Cilia, Part B

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