Cilia, Part A
- 1st Edition, Volume 524 - April 4, 2013
- Editor: Wallace F. Marshall
- Language: English
- Hardback ISBN:9 7 8 - 0 - 1 2 - 3 9 7 9 4 5 - 2
- eBook ISBN:9 7 8 - 0 - 1 2 - 3 9 8 4 8 6 - 9
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… Read more
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.
- 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
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists
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
- Edition: 1
- Volume: 524
- Published: April 4, 2013
- Language: English
WM
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, USARead Cilia, Part A on ScienceDirect