Motility Assays for Motor Proteins

Name: Motility Assays for Motor Proteins
Brand: Academic Press
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Academic Press

Erschienen am 17. November 1993

303 Seiten

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Inhalt

Front Cover
Motility Assays for Motor Proteins
Copyright Page
Contents
Contributors
Preface
Chapter 1. In Vitro Methods for Measuring Force and Velocity of the Actin-Myosin Interaction Using Purified Proteins
I. Introduction
II. Preparation of in Vitro Motility Assay Components
III. In Vitro Assay for Myosin Velocity
IV. In Vitro Assay for Myosin Force in the Motility Assay
V. Components of the Optical Trap System
VI. Future Directions
References
Chapter 2. Myosin-Specific Adaptations of the Motility Assay
I. Introduction
II. Description of Equipment
III. Basic Assay Procedure
IV. Adaptations of the Assay
V. Quantitation of Rate of Actin Filament Sliding
References
Chapter 3. Motility of Myosin I on Planar Lipid Surfaces
I. Introduction
II. Methods
III. Evaluation of the Method
References
Chapter 4. Microtubule and Axoneme Gliding Assays for Force Production by Microtubule Motor Proteins
I. Introduction
II. Microtubule Gliding Assay
III. Polarity of Force Production
References
Chapter 5. Analyzing Microtubule Motors in Real Time
I. Introduction
II. Configuration of the Computer-Video Microscope Setup
III. The Microtubule Motility Assay
IV. Overview of Typical Results Obtained with the Real-Time Motility Assay
V. Conclusions
Addendum
References
Chapter 6. Assays of Axonemal Dynein-Driven Motility
I. Introduction
II. Dark-Field Video Microscopy
III. Procedures and Analysis of ATP-Induced Microtubule Sliding in Flagellar Axonemes
IV. The Second Assay: Dynein-Driven Microtubule Translocation Using Purified Proteins
V. Summary: Future
References
Chapter 7. Preparation of Marked Microtubules for the Assay of the Polarity of Microtubule-Based Motors by Fluorescence Microscopy
I. Introduction
II. Preparation of Labeled Microtubules
III. Reagents
IV. Preparation of the Bright Microtubule Seeds
V. Preparation of the Polarity-Marked Microtubules
VI. Visualization of Marked Microtubules in Motor Polarity Assays
References
Chapter 8. Expression of Microtubule Motor Proteins in Bacteria for Characterization in in Vitro Motility Assays
I. Introduction
II. Construction of Plasmids for Expression in Bacteria
III. Vectors Used for Expression of Motor Proteins
IV. Expression of Proteins in Escherichia coli
V. Partial Purification of Proteins from Bacteria for in Vitro Motility Assays
VI. Brief Description of Motility Assays for Testing the Functional Properties of the Expressed Proteins
VII. Microtubule Binding Assays
VIII. Measurement of ATPase Activity as a Determinant of Motor Protein Function
IX. Problems Encountered with Prokaryotic Expression Systems and Some Solutions
References
Chapter 9. Tracking Nanometer Movements of Single Motor Molecules
I. Introduction
II. Movements by Single Motor Molecules
III. Microtubule Movements on Motor-Coated Glass
IV. Conclusion
References
Chapter 10. Assay of Microtubule Movement Driven by Single Kinesin Molecules
I. Introduction
II. Protein Preparation
III. Flow Cells and Microscopy
IV. Microscopy and Motion Analysis
V. In Vitro Motility Assay
VI. Evidence That Single Molecules Suffice for Microtubule-Based Motility
References
Chapter 11. In Vitro Motility Assays Using Microtubules Tethered to Tetruhymena Pellicles
I. Introduction
II. Isolation of Assay Components
III. In Vitro Motility Assay I: Inducing Chromosome and Vesicle Motions with Microtubule Depolymerization
IV. In Vitro Motility Assay II: Testing Microtubule Sliding Activity of Motor Enzymes
V. Other Procedures
VI. Additional Remarks
References
Chapter 12. Use of ATP Analogs in Motor Assays
I. Introduction
II. Purity Check and Purification of ATP Analogs
III. Chemistry of ATP Analogs
IV. Additional Precautions in Using ATP Analogs
V. Uses of ATP Analogs
References
Chapter 13. Myosin-Mediated Vesicular Transport in the Extruded Cytoplasm of Characean Algae Cells
I. Introduction
II. Extruded Algal Cytoplasm Preparations
III. Methods for the Direct Visualization of Myosin-Mediated Organelle Movements
IV. Observation of Myosin-Mediated Vesicular Movements in Vitro
V. Identification of a Soluble Pool of Myosins in Nitella Cytoplasm
VI. Concluding Remarks
References
Chapter 14. Assay of Vesicle Motility in Squid Axoplasm
I. Extrusion of Isolated Axoplasm
II. Physiological Buffers
III. Video Microscopy
IV. Pitfalls
V. Summary
References
Chapter 15. Assay of Membrane Motility in Interphase and Metaphase Xenopus Extracts
I. Introduction
II. Preparation of Xenopus Extracts and Membrane Fractions
III. Motility Assay
IV. Data Acquisition, Handling, and Analysis
V. Conclusions and Prospects
References
Chapter 16. Microtubule Motor-Dependent Formation of Tubulovesicular Networks from Endoplasmic Reticulum and Golgi Membranes
I. Introduction
II. Materials and Methods
III. Discussion
References
Chapter 17. Cytoplasmic Extracts from the Eggs of Sea Urchins and Clams for the Study of Microtubule-Associated Motility and Bundling
I. Introduction
II. Sea Urchin Egg Cytoplasmic Extracts
III. Clam Egg Cytoplasmic Extracts
IV. Visualizing the Microtubule-Associated Motility and Crosslinking
V. Example Results for Cytoplasmic Extracts
References
Chapter 18. Kinesin-Mediated Vesicular Transport in a Biochemically Defined Assay
I. Introduction
II. Assay Reagents and Components
III. Motility Assay Procedure
IV. Kinesin-Mediated Dynamics
V. Biological Implications of Kinesin-Supported Motility Assays
VI. Summary
References
Chapter 19. An Assay for the Activity of Microtubule-Based Motors on the Kinetochores of Isolated Chinese Hamster Ovary Chromosomes
I. Introduction
II. Methods for Assaying Motor Activity on the Kinetochore
III. Conclusions
References
Chapter 20. The Diatom Central Spindle as a Model System for Studying Antiparallel Microtubule Interactions during Spindle Elongation in Vitro
I. Introduction
II. Diatom Cultures
III. In Vitro Models
IV. Concluding Remarks
References
Index
Volumes in Series

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