Regulators and effectors of small GTPases: Rho family

Introduction......Page 39
Buffers......Page 40
Proteins......Page 41
MantGDP-Bound GTPases......Page 42
Fluorescence Spectroscopic Methods......Page 43
Nucleotide Binding......Page 44
Effector-Induced Nucleotide Dissociation Inhibition (GDI) Assay......Page 45
Biochemical Evaluation of the Rac Proteins......Page 46
References......Page 47
Biochemical Analyses of the Wrch Atypical Rho Family GTPases......Page 50
Molecular Constructs of Wrch-1......Page 51
Purification of GST-Wrch-1......Page 52
Purification of His6-Wrch-1......Page 55
Nucleotide Loading of Wrch-1......Page 56
GST-Wrch-1......Page 57
Measuring GTPase Activity of Wrch-1......Page 58
Measuring the Nucleotide Exchange Rate of Wrch-1......Page 59
Labeling of Palmitoylated Cysteine Residues in Wrch-1/2 Proteins......Page 61
Inhibition of Palmitoylation-Mediated Localization of Wrch-1......Page 62
References......Page 64
Purification of P-Rex1 from Neutrophils and Nucleotide Exchange Assay......Page 66
Preparation of Pig Leukocyte Cytosol......Page 67
Q-Sepharose Fast-Flow Column......Page 68
Desalting Column 2 and Heparin Sepharose Column......Page 69
Assessment of Purity......Page 70
Sf9 Cell Culture......Page 71
Amplification of Viral Particles......Page 72
Optimal Conditions for P-Rex and Rac Protein Production in Sf9 Cells......Page 74
Purification of Recombinant P-Rex1 Proteins from Sf9 Cells......Page 75
In Vitro Rac GEF Activity Assay......Page 77
Preparation of Liposomes......Page 78
Preparation of Gbetagamma Subunits......Page 79
References......Page 80
Introduction......Page 82
In Vitro GTPase Binding Assays Demonstrate That the DHR-2 Domains Form a Stable Complex with Nucleotide-free Rho GTPases......Page 84
Bacterial Expression and Purification of GST-Tagged DHR-2 Domains and Rho GTPases......Page 86
In Vitro Transcription and Translation of 35S-Labeled DHR-2 Domains and their Binding to Immobilized GST-Fusio......Page 88
Precipitation of Full-Length DOCK180 Proteins in Mammalian Cells with the Bacterially Produced GST-Rho GTPases......Page 90
Preparation of 3H-GDP-Loaded Rac and Cdc42......Page 92
In Vitro GEF Assays with the DHR-2 Domains......Page 93
The DHR-2 Domains of DOCK180 and DOCK2 Contain Rho-GEF Activity In Vivo......Page 94
Discussion......Page 96
References......Page 97
Introduction......Page 99
Expression and Purification of Cdc42/Rac from E. coli Expression Systems......Page 100
Expression and Purification of Cool/Pix and Pak Proteins in Insect Cells......Page 101
Expression and Immunoprecipitation of Cool/Pix and its Interacting Proteins in Mammalian Cells......Page 102
GDP Dissociation Assay......Page 103
Measurement of [3H]GDP Remaining Associated with the GTP-Binding Protein During the Exchange Assay......Page 104
Pak Assay......Page 106
Discussion......Page 108
References......Page 109
Introduction......Page 111
Liposomes of Defined Lipid Composition......Page 112
Buffers and Proteins......Page 113
General Description......Page 114
Optimal Lipid Composition......Page 115
Guanine Nucleotide Exchange on Liposome-Bound Rac......Page 117
Glucosylation of Liposome-Bound Rac......Page 118
References......Page 120
Phosphorylation of RhoGDI by p21-Activated Kinase 1......Page 122
Introduction......Page 123
In Vitro Phosphorylation of RhoGDI by Pak1......Page 125
In Vivo Phosphorylation of RhoGDI by Pak1......Page 126
Binding of Pak1 to RhoGDI......Page 127
Analysis of Phosphorylation Sites on RhoGDI......Page 128
In Vitro......Page 129
In Vivo......Page 130
References......Page 131
Introduction......Page 133
Competition-Based Phosphoinositide Binding Assay......Page 134
Identification of ARAP3......Page 135
Purification of Recombinant ARAP3......Page 136
Purification of Recombinant Rho or Ras GTPases from E. coli......Page 137
In Vitro Rho GAP Assays......Page 138
In Vivo Rho GAP Assay......Page 140
In Vitro Arf GAP Assay......Page 142
In Vivo Arf6 GAP Assay......Page 143
Acknowledgments......Page 144
References......Page 145
Introduction......Page 146
Expression of Rac and Rho......Page 148
Required Solutions......Page 149
Potential Experimental Error Associated with Separation on Nitrocellulose......Page 150
Modification of the Substrate Specificity by the Lipid Environment......Page 151
Effect of the Posttranslational Modification of the Small GTPase on the Substrate Specificity of GAPs......Page 154
Conclusions......Page 156
References......Page 157
Introduction......Page 160
The C2 Toxin as a Cell Delivery System......Page 161
Expression and Purification of Recombinant C2IN-C3 and C2II Proteins......Page 162
[32P]Labeling of Rho by the C3 Fusion Toxin In Vitro......Page 164
Inactivation of Rho in Cultured Monolayer Cells......Page 165
References......Page 168
Introduction......Page 171
Recombinant Protein Preparation......Page 172
Immunofluorescence......Page 175
Results and Discussion......Page 176
Conclusions......Page 181
References......Page 182
Imaging and Photobleach Correction of Mero-CBD, Sensor of Endogenous Cdc42 Activation......Page 184
Mero-CBD Preparation......Page 185
Cell Injection and Image Acquisition......Page 186
Bandpass Filters and Dichroic Selection......Page 187
Shading Correction......Page 188
Image Registration......Page 190
Image Masking......Page 192
Photobleach Correction......Page 193
Appendix A......Page 196
Acknowledgment......Page 199
References......Page 200
Cdc42 and PI(4,5)P2-Induced Actin Assembly in Xenopus Egg Extracts......Page 201
Introduction......Page 202
Preparation of Xenopus Egg Extracts for Actin Assembly Experiments......Page 203
Priming Frogs and Inducing Ovulation......Page 204
Collecting Eggs......Page 205
Making Cytoplasmic Extract......Page 206
Making High-Speed Supernatant......Page 208
Procedure......Page 209
Procedure......Page 211
Actin Polymerization Assays......Page 212
Kinetic Analysis of Actin Assembly Using Pyrene Actin......Page 213
Microscopic Analysis of Actin Assembly Using Rhodamine Actin......Page 214
Functional Analysis of Actin Assembly by Immunodepletion and Rescue......Page 215
Immunodepletion of Xenopus Egg Extracts......Page 216
References......Page 217
Introduction......Page 219
Preparation of Recombinant N-WASP......Page 220
Purification of Native N-WASP-WIP Complex from Xenopus Egg Extracts......Page 222
Preparation of Recombinant Toca-1......Page 226
Purification of the Arp2/3 Complex from Bovine Brain Extracts......Page 227
Cdc42 and PIP2 Vesicles......Page 229
Sample Reactions......Page 230
Properties of the Purified System......Page 233
References......Page 234
Biochemical Analysis of Mammalian Formin Effects on Actin Dynamics......Page 236
Introduction......Page 237
Basic Actin Polymerization Kinetics Description......Page 240
Actin Polymerization by Fluorescence Spectroscopy......Page 241
Method......Page 242
Calculations......Page 243
Special Considerations......Page 244
Variation......Page 245
Method......Page 246
Special Considerations......Page 247
Variation......Page 248
Fluorescence Microscopy (Single Filament Analysis)......Page 249
Method......Page 250
Special Considerations......Page 251
Special Considerations......Page 252
mDia1 Regulation......Page 253
Capping Protein......Page 254
Special Considerations......Page 255
Expression and Purification of Mammalian Formin FH2 Domains......Page 256
Method......Page 257
References......Page 258
Introduction......Page 261
Choosing the Tag and Expression System......Page 263
Monitoring Expression......Page 266
To Freeze, or Not To Freeze?......Page 267
Expression in Bacteria......Page 268
Purification from Bacterial Pellet......Page 269
Expression in Yeast......Page 270
Purification from Frozen Yeast Powder......Page 271
Preparation of Pyrene Actin......Page 272
Important Notes for Actin Assembly Reactions......Page 273
Tips for Filament Elongation Reactions......Page 274
Data Analysis and Formatting......Page 276
Procedure......Page 277
Procedure......Page 278
References......Page 279
Introduction......Page 281
Purification of PKN......Page 282
Purification of PKN1 from the Soluble Cytosolic Fraction of Rat Testes......Page 283
Rapid Purification of PKN1 from the Membrane Fraction of Bovine Brain......Page 286
Expression and Purification of Recombinant PKNs from Insect Cells......Page 287
Kinase Assay......Page 289
Modifiers of Kinase Activity......Page 293
References......Page 294
Introduction......Page 298
Baculovirus Expression Vector for the Kinase-SH3-CRIB Construct of ACK1......Page 299
Step 2. Source Q FPLC column chromatography......Page 300
Substrate Specificity of ACK1 Kinase Using Synthetic Peptide Substrates......Page 301
Autophosphorylation of ACK1......Page 304
Concluding Remarks......Page 306
References......Page 307
Direct Activation of Purified Phospholipase C Epsilon by RhoA Studied in Reconstituted Phospholipid Vesicles......Page 309
Introduction......Page 310
Cell Infection and Harvest of Cytosolic Fraction......Page 312
Metal Chelate Affinity Chromatography......Page 313
Sephacryl S-300 Gel-Filtration Chromatography......Page 315
Cell Infection and Membrane Protein Extraction......Page 316
PLC Assay......Page 317
Acknowledgments......Page 319
References......Page 320
Introduction......Page 321
Expression Constructs......Page 322
Protein Purification......Page 323
Immobilization of PLCbeta Ligands on Sensor Surfaces......Page 326
Regeneration of Sensor Surfaces......Page 327
Discussion......Page 328
References......Page 329
Introduction......Page 330
Buffers......Page 331
Overview......Page 332
Overview......Page 333
Overview......Page 334
Handling of Wiskostatin......Page 335
Guanidine Hydrochloride or Urea Denaturation of WASP Proteins......Page 337
Mant Fluorescence Experiments......Page 338
Fluorescence Competition Experiments......Page 339
Pyrene Actin Polymerization Assays......Page 340
Buffers......Page 341
NMR Analyses of Wiskostatin and its Interactions with WASP......Page 342
References......Page 343
The Use of GFP to Localize Rho GTPases in Living Cells......Page 346
Introduction......Page 347
Expression Constructs......Page 349
Primer Design......Page 350
Restriction Digests (Must be Applied to both PCR Product and GFP Expression Vector)......Page 351
Bacterial Transformation and DNA Isolation......Page 352
Transfection of Mammalian Cells......Page 354
Transfection Method......Page 355
Establishment of Stable Cell Lines......Page 356
Imaging......Page 357
Identification of Subcellular Compartments in Live Cells......Page 359
Indirect Immunofluorescence......Page 362
Subcellular Fractionation......Page 363
References......Page 364
Analysis of the Spatiotemporal Activation of Rho GTPases Using Raichu Probes......Page 366
Introduction......Page 367
Basic Structure of Raichu Probes......Page 368
Design Considerations......Page 369
Characterization of Candidate Probes......Page 371
Imaging System......Page 372
Image Acquisition......Page 374
Cell Migration......Page 376
Advantages and Disadvantages of Raichu Probes......Page 378
References......Page 381
Measurement of Activity of Rho GTPases During Mitosis......Page 384
Cell Cycle Synchronization of HeLa S3 Cells......Page 385
Purification of GST CRIB-Pak Fusion Protein......Page 387
Precipitation of GTP-Bound Cdc42 or Rac in HeLa S3 Cells in Mitosis......Page 388
Preparation of GST-mDia1-RBD......Page 389
Precipitation of GTP-Bound Rho in HeLa S3 Cells in Mitosis......Page 391
Effect of Ect2 Overexpression and Depletion on the Levels of GTP-RhoA and GTP-Cdc42 During Mitosis......Page 392
Transfection of Ect2-N1 During Cell Cycle Synchronization of HeLa S3 Cells......Page 393
Depletion of Ect2 by RNA Interference......Page 394
References......Page 396
Inhibition of Rho GTPases by RNA Interference......Page 398
Introduction......Page 399
Materials for siRNA Transfection......Page 400
Procedure for siRNA Transfection......Page 401
Alternative Procedure: Transfection of GD25 Cells with siRNA Using RNAiFect......Page 402
GTPase Assays and Immunoblotting......Page 403
Procedure for GTPase Assays......Page 404
Evaluation of Specificity......Page 406
Materials for Cotransfection Rescue......Page 410
Procedure for Cotransfection Rescue Experiment......Page 411
References......Page 413
Introduction......Page 415
Technical Background......Page 416
Annealing of Oligos......Page 418
Phosphorylation of Annealed Oligos......Page 419
Transient Transfection of RNAi Constructs by Nucleofection......Page 420
Analysis of Defects in Cell Adhesion and Polarity after Gene Suppression......Page 422
Rescue the RNAi Phenotype with Ectopic Protein Expression......Page 424
References......Page 426
Nucleofection of Primary Neurons......Page 428
Drawbacks......Page 429
Preparation and Nucleofection of Cerebellar Granule Neurons for Use in Migration Assays......Page 430
Dissociation Procedure......Page 431
Nucleofection......Page 432
Reagents and Materials......Page 433
Dissociation Procedure......Page 434
Nucleofection......Page 435
Reagents and Materials......Page 436
Preparation of Hippocampal Neurons......Page 437
Nucleofection......Page 438
RNAi Using Nucleofection......Page 440
References......Page 441
Dock180-ELMO Cooperation in Rac Activation......Page 443
Dock180 Family of Proteins......Page 444
CED-12/ELMO Proteins, Master Regulators of the CDM Proteins......Page 446
ELMO PH Domain-dependent Stabilization of the Complex Formation between Dock180 and Nucleotide-free Rac......Page 447
Binding of the PxxP Motif of ELMO to the SH3 Domain of Dock180 Relieves a Steric Inhibition of Dock180......Page 448
ELMO Facilitates Translocation of the Dock180–ELMO Complex to the Plasma Membrane......Page 449
Purification of GTS-Rac from E. coli......Page 451
Purification of the GST- Protein......Page 452
Purification of the Dock180 and ELMO Proteins from Transfected 293 T Cells......Page 453
Phagocytosis Assay......Page 454
References......Page 455
Rho GTPase Activation by Cell-Cell Adhesion......Page 458
Cell Culture and Induction of Cell-Cell Contacts......Page 459
Activation Assays......Page 460
GTP/GDP Loading......Page 461
Equipment and Reagents......Page 463
Controls for the Specificity of Activation of Rho Small GTPases by Cadherin-Dependent Adhesion......Page 465
Inhibition of Cadherin Adhesion......Page 466
Reagents......Page 467
Equipment and Reagents......Page 468
References......Page 470
Activation of Rap1, Cdc42, and Rac by Nectin Adhesion System......Page 472
Introduction......Page 473
Materials......Page 475
Affinity Purification of GST-PAK-CRIB......Page 476
Generation of Nectin-1-L Cells and Cell Culture......Page 477
Pull-down Assay for Cdc42 and Rac......Page 478
Cell-Spreading Assay......Page 479
References......Page 480
Introduction......Page 482
Expression and Purification of GST-Rho Constructs......Page 484
Pull-Down of GEFs or GAPs......Page 485
GAP and GEF Pull-Downs......Page 486
Stability of GST-Rho Proteins......Page 491
References......Page 492
Introduction......Page 495
Purification of Bacterially Expressed Smurf1 and RhoA......Page 496
In Vitro Interaction between Smurf1 and RhoA......Page 497
Cell Type, Growth Conditions, and Transient Transfection Method......Page 498
Reagents for Transfection......Page 499
Degradation Assay of RhoA In Vivo......Page 500
Ubiquitination Assay of RhoA In Vivo......Page 501
References......Page 504
Ubiquitin-Mediated Proteasomal Degradation of Rho Proteins by the CNF1 Toxin......Page 506
Introduction......Page 507
CNF1 Toxin Purification and Activity Normalization......Page 508
Recruitment of Rho Proteins to Cellular Membranes......Page 509
Measure of Rho Protein Activation by Effector-Binding Pull-Down......Page 510
In Vivo Ubiquitylation of Rho Proteins......Page 511
Comments......Page 512
References......Page 514
Regulation of Superoxide-Producing NADPH Oxidases in Nonphagocytic Cells......Page 516
Introduction: The Phagocyte NADPH Oxidase......Page 517
Regulation of Nox1, a Nonphagocytic NADPH Oxidase......Page 518
Other Nonphagocytic NADPH Oxidases......Page 519
Reconstitution of NADPH Oxidases in Cultured Cells......Page 520
Measurement of Superoxide Production......Page 523
Phorbol 12-Myristate 13-Acetate (PMA)......Page 524
References......Page 525
Activation of MEKK1 by Rho GTPases......Page 529
Introduction......Page 530
Purification of MEKK1 from Sf9 Cells......Page 531
Materials and Conditions......Page 532
Purification and GTP-Loading of RhoA......Page 533
Buffers and Reaction Mixture......Page 534
Immunoprecipitation and Kinase Assay......Page 535
Acknowledgment......Page 537
References......Page 538
Further Reading......Page 539
Introduction......Page 540
Immunoreagents......Page 541
Selective Small Molecule Inhibitors......Page 542
Loss-Of-Expression Strategies......Page 543
Western Immunoblotting of POSH and MLKs......Page 544
Immunoprecipitation (IP)/Co-IP......Page 545
In Vitro Binding Assays to Evaluate POSH-Interacting Proteins......Page 546
Evaluation of JNK Pathway Kinase Activities......Page 547
Cell Lysis Buffer......Page 548
References......Page 549
Quantification of Isozyme-Specific Activation of Phospholipase C-beta2 by Rac GTPases and Phospholipase C-epsiv by Rho GTPase......Page 551
Introduction......Page 552
Cell Culture......Page 553
Transfection......Page 554
Phosphoinositide Hydrolysis Detection by Scintillation Proximity Assay......Page 555
Application of Assay to Quantify Activation of PLC Isozymes by Rho Family GTPases......Page 556
References......Page 560
Introduction......Page 562
Preparation of Recombinant GST-PBD Fusion Protein......Page 567
GST-RBD......Page 568
RAC/CDC42 Assay......Page 569
Western Blotting......Page 570
Notes......Page 571
References......Page 572
Introduction......Page 574
Purification of Cell-Permeable C3 Proteins......Page 575
Assay Method Overview......Page 576
Method for Measurement of NAD+ Glycohydrolase Activity of Cell-Permeable C3 Proteins......Page 577
Measurements of GH Activity Using the Fluorescence-Based Assay......Page 578
Correlation of Glycohydrolase Activity with Biological Activity......Page 579
References......Page 581
Further Reading......Page 582
Use of TIRF Microscopy to Visualize Actin and Microtubules in Migrating Cells......Page 583
Introduction......Page 584
Principles of TIRF Microscopy......Page 585
Procedure for Setting up TIRF Illumination Mode......Page 586
Procedure......Page 588
Procedure......Page 589
Image Analysis and Quantification......Page 591
Materials......Page 592
Kymograph Analysis of Microtubule Dynamics (Metamorph Software)......Page 593
References......Page 594
Introduction......Page 596
Electroporation of COS7 Cells......Page 598
RhoE and ROCK I Microinjection in Swiss 3T3 Cells......Page 599
Microinjection of Swiss 3T3 Cells......Page 600
RhoE Inhibits Phosphorylation of Myosin Light Chain Phosphatase by ROCK I......Page 601
Purification of RhoE......Page 602
References......Page 603
Conditional Regulation of a ROCK-Estrogen Receptor Fusion Protein......Page 605
Introduction......Page 606
Generation of Constructs......Page 607
Cell Culture and Western Blotting......Page 608
In Vitro Kinase Assays......Page 609
Results and Discussion......Page 610
References......Page 616
Introduction......Page 618
Rational Design of Rac Activation-Specific Inhibitors......Page 619
In Vitro Screening and Examination......Page 620
Reversal of PC-3 Prostate Tumor Cell Phenotypes......Page 623
Inhibition of Platelet Activation......Page 625
Induction of Hematopoietic Stem Cell Mobilization......Page 627
References......Page 628
In Vitro Assay of Primary Astrocyte Migration as a Tool to Study Rho GTPase Function in Cell Polarization......Page 630
Introduction......Page 631
Dissection Procedure......Page 633
Tissue Culture Plate Coating......Page 634
Culture Purification and Differentiation......Page 635
Scratch-Induced Astrocyte Migration......Page 636
Procedure for Immunostaining......Page 637
Astrocyte Migration Assay......Page 638
Quantification of Astrocyte Protrusion......Page 640
Centrosome Reorientation Assay......Page 641
References......Page 642
Introduction......Page 644
Generation of a Stable Cell Line Expressing GFP-Tubulin......Page 646
Wound Healing Assay......Page 647
Preparation of Starved Cell Monolayers and Wounding......Page 650
Materials......Page 651
Live Cell Imaging of Centrosome Reorientation......Page 652
Image Acquisition......Page 654
Objectives......Page 655
References......Page 656
Introduction......Page 658
Lentiviral Vectors for shRNA Delivery......Page 659
Selection of siRNA Target Sequences and Design of shRNA Templates......Page 661
Production of Lentiviral Supernatants......Page 662
Titration of Lentiviral Supernatants......Page 664
Isolation of Primary Hippocampal Neurons......Page 665
Lentiviral Transduction of Hippocampal Neurons......Page 666
Effectiveness and Specificity of shRNA-Mediated Silencing......Page 667
References......Page 668
Introduction......Page 671
Preparation of Mouse Neutrophils......Page 673
Tracking Mouse Neutrophil Migration......Page 675
Directional Sensing Detection......Page 677
References......Page 678
Introduction......Page 680
Approaches for Examining EGFR Endocytosis and Degradation......Page 681
EGF Receptor Endocytosis Assays Using Texas-Red-Conjugated EGF......Page 682
Procedures......Page 683
Materials......Page 684
Procedures......Page 685
Materials......Page 686
Procedures......Page 687
References......Page 689
Introduction......Page 692
Collagen I......Page 693
Visualization of Collagen I in Gels......Page 695
Reagents......Page 696
Visualizing Matrix Degradation......Page 698
Invasion Assays Without Gradients......Page 699
Invasion Assays with Gradients......Page 700
Notes......Page 701
Protocols for Fixation and Staining of Invasion Assays......Page 702
Procedure for Imaging and Quantification Using a Laser‐Scanning Microscope\0......Page 704
Comparison of Different Cell Lines and Different Motility Assays......Page 705
Perspective......Page 708
References......Page 709
An In Vitro Model to Study the Role of Endothelial Rho GTPases During Leukocyte Transendothelial Migration......Page 711
Introduction......Page 712
Growth of HUVECs......Page 713
Analysis of ICAM-1-Induced RhoA Activation......Page 714
Materials......Page 715
Materials......Page 716
Preparation of High-titer Purified Adenoviruses Expressing Rho GTPase Mutants......Page 717
Titration of Adenovirus......Page 718
Infection of HUVECs......Page 719
Time‐lapse Digital Microscopy\0......Page 720
References......Page 721
Introduction......Page 724
Procedure......Page 725
Mitosis-Specific Expression of Rho GTPase Mutants......Page 726
Plasmids and siRNA Preparation......Page 727
Characterization of Knockdown of Cdc42......Page 728
Effect of Toxin B Treatment and Expression of Rho GTPase Mutants on Mitosis......Page 729
Time-Lapse Live Imaging......Page 732
References......Page 733
Plexin-Induced Collapse Assay in COS Cells......Page 734
Antibodies/Probes......Page 735
Preparation of Recombinant Semaphorins......Page 736
Sema3A-Fc-Induced Collapse......Page 737
Sema4D-Fc-Induced Collapse......Page 741
Discussion......Page 743
References......Page 744
Morphological and Biochemical Analysis of Rac1 in Three-Dimensional Epithelial Cell Cultures......Page 746
Background......Page 747
Plating in Collagen I......Page 748
Maintaining Cyst Cultures......Page 749
Background......Page 750
Reagents......Page 751
Permeabilization and Antibody Treatment......Page 752
Postfixation, Nuclear Staining, and Mounting......Page 753
Growing MDCK Cysts for Biochemical Analysis......Page 754
Solubilization of 3D Cultures......Page 755
Collagen I Digestion......Page 756
Preparing Samples for Immunoprecipitation......Page 757
Reagents......Page 758
Lysing Collagen I Cultures......Page 759
References......Page 760
Introduction......Page 762
Materials......Page 763
Materials......Page 764
Preparation of Protein Extracts......Page 765
Cell Cycle......Page 766
Cdk4 Kinase Assay......Page 767
Matrigeltrade/Collagen Invasion Assay......Page 769
References......Page 770
Further Reading......Page 771
Introduction......Page 772
Preparation of GST-Fusion Proteins......Page 773
GTP Loading of TC10......Page 774
DNA Preparation......Page 776
GST-Pak1 Pull-Down Assay to Determine Activity State of TC10......Page 777
Wortmannin Treatment......Page 778
Electroporation......Page 779
2-Deoxyglucose Uptake......Page 780
Immunofluorescence and Image Analysis......Page 781
Sucrose Density Gradient Centrifugation......Page 782
Preparation of Plasma Membrane Sheets......Page 783
Immunofluorescence......Page 784
References......Page 785
Introduction: The Development of Neuronal Polarity......Page 786
Preparation and Culture of Hippocampal Neurons......Page 788
Lipofection......Page 789
Calcium Phosphate Coprecipitation......Page 790
Immunofluorescence......Page 791
Analysis of Neuronal Polarity......Page 792
Expression of GTPase Mutants......Page 793
Knock-Down by RNA Interference......Page 794
References......Page 797
In Vitro Assembly of Filopodia-Like Bundles......Page 799
Introduction......Page 800
Reagents......Page 802
Light Microscopy......Page 804
Procedure for Immunofluorescent Staining......Page 806
Electron Microscopy......Page 807
Reagents......Page 808
Procedure......Page 809
References......Page 810