Reconstituting the Cytoskeleton

Contributors

Preface

Acknowledgment

Section I: Polymer Dynamics

Chapter One: Actin Filament Dynamics Using Microfluidics

Abstract

1 Introduction

2 Key Technical Aspects of the Microfluidic Method

3 Assets for the Study of Actin Dynamics

4 Perspectives

Chapter Two: Bacterial Actin-Like Proteins: Purification and Characterization of Self-Assembly Properties

Abstract

1 Introduction

2 Conclusion

Acknowledgments

Chapter Three: Quantitative Analysis of Microtubule Self-assembly Kinetics and Tip Structure

Abstract

1 Introduction

2 Tip-Tracking Algorithm Walkthrough

3 In Vivo Tip-Tracking Protocol

4 TipTracker Optimization Using Fixed Cells

5 Considerations for In Vitro Assays and Other Filament Systems

Acknowledgments

Section II: Polymer Nucleation and Regulation

Chapter Four: Biochemical Reconstitution of the WAVE Regulatory Complex

Abstract

1 Introduction

2 Overview of the In Vitro Reconstitution Method

3 Key Reagents

4 Reconstitution of the WAVE/Abi/HSPC300 Trimer

5 Reconstitution of the Sra1/Nap1 Dimer

6 Reconstitution of the WRC Pentamer

Acknowledgments

Chapter Five: Rotational Movement of Formins Evaluated by Using Single-Molecule Fluorescence Polarization

Abstract

1 Introduction

2 Theoretical Concepts and Optics for Single-Molecule Fluorescence Polarization

3 Proteins

4 Observation of Rotational Movement of TMR-F-Actin

5 Limiting Axial Rotation of F-Actin Using Biotinylated Actin

6 Data Analysis

7 Perspectives

Acknowledgments

Chapter Six: Single-Molecule Studies of Actin Assembly and Disassembly Factors

Abstract

1 Introduction

2 Protein Tags, Labeling, and Tethering

3 Labeling and Surface Confinement of Actin Filaments

4 Slide Surface Treatment

5 Requirements for Single-Molecule Detection

6 Microscope Considerations

7 Imaging Actin Filaments and Single Molecules

8 Dual-Color TIRF Imaging of Actin-Regulatory Mechanisms

9 Measuring Kinetics of Single-Molecule Interactions with Actin Filaments

10 Coordination of Multiple Actin-Regulatory Factors from Multicolor Imaging

Acknowledgments

Chapter Seven: Assaying Microtubule Nucleation by the γ-Tubulin Ring Complex

Abstract

1 Introduction

2 γTuRC Isolation

3 Electron Microscopy

4 Microtubule Nucleation

5 Summary

Acknowledgments

Chapter Eight: Reconstituting Dynamic Microtubule Polymerization Regulation by TOG Domain Proteins

Abstract

1 Introduction

2 Methods

3 Conclusion

Acknowledgments

Chapter Nine: Generation of Differentially Modified Microtubules Using In Vitro Enzymatic Approaches

Abstract

1 Introduction

2 Purification and Characterization of Unmodified Microtubules

3 Purification of Tubulin Modification Enzymes: α-TAT, TTLL7, and TTL

4 Generation of Differentially Modified Tubulin and Microtubules

5 Conclusions

Section III: Molecular Motor Ensembles on Natural and Engineered Cargoes

Chapter Ten: Engineering Defined Motor Ensembles with DNA Origami

Abstract

1 Introduction

2 Building the DNA Origami Structure

3 Covalent Attachment of Oligonucleotides to Motor Proteins

4 Forming the Motor–DNA Origami Complex

5 Single-Molecule Motility Analysis of Motor–DNA Origami Complexes

6 Data Analysis Considerations

7 Summary and Future Directions

Acknowledgments

Chapter Eleven: Construction and Analyses of Elastically Coupled Multiple-Motor Systems

Abstract

1 Introduction

2 Synthetic Strategy: A Modular Plug-and-Play Approach

3 Two-Motor Biophysical Assays

4 Concluding Remarks and Future Perspectives

Acknowledgments

Chapter Twelve: Reconstitution of Cortical Dynein Function

Abstract

1 Introduction

2 Reconstitution of Cortical Dynein Function in 1D Geometries

3 Reconstitution of Cortical Dynein Function in 2D Geometries

4 Reconstitution of Cortical Dynein Function in 3D Geometries

5 Discussion and Perspectives

Acknowledgments

Chapter Thirteen: Reconstitution of Microtubule-Dependent Organelle Transport

Abstract

1 Introduction

2 Isolation and Motility of LBPs from Dictyostelium

3 Isolation and Motility of LDs from Rat Liver

4 Conclusions

Acknowledgments

Chapter Fourteen: Reconstituting the Motility of Isolated Intracellular Cargoes

Abstract

1 Introduction

2 Isolation of Neuronal Transport Vesicles

3 Isolation of Latex Bead Compartments

4 In Vitro Motility Assays

5 Imaging and Analysis

6 Troubleshooting

7 Comparison to Measurements in Living Cells

8 Outlook

Acknowledgments

Section IV: Building Higher Order Networks and Interactions

Chapter Fifteen: Reconstitution of Contractile Actomyosin Arrays

Abstract

1 Introduction

2 Reagents

3 Reconstituted Actomyosin Bundles

4 Biomimetic Cortex

5 Future Directions

Acknowledgment

Chapter Sixteen: Directed Actin Assembly and Motility

Abstract

1 Introduction

2 Reconstitution of Actin-Based Motility in a G-Actin-Buffered Medium

3 Assembly of Branched Actin Networks on a Wide Range of Geometries

4 Reconstitution of 3D Connections of Structured Actin Networks

5 Concluding Remarks

Acknowledgments

Chapter Seventeen: In Vitro Reconstitution of Dynamic Microtubules Interacting with Actin Filament Networks

Abstract

1 Introduction

2 Experimental Methods

3 Outlook

4 Reagents and Stocks

Acknowledgments

Chapter Eighteen: Measuring Kinetochore–Microtubule Interaction In Vitro

Abstract

1 Introduction

2 The Leap to In Vitro: Purification of Intact, Functional Kinetochores

3 The Assays

4 Summary

Acknowledgments

Chapter Nineteen: Micropattern-Guided Assembly of Overlapping Pairs of Dynamic Microtubules

Abstract

1 Introduction

2 Reagents and Equipment

3 Glass Treatment and Surface Chemistry

4 Microtubule Overlap Assay on Micropattern

5 Discussion

Acknowledgments

Section V: Cell Extract Systems

Chapter Twenty: WAVE Regulatory Complex Activation

Abstract

1 Introduction

2 Generating Phospholipid Bilayer-Coated Silica Microspheres

3 Preparation of Brain Extract

4 Assay of WAVE-Dependent Motility by Phospholipid-Coated Microspheres

5 Reconstituting Activation of WRC by Cooperating Arf and Rac1 GTPases

6 Concluding Remarks

Acknowledgments

Chapter Twenty-One: Dissecting Principles Governing Actin Assembly Using Yeast Extracts

Abstract

1 Introduction

2 Yeast Culture and Extract Preparation

3 Surface Protein Activation

4 Actin Assembly from Yeast Extracts

5 Deducing Protein Functions with Mutant Strains

6 Perspectives

Acknowledgments

Chapter Twenty-Two: Xenopus Egg Cytoplasm with Intact Actin

Abstract

1 Introduction

2 Actin-Intact Extract Preparation

3 Cell Cycle-Regulated Actomyosin Contractility

4 Cell-Free Reconstitution of I-Phase Aster Growth and Interaction

Acknowledgments

Chapter Twenty-Three: Glycogen-Supplemented Mitotic Cytosol for Analyzing Xenopus Egg Microtubule Organization

Abstract

1 Introduction

2 Development of the Cytosol System

3 Preparation of Glycogen-Supplemented Cytosol

4 Assay of Ran-Promoted Microtubule Assembly

5 Self-Organization of Spindle-Like Microtubule Arrays

6 Antibody-Based Fluorescent Probes

7 Future Directions

Acknowledgments

Chapter Twenty-Four: Spindle Assembly on Immobilized Chromatin Micropatterns

Abstract

1 Introduction

2 Overview of the Method

3 Preparative Steps

4 Patterning

5 Spindle Arrays

6 Analysis

7 Conclusions

Acknowledgments

Author Index

Subject Index