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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