| 目录 |
| | Contributors | xiii |
| | Preface | xxi |
| ch. 1 | Imaging Centrosomes and Cilia in the Mouse Kidney / Moe R. Mahjoub | 1 |
| | Introduction | 2 |
| 1. | Methods | 4 |
| | Conclusion | 13 |
| | References | 14 |
| ch. 2 | Ependymal Cell Differentiation, from Monociliated to Multiciliated Cells / Nathalie Spassky | 19 |
| | Introduction | 20 |
| 1. | Methods | 20 |
| | Conclusion | 32 |
| | Acknowledgments | 32 |
| | References | 32 |
| ch. 3 | Observing Planar Cell Polarity in Multiciliated Mouse Airway Epithelial Cells / Jeffrey D. Axelrod | 37 |
| | Introduction | 38 |
| 1. | Acquisition or Generation of Multiciliated Airway Epithelial Cells | 41 |
| 2. | Assessment of Ultrastructural Planar Polarity of Motile Cilia | 42 |
| 3. | Localization of PCP Signaling Pathway Proteins in Multiciliated Airway Epithelial Cells | 48 |
| | Conclusions | 52 |
| | Acknowledgments | 52 |
| | References | 52 |
| ch. 4 | Imaging and Analyzing Primary Cilia in Cardiac Cells / Sigolene M. Meilhac | 55 |
| | Introduction | 56 |
| 1. | Imaging Cilia In situ, in Fixed Hearts | 57 |
| 2. | Imaging and Manipulating Cilia in Primary Cultures of Cardiac Cells | 61 |
| 3. | Quantitative Analysis of Cilium Orientation | 67 |
| | Conclusion | 72 |
| | Acknowledgments | 72 |
| | Supplementary Data | 72 |
| | References | 72 |
| ch. 5 | Cilia in Photoreceptors / Hemant Khanna | 75 |
| | Introduction | 76 |
| 1. | Methods | 77 |
| | Conclusions | 91 |
| | Acknowledgments | 91 |
| | References | 92 |
| ch. 6 | Analysis of Primary Cilia in the Developing Mouse Brain / Michaela Wilsch-Brauninger | 93 |
| | Introduction | 94 |
| 1. | Neurogenesis in the Developing Neocortex | 94 |
| 2. | Primary Cilia in the Developing Neocortex | 96 |
| 3. | Material | 97 |
| 4. | Methods | 103 |
| | Conclusion | 126 |
| | Acknowledgments | 126 |
| | References | 126 |
| ch. 7 | In Vivo Investigation of Cilia Structure and Function Using Xenopus / John B. Wallingford | 131 |
| | Introduction | 132 |
| 1. | A Brief Introduction to Xenopus Husbandry and Manipulation | 133 |
| 2. | Imaging of Cilia in Fixed Xenopus Embryos and Tissues | 134 |
| 3. | Methods for Live Cell Analysis of Cilia in Xenopus | 139 |
| 4. | Mounting Xenopus Embryos | 142 |
| 5. | Image Acquisition | 145 |
| 6. | Quantitative Analysis of Ciliary Properties | 151 |
| | Conclusion | 154 |
| | Acknowledgments | 155 |
| | References | 155 |
| ch. 8 | A Quantitative Approach to Study Endothelial Cilia Bending Stiffness During Blood Flow Mechanodetection In Vivo / Julien Vermot | 161 |
| | Introduction | 162 |
| 1. | The Challenge of Measuring Ciliary Mechanics | 162 |
| 2. | Live Imaging | 164 |
| 3. | Image Processing | 168 |
| 4. | Bending Rigidity Estimation through Modeling | 168 |
| 5. | Material | 172 |
| | Conclusion | 172 |
| | Acknowledgments | 173 |
| | References | 173 |
| ch. 9 | Quantitative Description of Fluid Flows Produced by Left---Right Cilia in Zebrafish / Jeffrey D. Amack | 175 |
| | Introduction | 176 |
| 1. | Methods | 177 |
| 2. | Application | 183 |
| | Conclusion | 185 |
| | Acknowledgments | 185 |
| | References | 185 |
| ch. 10 | Efficient Live Fluorescence Imaging of Intraflagellar Transport in Mammalian Primary Cilia / Wallace F. Marshall | 189 |
| | Introduction | 190 |
| 1. | Preparation of Cells for IFT Imaging | 191 |
| 2. | Live Imaging of IFT | 193 |
| 3. | Image Processing and Data Analysis | 195 |
| 4. | Discussion | 198 |
| | Acknowledgments | 199 |
| | Supplementary Data | 199 |
| | References | 199 |
| ch. 11 | Analysis of Soluble Protein Entry Into Primary Cilia Using Semipermeabilized Cells / Maxence V. Nachury | 203 |
| | Introduction | 204 |
| 1. | Methods | 205 |
| 2. | Conclusions and Future Outlook | 219 |
| | Acknowledgments | 219 |
| | References | 220 |
| ch. 12 | Methods for Imaging Individual Cilia in Living Echinoid Embryos / Elyse M. Doherty | 223 |
| | Introduction | 224 |
| 1. | Methods | 225 |
| | Conclusion | 240 |
| | Acknowledgments | 240 |
| | Supplementary Data | 240 |
| | References | 240 |
| ch. 13 | The planarian Schmidtea Mediterranea as a Model for Studying Motile Cilia and Multiciliated Cells / Juliette Azimzadeh | 243 |
| | Introduction | 244 |
| 1. | Maintaining Planarians in the Lab | 247 |
| 2. | RNA Interference | 251 |
| 3. | Whole-Mount Immunofluorescence Staining | 255 |
| 4. | Electron Microscopy | 258 |
| 5. | Live Imaging | 260 |
| | Acknowledgments | 261 |
| | References | 261 |
| ch. 14 | Quantitative Analysis of Flagellar Proteins in Drosophila Sperm Tails / Renata Basto | 263 |
| | Introduction | 264 |
| 1. | Methods | 266 |
| | Concluding Remarks | 276 |
| | Acknowledgments | 276 |
| | References | 276 |
| ch. 15 | Imaging Cilia in Drosophila melanogaster / Benedicte Durand | 279 |
| | Introduction | 280 |
| 1. | Immunodetection of Cilia | 283 |
| 2. | Observation of Cilia Ultrastructure by Transmission Electron Microscopy | 293 |
| | Acknowledgments | 298 |
| | References | 298 |
| ch. 16 | Studying G Protein-coupled Receptors: Immunoblotting, Immunoprecipitation, Phosphorylation, Surface Labeling, and Cross-linking Protocols / Saikat Mukhopadhyay | 303 |
| | Introduction | 305 |
| 1. | Reagents | 307 |
| 2. | Protocol for Immunoblotting GPCRs | 311 |
| 3. | Protocol for Immunoprecipitating GPCRs | 313 |
| 4. | GPCR Labeling and Cross-Linking Studies | 317 |
| 5. | Comments on Biochemical Assays Involving GPCRs and Multipass Transmembrane Proteins | 319 |
| | Conclusions | 320 |
| | Acknowledgments | 320 |
| | References | 320 |
| ch. 17 | Image Analysis of Caenorhabditis elegans Ciliary Transition Zone Structure, Ultrastructure Molecular Composition, and Function / Oliver E. Blacque | 323 |
| | Introduction | 324 |
| 1. | Cilium Compartmentalization and the Transition Zone | 324 |
| 2. | Caenorhabditis elegans Sensory Neuronal Cilia Model | 326 |
| 3. | Methods | 328 |
| | Concluding Remarks | 344 |
| | References | 344 |
| ch. 18 | Genetic and Genomic Approaches to Identify Genes Involved in Flagellar Assembly in Chlamydomonas Reinhardtii / Susan K. Dutcher | 349 |
| | Introduction | 350 |
| 1. | Identifying the Causative Genes Using Forward Genetics | 351 |
| 2. | Identifying Gene Function Using Reverse Genetics | 370 |
| 3. | Identifying Genes Using the RNA Transcriptome | 373 |
| 4. | Identifying Genes Using Genetic Interactions | 375 |
| 5. | Summary | 378 |
| | Acknowledgments | 378 |
| | References | 378 |
| ch. 19 | Axonemal Motility in Chlamydomonas / Ritsu Kamiya | 387 |
| | Introduction | 388 |
| 1. | Methods | 389 |
| | Acknowledgments | 400 |
| | References | 400 |
| ch. 20 | Posttranscriptional Control Over Rapid Development and Ciliogenesis in Marsilea / Corine M. van der Weele | 403 |
| | Introduction | 404 |
| 1. | The Development of Experimental Protocols in a Nonmodel System | 409 |
| 2. | Centrin Translation is Necessary for Formation of the Blepharoplast | 412 |
| 3. | Cell Fate in the Male Gametophyte of Marsilea: Only Two Types of Cells are Produced | 415 |
| 4. | Spermidine Provides a Driving Force behind Rapid Development in the Male Gametophyte | 417 |
| 5. | Spermidine Levels Change in the Gametophyte | 418 |
| 6. | Stored mRNAs are Transcribed during the Natural Process of Spore Desiccation | 420 |
| 7. | Mago Nashi and Speckle mRNA Dynamics | 423 |
| 8. | Emerging Themes from the Transcriptome of the Rapidly Developing Male Gametophyte of Marsilea | 423 |
| 9. | Many of the Stored Transcripts in the Spore Nucleus are Partially Processed Pre-mRNAs | 425 |
| 10. | NudC, an Archetype IRT in the Gametophyte | 426 |
| 11. | IRT Splicing as a Means to Regulate the Timing and Location of Translation | 427 |
| 12. | Polyadenylation as a Late Regulator of Translation | 430 |
| | Conclusions, Predictions, and a Working Model for the Control of Rapid Development | 430 |
| | Acknowledgments | 438 |
| | References | 438 |
| ch. 21 | Total Internal Reflection Fluorescence Microscopy of Intraflagellar Transport in Tetrahymena thermophila / Jacek Gaertig | 445 |
| | Introduction | 446 |
| 1. | TIRF Microscope Configuration and Data Analysis in Application to IFT in Tetrahymena | 447 |
| 2. | Using a TIRF microscope to Image Tetrahymena cilia | 447 |
| 3. | Limitations | 453 |
| | Acknowledgments | 454 |
| | Supplementary Data | 454 |
| | References | 454 |
| ch. 22 | Ciliary Heterogeneity within a Single Cell: The Paramecium Model / Michel Lemullois | 457 |
| 1. | The Paramecium Model | 458 |
| 2. | Cilia in Paramecium Organization | 460 |
| 3. | Staining the Cilia with Antitubulin Antibodies | 465 |
| 4. | Protocols | 470 |
| 5. | Conclusion | 478 |
| 6. | Chemical Reagents and Buffers | 479 |
| | Acknowledgments | 479 |
| | References | 479 |
| ch. 23 | Imaging Intraflagellar Transport in Trypanosomas / Philippe Bastin | 487 |
| | Introduction | 488 |
| 1. | Producing Trypanosomes Expressing Fluorescent IFT Fusion Proteins | 490 |
| 2. | Visualization of IFT and Acquisition of Time-Lapse Videos | 498 |
| 3. | Quantification of IFT | 500 |
| | Conclusion | 504 |
| | Acknowledgments | 504 |
| | Supplementary Data | 504 |
| | References | 504 |
| ch. 24 | Scanning and Three-Dimensional Electron Microscopy Methods for the Study of Trypanosoma brucei and Leishmania mexicana Flagella / Sue Vaughan | 509 |
| | Introduction | 510 |
| 1. | Rationale | 515 |
| 2. | Methods | 516 |
| 3. | Materials | 535 |
| 4. | Discussion | 537 |
| | Acknowledgments | 538 |
| | References | 538 |
| | Volumes in Series | 543 |
| | Index | 555 |
