Methods in cell biology. v.92, Cilia : motors and regulation / edited by Stephen M. King, Gregory J. Pazour. — Amsterdam : Academic Press, 2009. – (58.1574/M592/v.92) |
Contents
CONTENTS
Contributors
Preface
1. Bioinformatic Approaches to Dynein Heavy Chain Classification
I. Introduction
II. Methods
III. Materials
IV. Results and Discussion
References
2. Identification and Characterization of Dynein Genes in Tetrahymena
I. Introduction
II. Methods and Results
III. Discussion
References
3. Purification of Axonemal Dyneins and Dynein-Associated Components from Chlamydomonas
I. Introduction
II. Detachment and Isolation of Flagella
III. Flagellar Demembranation
IV. Dynein Extraction from Flagellar Axonemes
V. Fractionation of Flagellar Extracts
VI. Immunoprecipitation from Cell Body Extracts
VII. Conclusions
References
4. Purification of Dyneins from Sperm Flagella
I. Introduction
II. Short Protocol for Isolation of Sperm Outer Arm Dynein from the Ascidian C. intestinalis
III. Collection of Sperm
IV. Isolation of Sperm Flagella
V. Purification of Outer Arm Dyneins
VI. Regulation of Outer Arm Dynein
VII. Purification of Inner Arm Dyneins
VIII. Conclusion
References
5. Protein Engineering Approaches to Study the Dynein Mechanism using a Dictyostelium Expression System
I. Introduction
II. Preparation of Recombinant Dynein
III. FKET-Based Detection of Dynein's Conformational Changes
IV. Motility Assays
References
6. Biophysical Measurements on Axonemal Dyneins
I. Introduction
II. Materials and Methods
III. Results and Discussion
IV. Summary
References
7. Protein Electroporation into Chlamydomonas for Mutant Rescue
I. Introduction
II. Materials and Methods
III. Discussion
References
8. Analysis of the Role of Nucleotides in Axonemal Dynein Function
I. Introduction
II. Materials and Methods
III. Methods
IV. Results and Discussion
V. Outlook
References
9. The Regulation of Dynein-Driven Microtubule Sliding in Chlamydomonas Flagella by Axonemal Kinases and Phosphatases
I. Introduction
II. The Chlamydomonas Experimental System: Axoneme Isolation and the Flagellar Mutants
III. In Vitro Microtubule Sliding Assays: The Method and Discovery of Kinases and Phosphatases that Regulate Dynein
IV. Biochemical Analysis of Kinases and Phosphatases Localized to the Chlamydomonas Axoneme
V. Discussion of the Regulatory Model and Concluding Remarks
References
10. Analysis of Redox-Sensitive Dynein Components
I. Introduction
II. Materials and Reagents
III. Methods
References
11. Calcium Regulation of Ciliary Motility: Analysis of Axonemal Calcium-Binding Proteins
I. Introduction
II. How Does Calcium Enter Motile Cilia?
III. What Ciliary Proteins Serve as Calcium Sensors?
IV. How Does a Change in Calcium Concentration Modulate Motility?
V. Summary
References
12. Isolation and Analysis of Radial Spoke Proteins
I. Introduction
II. Isolation of Intact RS Complexes
III. Two-Dimensional Gel Electrophoresis of the RS Complexes
IV. Blue Native PAGE for Characterizing Subparticles in RS Complexes
V. Summary
References
13. Analysis of the Central Pair Microtubule Complex in Chlamydomonas reinhardtii
I. Introduction
II. Basic Methods for Central Pair Structure Analysis by Thin Section Electron Microscopy
III. Genetic Dissection of the Central Pair
IV. Biochemical Dissection of the Central Pair
V. Methods to Study Central Pair Structural Conformation
VI. Summary
References