Cilia: Motors and Regulation

1. Auflage 2009
ISBN: 978-0-08-096107-1
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 92, 400 Seiten

Reihe: Methods in Cell Biology

Along with its companion volume on axonemal dynein-mediated motility, this book provides researchers with a comprehensive and up-to-date source of methods for the analysis cilia and flagella, focusing primarily on approaches that have been devised or significantly extended since the last volume of Methods in Cell Biology on this topic (volume 47, 1995). Edited by Stephen M. King and Gregory J. Pazour, the newest installment of this highly acclaimed serial will serve as an essential addition to the study of cilia and flagella. * Covers protocols for cilia and flagella across systems and species * Both classic and state-of-the-art methods readily adaptable across model systems, and designed to last the test of time * Relevant to clinicians interested in respiratory disease, male infertility, and other syndromes who need to learn biochemical, molecular, and genetic approaches to studying cilia, flagella, and related structures

Autoren/Hrsg.

Inhaltsverzeichnis

1;Cover;1
2;Methods in Cell Biology;4
3;Copyright Page;5
4;Contents;6
5;Contributors;10
6;Preface;14
7;Chapter 1 Bioinformatic Approaches to Dynein Heavy Chain Classification;16
7.1;I. Introduction;17
7.2;II. Methods;18
7.3;III. Materials;20
7.4;IV. Results and Discussion;21
7.5;Acknowledgments;23
7.6;References;23
8;Chapter 2 Identification and Characterization of Dynein Genes in;26
8.1;I. Introduction;27
8.2;II. Methods and Results;29
8.3;III. Discussion;41
8.4;Acknowledgments;42
8.5;References;42
9;Chapter 3 Purification of Axonemal Dyneins and Dynein-Associated Components from;46
9.1;I. Introduction;47
9.2;II. Detachment and Isolation of Flagella;49
9.3;III. Flagellar Demembranation;50
9.4;IV. Dynein Extraction from Flagellar Axonemes;51
9.5;V. Fractionation of Flagellar Extracts;51
9.6;VI. Immunoprecipitation from Cell Body Extracts;59
9.7;VII. Conclusions;61
9.8;Acknowledgments;61
9.9;References;61
10;Chapter 4 Purification of Dyneins from Sperm Flagella;64
10.1;I. Introduction;65
10.2;II. Short Protocol for Isolation of Sperm Outer Arm Dynein from the Ascidian;66
10.3;III. Collection of Sperm;67
10.4;IV. Isolation of Sperm Flagella;69
10.5;V. Purification of Outer Arm Dyneins;70
10.6;VI. Regulation of Outer Arm Dynein;76
10.7;VII. Purification of Inner Arm Dyneins;76
10.8;VIII. Conclusion;77
10.9;References;77
11;Chapter 5 Protein Engineering Approaches to Study the Dynein Mechanism using a Dictyostelium Expression System;80
11.1;I. Introduction;81
11.2;II. Preparation of Recombinant Dynein;82
11.3;III. FRET-Based Detection of Dynein’s Conformational Changes;87
11.4;IV. Motility Assays;90
11.5;Acknowledgments;95
11.6;References;95
12;Chapter 6 Biophysical Measurements on Axonemal Dyneins;98
12.1;I. Introduction;99
12.2;II. Materials and Methods;100
12.3;III. Results and Discussion;115
12.4;IV. Summary;117
12.5;Acknowledgments;118
12.6;References;118
13;Chapter 7 Protein Electroporation into Chlamydomonas for Mutant Rescue;122
13.1;I. Introduction;122
13.2;II. Materials and Methods;123
13.3;III. Discussion;125
13.4;References;125
14;Chapter 8 Analysis of the Role of Nucleotides in Axonemal Dynein Function;128
14.1;I. Introduction;129
14.2;II. Materials and Methods;132
14.3;III. Methods;134
14.4;IV. Results and Discussion;137
14.5;V. Outlook;144
14.6;Acknowledgments;145
14.7;References;145
15;Chapter 9 The Regulation of Dynein-Driven Microtubule Sliding in Chlamydomonas Flagella by Axonemal Kinases and Phosphatases;148
15.1;I. Introduction;149
15.2;II. The Chlamydomonas Experimental System: Axoneme Isolationand the Flagellar Mutants;152
15.3;III. In Vitro Microtubule Sliding Assays: The Method andDiscovery of Kinases and Phosphatases that Regulate Dynein;153
15.4;IV. Biochemical Analysis of Kinases and Phosphatases Localizedto the Chlamydomonas Axoneme;157
15.5;V. Discussion of the Regulatory Model and Concluding Remarks;160
15.6;References;162
16;Chapter 10 Analysis of Redox-Sensitive Dynein Components;168
16.1;I. Introduction;169
16.2;II. Materials and Reagents;170
16.3;III. Methods;170
16.4;Acknowledgments;175
16.5;References;175
17;Chapter 11 Calcium Regulation of Ciliary Motility: Analysis of Axonemal Calcium-Binding Proteins;178
17.1;I. Introduction;179
17.2;II. How Does Calcium Enter Motile Cilia?;179
17.3;III. What Ciliary Proteins Serve as Calcium Sensors?;180
17.4;IV. How Does a Change in Calcium Concentration Modulate Motility?;188
17.5;V. Summary;191
17.6;References;191
18;Chapter 12 Isolation and Analysis of Radial Spoke Proteins;196
18.1;I. Introduction;197
18.2;II. Isolation of Intact RS Complexes;198
18.3;III. Two-Dimensional Gel Electrophoresis of the RS Complexes;203
18.4;IV. Blue Native PAGE for Characterizing Subparticles in RS Complexes;207
18.5;V. Summary;210
18.6;References;210
19;Chapter 13 Analysis of the Central Pair Microtubule Complex in;212
19.1;I. Introduction;213
19.2;II. Basic Methods for Central Pair Structure Analysis by Thin Section Electron Microscopy;214
19.3;III. Genetic Dissection of the Central Pair;216
19.4;IV. Biochemical Dissection of the Central Pair;220
19.5;V. Methods to Study Central Pair Structural Conformation;222
19.6;VI. Summary;226
19.7;References;227
20;Index;230
21;Volumes in series;238