of the field and an account of the phylogenetics of the Rho family, general biochemical methods, and functional assays that allow monitoring the consequences of manipulating Rho GTPases in a variety of contexts. Additionally, the volume devotes a section to advanced imaging methods and to recently developed high throughput methods, closing with techniques specifically designed for studies in selected non-mammalian model organisms. Written in the successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.   Authoritative and easily accessible, Rho GTPase: Methods and Protocols provides techniques that are standard for researchers in the field but also includes protocols for those who, being already familiar with some of the techniques, wish to explore additional aspects.

✦ Table of Contents

Cover......Page 1
Frontmatter......Page 4
Preface......Page 6
Contents......Page 8
Contributors......Page 12
Part I: History and Phylogenesis
......Page 16
1. Introduction......Page 18
2. Identification of Rho GTPases......Page 19
3. Rho GTPases as Regulators of the Actin Cytoskeleton......Page 20
4. Regulation of Rho GTPases......Page 21
6. Rho GTPases and Transcription......Page 22
8. Rho GTPases as Targets of Bacterial Toxins and Viral Proteins......Page 23
References......Page 24
1. Introduction: The Rho Family and Its Origin......Page 28
2. Challenges of Reconstructing the History of Gene Duplications and Losses Within the Rho Family......Page 33
3.1. Assembling a Sequence Dataset......Page 36
3.2. Building a Multiple Alignment......Page 39
3.3. Inferring a Tree......Page 40
3.4. From a Phylogenetic Tree to an Evolutionary Scenario......Page 41
3.4.2. Rac......Page 42
3.4.4. Rho1......Page 43
3.4.6. Novel Fungal Rho Groups......Page 44
3.4.7. Conclusions......Page 45
4. Outlook......Page 46
References......Page 47
Part II: Biochemistry of Rho GTPases
......Page 50
1. Introduction......Page 52
2.2. Chemicals and Reagents......Page 53
2.3. Nucleotides......Page 54
2.5. Chromatography Columns......Page 55
3.1. Gene Expression and Bacterial Culture Conditions......Page 56
3.2. Bacterial Lysis......Page 57
3.3.1. Purification Steps as GST Fusion......Page 58
3.3.2. Removal of the GST Tag......Page 59
3.4. Preparation of Nucleotide-Free Forms of Rho GTPases......Page 60
3.6. Preparation of GppNHp-Bound and mantGppNHp-Bound GTPases......Page 61
3.7.1. Measurement of Slow Reactions......Page 62
3.7.2. Measurement of Fast Reactions......Page 63
3.8. Intrinsic and GAP-Stimulated GTP Hydrolysis Reaction......Page 64
3.8.2. Measurement of Fast Reactions......Page 65
3.9.1. Kinetic Measurements (Direct)......Page 66
3.9.3. Measurements of Nucleotide Dissociation Inhibition Through Effector Binding (Indirect)......Page 68
3.10. Anticipated Results......Page 69
4. Notes......Page 70
References......Page 73
1. Introduction......Page 74
1.1. Inactivating Toxins......Page 75
1.2. Activating Toxins......Page 76
1.3. Toxin Uptake......Page 77
2.1. Buffers......Page 79
2.3. Cell Biology......Page 80
3.1. Protein Expression and Purification......Page 81
3.2. Intoxication of Eukaryotic Cells......Page 84
3.3. Anticipated Results......Page 85
4. Notes......Page 87
References......Page 89
1. Introduction......Page 92
2.2. Cell Transfection......Page 93
2.4. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis......Page 94
2.6. Antibodies and Conjugates......Page 95
3.3.1. Preparation of the Cobalt Resin......Page 96
3.4. SDS-PAGE Sample Resolution......Page 97
4. Notes......Page 99
References......Page 100
1. Introduction......Page 102
2.3. Chemicals, Antibodies, and Pharmacological Inhibitors......Page 105
3.1. Transfection and Microscopy......Page 107
3.2. Biotin–BMCC Labeling......Page 108
4. Notes......Page 109
References......Page 110
1. Introduction......Page 112
2.3. Lysis and Western Blotting......Page 114
3.1. Transfection and Lovastatin Treatment......Page 115
3.3. SDS-PAGE and Western Blotting......Page 116
4. Notes......Page 117
References......Page 119
1. Introduction......Page 122
2.1. Buffers, Reagents, and Equipment......Page 123
2.2. Proteins......Page 125
3.1.1. Purification of His-Tagged Proteins......Page 126
3.2. Preparation of Lipid Vesicles for Fluorescence Spectroscopy......Page 127
3.3. FRET Measurements for Monitoring the Interaction of Rho GTPases with Lipid Membranes and RhoGDI......Page 128
3.3.2. Monitoring the Rate of Exchange of Rho GTPases Between Lipids......Page 129
4. Notes......Page 130
References......Page 133
Part III: Functional Assays
......Page 136
1. Introduction......Page 138
2.1. Cell Culture, Transfection, and Staining......Page 140
2.2. Confocal Microscopy......Page 141
3.1. HUVEC Culture......Page 142
3.3. PC3 Cell Culture......Page 143
3.5. PC3 Cell Labeling......Page 144
3.6.2. PC3 Cell Adhesion Assay to ECM Components......Page 145
3.6.3. Normalization of Cell Number and Analysis of Adhesion Results......Page 146
3.7.1. Time-Lapse Microscopy Analysis of PC3 Cell Intercalation Between EC......Page 147
3.8.1. Immunofluorescence After PC3 Cell Adhesion Assays to EC......Page 148
3.8.2. Analysis of the Results......Page 150
3.9.1. TEM Assay......Page 152
4. Notes......Page 154
References......Page 157
1. Introduction......Page 158
2. Materials......Page 161
3. Methods......Page 162
3.1. Aggregation Assay......Page 165
3.2.1. Analysis Using ImageJ......Page 166
3.2.2. Analysis in Microsoft Excel......Page 167
4. Notes......Page 168
References......Page 169
1. Introduction......Page 172
2.2. Isolation of Keratinocytes......Page 175
3.2. Isolation of Keratinocytes......Page 176
3.3. Transfection......Page 177
3.4. Sorting......Page 178
4. Notes......Page 179
References......Page 181
1. Introduction......Page 182
2.2. In Vitro Osteoclast Culture......Page 184
2.3. Rho GTPase Activation Assay......Page 185
3.1. Mouse Dissection, Preosteoclast Isolation, and In Vitro Osteoclast Culture......Page 186
3.2. Rho-GTPase Activation Assay......Page 188
3.3. TAT Protein Constructs and Migration Rescue......Page 190
3.3.2. Studying the Effects of TAT Fusion Proteins on Migration......Page 191
4. Notes......Page 192
References......Page 193
1. Introduction......Page 196
2.3. Lysis Buffers and Rho GTPase Pulldown......Page 198
3.1. N1E-115 Cell Culture and Differentiation......Page 199
3.2. Neurite and Soma Purification on Microporous Transwell Filters......Page 200
3.3. Transfection of siRNAs in N1E-115 Cells......Page 202
3.4.1. F-Actin/Tubulin Immunostaining......Page 203
3.4.2. Neurite Outgrowth Analysis and High Resolution Imaging......Page 204
4. Notes......Page 206
References......Page 209
1. Introduction......Page 210
2.1. Buffers......Page 213
2.2. Chemicals and Reagents......Page 214
2.3. Equipment......Page 215
3.1. Estimation of Binding of Rac to p67phox or Noxa1
......Page 216
3.2.1. Preparation of Human Neutrophil Membrane Fraction as a Source of Nox2......Page 217
3.2.2. Reconstitution of a Cell-Free System for Nox2 Activation......Page 219
3.3.2. Estimation of Oxidase Proteins Ectopically Expressed in Cultured Cells......Page 220
3.3.4. Estimation of the Role for Rac in Membrane Localization of p67  phox in HEK293 Cells......Page 222
4. Notes......Page 223
References......Page 225
Part IV: Imaging and High Throughput Methods
......Page 228
1. Introduction......Page 230
1.2. General Considerations for the Single-Mode Imaging of Rho GTPase Biosensors......Page 231
1.3. General Considerations for the Multiplex-Mode Imaging of Rho GTPase Biosensors......Page 234
2.1. Cell Culture, Transfection, and Viral Transduction......Page 236
2.3. Imaging......Page 237
3.1. Dye Labeling of the Multiplex-Compatible Biosensor for Cdc42......Page 239
3.2. Viral Transduction and Stable tet-OFF Cell Line Production......Page 240
3.3. Viral Transduction and Stable Biosensor Cell Line Production......Page 241
3.4. A Typical Procedure and Key Considerations for the Single-Mode Imaging of the Rho GTPase Biosensor......Page 242
3.5. A Typical Procedure and Key Considerations for the Multiplex-Mode Imaging of Rho GTPase Biosensors......Page 244
4. Notes......Page 246
References......Page 249
1. Introduction......Page 250
2.1. Cells, Media, and Opsonin......Page 252
2.3. Microscope for FRET Microscopy and Leiden Chamber......Page 253
2.4. Data Analysis Software and the FRET Calculator......Page 255
3.3. Image Alignment......Page 256
3.4. Calibration of the FRET Microscope......Page 257
3.7. Data Analysis of Rac/Cdc42 Activity During Phagocytosis......Page 258
3.8. Interpreting sFRET Data of Rac and Cdc42 Activation and Conversion of these Images to Represent the Fractions of Active GTPase......Page 259
4. Notes......Page 263
References......Page 265
1.1. Rho GTPase Inhibitors......Page 268
1.2. Fluorescence-Based Assay......Page 269
1.3. High-Throughput Screening......Page 270
2.1. Reagents and Buffers......Page 271
3.1. Primary Screening of 384-Well Plates......Page 273
3.1.1. Coating of the Bead Sets with Small GTPases and Preparation of 384-Well Plates for Primary Screening......Page 274
3.1.3. Data Analysis......Page 276
3.2. Dose–Response Measurements......Page 278
3.2.2. Dose–Response Assay......Page 279
4. Notes......Page 283
References......Page 284
1. Introduction......Page 286
2.1. Proteins and Conjugates......Page 287
2.3. Protein–Protein Interaction Assay......Page 288
3.1. Coupling of Capture Antibodies to Microspheres......Page 289
3.2. Competitive Nucleotide Exchange: Complex Formation......Page 290
3.3. Effect of Prenylmimetic Compounds......Page 292
3.4. Optional Multiplex Setup......Page 293
3.5. YopT Cleavage Assay......Page 294
4. Notes......Page 295
References......Page 296
1. Introduction......Page 298
2.1. Rho GTPases Purification......Page 299
2.2. Phage Display......Page 300
2.4. Production and Purification of Soluble Monoclonal scFv......Page 301
3.1.1. Growth of Plasmid-Bearing Bacteria......Page 302
3.1.4. Elution of GST Fusion Protein......Page 303
3.2.1. Helper Phage Production......Page 304
3.2.2. Library Packaging......Page 305
3.2.3. Selection by Panning......Page 306
3.3.1. Polyclonal Phage ELISA......Page 309
3.3.3. Monoclonal Phage ELISA......Page 310
3.3.4. Production of Monoclonal Phage in Large Quantities......Page 311
3.4. Production and Purification of Soluble Monoclonal scFv from Selected Clones......Page 312
4. Notes......Page 314
References......Page 317
1. Introduction......Page 320
2.1. Transfection......Page 323
2.4. Silver Staining (see Note 9 )......Page 324
3.1. Transfection......Page 325
3.3. Cell Lysis, Co-immunopreci pitation, and Gel Electrophoresis......Page 326
3.5. Mass Spectrometry Sample Preparation......Page 327
4. Notes......Page 328
References......Page 330
Part V: Rho GTPases in Non-mammalian Model Organisms
......Page 334
1.1. The Zebrafish Model......Page 336
1.2. Rho Small GTPases in Zebrafish......Page 337
1.4. Functional Rescue by mRNA Overexpression......Page 338
1.6. In Situ Hybridization with Riboprobes......Page 339
2.3. mRNA In Vitro Synthesis for Functional Rescue......Page 340
2.5. Whole-Mount In Situ Hybridization......Page 341
3.1. MO Design and Preparation for Gene Knockdown......Page 342
3.3. Microinjection......Page 343
3.4. Whole-Mount In Situ Hybridization......Page 344
4. Notes......Page 348
References......Page 351
1. Introduction......Page 354
1.2. C. elegans Rho Family GTPases......Page 355
1.3. Genetics of Rho GTPases in C. elegans......Page 356
1.4. Constitutively Active and Dominant Negative Rho GTPases......Page 357
1.5. Transgenic Manipulation of Rho GTPases in C. elegans......Page 358
2.1. Buffers, Media, and Equipment......Page 360
2.2.2. “Chunking” Worms......Page 361
3.1.2. Preparing Agarose Pads for Injection......Page 362
3.1.6. Breaking the Needle for Injection......Page 363
3.1.9. Recovering the Worm......Page 364
3.2.1. Heat Shocking to Generate Males for C. elegans Crosses......Page 365
3.2.3. Strategies to Introduce Promoter ::gfp::gtpase Transgenes into Different Mutant Backgrounds......Page 366
3.3.3. Recovering Animals from the Agarose Pad......Page 368
3.4.2. Strategies for Axon Pathfinding Scoring Defects in C. elegans Neurons......Page 369
4. Notes......Page 370
References......Page 372
1. Introduction......Page 374
2.1. Drosophila Maintenance......Page 375
2.2. Reagents and Buffers......Page 376
3. Methods......Page 377
4. Notes......Page 380
References......Page 381
1. Introduction......Page 384
2.1. Buffers and Solutions......Page 386
2.2. Expression of Activation Probes in Escherichia coli......Page 387
2.4. Immunoblot, Antibodies, and Proteins......Page 388
3.1. Preparation of Cdc42p and Rho1p Activation Probes......Page 389
3.3. Preparation of Yeast Vacuoles for Activation Assays......Page 390
3.4. Preparation of In Vitro Reactions for Rho GTPase Activation......Page 391
3.5. Affinity Isolation of Activated Cdc42p-GTP and Rho1p-GTP from Lysates and Reactions......Page 392
4. Notes......Page 393
References......Page 395
1.1. Dictyostelium discoideum as a Model Organism......Page 396
1.2. Rho GTPases in D. discoideum......Page 397
1.3. Assaying Rac-Dependent Processes in D. discoideum......Page 398
2.2. Determination of F-Actin......Page 400
3.1. Determination of the Relative F-Actin Content......Page 401
3.2. Actin Polymerization Response upon cAMP Stimulation......Page 402
3.3.1. Production of GST-WASP CRIB......Page 403
3.3.2. Pull-Down of Activated Rac upon cAMP Stimulation......Page 404
4. Notes......Page 405
References......Page 407
1. Introduction......Page 408
3.1. Transient Expression in Tobacco Pollen Tubes......Page 410
3.3. Image Analysis......Page 412
References......Page 415
Index......Page 418