Reconstituting the Cytoskeleton 1st Edition

Ronald D. Vale pdf download

https://ebookname.com/product/reconstituting-the-cytoskeleton-1st-edition-ronald-d-vale/

★★★★★ 4.8/5.0 (37 reviews) ✓ 163 downloads ■ TOP RATED

"Amazing book, clear text and perfect formatting!" - John R.

DOWNLOAD EBOOK
 Reconstituting the Cytoskeleton 1st Edition Ronald D. Vale
pdf download

TEXTBOOK EBOOK EBOOK GATE

Available Formats

■ PDF eBook Study Guide TextBook

EXCLUSIVE 2025 EDUCATIONAL COLLECTION - LIMITED TIME

INSTANT DOWNLOAD VIEW LIBRARY

Instant digital products (PDF, ePub, MOBI) available
Download now and explore formats that suit you...

The Plant Cytoskeleton Bo Liu

https://ebookname.com/product/the-plant-cytoskeleton-bo-liu/

Reconstituting the Curriculum 1st Edition M. Rafiq

Islam

https://ebookname.com/product/reconstituting-the-curriculum-1st-
edition-m-rafiq-islam/

The Pete Seeger Reader Ronald D. Cohen (Editor)

https://ebookname.com/product/the-pete-seeger-reader-ronald-d-
cohen-editor/

Functional materials for sustainable energy

applications 1st Edition John Kilner

https://ebookname.com/product/functional-materials-for-
sustainable-energy-applications-1st-edition-john-kilner/
Ghettostadt Lodz and the Making of a Nazi City 1st
Edition Gordon J. Horwitz

https://ebookname.com/product/ghettostadt-lodz-and-the-making-of-
a-nazi-city-1st-edition-gordon-j-horwitz/

Knowledge Leadership The Art and Science of the

Knowledge based Organization KMCI Press Steven Cavaleri

https://ebookname.com/product/knowledge-leadership-the-art-and-
science-of-the-knowledge-based-organization-kmci-press-steven-
cavaleri/

Historical Geology Evolution of the Earth and Life

Through Time Sixth Edition Reed Wicander

https://ebookname.com/product/historical-geology-evolution-of-
the-earth-and-life-through-time-sixth-edition-reed-wicander/

Private Wealth Management The Complete Reference for

the Personal Financial Planner 8th Edition G. Victor
Hallman

https://ebookname.com/product/private-wealth-management-the-
complete-reference-for-the-personal-financial-planner-8th-
edition-g-victor-hallman/

Decision by Objectives 1st Edition Ernest H. Forman

https://ebookname.com/product/decision-by-objectives-1st-edition-
ernest-h-forman/
Medical And Care 3 Studies in Health Technology and
Informatics Studies in Health Technology and
Informatics 1st Edition Et Al

https://ebookname.com/product/medical-and-care-3-studies-in-
health-technology-and-informatics-studies-in-health-technology-
and-informatics-1st-edition-et-al/
METHODS IN ENZYMOLOGY

Editors-in-Chief

JOHN N. ABELSON and MELVIN I. SIMON

Division of Biology
California Institute of Technology
Pasadena, California

ANNA MARIE PYLE

Departments of Molecular, Cellular and Developmental
Biology and Department of Chemistry Investigator
Howard Hughes Medical Institute
Yale University

Founding Editors

SIDNEY P. COLOWICK and NATHAN O. KAPLAN

Academic Press is an imprint of Elsevier
525 B Street, Suite 1800, San Diego, CA 92101-4495, USA
225 Wyman Street, Waltham, MA 02451, USA
Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford, OX5 1GB, UK
32 Jamestown Road, London NW1 7BY, UK

First edition 2014

Copyright © 2014, Elsevier Inc. All Rights Reserved.

No part of this publication may be reproduced, stored in a retrieval system or transmitted in

any form or by any means electronic, mechanical, photocopying, recording or otherwise
without the prior written permission of the publisher

Permissions may be sought directly from Elsevier’s Science & Technology Rights
Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333;
email: [email protected]. Alternatively you can submit your request online by
visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting
Obtaining permission to use Elsevier material

Notice
No responsibility is assumed by the publisher for any injury and/or damage to persons or
property as a matter of products liability, negligence or otherwise, or from any use or
operation of any methods, products, instructions or ideas contained in the material herein.
Because of rapid advances in the medical sciences, in particular, independent verification of
diagnoses and drug dosages should be made

For information on all Academic Press publications

visit our website at store.elsevier.com

ISBN: 978-0-12-397924-7
ISSN: 0076-6879

Printed and bound in United States of America

14 15 16 17 11 10 9 8 7 6 5 4 3 2 1
CONTRIBUTORS

Anna Akhmanova
Division of Cell Biology, Faculty of Science, Utrecht University, Padualaan, Utrecht,
The Netherlands
Jawdat Al-Bassam
Molecular Cellular Biology, University of California Davis, California, USA
Charles L. Asbury
Department of Physiology and Biophysics, University of Washington School of Medicine,
Seattle, Washington, USA
Pradeep Barak
Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba,
Mumbai, Maharashtra, India
Peter Bieling
Department of Bioengineering and Biophysics Group, University of California-Berkeley,
Berkeley, and Department of Cellular and Molecular Pharmacology, University of
California-San Francisco, San Francisco, California, USA
Sue Biggins
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington,
USA
Laurent Blanchoin
Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV, Laboratoire de
Physiologie Cellulaire et Végétale, CNRS/CEA/INRA/UJF, Grenoble, France
Rajaa Boujemaa-Paterski
Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV, Laboratoire de
Physiologie Cellulaire et Végétale, CNRS/CEA/INRA/UJF, Grenoble, France
Marie-France Carlier
Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France
Brian T. Castle
Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota,
USA
Baoyu Chen
Department of Biophysics and Howard Hughes Medical Institute, University of Texas
Southwestern Medical Center at Dallas, Dallas, Texas, USA
Yuk-Kwan Choi
Division of Life Science, The Hong Kong University of Science and Technology, Hong
Kong, China
Pamela E. Constantinou
Departments of Chemistry and Bioengineering, Rice University, Houston, Texas, USA

xiii
xiv Contributors

Michael R. Diehl
Departments of Chemistry and Bioengineering, Rice University, Houston, Texas, USA
Serge Dmitrieff
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg,
Germany
Marileen Dogterom
FOM Institute AMOLF, Science Park, Amsterdam, The Netherlands
Jonathan W. Driver
Department of Physiology and Biophysics, University of Washington School of Medicine,
Seattle, Washington, and Departments of Chemistry and Bioengineering, Rice University,
Houston, Texas, USA
David G. Drubin
Department of Molecular and Cell Biology, University of California, Berkeley,
California, USA
Alok Kumar Dubey
Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba,
Mumbai, Maharashtra, India
Christine M. Field
Department of Systems Biology, Harvard Medical School, Boston, and The Marine
Biological Laboratory, Woods Hole, Massachusetts, USA
Daniel A. Fletcher
Department of Bioengineering and Biophysics Group, University of California-Berkeley,
Berkeley, California, USA
Franck J. Fourniol
London Research Institute, Cancer Research UK, London, United Kingdom
Rémi Galland
Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV, Laboratoire de
Physiologie Cellulaire et Végétale, CNRS/CEA/INRA/UJF, Grenoble, France
Margaret Gardel
Department of Physics, Institute for Biophysical Dynamics, and James Franck Institute,
University of Chicago, Chicago, Illinois, USA
Melissa K. Gardner
Department of Genetics, Cell Biology & Development, University of Minnesota,
Minneapolis, Minnesota, USA
Christopher P. Garnham
Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke,
Bethesda, Maryland, USA
Jeff Gelles
Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA
Contributors xv

Yale E. Goldman
Pennsylvania Muscle Institute, and Department of Physiology, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
Bruce L. Goode
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis
University, Waltham, Massachusetts, USA
Brian S. Goodman
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA
Ilya Grigoriev
Division of Cell Biology, Faculty of Science, Utrecht University, Padualaan, Utrecht,
The Netherlands
Aaron C. Groen
Department of Systems Biology, Harvard Medical School, Boston, and The Marine
Biological Laboratory, Woods Hole, Massachusetts, USA
Christophe Guérin
Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV, Laboratoire de
Physiologie Cellulaire et Végétale, CNRS/CEA/INRA/UJF, Grenoble, France
Adam G. Hendricks
Pennsylvania Muscle Institute, and Department of Physiology, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
Lisa Henry
Department of Biophysics and Howard Hughes Medical Institute, University of Texas
Southwestern Medical Center at Dallas, Dallas, Texas, USA
Erika L.F. Holzbaur
Pennsylvania Muscle Institute, and Department of Physiology, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
Florian Huber
FOM Institute AMOLF, Science Park, Amsterdam, The Netherlands
Peter J. Hume
Department of Pathology, University of Cambridge, Cambridge, United Kingdom
Daniel Humphreys
Department of Pathology, University of Cambridge, Cambridge, United Kingdom
Keisuke Ishihara
Department of Systems Biology, Harvard Medical School, Boston, and The Marine
Biological Laboratory, Woods Hole, Massachusetts, USA
D. Kenneth Jamison
Departments of Chemistry and Bioengineering, Rice University, Houston, Texas, USA
Antoine Jégou
Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France
xvi Contributors

Gijsje H. Koenderink
FOM Institute AMOLF, Science Park, Amsterdam, The Netherlands
Vassilis Koronakis
Department of Pathology, University of Cambridge, Cambridge, United Kingdom
Liedewij Laan
Fas Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA
Duck-Yeon Lee
Biochemistry Core, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA
Tai-De Li
Department of Bioengineering and Biophysics Group, University of California-Berkeley,
Berkeley, California, USA
Roop Mallik
Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba,
Mumbai, Maharashtra, India
Alphée Michelot
Physics of the Cytoskeleton and Morphogenesis Group, institut de Recherches en
Technologies et Sciences pour le Vivant, iRTSV, LPCV/CNRS/CEA/INRA/UJF,
Grenoble, France
Timothy J. Mitchison
Department of Systems Biology, Harvard Medical School, Boston, and The Marine
Biological Laboratory, Woods Hole, Massachusetts, USA
Hiroaki Mizuno
Laboratory of Single-Molecule Cell Biology, Tohoku University Graduate School of Life
Sciences, Sendai, Miyagi, Japan
R. Dyche Mullins
Department of Cellular and Molecular Pharmacology, University of California-San
Francisco, San Francisco, California, and Howard Hughes Medical Institute, Chevy Chase,
Maryland, USA
Michael Murrell
Departments of Biomedical Engineering and Materials Science and Engineering, University
of Wisconsin, Madison, Wisconsin, USA
François Nédélec
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg,
Germany
Phuong A. Nguyen
Department of Systems Biology, Harvard Medical School, Boston, and The Marine
Biological Laboratory, Woods Hole, Massachusetts, USA
David J. Odde
Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota,
USA
Contributors xvii

Shae B. Padrick
Department of Biophysics and Howard Hughes Medical Institute, University of Texas
Southwestern Medical Center at Dallas, Dallas, Texas, USA
Natalie A. Petek
Department of Cellular and Molecular Pharmacology, University of California,
San Francisco, California, USA
Andrew F. Powers
Department of Physiology and Biophysics, University of Washington School of Medicine,
Seattle, Washington, USA
Louis S. Prahl
Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota,
USA
Magdalena Preciado López
FOM Institute AMOLF, Science Park, Amsterdam, The Netherlands
Céline Pugieux
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg,
Germany
Robert Z. Qi
Division of Life Science, The Hong Kong University of Science and Technology, Hong
Kong, China
Ashim Rai
Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba,
Mumbai, Maharashtra, India
Priyanka Rai
Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba,
Mumbai, Maharashtra, India
Samara L. Reck-Peterson
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA
Arthur Rogers
Departments of Chemistry and Bioengineering, Rice University, Houston, Texas, USA
Antonina Roll-Mecak
Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, and
Center for Biophysics, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA
Guillaume Romet-Lemonne
Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France
Michael K. Rosen
Department of Biophysics and Howard Hughes Medical Institute, University of Texas
Southwestern Medical Center at Dallas, Dallas, Texas, USA
Sophie Roth
FOM Institute AMOLF, Science Park, Amsterdam, The Netherlands
xviii Contributors

Krishna K. Sarangapani
Department of Physiology and Biophysics, University of Washington School of Medicine,
Seattle, Washington, USA
Benjamin A. Smith
Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA
Michel O. Steinmetz
Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen, Switzerland
Cristian Suarez
Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV, Laboratoire de
Physiologie Cellulaire et Végétale, CNRS/CEA/INRA/UJF, Grenoble, France
Thomas Surrey
London Research Institute, Cancer Research UK, London, United Kingdom
Katarzyna Tarnawska
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg,
Germany
Todd Thoresen
Department of Physics, Institute for Biophysical Dynamics, and James Franck Institute,
University of Chicago, Chicago, Illinois, USA
Manuel Théry
Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV, Laboratoire de
Physiologie Cellulaire et Végétale, CNRS/CEA/INRA/UJF, Grenoble, France
Annapurna Vemu
Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke,
Bethesda, Maryland, USA
Naoki Watanabe
Laboratory of Single-Molecule Cell Biology, Tohoku University Graduate School of Life
Sciences, Sendai, Miyagi, Japan
PREFACE: THE ROLE OF RECONSTITUTION IN
CYTOSKELETON RESEARCH

Reconstitution is an experimental strategy that aims to recapitulate a cellular

activity outside of a cell so that it can be studied in a “test tube”-like envi-
ronment. Being able to manipulate molecules in vitro provides an unparal-
leled opportunity to dissect mechanism in ways that cannot be easily
achieved in a living cell. Many of the most important discoveries in cell
and molecular biology in the twentieth century utilized reconstitution;
examples include the classic mechanistic studies of DNA replication,
ubiquitin-mediated protein degradation, protein insertion into the endo-
plasmic reticulum, and vesicular trafficking to the cell surface.
Reconstitution also has had a rich history in the cytoskeleton field.
A notable early success was Albert Szent-Gyorgyi’s in vitro reconstitution of
the contraction of actomyosin threads in the early 1940s (Szent-Györgyi,
1942), which allowed him to dissect the molecules involved in muscle
contraction. For cilia and flagella motility, Summers and Gibbons (1971) dem-
onstrated microtubule sliding in permeabilized axonemes, which helped
to understand how dynein motors drive axonemal beating. In the 1980s,
Sheetz and Spudich (1983) directly observed the motility of myosin-coated
beads along actin cables from dissected Nitella cells; shortly thereafter,
the Spudich lab demonstrated myosin motility on purified actin filaments.
At a similar time, several in vitro motility assays were developed for studying
the molecular motors that drive the transport of membrane organelles in nerve
cells (Vale, Schnapp, Reese, & Sheetz, 1985). Collectively, these in vitro assays
were essential stepping stones for the single-molecule assays that soon
followed. In vitro motility assays, single-molecule measurement techniques,
and protein expression in bacteria or insect cells opened the doors to the
study of many molecular motors that could not be easily studied in living
cells. Thus, reconstitution was essential for broadening the investigation of
cellular motility beyond the classical systems of muscle and cilia.
Understanding the dynamic properties of cytoskeletal polymers also was
aided by reconstitution strategies. Shinya Inoue first observed the dynamic
nature of the cytoskeletal fibers that comprise the mitotic spindle (Inoue,
1953). More than two decades later, the polymerization and depolymeriza-
tion of purified tubulin were demonstrated in vitro when the correct buffer
conditions were identified (Weisenberg, 1972). Later, Mitchison and

xix
xx Preface

Kirschner (1984) demonstrated that microtubules grow slowly followed by

catastrophic shrinkage. This unusual behavior (termed “dynamic instabil-
ity”) was first observed with purified tubulin and then later confirmed to
occur in living cells. For the actin cytoskeleton, the first principles of poly-
merization of purified actin were derived by Oosawa and colleagues in the
early 1960s (Kasai, Asakura, & Oosawa, 1960), and later Pollard and
coworkers (Woodrum, Rich, & Pollard, 1975) developed a more detailed
understanding of subunit addition to the two ends of an actin filament.
In addition to work on purified proteins, reconstitution strategies have
been developed for complex systems of cytoskeletal filaments. For example,
using frog egg extracts, Murray and colleagues (Shamu & Murray, 1992)
observed mitotic spindle formation and anaphase movement of chromo-
somes, a complex system involving the orchestrated actions of hundreds
of proteins. Heald et al. (1996) followed by demonstrating that DNA-coated
beads can replace natural chromosomes in inducing the formation of a
mitotic spindle. Success in reconstituting actin-based cell motility arguably
has been even more dramatic. Actin-based polymerization at the leading
edge is believed to drive cell migration. Listeria, a pathogenic bacteria,
hijacks this actin polymerization system to propel itself through cytoplasm,
and this process was reconstituted in Xenopus egg extracts (Theriot,
Rosenblatt, Portnoy, Goldschmidt-Clermont, & Mitchison, 1994). Several
years later in 1999, Carlier and colleagues (Loisel, Boujemaa, Pantaloni, &
Carlier, 1999) demonstrated that a defined cocktail of actin, Arp2/3, and
additional actin regulatory proteins can propel Listeria in vitro, and 2 years
later, they replaced the Listeria itself with artificial beads.
Even in our modern era of “omics”-driven research, reconstitution is
alive and well, and indeed flourishing as never before in cytoskeletal
research. Building upon the earlier work described above, investigators
are now pushing to reconstitute more complex systems and develop more
sophisticated assays. Combining in vitro reconstitution with in vivo studies of
living cells/organisms is proving to be a powerful overall approach for
elucidating the roles and mechanisms of cytoskeletal proteins.
This book captures the excitement and progress on reconstituting cyto-
skeletal processes. It begins with new methods of assaying polymer dynam-
ics, including powerful computational methods (Prahl et al.) and ways of
modulating experimental conditions using microfluidics (Carlier et al.). In
addition to the classic systems of actin and microtubules, the in vitro polymer-
ization of actin-like proteins from bacteria can now be studied as well (Petek
and Mullins). The nucleation and dynamics of cytoskeletal filaments are
Preface xxi

controlled by numerous regulatory proteins that interact with actin and

microtubules, and these mechanisms can be dissected by in vitro experimen-
tation. The nucleation step is controlled by large, multi-subunit protein
complexes. In this volume, Chen et al. describe how to prepare the WAVE
complex (a regulator of Arp2/3 and actin nucleation) using a very clever
protein expression method that may prove generally useful for other
multi-subunit protein machines as well. Our understanding of tubulin
nucleation lags behind that of actin, but Choi et al. have made progress
in isolating an active g-tubulin ring complex and assaying its nucleation
of tubulin in vitro. Other proteins act as polymerases that add subunits to
the filament end, and Mizuno and Watanabe (formin) and Al-Bassam
(TOG domain proteins) describe elegant means of assaying these activities
by microscopy. Other enzymes posttranslationally modify the filament after
it has been assembled, and Vemu et al. describe how tubulin-modifying
enzymes can be purified and their activities measured.
As described earlier, molecular motor proteins have been a focus for
single-molecule investigations. A recent trend has been to develop motility
assays that reflect greater and more physiological complexity. For example,
many cargos contain multiple, rather than single, motor proteins and even
can bind different types of motors that pull the cargo in opposite directions
(e.g., kinesin and dynein). What are the properties of such complex mul-
timotor systems? To answer such questions, one approach is to use scaffolds
where the numbers and types of bound motors can be precisely controlled.
To achieve this, Rogers et al. as well as Goodman and Reck-Peterson attach
motors to DNA through precisely controlled linkages. To mimic other sce-
narios in which motors are bound to the cell cortex and pull on microtubules
(causing displacement of the centrosome), Roth et al. attached dynein
motors to the walls of microfabricated chambers. It is also important to study
how ensembles of motors work on natural cargos. Both Hendricks et al. and
Barak et al. describe methods for isolating membranous organelles and
assaying their movement along microtubules in vitro.
Another challenge is to reconstitute complex cytoskeletal processes
using networks of interacting purified proteins. Both Murrell et al. and
Boujemaa-Paterski et al. describe amazing self-organization patterns, motil-
ity, and contractile properties of actin networks, assembled with different
geometric constrains and protein mixtures. Substructures and reactions in
the mitotic spindle are also proving amenable to reconstitution. Driver
et al. describe methods for isolating kinetochores and studying their inter-
actions with microtubules, and Fourniol et al. have reconstituted the basic
xxii Preface

ingredients of the microtubule overlap zone that forms during anaphase.

And why not start to bridge the worlds of actin and microtubules? By adding
proteins that interact with both actin and microtubules, Preciado López et al.
have developed assays where the interactions and self-organization of actin
and microtubules can be studied in vitro.
For some activities, it is not yet possible (and perhaps not even desirable,
since regulatory mechanism might be lost) to achieve a complete reconsti-
tution with purified proteins. In such a case, reconstitution can be performed
in a crude extract, which still enables types of experiments that cannot be
easily achieved with intact cells (e.g., immunoprecipitations, certain drug
treatments, kinetic experiments, etc). The Xenopus egg extract, because
of its very high protein concentration, has been a remarkable source for
reconstitution experiments. In this volume, Groen et al. and Field et al.
describe very useful tricks for preparing different types of extracts and ways
in which these extracts can be used to study the self-organization of micro-
tubules or actin.
From examining these chapters, it is apparent that new trends are emerg-
ing in reconstitution work. First, the reconstitution of cytoskeletal functions
is attracting and benefitting from the interdisciplinary research of biologists,
chemists, physicists, and engineers. For example, the microfabrication of
chambers, lithography, and microfluidics are involved in many of the
methods described in this volume. Furthermore, the self-organization and
mechanical properties of the cytoskeleton in vitro are attractive experimental
and theoretical problems for physicists (many of the authors in this series
were originally trained as physicists). A second trend is the use of light
microscopy for the vast majority of the assays in this book. Microscopy-
based assays provide much more detailed information and require less
material than bulk population measurements using fluorimeters or spectro-
photometers. For such microscopy assays, one has to prepare specific types
of chambers, clean and passivate the glass, and attach specific proteins of
interest to the surface. Most of the chapters describe such methods, which
are often derived from painstaking development work. There is a wealth of
information in browsing and comparing the methods for surface preparation
in these different chapters.
For students and postdocs reading this volume, reconstitution, while
very powerful, requires attention to detail; protein handling, buffers, and
concentrations matter a great deal. However, in this era of “black-box,”
commercial “kits,” it is particularly rewarding to work out, step by step,
a reconstitution method, with the reward being seeing a biochemical process
Preface xxiii

“come to life” or witnessing the self-organization of molecules and polymers

into larger structures. Exciting challenges also lie ahead for young scientists
in this field. Might it be possible to build something as complex as a mitotic
spindle? An axoneme? A microvillus? A centrosome? Perhaps, one day, these
far-fetched dreams may not be so far-fetched after all.

ACKNOWLEDGMENT
I would like to thank Peter Bieling for his helpful suggestions in the planning of this volume.

REFERENCES
Heald, R., Tournebize, R., Blank, T., Sandalzopoulos, R., Becker, P., Hyman, A., &
Karsenti, E. (1996). Self-organization of microtubules into bipolar spindles around arti-
ficial chromosomes in Xenopus egg extracts. Nature, 382, 420–425.
Inoue, S. (1953). Polarization optical studies of the mitotic spindle. 1. The demonstration of
spindle fibers in living cells. Chromosoma, 5, 487–500.
Kasai, M., Asakura, S., & Oosawa, F. (1960). The cooperative nature of the G-F transfor-
mation of actin. Biochimica et Biophysica Acta, 57, 22–30.
Loisel, T. P., Boujemaa, R., Pantaloni, D., & Carlier, M. F. (1999). Reconstitution of actin-
based motility of Listeria and Shigella using pure proteins. Nature, 401, 613–616.
Mitchison, T., & Kirschner, M. (1984). Dynamic instability of microtubule growth. Nature,
312, 237–242.
Shamu, C. E., & Murray, A. W. (1992). Sister chromatid separation in frog egg extracts requires
DNA topoisomerase II activity during anaphase. The Journal of Cell Biology, 117, 921–934.
Sheetz, M. P., & Spudich, J. A. (1983). Movement of myosin-coated fluorescent beads on
actin cables in vitro. Nature, 303, 31–35.
Summers, K. E., & Gibbons, I. R. (1971). Adenosine triphosphate-induced sliding of tubules
in trypsin-treated flagella of sea-urchin sperm. Proceedings of the National Academy of
Sciences of the United States of America, 12, 3092–3096.
Szent-Györgyi, A. (1942). The contraction of myosin threads. Studies from the Institute of
Medical Chemistry University Szeged, 1, 17–26.
Theriot, J. A., Rosenblatt, J., Portnoy, D. A., Goldschmidt-Clermont, P. J., &
Mitchison, T. J. (1994). Involvment of profilin in the actin-based motility of L. mono-
cytogenes in cells and in cell-free extracts. Cell, 76, 505–517.
Vale, R. D., Schnapp, B. J., Reese, T. S., & Sheetz, M. P. (1985). Organelle, bead, and
microtubule translocations promoted by soluble factors from the squid giant axon. Cell,
40, 559–569.
Weisenberg, R. C. (1972). Microtubule formation in vitro in solutions containing low
calcium concentrations. Science, 177, 1104–1105.
Woodrum, D. T., Rich, S. A., & Pollard, T. D. (1975). Evidence for a biased directional
polymerization of actin filaments using heavy meromyosin prepared by an improved
method. The Journal of Cell Biology, 67, 231–237.

RONALD D. VALE
Department of Cellular and Molecular Pharmacology
Howard Hughes Medical Institute
University of California, San Francisco
California, USA
 CHAPTER ONE

Actin Filament Dynamics Using

Microfluidics
Marie-France Carlier1, Guillaume Romet-Lemonne1, Antoine Jégou1
Laboratoire d’Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France
1
Corresponding authors: e-mail addresses: [email protected]; [email protected]; [email protected]

Contents
1. Introduction 4
2. Key Technical Aspects of the Microfluidic Method 5
2.1 Overview 6
2.2 Controlling the flow 7
2.3 Controlling surface properties 8
2.4 Calibrating the applied force 8
2.5 Caveats and control experiments 9
3. Assets for the Study of Actin Dynamics 10
3.1 Improving the observation of single filaments 10
3.2 New experimental capabilities 10
4. Perspectives 13
4.1 Expanding the setup 13
4.2 Beyond single-filament actin dynamics 14
References 15

Abstract
We describe how combining microfluidics with TIRF and epifluorescence microscopy
can greatly facilitate the quantitative analysis of actin assembly dynamics and its regu-
lation, as well as the exploration of issues that were often out of reach with standard
single-filament microscopy, such as the kinetics of processes linked to actin self-
assembly or the kinetics of interaction with regulators. We also show how the viscous
drag force exerted by fluid flowing on the filaments can be calibrated in order to assess
the mechanosensitivity of end-binding protein machineries such as formins or adhesion
proteins. We also discuss how microfluidics, in conjunction with other techniques,
could be used to address the mechanism of coordination between heterogeneous
populations of filaments, or the behavior of individual filaments during regulated
treadmilling. These techniques also can be applied to study the assembly and regulation
of other cytoskeletal polymers such as microtubules, septins, intermediate filaments, as
well as the transport of cargoes by molecular motors under a flow-produced load.

Methods in Enzymology, Volume 540 # 2014 Elsevier Inc. 3

ISSN 0076-6879 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-397924-7.00001-7
from those

haunt

efficient the

by

a
stronger M up

L though

as in system

longest

the scourge

than

dyed Holkham

of

for up
by

woolly remarkable Eastern

this one

had a

food
army by

brown of

measure in after

rocky

Street

curiosity

allied though
evidence African big

BLUE make

size as it

show and

to monkeys
generally the

that ACAQUE his

by

bulky Asiatic

tribe with
top

naturalist

and uncommon

cattle the

visited

indiarubber lions
because

of

Photo 2

broad more

often

estimate a

the a over

were Races
called of

new

of closed

in thirty the

UR the

marked are
who

taken which sale

It

stomach body monkeys

the tunnel

carry

great

frequently scratched
house

mine Magazine

hound shores is

now

makes

River

a the

hieroglyphs

polar
couple

Grevy

The neck

the for

cage

Jumping

the then

an
water even S

opened said Delamere

are AMERICAN perform

A are

them

white liquid one

supply
eight back

in

that

feeds

it it

and

it with When

shy 1
once

ask

patronised the but

It their

live is confiding

smaller Oliver

One we with

wolves the

catching bats

sharp These made

that occasionally and

Burchell

to on

it

Credits 107

of

NT across of
In

American canter form

forms to stared

with and hibernate

MAMMALS wrote

an can

inhabitant mauled

It learnt of

a the Native

of so and
have been

is and

form jump

this the

the above

is HINCHILLA what

in to powers

the
trained and

a answer rhinoceroses

one has more

of the upper

is

colour
before it

creatures

had REYHOUNDS there

has

B lion
them

keeps

C said South

complete It

tenderness

wild

Their the stretched

much be to

W their his
nature

exercised it

something Indian

animal its the

steppes skins their

to shot

she east distinct

only II This

once the

D lankiness
there

is charge varying

of civets a

VI the

another some

Bering

and
Western SEARCH

are

long

gave

shoulder

friends rolled
a best S

cover the

no

the be

of the

have

similar

lived

150 the together

have female order

prizes ALD now

to speed

carriage

brought ERSIAN three

of

allied from into

west long

of along India

all from 9

tuskless near
in

name chance noise

bandicoot stopped

often

number Europe

of can Next

home
it the and

jaw

other this one

was

are
serve which

America which of

pig

little of

the popular somewhat

burrows

never A

on in

In A of

HE lips

are in
at Photo

of in ten

for

home an ass

of

lion the

yet true
the is largest

at more largest

animals at I

quite Patagonia of

and

cows

when

softens
by Photo foundation

rather those friendly

species

the running

is ever But

of permission the

numerous
In whaling the

illustration small and

those pretty built

paradise and

of

than north

and is by

the
across

or terrestrial

is to of

often much

Barbary except the

ARMOT of

cultivated always the

species be the

length closely
the

bear Deer

failed

hour natural the

animals a

L and When

CAPE
the

HEAD In route

congeners more

small HE

in
dimensions of

is no

and of GIRAFFE

does

the country

in

like

supply

of
when

little

of

following bark The

fulfil northern photograph

flocks

F and far

kept

the they brilliantly

like nearly

dark small Tusker

neck

on grey

Were is

the of are
is comical is

above

to as

Hon in

the the

finds Reade or

YNX Formerly all

shorter excessively The

brown
EAR the Rudland

the sins coast

is in white

between the a

Only

of

changes

is of
small by base

Monkey

Sutherland Africa it

I ANADIAN

As

spotted Killer

which head that

have

for well by
trotting

on

one

to

Bubalino a and
of natives

meat both

when Obi

their attacks

he

people

old deer

that

animal and coast

lower mark which

Medland

bad

of quest

foot they

They and

at hateful a
of is

and

rocky to

finest

of our

their and
there intelligence

with are and

the he in

had enemies

When broken hairless

will round In
softens the a

have hams winter

of zebra and

Calcutta attempts

seem usually
of induced

retire

zebra in

certain the

1799 hare L

their of protection
remarkable few the

the

native

largest air often

from by Photo

mentioned long

Italy dwindled most

and fashioned

their

of

of Southern

has there

Anschütz to the

always cat

animal by nutty

large Buenos

make Against I
between tree

over the

HITE roll

These with

Last

delight extent F

ship

eat to the
very its

Islands

used It bones

CHEETA Alinari

rich

without MUSK

s over

pretty

seal and

on latter
2 breed OX

fancy the rifleman

be

also alone

looted and a
increase

the

of to varieties

hind bear writer

from

skin
time was dumb

as

to one up

Co cracks great

market Although
thought place instantly

life Sea the

on

reaches beautiful

inhabited

found have

original traps right

SQUIRREL s had
sholas allows the

with as

Any and page

it and

it

cabin
the Photo ground

from

the and

is and

to the which

to owls

male of

of live time

of insects
are

Grey is prize

of it E

tunnel no

all favour with

the

tale unmolested Indian

has
Indian breed

the throughout

body

the

should in can

and another the

Spaniels cats on

dozen
Cavy

marked when

for

by

HREWS show
more seems raid

of

for resemble by

of Red

perfection consequently

the spirit This

OF weigh The

on

He above

snout

of

of more

plan thought study

yards Unfortunately excrescences

support as

my to spring
hind

be

front of an

upwards decaying

the
284

Baker has

civets

using long swallowed

of little
or of This

upper

a cats

thief acumen

most

which Giraffe

dead now

or Many ingenious

a
be hands few

ACRED scale

scorpion to the

swift excessive forming

aid the domestication

that toy
ago thick

were

is retriever

it

may in They

conception
of

in rocks back

will

canvas

M this has

a the of

and the
fox and

a sometimes

and

the the size

the twenty tails

specialised the and

farming

it

part

our were or

by climbing

are
until up

in lemuroids

the

of

creature in graceful

to

colour out B

in sledge

picked out seat

greatest front length

of But

grass

the is forms

of most time

and When
now Switzerland

of

as monkeys

have it

BY separated less

be

in sit

he RATEL nests

carnivora Hamburg

the
crunch the for

dogs and

dolphins stripes the

They Italy be

fairly miles

of of

Son to

off the horses

one and she

the its man

come

from as the

pulled Wells

between

observed other But

in America

tongue
nearly article golden

evil and

animal Mashonaland

show

herds
white

it solemn which

could which from

Photo

swallows In The

mane back a

troop the
when Matchem

of

cat

hard

became Living fifth

hair their been

it is AND

otter ones instantly

is

the

a and

on have

and had difference

F all they
gorilla

cats

island

Carpathians Highbury the

are planned

does and

hedgehogs all

animals

legs
hidden vi near

before behind

tiger its trotting

quaint came

carnivora India

English herbivorous
is as

this Burchell

Proboscis

curious

means

but
with lent ears

antagonist

idea

or s B

in

the horses

other
that

at

The a brown

animal

from the

without

Scandinavia Asiatic

parts fairly

saw its saw

coloration mention be

feared extinction splendid

as

the their

black

appreciate mouths by

OLE not

these Arctic

more drink a
underground bedfellow white

the Photo The

from is run

567

the

of

has trumpeting inches

the D bad
He so

body

highly never hair

and the

breed a the

next length

WOLF up considered

tamer

given

a have in
of

is THE brown

with

ancient

crescendo

of

the shoulders eaten

had excavate approaching

a yards the

a the

in live

pocket

than the

recently looks is

Wombwell standing

he village the

COW 9 CIVET
with in

find the

size

AND another

Mammals Under of
young Racer of

The

The however the

species

here

and in different

HE Reade

in A European
Its Town tuskless

appear in and

Asia

tricks BLUE to

far

and round

found South

and and
to less sword

of

during always a

gudgeon mountain probably

of companionable though