Cilia: Motors and Regulation (eBook)

Cover 1
Methods in Cell Biology 4
Copyright Page 5
Contents 6
Contributors 10
Preface 14
Chapter 1 Bioinformatic Approaches to Dynein Heavy Chain Classification 16
I. Introduction 17
II. Methods 18
III. Materials 20
IV. Results and Discussion 21
Acknowledgments 23
References 23
Chapter 2 Identification and Characterization of Dynein Genes in 26
I. Introduction 27
II. Methods and Results 29
III. Discussion 41
Acknowledgments 42
References 42
Chapter 3 Purification of Axonemal Dyneins and Dynein-Associated Components from 46
I. Introduction 47
II. Detachment and Isolation of Flagella 49
III. Flagellar Demembranation 50
IV. Dynein Extraction from Flagellar Axonemes 51
V. Fractionation of Flagellar Extracts 51
VI. Immunoprecipitation from Cell Body Extracts 59
VII. Conclusions 61
Acknowledgments 61
References 61
Chapter 4 Purification of Dyneins from Sperm Flagella 64
I. Introduction 65
II. Short Protocol for Isolation of Sperm Outer Arm Dynein from the Ascidian 66
III. Collection of Sperm 67
IV. Isolation of Sperm Flagella 69
V. Purification of Outer Arm Dyneins 70
VI. Regulation of Outer Arm Dynein 76
VII. Purification of Inner Arm Dyneins 76
VIII. Conclusion 77
References 77
Chapter 5 Protein Engineering Approaches to Study the Dynein Mechanism using a Dictyostelium Expression System 80
I. Introduction 81
II. Preparation of Recombinant Dynein 82
III. FRET-Based Detection of Dynein’s Conformational Changes 87
IV. Motility Assays 90
Acknowledgments 95
References 95
Chapter 6 Biophysical Measurements on Axonemal Dyneins 98
I. Introduction 99
II. Materials and Methods 100
III. Results and Discussion 115
IV. Summary 117
Acknowledgments 118
References 118
Chapter 7 Protein Electroporation into Chlamydomonas for Mutant Rescue 122
I. Introduction 122
II. Materials and Methods 123
III. Discussion 125
References 125
Chapter 8 Analysis of the Role of Nucleotides in Axonemal Dynein Function 128
I. Introduction 129
II. Materials and Methods 132
III. Methods 134
IV. Results and Discussion 137
V. Outlook 144
Acknowledgments 145
References 145
Chapter 9 The Regulation of Dynein-Driven Microtubule Sliding in Chlamydomonas Flagella by Axonemal Kinases and Phosphatases 148
I. Introduction 149
II. The Chlamydomonas Experimental System: Axoneme Isolationand the Flagellar Mutants 152
III. In Vitro Microtubule Sliding Assays: The Method andDiscovery of Kinases and Phosphatases that Regulate Dynein 153
IV. Biochemical Analysis of Kinases and Phosphatases Localizedto the Chlamydomonas Axoneme 157
V. Discussion of the Regulatory Model and Concluding Remarks 160
References 162
Chapter 10 Analysis of Redox-Sensitive Dynein Components 168
I. Introduction 169
II. Materials and Reagents 170
III. Methods 170
Acknowledgments 175
References 175
Chapter 11 Calcium Regulation of Ciliary Motility: Analysis of Axonemal Calcium-Binding Proteins 178
I. Introduction 179
II. How Does Calcium Enter Motile Cilia? 179
III. What Ciliary Proteins Serve as Calcium Sensors? 180
IV. How Does a Change in Calcium Concentration Modulate Motility? 188
V. Summary 191
References 191
Chapter 12 Isolation and Analysis of Radial Spoke Proteins 196
I. Introduction 197
II. Isolation of Intact RS Complexes 198
III. Two-Dimensional Gel Electrophoresis of the RS Complexes 203
IV. Blue Native PAGE for Characterizing Subparticles in RS Complexes 207
V. Summary 210
References 210
Chapter 13 Analysis of the Central Pair Microtubule Complex in 212
I. Introduction 213
II. Basic Methods for Central Pair Structure Analysis by Thin Section Electron Microscopy 214
III. Genetic Dissection of the Central Pair 216
IV. Biochemical Dissection of the Central Pair 220
V. Methods to Study Central Pair Structural Conformation 222
VI. Summary 226
References 227
Index 230
Volumes in series 238