Reconstituting the Cytoskeleton - 1st Edition - ISBN: 9780123979247, 9780123984807

Reconstituting the Cytoskeleton, Volume 540

1st Edition

Serial Volume Editors: Ron Vale
eBook ISBN: 9780123984807
Hardcover ISBN: 9780123979247
Imprint: Academic Press
Published Date: 27th March 2014
Page Count: 512
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Table of Contents




Section I: Polymer Dynamics

Chapter One: Actin Filament Dynamics Using Microfluidics


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


1 Introduction

2 Conclusion


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


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


Section II: Polymer Nucleation and Regulation

Chapter Four: Biochemical Reconstitution of the WAVE Regulatory Complex


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


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


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


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


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


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


1 Introduction

2 γTuRC Isolation

3 Electron Microscopy

4 Microtubule Nucleation

5 Summary


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


1 Introduction

2 Methods

3 Conclusion


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


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


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


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


1 Introduction

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

3 Two-Motor Biophysical Assays

4 Concluding Remarks and Future Perspectives


Chapter Twelve: Reconstitution of Cortical Dynein Function


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


Chapter Thirteen: Reconstitution of Microtubule-Dependent Organelle Transport


1 Introduction

2 Isolation and Motility of LBPs from Dictyostelium

3 Isolation and Motility of LDs from Rat Liver

4 Conclusions


Chapter Fourteen: Reconstituting the Motility of Isolated Intracellular Cargoes


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


Section IV: Building Higher Order Networks and Interactions

Chapter Fifteen: Reconstitution of Contractile Actomyosin Arrays


1 Introduction

2 Reagents

3 Reconstituted Actomyosin Bundles

4 Biomimetic Cortex

5 Future Directions


Chapter Sixteen: Directed Actin Assembly and Motility


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


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


1 Introduction

2 Experimental Methods

3 Outlook

4 Reagents and Stocks


Chapter Eighteen: Measuring Kinetochore–Microtubule Interaction In Vitro


1 Introduction

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

3 The Assays

4 Summary


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


1 Introduction

2 Reagents and Equipment

3 Glass Treatment and Surface Chemistry

4 Microtubule Overlap Assay on Micropattern

5 Discussion


Section V: Cell Extract Systems

Chapter Twenty: WAVE Regulatory Complex Activation


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


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


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


Chapter Twenty-Two: Xenopus Egg Cytoplasm with Intact Actin


1 Introduction

2 Actin-Intact Extract Preparation

3 Cell Cycle-Regulated Actomyosin Contractility

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


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


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


Chapter Twenty-Four: Spindle Assembly on Immobilized Chromatin Micropatterns


1 Introduction

2 Overview of the Method

3 Preparative Steps

4 Patterning

5 Spindle Arrays

6 Analysis

7 Conclusions


Author Index

Subject Index


This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers cytoskeletal structure, including such topics as rotational movement of formins studied by fluorescence polarization microscopy, in vitro assembly assays for bacterial actin filaments, and modulators of microtubule plus end growth.

Key Features

  • Continues the legacy of this premier serial with quality chapters authored by leaders in the field
  • Covers cytoskeletal structure
  • Contains chapters with such topics as reconstitution of organelle transport and assaying microtubule nucleation by gamma-tubulin ring complex.


Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists


No. of pages:
© Academic Press 2014
Academic Press
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Ratings and Reviews

About the Serial Volume Editors

Ron Vale Serial Volume Editor

Ronald D. Vale is Professor of the Dept. of Cellular and Molecular Pharmacology at the University of California, San Francisco and is an Investigator in the Howard Hughes Medical Institute.

Vale received B.A. degrees in Biology and Chemistry from the University of California, Santa Barbara in 1980. Dr. Vale received his Ph.D. in Neuroscience from Stanford University in 1985 where he trained with Dr. Eric Shooter. He was a Staff Fellow with the N.I.H. stationed at the Marine Biological Laboratory in 1985-6 (with Tom Reese) and began his faculty appointment at UCSF in 1987. During his time at UCSF, he has served as the Director of the Cell Biology Program (4 years) and Vice-Chair (10 years) and Chair of the Dept. of Cellular and Molecular Pharmacology (5 years). He also holds an Adjunct Senior Scientist appointment with the Marine Biological Laboratory at Woods Hole.

Vale has served the scientific community by his participation on an NIH study section (1996-2002; chairing from 2000-2002). He served as President of the American Society of Cell Biology and on their International Affairs Committee. Vale is active in many educational activities. He founded and produces iBioSeminars, lectures by leading biologists which are made freely available on the web. Vale co-directed the 7 week Physiology Course at the Marine Biological Laboratory, transforming it to a venue of interdisciplinary research in physics and biology (2004-2008). His laboratory developed MicroManager, a widely used open source and freely available software package for microscopy. He also developed an educational web site on microscopy for elementary school children. Vale is active in helping young scientists in India by starting the very popular Young Investigator Meetings as well as an interactive web site ( for India biologists to obtain information on jobs/grants/collaborations.

Dr. Vale’s awards include the Pfizer Award in Enzyme Chemistry (1991), the Young Investigator Award from the Biophysical Society (1993), the Wiley Prize in Biomedical Sciences (2012), and the Albert Lasker Award for Basic Medical Research (2012). He was elected to the National Academy of Sciences in 2001 and to the American Academy of Sciences in 2002.

Affiliations and Expertise

Dept. of Cellular and Molecular Pharmacology at the University of California, San Francisco, USA