Bidirectional transport between the cytoplasm and the nucleoplasm is a finely tuned process, whose regulation is critical for the proper function of numerous biological pathways. Exchanges between these two compartments take place through nuclear pore complexes (NPCs), huge macromolecular assemblies embedded within the nu clear envelope (NE). Since the unearthing of this research field, more than 50 years ago, the combination of a large variety of approaches, performed in a broad range of model organisms, has permitted the deciphering of the main features of NPCs, their assembly and dynamics, as well as the precise rules governing and regulating nucleo-cytoplasmic exchanges. Although this field of research is extremely dynamic and has both contributed to and benefited from a wealth of innovating methods over recent years, no dedicated volume had been devoted to this topic since Elsevier published the Methods volume "Nuclear cytoplasmic transport and nuclear pore complexes" in 2006 (Volume 39(4): 275-380, ed. B.M.A. Fontoura). As this topic impinges on multiple fields, scattered protocols could however be found in volumes devoted to nuclear architecture, gene expression, small GTPases, or specific model systems. It was thus timely to set up a dedicated volume, and I am grateful to Phong Tran, senior editor of this series, for stimulating me to assemble this book, and to the many leaders in the field who have devoted some of their precious time to contribute to specific chapters.
Following the introduction, that presents an overview of the history and of the main tools and rules governing nuclear transport, the 21 chapters in this volume provide protocols developed in the most widespread model systems in the field, namely mammalian cells in culture (Chapters 2,6,10-12, 15,16, 18,19, and 21), yeast Sac-charomyces cerevisiae (Chapters 3,5,7,14, and 19-21), Xenopus laevis oocytes and eggs extracts (Chapters 2,4,8,9, and 18), and Caenorhabditis elegans (Chapter 13). The first chapters of this volume mainly focus on methods that have enabled impressive progress in our understanding of NPC structure and dynamics. These include the visualization of the NPC architecture and the localization of its constituents (the nucleoporins, Nups) that have largely benefited from transmission and scanning electron microscopy approaches (Chapters 2-4 and 13) and more recently from super-resolution imaging (Chapter 10), and recent protocols designed to study large Nups or NPC subcomplexes by negative staining (Chapter 5), to allow the precise determination of Nup stoichiometry (based on NE purifications and targeted prote-omics, Chapter 6), or to follow the in vivo assembly of newly synthesized proteins within complexes (Chapter 7). While such approaches have contributed to refine our view of NPC organization, they no doubt could be usefully applied to improve our understanding of any other complex macromolecular assemblies. NPCs are dynamic structures whose disassembly and reassembly upon open mitosis (Chapters 10 and 13) or de novo assembly during interphase (Chapter 11) can be monitored in a quantitative manner using live cell imaging. In addition, in vitro assays based on nuclei assembled using cell-free extracts of Xenopus eggs (Chapters 2, 8, and 9) or on semi-permeabilized mammalian cells (Chapters 12 and 15) have been adapted and refined to decipher the mechanisms that underlie these dynamic changes. To assess the critical function of nuclear pores in bidirectional transport of macromolecules, specific reporters and methods have been designed in each model organism to measure nuclear permeability (Chapters 10, 12, and 13), protein import or export (Chapters 8, 9, 13, and 14), or the fate of the various classes of RNAs that are assembled into ribonucleoparticles (Chapters 18—20). With respect to nuclear protein transport, this volume includes recent adaptations to the well-established in vitro assay that relies on digitonin-permeabilized cells (Chapter 15), quantitative mass-spectrometry approaches to identify specific import or export substrates (Chapter 16), and an example of nanodevices that mimic functional NPCs (Chapter 17). Dedicated tools to follow and characterize RNA transport mechanisms are provided in Chapter 18 (that details microinjections in Xenopus oocytes and FISH studies in vertebrates), Chapter 19 (dedicated to tRNA dynamics), and Chapter 20 (ribosome assembly and transport). While not covered by specific chapters, all these tools can be adapted to study nucleocytoplasmic trafficking of a wealth of other macromolecular assemblies, notably viral particles. Finally, the nuclear transport machinery further plays a critical role in multiple cellular processes, including cell cycle regulations (see for instance Chapters 14 and 15), maintenance of genetic integrity, and gene expression. For the latter topic, protocols developed to analyze gene positioning and to evaluate the association of Nups with transcribed genes are detailed in Chapter 21 (and referred to in Chapter 13).
Twenty one chapters cannot entirely cover the multiplicity of model systems and approaches so far used in this ever-expanding field, and I apologize to those whose protocols could not be included in this volume. However, all authors have clearly made considerable efforts to provide, in addition to step-by-step protocols that should enable scientists to reproduce their preferred methods, critical references to related or alternative techniques that have been developed to tackle similar questions. Other currently arising model organisms in the field (such as flies, plants, fungi, ciliates, or protozoans, to cite but a few) are unfortunately not included in this volume, and will clearly deserve specific chapters in the future.
I hope that all Teaders, junior scientists joining the nuclear transport field or already acquainted with it as well as outsiders of other fields, will appreciate, as much as I did, going through these carefully detailed methods chapters. I am particularly grateful to all authors for their commitment to this collective project. I would also like to thank the editors at Elsevier, Sarah Lay and Zoe Kruze, for their efficient help during this process, and all my collaborators at the Institut Jacques Monod for their constant support and understanding over these last busy months. Valerie Doye

Contributors xiii

Preface xix

CHAPTER 1 Fifty Years of Nuclear Pores and Nucleocytoplasmic Transport Studies: Multiple Tools Revealing Complex Rules 1

Aurelie G. Floch, Benoit Palancade, Valerie Doye

Introduction 2

1.1 The NPCs: A Modular Macromolecular Assembly 3

1.2 Nucleocytoplasmic 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

Boris Fichtman, Lihi Shaulov, Amnon Harel

Introduction 42

2.1 Rationale 44

2.2 Materials 48

2.3 Anchored Nuclei 49

2.4 Mammalian Cell Nuclei 51

2.5 Immunogold Labeling 52

2.6 Sample Preparation for FESEM 54

Acknowledgments 56

References 56

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

Elena Kiseleva, A. Christine Richardson, Jindriska Fiserova, Anton A. Strunov, Matthew C. Spink, Simeon R. Johnson, Martin W. Goldberg

Introduction 60

3.1 Conventional TEM 61

3.2 SEM and Immuno-SEM of Yeast Nuclei 65

3.3 Immunogold Labeling of Yeast Ultrathin Cryosections 69

Conclusions and Perspectives 76

Acknowledgments 77

References 77

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

Nelly Pante, Birthe Fahrenkrog

Introduction 82

4.1 Materials 86

4.2 Experimental Strategies 87

4.3 Preparation of Antibodies Conjugated with Colloidal Gold Particles 89

4.4 Immunogold Labeling of Nucleoporins Using Anti-Nucleoporin Antibodies 90

4.5 Immunogold Labeling of Nucleoporins Using Epitope-Tagged Nucleoporins 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

Dirk Flemming, Philipp Stelter, Ed Hurt

Introduction 100

5.1 Common Preparatory Steps 101

5.2 Dyn2 as an EM Label 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

Acknowledgment 114

References 114

CHAPTER 6 The Use of Targeted Proteomics to Determine the Stoichiometry of Large Macromolecular Assemblies 117

Alessandro Ori, Amparo Andres-Pons, Martin Beck

Introduction 118

6.1 Isolation of Nuclei and Nuclear Envelopes 119

6.2 Targeted Proteomics 130

Conclusions 143

Acknowledgments 144

References 144

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

Philipp Stelter, Ed Hurt

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

Cyril Bernis, Douglass J. Forbes

Introduction 166

8.1 Materials 168

8.2 Xenopus Egg Extracts 170

8.3 Demembianated 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

Conclusion 187

References 188

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

Nathalie Eisenhardt, Allana Schooley, Wolfram Antonin

Introduction 195

9.1 Preparation of Xenopus 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

Shotaro Otsuka, Anna Szymborska, Jan Ellenberg

Introduction 220

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

10.2 Monitoring NE Permeability in Living 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 Visualize Interphase Nuclear Pore Formation 239

Kazuhiro Maeshima, Tomoko Funakoshi, Naoko Imamoto

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 Drag 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

Joseph Marino, Lysie Champion, Cornelia Wandke, Peter Horvath, Monika I. Mayr, Ulrike Kutay

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 277

Peter Askjaer, Vincent Galy, Peter Meister

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

13.5 Immunofluorescence 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

Christopher Ptak, Richard W. Wozniak

Introduction 312

14.1 Observing Steady-State Localization 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

Maiko Furuta, Shingo Kose, Ralph H. Kehlenbach, Naoko Imarnoto

Introduction 332

15.1 Equipment, Material, Reagents, and Buffers 335

15.2 Purification of Recombinant Transport Factors 339

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

15.4 Interphase Nucleocytoplasmic Transport and Mitotic Chromosome Loading of Chromokinesin hKid 346

Conclusions and Perspectives 349

Acknowledgments 349

References 349

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

Makoto Kimura, Ketan Thakar, Samir Karaca, Naoko Imamoto, Ralph H. Kehlenbach

Introduction 354

16.1 Identification of CRM1-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 Nucleocytoplasmic Transport 379

Tijana Jovanovic-Talisman, Brian T. Chait, Michael P. Rout

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 391

References 391

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

Ichiro Taniguchi, Asako McCloskey, Mutsuhito Ohno

Introduction 396

18.1 Materials 398

18.2 Nuclear Export of Radiolabeled RNAs in Xenopus Oocytes 401

18.3 Nuclear Localization of Fluorescently Labeled RNAs in Xenopus Oocytes 405

18.4 Nuclear Export of Endogenous 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-Cytoplasmic tRNA Dynamics in Yeast and Mammalian Cells 415

Jacqueline B. Pierce, Shawn C. Chafe, Manoja, B. K. Eswara, George van der Merwe, Dev Mangroo

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 Location 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

Martin Altvater, Sabina Schutz, Yiming Chang, Vikram Govind Panse

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 Localization 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

Acknowledgments 458

References 458

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

Defne Emel Egecioglu, Agustina D'Urso, Donna Garvey Brickner, William H. Light, Jason H. Brickner

Introduction 464

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

21.2 Monitoring Interchromosomal Clustering of Genes at the NPC 471

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

21.4 List of Plasmids and Strains 481

Concluding Remarks, Possible Caveats, and Troubleshooting 482

Acknowledgments 483

References 483

Index 485

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