Cilia, Part B

Cilia, Part B

First published on March 16, 2013

Write a review

  • Editor: Wallace F. Marshall
  • Hardcover ISBN: 9780123979445
  • eBook ISBN: 9780123984852

Purchase options

Purchase options
Available
DRM-free (EPub, Mobi, PDF)
Sales tax will be calculated at check-out

Institutional Subscription

Free Global Shipping
No minimum order

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

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

Product details

  • No. of pages: 498
  • Language: English
  • Copyright: © Academic Press 2013
  • Published: March 16, 2013
  • Imprint: Academic Press
  • Hardcover ISBN: 9780123979445
  • eBook ISBN: 9780123984852

About the Serial Volume Editor

Wallace F. Marshall

Wallace F. Marshall
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

Ratings and Reviews

Write a review

There are currently no reviews for "Cilia, Part B"