Cilia, Part A - 1st Edition - ISBN: 9780123979452, 9780123984869

Cilia, Part A, Volume 524

1st Edition

Serial Volume Editors: Wallace F. Marshall
eBook ISBN: 9780123984869
Hardcover ISBN: 9780123979452
Imprint: Academic Press
Published Date: 4th April 2013
Page Count: 472
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Table of Contents

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


Description

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers cilia and includes chapters on such topics as electron microscopy of IFT in cilia and flagella, radial spoke isolation and assays, and biomechanical measurements of kinocilium.

Key Features

  • Continues the legacy of this premier serial with quality chapters authored by leaders in the field
  • Covers cilia
  • Contains chapters on such topics as electron microscopy of IFT in cilia and flagella, radial spoke isolation and assays, and biomechanical measurements of kinocilium

Readership

Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists


Details

No. of pages:
472
Language:
English
Copyright:
© Academic Press 2013
Published:
Imprint:
Academic Press
eBook ISBN:
9780123984869
Hardcover ISBN:
9780123979452

About the Serial Volume Editors

Wallace F. Marshall Serial Volume Editor

Wallace Marshall is an electrical engineer by training, who became interested in biology out of a desire to understand how cells solve engineering problems, such as determining the size of organelles. He received his Ph.D. at UCSF with John Sedat, where he studied the diffusional of motion of interphase chromatin using live cell imaging and computational image analysis. He then trained as a postdoc with Joel Rosenbaum at Yale, where he began studying the mechanisms regulating the length of cilia and flagella. He is now Profess of Biochemistry at UCSF, where he lab continues to study the assembly and length regulation of cilia and flagella, as well as the mechanisms that regulate the size of other organelles. His work takes advantage of an integrated combination of methods including genetics, microscopy, and computational modeling, as well as a wide variety of model organisms including Chlamydomonas reinhardtii, Stentor coeruleus, yeast, flatworms, and mammalian cells.

Affiliations and Expertise

Department of Biochemistry & Biophysics, University of California San Fransisco at Mission Bay, USA