GTPases regulating membrane dynamics

01 B7CV2-4J1JDCM-fm.pdf......Page 2
02 preface.pdf......Page 3
03 TOC.pdf......Page 5
04 contibutors.pdf......Page 10
Introduction......Page 17
Preparation of Transfection Solutions......Page 18
Spotting the Transfection Solution on Lab-Tek Dishes......Page 20
Plating of Cells on Lab-Tek Dishes with Dried Transfection Solution......Page 21
Additional Comments......Page 22
References......Page 23
Imaging Platforms for Measurement of Membrane Trafficking......Page 25
Methodology......Page 26
Expression and Transport of ts-O45-G to the Plasma Membrane......Page 27
Measurement of ts-O45-G Transport to the Plasma Membrane......Page 29
Comments......Page 30
Analyzing the Kinetics of Transport Carriers Using Kymographs......Page 32
Installation of the Macros and Plugins in Image J......Page 33
Summary and Perspectives......Page 34
References......Page 35
Introduction......Page 36
How Does the Harvester Search Engine Work?......Page 37
Examples of How to Query the Harvester Search Engine......Page 38
Database Identifier Cross-links......Page 39
A Closer Look at the Search Results......Page 40
References......Page 42
Introduction......Page 44
Example Uses......Page 46
Some Practical Comments and Suggested Good Practice Standards for Microinjection......Page 51
A Suggested Equipment Setup and Materials List......Page 53
Detailed Protocols......Page 54
References......Page 59
Introduction......Page 61
Materials......Page 65
Immuno-labeling for EM with NANOGOLD......Page 68
Materials......Page 69
Materials and Equipment......Page 70
Electron Microscopy Analysis......Page 72
Acknowledgments......Page 73
References......Page 74
Introduction......Page 76
Recombinant Expression......Page 78
Protocol......Page 79
Further Highlights......Page 81
References......Page 82
Purification and Properties of Mammalian Sec23/24 from Insect Cells......Page 85
Genomics-Practical Considerations......Page 86
General Considerations for Expression in Tn5 Cells......Page 87
Overview......Page 88
Lysis of the Harvested Cells......Page 89
Mono Q Anion Exchange Chromatography......Page 90
Analysis of the Final Product......Page 91
References......Page 92
Purification and Functional Properties of Yeast Sec12 GEF......Page 94
Protein Purification......Page 95
Assay of Functional Sec12p......Page 97
Kinetic Measurements......Page 98
Acknowledgements......Page 101
References......Page 102
Introduction......Page 103
Materials......Page 104
Construction of Expression Vectors......Page 105
Purification of Sec12Deltalum......Page 107
Comment......Page 108
Preparation of Proteoliposomes......Page 109
Real-time Measurement of Prebudding Complex Formation and Dissociation Monitored by FRET......Page 110
Real-time Measurement of Sar1p GTP Hydrolysis Monitored by Tryptophan Fluorescence......Page 111
References......Page 113
Introduction......Page 115
Buffers......Page 116
Liposomes Preparation......Page 117
Tryptophan Fluorescence Measurements of the GDP/GTP Cycle of the Small G Protein Arf and Sar......Page 120
Light Scattering Measurements of the Assembly-Disassembly Cycle of the COPI and COPII Coat on Liposomes......Page 122
A Flotation Assay for the Binding of ArfGAP1 to Liposomes of  Various Diameters......Page 124
References......Page 126
Introduction......Page 128
Microsome Membranes Preparation......Page 130
Assaying the Activation of PLD by Sar1......Page 131
PLD Assay......Page 132
References......Page 133
Use of Acyltransferase Inhibitors to Block Vesicular Traffic Between the ER and Golgi Complex......Page 136
Introduction......Page 137
Sources of CI-976 and Other Acyltransferase Inhibitors......Page 139
Adding CI-976 to Cultured Cells......Page 140
In Vivo Studies of COPII Budding......Page 141
In Vitro Studies of COPII Budding......Page 143
References......Page 144
Introduction......Page 147
Materials......Page 148
In Vitro Tethering of COPI Vesicles to Golgin-Coated Glass Slides......Page 149
Tethering of COPI Vesicles to Golgi Membranes Bound to Glass Slides......Page 150
Purification of Labeled COPI Vesicles......Page 151
Methods......Page 153
References......Page 155
Investigating the Role of ADP-Ribosylation Factor 6 in Tumor Cell Invasion and Extracellular Signal-Regulated......Page 157
Introduction......Page 158
Transfection by Metafectene Reagent......Page 159
CFDSE and TRSE Labeled Gelatin Coated Coverslips......Page 160
Fluorescent Labeling of Gelatin with TRSE......Page 161
Preparation of FITC-Gelatin Coated Coverslips......Page 162
Immunostaining for HA-Tagged ARF6 Mutants......Page 163
Image Capture and Confocal Microscopy......Page 164
Measurement of Endogenous ARF6-GTP Levels......Page 166
Measurement of Endogenous ERK Kinase Activity......Page 167
Acknowledgments......Page 168
References......Page 169
Assays and Properties of the Arf GAPs AGAP1, ASAP1, and Arf GAP1......Page 171
Introduction......Page 172
Properties of Arf GAP1, ASAP1 and AGAP1 Relevant to Determination of GAP Activity......Page 174
Loading Arf1 with GTP......Page 175
AGAP1......Page 177
Handling purified proteins......Page 178
Conversion of [alpha32 P]GTP to [alpha32 P]GDP......Page 179
Loss of [gamma32 P]GTP bound to Arf1......Page 181
Detection of change in tryptophan fluorescence of Arf1 on GTP to GDP conversion......Page 183
References......Page 185
Introduction......Page 188
Bacterial Expression of Myristoylated Arf1......Page 190
Purification of Myristoylated Arf1 from Bacteria......Page 191
Bacterial Lysis and Protein Purification......Page 194
References......Page 195
BIG1 and BIG2, Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factors for ADP-Ribosylation Factors......Page 199
Background......Page 200
Cellular Localization of BIG1 and BIG2......Page 201
Functional Interactions of BIG1......Page 203
Functional Interactions of BIG2......Page 204
Methods......Page 206
References......Page 207
Organization of Cytohesin Genes......Page 210
Coiled-coil domain......Page 211
Sec7 Domain......Page 212
PH Domain......Page 214
Assay of Cytohesin-1......Page 215
References......Page 218
ADP-Ribosylation Factor Domain Protein 1 (ARD1), a Multifunctional Protein with Ubiquitin E3 Ligase, GAP, and ARF.........Page 222
Introduction......Page 223
ARF and ARF Regulatory Domains of ARD1......Page 224
The RING Domain and E3 Ubiquitin Ligase Activity of ARD1......Page 225
Synthesis and Purification of Recombinant ARD1 and Its Fragments......Page 226
Assays of GTP Binding and GAP Activities......Page 229
References......Page 231
Expression of BIG2 and Analysis of Its Function in Mammalian Cells......Page 234
Introduction......Page 235
Large-Scale Plasmid Preparation......Page 236
Effects of BIG2 Mutant on the Endosomal Integrity......Page 237
Tfn Recycling......Page 238
GEF Activity In Vivo......Page 240
Acknowledgments......Page 242
References......Page 243
Introduction......Page 244
Preparation of GST-AMAP2 ArfGAP Domain......Page 246
Binding of the GST-AMAP2 ArfGAP Domain with Arf In Vitro......Page 247
Intracellular Binding of Full Length AMAP2 with Wild Type Arf6......Page 249
Selective Binding of AMAP2 to Proteins Related to Arf6 Function......Page 250
Subcellular Colocalization of AMAP2 and its Auxiliary Proteins with GTP-Arf6......Page 251
Small Interfering RNA (siRNA)-mediated Interference......Page 253
Tac and Tfn Uptake......Page 254
References......Page 257
Introduction......Page 260
Construction of Expression Vectors......Page 262
Procedure......Page 264
Procedure......Page 266
Results and Discussion......Page 267
References......Page 269
Functional Assay of ARNO and ARF6 in Neurite Elongation and Branching......Page 271
Introduction......Page 272
Coverslip Preparation......Page 273
Establishment of Hippocampal Cell Cultures......Page 274
Transfection of Primary Neuronal Cultures with ARF6 and ARNO Plasmids......Page 275
Morphological Analysis of Hippocampal Neurons Expressing ARNO, ARF6, and Mutant Forms of ARNO and ARF6......Page 276
Fixation and Staining of Cells......Page 277
Imaging Parameters......Page 278
References......Page 279
Introduction......Page 282
Yeast Two-Hybrid Analysis of Cytohesin 2 and IPCEF1 Interaction......Page 284
Detection of Cytohesin 2 and IPCEF1 Interaction Using GST-fusion Protein Pull-Down Assay......Page 287
IPCEF1 Modulates Cytohesin 2 GEF Activity In Vivo and In Vitro......Page 290
References......Page 295
Introduction......Page 297
Expression and Purification of Rac GTPases......Page 298
Preparation of Nucleotide-Loaded GTPases......Page 299
Isolation of the Endogenous GIT1/p95-APP1 Complex......Page 300
Analysis of the Eluted Fractions by Silver Staining......Page 301
Construction of Plasmids and Expression in Cells......Page 302
Analysis of GIT1/p95-APP1......Page 303
Acknowledgments......Page 306
References......Page 307
Introduction......Page 309
Purification of Recombinant GM130......Page 310
Purification of GM130DeltaN73......Page 313
Purification of Recombinant p115......Page 315
Purification of his-TA and other Truncations within the p115 Tail-Acidic Domain......Page 317
Purification of Recombinant Giantin Fragments......Page 318
Purification of Recombinant CASP......Page 321
Purification of Recombinant Golgin-84......Page 323
References......Page 325
Introduction......Page 327
Experimental Approaches to Study CtBP3/BARS Function......Page 329
Protein Purification......Page 331
Purification of GST-BARS and Mutants......Page 332
Purification of His-tagged BARS and Mutants......Page 333
Acyl-CoA and NAD Binding Experiments......Page 335
Acyltransferase Activity Assay......Page 336
Negative Staining......Page 337
Cellular Studies......Page 338
Cell Transfection......Page 339
Microinjection......Page 341
Preparation of Antibodies......Page 342
BARS Localization......Page 344
References......Page 345
Introduction......Page 348
Preparation of Recombinant GGA Domains......Page 350
Loading Arf1 with [35S]GTPgammaS and [alpha32P]GTP......Page 352
Three Assays for GGAbullArf1 Interactions......Page 353
Binding Determined by Inhibition of Arf GAP Activity......Page 355
Binding Determined by Slowing GTPgammaS Dissociation......Page 358
Use of GGA for Determining Cellular Levels of Arf1bullGTP......Page 360
References......Page 361
The Role of EFA6, Exchange Factor for Arf6, for Tight Junction Assembly, Functions, and Interaction with the Actin Cytoskelet......Page 365
Introduction......Page 366
Cells......Page 367
Confocal Immunofluorescence of Polarized Cells......Page 368
Transepithelial Electrical Resistance (TER)......Page 369
Calcium Switch......Page 370
EFA6 Effects on TJ Permeability and Actin Cytoskeleton......Page 372
Endocytosis Assay......Page 373
Cell Surface Delivery and Half-Life Assay......Page 374
Detergent Partitioning......Page 376
References......Page 377
In Vitro Reconstitution of ARF-Regulated Cytoskeletal Dynamics on Golgi Membranes......Page 379
Introduction......Page 380
Incubation Conditions for Golgi-Membrane Binding and Vesicle Budding Reactions......Page 381
Incubation Conditions......Page 382
Solutions......Page 383
Solutions......Page 384
Solutions......Page 385
Procedures......Page 386
Using Liposomes to Study Membrane-Associated Cytoskeletal.Dynamics......Page 387
Additional Materials and Solutions......Page 388
Materials and Solutions......Page 389
Conclusions......Page 390
References......Page 391
Identification of Arfaptin 1 and 2 as Binding Proteins for GTP-ARFs......Page 393
Preparation of Rac1 Plasmids......Page 394
Procedure for Yeast Two-Hybrid Interaction Assay Between Rac1 and Arfaptins......Page 395
Preparation of Recombinant Rac1 and ARFs in CHO-K1 Cells......Page 396
Procedure for GST Pull-Downs......Page 397
Differential Binding of Arfaptin 2 to Arfs and Rac1......Page 398
Discussion......Page 399
References......Page 400
Analysis of Arf Interaction with GGAs In Vitro and In Vivo......Page 402
Introduction......Page 403
Plasmid Construction......Page 404
Pull-Down Assay......Page 405
Error-Prone PCR and Reverse Two-Hybrid Screening......Page 408
DNA Transfection and Immunofluorescence Analysis......Page 409
References......Page 411
Arf6 Modulates the beta-Actin Specific Capping Protein, betacap73......Page 413
Introduction......Page 414
Isoactin Assembly Assays......Page 417
Expression and Purification of GST-betacap73......Page 419
Analysis of Endothelial Cell Motility Following Injury In Vitro......Page 420
Analysis of Protein Interaction by Co-Immunoprecipitation......Page 421
References......Page 422
Introduction......Page 424
Purification of recombinant myristoylated Arf1 and Arf6 mutants......Page 427
Preparation of Rat Brain Cytosol and LP2 Membranes for Recruitment Experiments......Page 429
Arf6-Mediated Recruitment of Clathrin/AP-2 Coats onto LP2 Membranes......Page 430
Identification of Binding Partners of Recombinant Myristoylated  Arf6 by Chemical Cross-Linking......Page 431
Pull-Down Experiments......Page 432
References......Page 433
Assays to Study Phospholipase D Regulation by Inositol Phospholipids and ADP-Ribosylation Factor 6......Page 435
Introduction......Page 436
Protocol......Page 439
Whole Cell PLD Assay......Page 441
Protocol......Page 442
Protocol......Page 444
References......Page 445
Introduction......Page 448
Buffers......Page 449
Expression and Purification of Flag-ASAP1 from Transfected HEK293T Cells......Page 450
Overlay Assay to Analyze the Effect of ASAP1 Tyrosine Phosphorylation on Phospholipid Binding......Page 452
Liposomes Preparation......Page 454
Cellular Arf1-GTPase Assay......Page 455
References......Page 458
Background on Arf Family......Page 460
Studies Utilizing Arf6 Mutants......Page 461
Arf6 Regulates Endocytic Membrane Trafficking......Page 462
Arf6 Regulates Plasma Membrane PIP 5-Kinase and PIP2 Synthesis......Page 465
Detection of Cellular PIP2 by Immunocytochemistry......Page 466
References......Page 467
Introduction......Page 470
Expression of GST-Arl1 Fusion Protein......Page 471
In Vitro Guanine Nucleotide Exchange Reactions of GST-Arl1......Page 472
Cloning and Expression of GST-GRIP......Page 473
GST-GRIP Selectively Pulls-Down Cytosolic Active Arl1......Page 474
Methods......Page 476
Results......Page 477
References......Page 478
Introduction......Page 480
Media and buffers......Page 481
Purification of STxB by Q-sepharose......Page 482
Sulfate Starvation......Page 483
Re-Constitution of EE/RE-to-TGN Transport......Page 484
Result and Discussion......Page 485
siRNA Treatment......Page 486
In Vivo STxB Transport Assay-Biochemical Analysis......Page 487
Result and Discussion......Page 488
Effect of Arl1-Q71L Overexpression on In Vivo STxB Transport as Revealed by a Morphological Approach......Page 489
References......Page 490
Assays Used in the Analysis of Arl2 and Its Binding Partners......Page 492
Introduction......Page 493
Purification of Recombinant Human Arl2 and BART......Page 494
Nucleotide Binding to Arl2......Page 495
Gel Overlay Assay......Page 498
Comments......Page 499
Arl2 GAP Assays......Page 500
TLC-Based Assay......Page 501
Arl2 GAP Assay Using Charcoal-Based Separation......Page 502
Acknowledgments......Page 504
References......Page 505
Introduction......Page 507
Using Yeast-2-Hybrid to Study the Arl3-RP2 Interaction......Page 509
Cloning RP2 and Arl3......Page 510
Yeast Transformation......Page 511
Verification of Bait Protein Expression......Page 512
Inhibition of Myristoylation......Page 513
Sucrose Gradient Centrifugation......Page 514
Defining the Localization of RP2 in the Retina......Page 515
Future Directions......Page 517
References......Page 518
Reconstitution of Transport to Recycling Endosomes In Vitro......Page 520
General Considerations......Page 521
Cells and Cell Culture......Page 523
Labeling of Transferrin with Acridinium Ester......Page 524
Preparation of Endosome-Enriched Fractions......Page 525
Immunoadsorption of Recycling Endosomes......Page 526
In Vitro Fusion Assay......Page 527
References......Page 529
Robust Colorimetric Assays for Dynamin's Basal and Stimulated GTPase Activities......Page 531
Introduction......Page 532
Dynamin and GED Purification and Storage......Page 533
Lipid Nanotubule Preparation and Storage......Page 534
Basal GTPase Activity......Page 535
Liposome-Stimulated GTPase Assays......Page 538
Lipid Nanotubule-Stimulated GTPase Activity......Page 540
GED-Stimulated GTPase Activity......Page 541
References......Page 543
Introduction......Page 545
Preparation and Storage of Cytosol, Clathrin, AP2, and Dynamin......Page 546
Isolation of Rat Liver PMs......Page 547
In Vitro CCV Formation Assay from Isolated PMs......Page 548
Inhibition of Membrane Fusion Increases Efficiency......Page 550
Endocytosis of Different Endocytic Receptors......Page 551
Inhibition of LRP Uptake by a Kinase Inhibitor A3......Page 552
References......Page 553
Introduction......Page 554
Experimental Procedure......Page 555
Results......Page 557
Nucleotide Association and Dissociation Kinetics......Page 559
Results......Page 561
Nucleotide Hydrolysis Activity......Page 562
Experimental Procedure......Page 563
Nucleotide-Dependent Self-Association......Page 565
Results......Page 566
References......Page 568
Introduction......Page 570
Purification of Dynamin from Bovine Brain......Page 572
Expression and Purification of Amphiphysin and Its Truncation Constructs......Page 573
Preparation of Large Unilamellar Liposomes......Page 574
Stimulation of Dynamin GTPase Activity by Amphiphysin......Page 575
References......Page 577
Introduction......Page 580
Constructs......Page 584
Binding Assay......Page 585
Preparation of Brain TritonX100 Extract......Page 586
References......Page 587
Expression and Properties of Sorting Nexin 9 in Dynamin-Mediated Endocytosis......Page 589
Introduction......Page 590
Recombinant SNX9......Page 592
SNX9-Complex in Cytosol......Page 593
Pull-Downs......Page 595
Immunodetection......Page 596
Recruitment to Cellular Membranes......Page 597
Recruitment of SNX9 and Dyn2 to Membranes In Vivo......Page 599
References......Page 600
Rapid Purification of Native Dynamin I and Colorimetric GTPase Assay......Page 601
Purification of Native Dynamin I......Page 602
Expression and Purification of Recombinant GST-Amphiphysin II-SH3 Domain Protein......Page 603
Buffers and Reagents.......Page 605
Day 1......Page 606
Day 2......Page 607
Colorimetric GTPase Assay......Page 608
Buffers and Reagents......Page 609
Procedure......Page 610
Essential Controls......Page 612
Acknowledgments......Page 613
References......Page 614
Introduction......Page 615
Expression and Purification of His6-tagged Auxilin-1......Page 616
Expression and Purification of GST-dynamin......Page 617
Nucleotide-Specific Dynamin Affinity Columns......Page 619
Nucleotide-Specific Pull-Down Assays......Page 622
Auxilin-Inhibited GAP-stimulated GTPase Activity......Page 623
Fluorescence Lifetime Imaging Microscopy of Auxilin-Dynamin Interactions......Page 625
References......Page 629
Introduction......Page 631
Method......Page 632
Method......Page 634
Background......Page 635
Method......Page 638
Method......Page 639
Analysis of other Biochemical Properties of DLP1......Page 640
References......Page 641
Introduction......Page 643
Introduction......Page 644
Lipid Stocks.......Page 646
Making Liposomes of a Defined Size......Page 647
Purification of Dynamin and Its Mutants from E. coli......Page 648
Introduction......Page 649
Studying the Conformational Stage of Dynamin Spirals......Page 650
Introduction......Page 651
Carbon Coated Electron Microscopy Grids......Page 652
Analysis of Coat Formation......Page 654
Conclusions and Future Directions......Page 655
References......Page 656
Introduction......Page 658
Continuous GTPase Assay......Page 661
Procedure......Page 662
Data Analysis......Page 664
References......Page 665
Introduction......Page 667
Procedure......Page 668
Digitonin Fractionation of Mitochondria......Page 669
Procedure......Page 670
Comments......Page 671
Procedure......Page 672
Comments......Page 673
Comments......Page 674
Bacterial Expression and Purification......Page 675
GTP Hydrolysis......Page 676
References......Page 678
Introduction......Page 679
Expression and Purification of MxA......Page 681
MxA Oligomerization in Living Cells......Page 683
Structural Analysis of MxA Oligomers by Cryo-transmission Electron Microscopy......Page 684
Membrane Association of MxA......Page 685
MxA Interaction with Viral Target Structures......Page 687
Coimmunoprecipitation of MxA with Viral N Protein......Page 688
References......Page 689
x author index.pdf......Page 691
x mie index.pdf......Page 722
x subject index.pdf......Page 745