Cilia, Part B - 1st Edition - ISBN: 9780123979445, 9780123984852

Cilia, Part B, Volume 525

1st Edition

Serial Volume Editors: Wallace F. Marshall
eBook ISBN: 9780123984852
Hardcover ISBN: 9780123979445
Imprint: Academic Press
Published Date: 16th April 2013
Page Count: 498
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Table of Contents

Contributors

Preface

Methods in Enzymology

Chapter One. Non-Motile Primary Cilia as Fluid Shear Stress Mechanosensors

1 Introduction

2 Identification of Primary Cilium

3 Mechanosensory Cilia

4 Summary

Acknowledgments

References

Chapter Two. Biomechanical Measurement of Kinocilium

1 Introduction

2 Sample Preparation and Measurement

3 Determining Mechanical Properties from Experimental Measurements

4 Method Discussion

Acknowledgments

References

Chapter Three. Analysis of Primary Cilia in Directional Cell Migration in Fibroblasts

1 Introduction

2 Localization of Primary Cilia by Immunofluorescence Microscopy

3 Cell Migration and Orientation of Primary Cilia in Scratch Assays

4 Cell Migration and Ciliary Signaling in Micropipette Assays

5 Summary

Acknowledgments

References

Chapter Four. Sperm Patch-Clamp

1 Introduction

2 Equipment

3 Isolation of Spermatozoa for Patch-Clamp Experiments

4 Patch-Clamp Recording

5 Potential Pitfalls Associated with Sperm Patch Clamp

6 Conclusions

Acknowledgments

References

Chapter Five. Laser Trap Measurements of Flagellar Membrane Motility

1 Introduction

2 Laser Trap Overview

3 Measuring Flagellar Membrane Transport

Acknowledgments

References

Chapter Six. Probing Ciliogenesis Using Micropatterned Substrates

1 Introduction

2 Micropatterned Substrates Fabrication

3 Cells Plating on Micropatterned Slides

4 Cell Fixation and Immunofluorescence

5 Automated Image Acquisition

6 Automated Image Analysis

Acknowledgments

References

Chapter Seven. Fluorescent Imaging of Drosophila melanogaster Sperm in the Reproductive Tract

1 Introduction

2 Imaging the Fly Sperm

3 The Fly Sperm as a Model for Studying Flagellar Motility

Acknowledgments

References

Chapter Eight. Visualization of Mouse Nodal Cilia and Nodal Flow

1 Introduction and Historical Overview

2 Tools and Solutions

3 Dissection Procedures

4 Observation by Light Microscopy

5 Discussion

References

Chapter Nine. Dissecting the Functional Interplay Between the TOR Pathway and the Cilium in Zebrafish

1 Introduction

2 Manipulation of the TOR Pathway in Zebrafish

3 Analysis of Cilia Length in Zebrafish

4 Cilia Motility and Fluid Flow Dynamics in the KV

5 Assessing for Laterality Defects in Zebrafish

6 Utilizing Chlamydomonas to Assay for TOR-Mediated Cilia Length Control

7 Summary

Acknowledgments

References

Chapter Ten. Using Xenopus Skin to Study Cilia Development and Function

1 Introduction

2 Molecular Manipulation of Xenopus Ciliated Epithelia

3 Visualization of Cilia in Xenopus Embryos

4 Embryological Manipulations Useful for the Analysis of Ciliated Epithelia

5 Conclusion

Acknowledgments

References

Chapter Eleven. Zebrafish Cilia

1 Introduction

2 Molecular Approaches to Ciliogenesis in Zebrafish

3 Wholemount Confocal Imaging of Zebrafish Cilia

4 Electron Microscopy Methods for Fish Cilia

5 Live Imaging of Zebrafish Cilia Using arl13b:GFP Expression

6 Summary

Acknowledgments

References

Chapter Twelve. Analysis of Ciliary Assembly and Function in Planaria

1 Introduction

2 Maintaining Planarians in the Lab

3 Genetic Manipulation of Planaria

4 Imaging Planarian Cilia

5 Planaria Gliding and Ciliary Motility Assays

6 Summary

References

Chapter Thirteen. Discovery and Functional Evaluation of Ciliary Proteins in Tetrahymena thermophila

1 Introduction

2 Cell Culture

3 Deciliation, Purification, and Fractionation of Cilia

4 Localization of Proteins in Tetrahymena

5 Phenotypic Studies on Live Ciliary Mutants

6 Summary

Acknowledgments

References

Chapter Fourteen. Analysis of Ciliogenesis in Primary Culture Mouse Tracheal Epithelial Cells

1 Introduction

2 Generation of MTEC Cultures

3 Staging Ciliogenesis in MTEC Preparations

4 Assessment of Ciliogenesis and MCCs by Transmission Electron Microscopy

5 Gene Transfer, Drug Treatment, and FACS of MTEC Preparations

6 Summary

Acknowledgments

References

Chapter Fifteen. Isolation of Mammalian Primary Cilia

1 Introduction

2 Cell Culture

3 Isolation of Mammalian Primary Cilia

4 Protein Correlation Profiling

5 Immunofluorescence Microscopy of Primary Cilia

6 Summary

Acknowledgments

References

Chapter Sixteen. Finding Ciliary Genes

1 Introduction

2 Choice of Bioinformatics Software

3 Retrieving Genome (Sequence) Data

4 Finding Orthologous Genes

5 Searching Sequences for Regulatory Elements

6 Scoring Candidate Genes

7 Summary and Discussion

Acknowledgments

References

Chapter Seventeen. Chemical Screening Methods for Flagellar Phenotypes in Chlamydomonas

1 Introduction

2 Screen Design Considerations

3 Flagellar Length Assay

4 Deflagellation Assay

5 Motility Assay

6 Nonflagellar Phenotypes

7 Data Analysis

8 Target Validation and Identification

9 Summary

Acknowledgments

References

Chapter Eighteen. Centrosome Isolation and Analysis by Mass Spectrometry-Based Proteomics

1 Introduction

2 Centrosome Isolation

3 Centrosome Protein Identification

4 Probing Centrosome Protein Properties by Quantitative Proteomics

5 Summary

Acknowledgments

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 methods for studying ciliary polarity in Xenopus, analysis of signaling pathways in mammalian spermatozoa, and biochemical and physiological analysis of axonemal dyneins.

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 methods for studying ciliary polarity in Xenopus,
    analysis of signaling pathways in mammalian spermatozoa, and biochemical and physiological analysis of axonemal dyneins

Readership

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


Details

No. of pages:
498
Language:
English
Copyright:
© Academic Press 2013
Published:
Imprint:
Academic Press
eBook ISBN:
9780123984852
Hardcover ISBN:
9780123979445

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