Volume 122 of Methods in Cell Biology describes modern tools and techniques used to study nuclear pore complexes and nucleocytoplasmic transport in diverse eukaryotic model systems (including mammalian cells, Xenopus, C. elegans, yeast).
The volume enables investigators to analyze nuclear pore complex structure, assembly, and dynamics; to evaluate protein and RNA trafficking through the nuclear envelope; and to design in vivo or in vitro assays appropriate to their research needs.
Beyond the study of nuclear pores and transport as such, these protocols will also be helpful to scientists characterizing gene regulation, signal transduction, cell cycle, viral infections, or aging. The NPC being one of the largest multiprotein complexes in the cell, some protocols will also be of interest for people currently characterizing other macromolecular assemblies. This book is thus designed for laboratory use by graduate students, technicians, and researchers in many molecular and cellular disciplines.

Contributors xiii

Preface xix

CHAPTER 1 Fifty Years of Nuclear Pores and Nucleocytoplasmic Transport Studies:

Multiple Tools Revealing Complex Rules 1

Introduction 2

1.1 The NPCs: A Modular Macromolecular Assembly 3

1.2 Nucleocytop1asmic Trafficking : The Rules of the Road 12

1.3 The Nuclear Transport Machinery: A Dynamic and Versatile Device 23

Concluding Remarks 31

Acknowledgments 31

References 32

CHAPTER 2 Imaging Metazoan Nuclear Pore Complexes by Field Emission Scanning Electron Microscopy 41

Introduction 42

2.1 Rationa1e 44

2.2 Material s 48

2.3 Anchored Nuclei 49

2.4 Mammalian Cell Nuclei 51

2.51mmunogold1abeling 52

2.6 Sample Preparation for FESEM 54

Acknow1edgments 56

References 56

CHAPTER 3 Imaging Yeast NPCs: From Classical Electron Microscopy to 1mmuno-SEM 59

Introduction 60

3.1 Conventional TEM 61

3.2 SEM and Immuno-SEM of Yeast Nuclei 65

3.3 Immunogold 1abeling of Yeast Ultrathin Cryosections 69

Conclusions and Perspectives 76

Acknowledgments 77

References 77

CHAPTER 4 Exploring Nuclear Pore Complex Molecular Architecture by1mmuno-Electron Microscopy Using Xenopus Oocytes 81

Introduction 82

4.1 Materials 86

4.2 Experimental Strategies 87

4.3 Preparation of Antibodies Conjugated with Colloidal Gold Particles 89

4.4 Immunogold1abeling of Nucleoporins Using Anti-Nucleoporin Antibodies 90

4.5 Immunogold1abeling of Nucleoporins Using Epitope-Tagged Nucleopori ns 93

Concluding Remarks 96

Acknowledgments 96

References 96

CHAPTER 5 Utilizing the Dyn2 Dimerization-Zipper as a Tool to Probe NPC Structure and Function 99

Introduction 100

5.1 Common Preparatory Steps 101

5.2 Dyn2 as an EM1abel to Map Subcomplexes and Single Nups 107

5.3 Probing the FG Network by Selective Insertion of the eDID-Dyn2 Complex into FG Repeat Domains 111

5.4 Materials and Reagents 113

Conclusion 114

Acknow1edgment 114

References 114

CHAPTER 6 The Use of Targeted Proteomics to Determine the Stoichiometry of arge Macromolecular Assemblies 111

Introduction 118

6.1 Isolation of Nuclei and Nuclear Envelopes 119

6.2 Targeted Proteomics 130

Conclusions 143

Acknowled gments 144

References 144

CHAPTER 7 A Pulse-Chase Epitope 1abeling to Study Cellular Dynamics of Newly Synthesized Proteins: A Novel Strategy to Characterize NPC Biogenesis and Ribosome Maturation/Export 147

Introduction 148

7.1 Cloning into the Saccharomyces cerevisiae Pulse-Chase Vectors 150

7.2 Epitope Pulse-Chase Protocols 151

7.3 Material 160

Conclusion and Perspectives 161

Acknowledgments 162

References 162

CHAPTER 8 Analysis of Nuclear Reconstitution, Nuclear Envelope Assembly, and Nuclear Pore Assembly Using Xenopus In Vitro Assays 165

Introduction 166

8.1 Materials 168

8.2 Xenopus Egg Extracts 170

8.3 Demembran ated Sperm Chromatin 177

8.4 In Vitro Reconstitution of Nuclei 180

8.5 Assaying Assembly and Integrity of the Nuclear Envelope .182

8.6 A Nuclear Pore Complex Assembly Assay Using Pore-Free Nuclear Intermediates 184

Conclu sion 187

References 188

CHAPTER 9 Xenopus In Vitro Assays to Analyze the Function of Transmembrane Nucleoporins and Targeting of Inner Nuclear Membrane Proteins 193

Introduction 195

9.1 Preparation of Xe11opus Egg Extract Cytosol and Membranes 196

9.2 Protein Expression 201

9.3 Biochemical Procedures 204

9.4 Nuclear Assembly Reactions 210

Conclusion 216

References 217

CHAPTER 10 Imaging the Assembly, Structure, and Function of the Nuclear Pore Inside Cells 219

Introduction 220

10.1 Measuring the Kinetics of Postmitotic NPC Assembly in1iving Cells by Multicolor 4D Imaging 221

10.2 Monjtoring NE Permeability in1iving Cells by Sequential Photoswitching 225

10.3 Structural Analysis of the NPC by Super-Resolution Microscopy 228

10.4 Future Perspective 234

10.5 Materials and Instruments 234

Acknowledgments 235

References 236

CHAPTER 11 Cell-Fusion Method to Visualize1nterphase Nuclear Pore Formation 239

Introduction 240

11.1 Materials and Equipment 246

11.2 Quantitative Analysis of Interphase NPC Formation using Cell-Fusion Method 247

11.3 Combining the Cell-Fusion Method with Drug and siRNA Treatments 249

11.4 Visualization of Interphase NPC Formation using Photobleaching 250

Conclusions 252

Acknowledgments 252

References 252

CHAPTER 12 An In Vitro System to Study Nuclear Envelope Breakdown 255

Introduction 256

12.1 Preparative Steps 258

12.2 NEBD Assay 264

12.3 Special Treatments 267

12.4 Data Analysis 270

12.5 Future Directions 273

12.6 Material and Reagents 273

Acknowledgments 275

References 275

CHAPTER 13 Modern Tools to Study Nuclear Pore Complexes and Nucleocytoplasmic Transport in Caenorhabditis elegans 211

Introduction 278

13.1 Forward and Reverse Genetics 279

13.2 Transgenesis 287

13.3 Live Imaging of Embryos 290

13.4 In Vivo Methods to Evaluate Structural and Functional Integrity of the NE 296

13.51mmunofl uorescence and Electron Microscopy 298

13.6 Interaction of Nups with Chromatin 303

Summary and Future Perspectives 304

Acknowledgments 304

References 305

CHAPTER 14 Assessing Regulated Nuclear Transport in Saccharomyces cerevisiae 311

Introduction 312

14.1 Observing Steady-State1ocalization of Kap Cargo Proteins 314

14.2 Perturbing Nuclear Transport 319

14.3 Materials and Reagents 327

Acknowledgments 328

References 328

CHAPTER 15 Analysis of Nucleocytoplasmic Transport in Digitonin-Permeabilized Cells Under Different Cellular Conditions 331

Introduction 332

15.1 Equipment, Material, Reagents, and Buffers 335

15.2 Purification of Recombinant Transport Factors 339

15.3 Use of Digitonin-Permeabilized Cells to Study Nuclear Transport Under Normal and Heat-Shock Conditions 342

15.4 Interphase Nucleocytoplasmic Transport and Mitotic Chromosome1oading of Choromokinesin hKid 346

Conclusions and Perspectives 349

Acknowledgments 349

References 349

CHAPTER 16 Novel Approaches for the Identification of Nuclear Transport Receptor Substrates 353

Introduction 354

16.1 Identification of CRMI-Dependent Export Cargos 357

16.2 Identification of Nuclear Import Cargos 364

Conclusions 375

Acknowledgments 375

References 376

CHAPTER 17 NPC Mimics: Probing the Mechanism of Nucleocytoplasm ic Transport 379

Introduction 380

17.1 Protein Production and Purification 381

17.2 Preparation of Nanoselective Filters 384

17.3 Device Setup 385

17.4 Flux Measurements 387

17.5 Materials and Reagents (Listed by Alphabetical Order) 389

Conclusions 39 1

References 391

CHAPTER 18 Analys is of RNA Transport in Xenopus Oocytes and Mammalian Cells 395

Introduction 396

18.1 Materials 398

18.2 Nuclear Export of Radiolabeled RNAs in Xenopus Oocytes 401

18.3 Nuclear1ocalization of Fluorescently1abeled RNAs in Xenopus Oocytes 405

18.4 Nuclear Export of Endogenou s poly(A) +RNA in Mammalian Cells 407

18.5 Nuclear Export of Exogenously Expressed GFP mRNA in Mammalian Cells 409

Concluding Remarks 411

Acknowledgment 411

References 412

CHAPTER 19 Strategies for Investigating Nuclear-Cytoplasmi c tRNA Dynamics in Yeast and Mammalian Cells 415

Introduction 416

19.1 Identification of tRNA-Interacting Proteins Using a Yeast Three-Hybrid Interaction Screen 419

19.2 Amber Suppression In Vivo Nuclear tRNA Export Assay 423

19.3 Fluorescence In Situ Hybridization Detection of the Cellular 1ocation of tRNA 426

19.4 Analysis of tRNA Aminoacylation Status in the Nucleus 431

Concluding Remarks 435

References 435

CHAPTER 20 Dissecting Ribosome Assembly and Transport in Budding Yeast 437

Introduction 438

20.1 Localization of Preribosomal Subunits by Fluorescence Microscopy 441

20.2 Fractionation of Cell Extracts by Sucrose Gradient Sedimentation 443

20.3 Isolation of Preribosomal Particles by TAP 450

20.4 Monitoring 1ocalization of the 40S Preribosome by Fluorescence In situ Hybridization 453

20.5 Analysis of Shuttling Trans-Acting Factors by Heterokaryon Assays 454

20.6 Material, Reagents and Yeast Media 456

Conclusions 458

Acknowled gments 458

References 458

CHAPTER 21 Approaches to Studying Subnuclear Organization and Gene-Nuclear Pore Interactions 463

Introduction 464

21.1 A Quantitative Assay for Gene1ocalization to the Nuclear Pore Complex in Yeast 465

21.2 Monitoring Interchromosomal Clustering of Genes at the NPC 471

21.3 Using Chromatin Immunoprecipitation to Probe Nuclear Organization, Transcription, and Chromat in Structure in Yeast and Human Cells 474

21.41ist of Plasmid s and Strains 481

Concluding Remarks, Possible Caveats, and Troubleshooting 482

Acknowledgments 483

References 483

lndex 485

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