Cilia, Part A

Series Page

Contributors

Preface

Methods in Enzymology

Chapter One. Isolation of Intraflagellar Transport Particle Proteins from Chlamydomonas reinhardtii

Abstract

1 Introduction

2 Media

3 Isolating Flagella

4 Preparing Soluble Flagellar Proteins

5 Isolating IFT Particle Proteins by Fractioning Soluble Flagellar Proteins Through Sucrose Density Gradient

6 Isolating IFT Particle Proteins by Immunoprecipitation from Soluble Flagellar Proteins

7 Conclusions

Acknowledgments

References

Chapter Two. Molecular Tools for Studying the Radial Spoke

Abstract

1 Introduction

2 Recovery of Genomic DNA

3 Slot Lysis Assay for Large-Scale Screenings

4 DNA Alterations

5 Tagging

6 Ni-NTA Affinity Purification of the Radial Spoke Complex

7 Flagella Miniprep

8 Summary

Acknowledgments

References

Chapter Three. Methods for Analysis of Calcium/Calmodulin Signaling in Cilia and Flagella

Abstract

1 Introduction

2 Calcium Entry into the Intraflagellar Space

3 Calcium-Binding Proteins

4 Calcium-Induced Modulation of Motility

5 Summary

References

Chapter Four. Analysis of Flagellar Protein Ubiquitination

Abstract

1 Introduction

2 Summary of Putative Ubiquitin, Ubiquitin-Like Proteins, E1, E2, and E3 in Flagella/Cilia

3 Determination of the Flagellar Localization of Candidate Proteins with Fluorescence Proteins

4 Analysis of the Free Ubiquitin in Flagella

5 In Vitro Ubiquitination System of Flagella

6 Analysis of Flagellar Ubiquitination During Flagellar Shortening

7 Analysis of Flagellar Ubiquitination During Mating

8 Summary

Acknowledgment

References

Chapter Five. Analysis of Ciliary Import

Abstract

1 Introduction

2 General Considerations

3 Analysis of the Permeability Barrier to Ciliary Entry Using Microinjection

4 Analysis of Ciliary Import by Fluorescence Recovery After Photobleaching

Acknowledgments

References

Chapter Six. Analysis of Signaling Pathways Controlling Flagellar Movements in Mammalian Spermatozoa

Abstract

1 Introduction

2 Spermatozoa Preparations

3 Expression of sAC in a Heterologous System

4 Measurement of Soluble Adenylyl Cyclase Activity

5 Measurement of cAMP Accumulation in Spermatozoa

6 Isolation of Macromolecular Complexes Including sAC

7 Summary

Acknowledgment

References

Chapter Seven. Analysis of Microtubule Plus-End-Tracking Proteins in Cilia

Abstract

1 Introduction

2 Detection of EB Proteins in Cilia by Fluorescence Microscopy

3 Functional Analysis of EB Proteins in Cultures of Ciliated Mammalian Cells

4 Identification of EB-Binding Partners in Cilia

5 Summary

Acknowledgments

References

Chapter Eight. Biochemical and Physiological Analysis of Axonemal Dyneins

Abstract

Abbreviations

1 Introduction

2 Axonemal Dynein Subcomplexes from Cytoplasm

3 Localization of Axonemal Dynein Components in Chlamydomonas Flagella

4 Analysis of Dynein-Driven Motility in Chlamydomonas

5 Conclusions

Acknowledgment

References

Chapter Nine. Measuring the Regulation of Dynein Activity During Flagellar Motility

Abstract

1 Introduction

2 Materials and Methods

3 Important Parameters in Beating Flagella

4 Microtubule Sliding Induced by Dynein

5 Dynein Activation by Mechanical Force of Bending

6 Outlook

Acknowledgments

References

Chapter Ten. Analysis of Interactions Between Intraflagellar Transport Proteins

Abstract

1 Introduction

2 Isolation of IFT Complexes from Isolated Flagella

3 Isolation of IFT Complex A from Whole Cell Extracts

4 Architectural Analysis of IFT Complex B

5 Bacterially Based Interaction Analysis of IFT B Proteins

6 Yeast-Based Interaction Analysis of IFT A Proteins

7 Summary

Acknowledgments

References

Chapter Eleven. Analysis of Ciliary Membrane Protein Dynamics Using SNAP Technology

Abstract

1 Introduction

2 Generation of SNAP-Tagged Ciliary Targeted Proteins

3 Pulse-Chase Analysis of the Trafficking of Fibrocystin-SNAP to the Cilium

4 Example Protocol

5 Future Prospects

Acknowledgment

References

Further Reading

Chapter Twelve. Engineering Oscillating Microtubule Bundles

Abstract

1 Introduction

2 Purification and Preparation of Proteins

3 Depletion-Driven Bundling of MTs

4 Glass Surface Treatment, Acrylamide Coating, and Flow Cell Construction

5 Active MT Bundle Samples

6 Protocol Qualifications and Outlook

Acknowledgments

References

Chapter Thirteen. Purification of Exosome-Like Vesicles from Urine

Abstract

1 Introduction

2 Materials

3 Collecting Crude ELVs

4 Removing the Tamm–Horsfall Protein

5 Fractionation

6 Downstream Processing

7 Conclusions

References

Chapter Fourteen. Electron Microscopy of Flagella, Primary Cilia, and Intraflagellar Transport in Flat-Embedded Cells

Abstract

1 Introduction

2 Preparing Chlamydomonas reinhardtii and Monolayers of Mammalian Culture Cells for Flat Embedding

3 Dehydration, Infiltration, and Flat Embedding

4 Preparation of Blocks and Ultramicrotomy

5 Summary

Acknowledgments

References

Chapter Fifteen. In vivo Imaging of IFT in Chlamydomonas Flagella

Abstract

1 Introduction

2 Sample Preparation

3 Visualizing IFT by DIC

4 Visualizing IFT by TIRFM

5 Analysis of IFT Using Kymograms

6 Open Questions and Future Directions

7 Useful Links

8 Summary

Acknowledgements

References

Chapter Sixteen. Measuring Rates of Intraflagellar Transport Along Caenorhabditis elegans Sensory Cilia Using Fluorescence Microscopy

Abstract

1 Introduction

2 Preparation of Living Transgenic C. elegans Expressing Fluorescent IFT Proteins

3 Direct Tracking of IFT Proteins Using Spinning Disc Confocal Microscopy and Kymography (Figs. 16.3, steps 3–7 and 16.4)

4 Indirect Estimation of the Rates of IFT Using Fluorescence Recovery After Photobleaching and Mathematical Modeling

5 Summary

Acknowledgments

References

Chapter Seventeen. 3D Structural Analysis of Flagella/Cilia by Cryo-Electron Tomography

Abstract

1 Introduction

2 Purification of Flagella

3 Preparation of Specimens for Cryo-ET

4 Cryo-Electron Microscopy and Data Acquisition

5 Tomogram Reconstruction

6 Subtomogram Averaging

7 Classification

8 Studying the Interaction of the Flagellar Component

9 Outlook

References

Chapter Eighteen. Electron Tomography of IFT Particles

Abstract

1 Introduction

2 Overview of Electron Tomography

3 Sample Preparation

4 Tomography Data Collection

5 Tomographic Reconstruction

Acknowledgments

References

Chapter Nineteen. Reconstitution of Flagellar Sliding

Abstract

1 Introduction

2 Culturing Chlamydomonas Cells

3 Purifications

4 Reconstitution Assays

5 Summary

References

Author Index

Subject Index