Molecular Motors

Edited byManfred Schliwa

InnodataFile Attachment3527605657.jpg

Also of Interest

W. Ehrfeld, V. Hessel, H. Löwe

Microreactors – New Technologyfor Modern Chemistry (2000)ISBN 3-527-29590-9

S. P. Nunes and K.-V. Peinemann (Eds.)

Membrane Technology – the Chemical Industry (2001)SBN 3-527-28485-0

J. G. Sanchez Marcano and Th. T. Tsotsis

Catalytic Membranes and Membrane Reactors (2002)ISBN 3-527-30277-8

H. Schmidt-Traub (Ed.)

Chromatographic Separation –Fine Chemicals and Pharmaceutical Agents (planned 2003)

Molecular Motors

Edited byM. Schliwa

Also of Interest

W. Ehrfeld, V. Hessel, H. Löwe

Microreactors – New Technologyfor Modern Chemistry (2000)ISBN 3-527-29590-9

S. P. Nunes and K.-V. Peinemann (Eds.)

Membrane Technology – the Chemical Industry (2001)SBN 3-527-28485-0

J. G. Sanchez Marcano and Th. T. Tsotsis

Catalytic Membranes and Membrane Reactors (2002)ISBN 3-527-30277-8

H. Schmidt-Traub (Ed.)

Chromatographic Separation –Fine Chemicals and Pharmaceutical Agents (planned 2003)

Molecular Motors

Edited byManfred Schliwa

Edited by

Prof. Dr. Manfred SchliwaLudwig-Maximilians-UniversitätAdolf-Butenandt-InstitutZellbiologieSchillerstrasse 4280336 MünchenGermany

This book was carefully produced. Never-theless, editors, authors and publisher do notwarrant the information contained therein tobe free of errors. Readers are advised to keepin mind that statements, data, illustrations,procedural details or other items mayinadvertently be inaccurate.

Library of Congress Card No.: applied forA catalogue record for this book is availablefrom the British Library.

Bibliographic information published byDie Deutsche BibliothekDie Deutsche Bibliothek lists this publicationin the Deutsche Nationalbibliografie;detailed bibliographic data is available in theinternet at http://dnb.ddb.de.

� 2003 Wiley-VCH Verlag GmbH & Co. KGaA,WeinheimAll rights reserved (including those oftranslation in other languages). No part ofthis book may be reproduced in any form –nor transmitted or translated into a machinelanguage without written permission fromthe publishers. Registered names, trade-marks, etc. used in this book, even when notspecifically marked as such, are not to beconsidered unprotected by law.

Printed in the Federal Republic of Germany.Printed on acid-free paper.

Composition Hagedorn Kommunikation,ViernheimPrinting Druckhaus Darmstadt GmbH, DarmstadtBookbinding Buchbinderei Schaumann GmbH,Darmstadt

ISBN 3-527-30594-7

Preface

Editors of compilations such as this tend to stress in their prefaces that significantconceptual advances have been made recently, that novel technical developmentshave opened extraordinary opportunities for unprecedented discoveries and thatthe time seemed ripe to take stock and to point out developments which will ad-vance the field in the near future. Well, all of this is true for this book too. It isalso true that on such occasions we realize how much we have learned and yethow little we know. Since the publication nearly 40 years ago, of the landmark trea-tise on cell movement edited by Robert D. Allen and Noburô Kamiya and entitledPrimitive Motile Systems in Cell Biology, the field has moved from the phenomeno-logical to the mechanistic and from the largely structural to the primarily molecu-lar. We have come to appreciate that at every level of complexity the cell operatesthrough molecular machines. Some of these machines are single molecules thatcarry out one specific task, undergoing only small structural changes in the pro-cess. Others are macromolecular complexes composed of dozens, even hundredsof different components engaged in elaborate biochemical operations. Amongthe multitude of molecular machines of a cell, one group stands out owing toits ability to generate one of the hallmark characteristics of living systems: move-ment. The chapters of this book offer insights into the workings, interactions andfunctions of these remarkable molecules which are responsible for various formsof movement encountered in cells. The subdivision of the book into five sectionsdeveloped naturally. First we learn about the basic designs of some of the most pro-minent cellular motors before considering their mechanochemistry; the role of mo-tors in the context of elaborate cellular activities is considered next, followed by ex-amples of defects which result when motors run ‘wild’; finally, biomotors are putinto perspective with regard to nanobiotechnological applications and other typesof molecular motors. The outcome is a pretty sizeable book, as can plainly beseen. Nevertheless, it is but an introduction to the subject, as other types of biolo-gical machines exist that could also, with some justification, be called motors butare not considered here for reasons of space. It is my hope, however, that salientfeatures of cellular motors are covered even though gaps undoubtedly remain.

I would like to express my sincere gratitude first and foremost to the authorswho have managed to complete their chapters under pretty tight time constraints.I would also like to thank the staff at Wiley-VCH, in particular Dr. Andreas

VPreface

Sendtko, who have helped me in every respect and to Ursula Euteneuer for criticalreading and helpful comments and discussions. Thanks to the efforts of everyoneconcerned less than one year has elapsed between conception of the book and thecompletion of the printed product. You might say all of us have motored along justfine.

Manfred SchliwaSeptember 2002

VI Preface

Contents

Preface V

List of Contributors XIX

Part 1 Basic Principles of Motor Design

1 The Myosin Superfamily: An Overview 31.1 An Introduction to the Myosin Superfamily 31.2 Functional Properties of Myosins 71.2.1 Directionality and Processivity 71.2.2 Protein Motifs Found in Myosins 81.2.3 Myosin Regulation 101.3 Diverse Functions for Myosins 111.3.1 Non-muscle Contractile Structures 141.3.2 Cell Motility and Adhesion 151.3.3 Organelle/Cellular Component Transport 161.3.4 Maintenance of Actin-rich Extensions 211.3.5 Membrane Trafficking 241.3.6 Signal Transduction 261.4 Myosins in Disease 281.4.1 Griscelli Syndrome 281.4.2 Roles for Myosins in Hearing 291.5 New Myosins and Myosin Functions on the Horizon 311.6 Conclusions 32

References 33

2 Dynein Motors: Structure, Mechanochemistry and Regulation 452.1 Introduction 452.2 Structural Organization of the Motor, Cargo-binding and Regulatory

Components 462.2.1 Heavy Chains 482.2.2 Intermediate Chains 53

VIIContents

2.2.3 Light Intermediate Chains 562.2.4 The LC8 Light Chain Class 572.2.5 The Tctex1/Tctex2 Light Chain Class 592.2.6 The LC7/roadblock Light Chain Class 612.2.7 Heavy Chain-associated Regulatory Light Chains 622.2.7.1 Light chain 1 622.2.7.2 Calmodulin-related light chains 632.2.7.3 Thioredoxins 642.2.7.4 p29 (cAMP-dependent phosphoprotein) 642.2.8 Light Chains Associated with Inner Arms I2/3 652.3 Mechanochemistry and Motility 652.4 Dynein Deficiencies and Disease 672.5 Conclusions 69

References 70

3 Kinesin Superfamily Proteins 793.1 Introduction 793.2 The Kinesin Superfamily Proteins 823.3 N-Kinesins 873.3.1 N-1 Kinesins 873.3.2 N-2 Kinesins 913.3.3 N-3 Kinesins 913.3.3.1 The Unc104/KIF1 family 913.3.3.2 The KIF13 family 923.3.3.3 The KIF16 family 923.3.4 N-4 Kinesins 923.3.4.1 The KIF3 family 933.3.4.2 The Osm3/KIF17 family 943.3.5 N-5 Kinesins 943.3.6 N-6 Kinesins 943.3.6.1 The CHO1/KIF23 family 953.3.6.2 The KIF20/Rab6 kinesin family 953.3.7 N-7 Kinesins 953.3.8 N-8 Kinesins 953.3.8.1 The Kid/KIF22 family 953.3.8.2 The KIF18 family 963.3.9 N-9 Kinesins 963.3.10 N-10 Kinesins 963.3.11 N-11 Kinesins 963.4 M-Kinesins 963.5 C-Kinesins 973.5.1 C-1 Kinesins 973.5.2 C-2 Kinesins 973.6 Orphans 983.7 Cargoes of KIFs; Specificity and Redundancy 98

VIII Contents

3.8 Recognition and Binding to Cargoes 993.9 How to Determine the Direction of Transport 100

References 100

4 The Bacterial Flagellar Motor 1114.1 Introduction 1114.2 Structure 1144.2.1 Propeller and Drive-shaft 1174.2.2 Rotor 1174.2.3 Stator 1184.2.4 Rotor�Stator Interactions 1194.3 Function 1204.3.1 Motor Driven by H� and Na� Ion Flux 1214.3.2 Torque versus Speed 1224.3.3 Independent Torque Generators 1264.3.4 Proton Motive Force, Sodium-motive Force, Ion Flux 1284.3.5 Reversibility 1314.3.6 Steps? 1314.4 Models 1324.4.1 Conceptual Models 1334.4.2 Kinetic Models 1354.5 Summary 136

References 137

5 F1-Motor of ATP Synthase 1415.1 Introduction 1415.2 ATP Synthase 1415.3 F1-Motor 1425.4 Imaging of Rotation of F1-Motor 1445.5 High-speed Imaging of F1 Rotation 1455.6 New Crystal Structure for the F1-Motor 1465.7 Catalysis and Rotation of F1-Motor 1485.8 Perspectives 150

References 151

6 RNA and DNA Polymerases 1536.1 Introduction 1536.2 NTP Polymerization Mechanism 1556.3 Basic Methods used to Study Polymerase Movement during

Transcription 1586.3.1 The Tethered Particle Motion Approach 1586.3.2 The Surface Force Microscopy Technique 1586.3.3 The Optical Tweezer Method 1596.3.4 Method for Visualization of DNA Rotation during Transcription 1616.3.5 Footprinting Approach 161

IXContents

6.3.6 Single Molecule Assay for DNA Polymerase 1626.4 Mechanism of Force Generation for RNAP and DNAP 1646.5 Molecular Model for RNAP Translocation 1686.6 Possible Utilization of the Energy Released upon NTP Cleavage 1716.7 Single-Molecule Studies and Molecular Mechanisms of Transcription

Pausing and Arrest 1726.8 Concluding Remarks 174

References 175

7 Helicases as Molecular Motors 1797.1 Introduction 1797.2 Basic Properties of Helicases 1827.3 Mechanism of Helicase Activity 1887.3.1 Unidirectional Translocation 1887.3.2 Step Size of the Helicase 1927.3.3 NA Strand Separation 1927.4 HCV Helicase 1947.5 Bacteriophage T7 gp4 Helicase 1967.6 Conclusions 197

References 198

Part 2 Mechanochemistry

8 How Protein Motors Convert Chemical Energy into Mechanical Work 2078.1 Introduction 2078.2 A Brief Description of ATP Synthase Structure 2088.3 The F1 Motor: A Power Stroke 2098.4 The F0 Motor: A Brownian Ratchet 2128.4.1 A Pure Brownian Ratchet 2128.4.2 A Pure Power Stroke 2148.5 Coupling and Coordination of Motors 2168.6 Measures of Efficiency 2188.7 Discussion 220

A1 Example Models to Illustrate the Difference between Ratchets and PowerStrokes 221

A1.1 Example 1: A power stroke without Brownian fluctuations 221A1.2 Example 2: A power stroke with Brownian fluctuations 222A1.3 Example 3: A Brownian ratchet that biases fluctuations 223A1.4 Example 4: A Brownian ratchet that rectifies fluctuations 224

A2 A Closer Look at Binding Free Energy 225References 227

X Contents

9 Molecular Motor Directionality 2299.1 Introduction 2299.2 Reversed Kinens 2299.2.1 Chimeric Kinesin Motors 2319.2.2 A Neck Mutant 2339.3 Backwards Myosins 2349.3.1 Chimeric Myosin Motors 2359.4 Bidirectional Dyneins? 2379.5 Perspectives 238

References 239

10 Kinesins: Processivity and Chemomechanical Coupling 24310.1 Introduction 24310.2 Kinesin Motility and Processivity 24410.3 Biochemical Evidence for Kinesin Processivity 24610.4 Step Size of Kinesin and its Path along the Microtubule 24610.5 Kinesin Stoichiometry 24710.6 Coordination between the Two Heads of Kinesin 24810.7 Testing Processivity with One-headed Kinesin Mutants 24910.8 ATP Hydrolysis Cycle of One-headed Kinesin 25010.9 Structural Studies on Dimeric Kinesin 25310.10 Two-headed Kinesin ATP Hydrolysis Cycle 25410.11 Load Dependent Transitions 25710.12 Ncd is a Non-processive Kinesin Family Member 25910.13 A Processive Monomeric Kinesin, KIF1A 26210.14 Unresolved Questions 264

References 266

11 Quantitative Measurements of Myosin Movement In Vitro:The Reductionist Approach Carried to Single Molecules 271

11.1 Introduction 27111.2 Quantitative In Vitro Assays for Myosin Movement Established the Motor

Domain of Myosin 27211.3 Structural Studies Revealed Putative Pre-stroke and Post-stroke States of

the Myosin Head 27311.4 Single Molecule Analysis Revealed a Unitary Small Step in Motion as

Myosin Interacts with Actin 27511.5 Molecular Genetic Approaches Have Indicated Roles of Various Domains

and Specific Residues of the Myosin Motor 27711.6 Myosin V uses its Longer Lever Arm to Take a Larger Step along Actin 27811.7 Conclusions and Perspectives 282

References 283

XIContents

12 Structures of Kinesin Motor Domains: Implications for ConformationalSwitching Involved in Mechanochemical Coupling 287

12.1 Introduction 28712.2 Structures of Kinesin Motor Domains 28812.2.1 General Features of the Catalytic Core 29012.2.2 The Nucleotide-Binding Active Site 29112.2.2.1 N1, also called P-loop

(G86xxxGKS/T, residue numbering according to rat kinesin) 29112.2.2.2 N2 � Switch 1 (N199xxSSR) 29112.2.2.3 N3 � Switch 2 (D232LAGSEKVGKT) 29212.2.2.4 N4, (R14xRP) 29212.2.3 Neck Linker, Neck and Hinge 29212.3 Comparison with G-Proteins and Myosin 29312.4 Mechanochemical Coupling from a Structural Point of View 29412.5 Perspectives 300

References 301

13 Single Molecule Measurements and Molecular Motors 30513.1 Introduction 30513.2 Manipulation of Actin Filaments 30613.3 Nanometry of Actin Filaments 30813.4 Movement of Actin Filaments Caused by Single Myosin Molecules 30913.5 Visualization of Single Molecules 31013.6 Visualization of ATP Turnover and Mechano-chemical Coupling 31213.7 Visualization of the Movement of Single Kinesin Motors 31413.8 Visualization of the Processive Movement of Single Myosin Motors 31713.9 Manipulation of Single Myosin Molecules with a Scanning Probe and

Nanometry 31913.10 Biased Brownian Movement 32013.11 Concluding Remarks 321

References 322

Part 3 Functional Implications

14 Mitotic Spindle Motors 32714.1 Microtubules, Motors and Mitosis 32714.2 The Physical Nature of Mitotic Movements 32914.3 MT Polymerization and Depolymerization as Mitotic Motors 33114.4 Kinesins and Dyneins as Mitotic Motors 33414.5 Functional Coordination of Mitotic Motors 33814.6 Motor Action and Force-Generation during Mitosis 33914.6.1 Mitotic Motors and Spindle Formation at Early Stages of Mitosis 33914.6.2 Mitotic Motors and Force Generation in Prometaphase�Metaphase 34014.6.3 Mitotic Motors and Force Generation in Anaphase 343

XII Contents

14.7 Does a Spindle Matrix Facilitate the Function of Mitotic Motors? 34514.8 Mitotic Motors and Intracellular Transport Systems 34614.9 Mitotic Motors and the Spindle Assembly Checkpoint 34914.10 Conclusions and Future Studies 350

References 351

15 The Roles of Molecular Motors in Generating Developmental Asymmetry 35715.1 Introduction 35715.2 Localization of a L/R Determinant by Asymmetric Flow of Extraembryonic

Fluid 35715.2.1 Situs Inversus in Humans 35715.2.2 Mice with Mutations in kif3a, kif3b, or lrd Lead to Nodal Flow Model 35815.2.3 Inv Mutants Challenge the Nodal Flow Model 36015.3 Asymmetric RNA Localization 36115.3.1 A/P Patterning in Drosophila Oocytes and Embryos 36115.3.1.1 Anterior localization of bcd mRNA in the oocyte 36215.3.1.2 Posterior localization of osk mRNA within the oocyte by kinesin 36415.3.1.3 Localization of mRNAs in blastoderm embryos 36515.3.2 Yeast Mating Type Switching 36615.4 Asymmetric Organelle Localization 36815.4.1 Localization of the Fusome and Drosophila Oocyte Selection 36815.4.2 Drosophila Oocyte Nuclear Migration 36815.4.3 Lipid Droplet Migration in Drosophila Embryos 36915.4.4 Nuclear Migration in Drosophila Photoreceptors 37115.5 Future Directions 372

References 372

16 Motors and Membrane Trafficking 37716.1 Introduction 37716.2 The Logic and Order of Membrane Trafficking 37916.3 The Cytoskeleton and Motor Proteins in Membrane Trafficking 38016.3.1 Role of the Cytoskeleton and Motor Proteins in Organelle

Localization 38016.3.2 Role of the Cytoskeleton in Membrane Trafficking Events 38016.3.3 Role of Motor Proteins in Membrane Trafficking Events 38216.4 Cooperation between Motors 38316.4.1 Coordination of Movement along Microtubule and Actin Tracks 38316.4.2 Coordination of Bidirectional Movement along Microtubule Tracks 38516.4.3 Molecular Mechanisms for the Coordination of Motors on the Same

Transport Cargo 38716.5 Molecular Mechanisms of Motor–Cargo Linkage 38816.5.1 Soluble Adaptor or Scaffolding Proteins as Motor�Cargo Linkers 38816.5.1.1 Other scaffolding complexes 39216.5.2 Motor�Cargo Linkage via Members of the Rab Family of Small

G-proteins 393

XIIIContents

16.5.3 Other Mechanisms for Linking Microtubule-based Motors totheir Cargoes 395

16.5.3.1 Attachment to the membrane cytoskeleton 39516.5.3.2 Attachment via integral membrane proteins 39616.6 Regulation of Motor Activity 39716.6.1 Motor Proteins must be Regulated at Several Steps of their Transport

Cycle 39716.6.2 Molecular Mechanisms for Regulating Motor Activity 39916.7 Concluding Remarks 400

References 401

17 Regulation of Molecular Motors 41117.1 Introduction 41117.2 The Role of Phosphorylation in Regulating Molecular Motors 41117.2.1 Phosphorylation can Control Motor�Organelle or Motor�Spindle

Binding 41217.2.1.1 Interaction of dynein with organelles can be regulated by

phosphorylation 41217.2.1.2 Interaction of dynein with dynactin can be regulated by

phosphorylation 41217.2.1.3 Interaction of kinesin with organelles can be regulated by

phosphorylation 41317.2.1.4 Interaction of kinesin family members with the spindle can be regulated

by phosphorylation 41417.2.1.5 The binding of myosin V to melanosomes is regulated by

phosphorylation 41417.2.2 The Activity of Motors may be Regulated by Phosphorylation 41617.2.2.1 Phosphorylation of axonemal dynein inhibits its activity 41617.2.2.2 Phosphorylation can activate or inhibit cytoplasmic dynein 41617.2.2.3 Phosphorylation can activate or inhibit the activity of members

of the kinesin family 41717.3 The Role of G Proteins in Regulating Molecular Motors 41917.3.1 G Proteins Mediate Motor�Cargo Interactions 41917.3.1.1 Rab27a recruits myosin Va to melanosomes 41917.3.1.2 G proteins may recruit microtubule motors to organelles 42117.3.2 G Proteins may Activate Motors 42217.3.3 Motors may Bind Directly to G Proteins but the Function of these

Interactions Remains Unclear 42217.3.4 A Light Chain of Dynein may be Involved in Regulating G Protein GTPase

Activity 42317.4 Other Mechanisms of Regulation 42417.4.1 Kinesin Folding 42417.4.2 Lis 1 Interaction with Cytoplasmic Dynein 42517.4.3 Motor Protein Regulation during the Cell Cycle 42517.4.4 Motor Complexes and Coordination 426

XIV Contents

17.5 Summary 427References 427

18 Molecular Motors in Plant Cells 43318.1 Introduction 43318.2 Microtubule-based Motors 43418.2.1 Kinesin-like Proteins 43418.2.1.1 Phylogenetic analysis 44318.2.2 Dyneins 44718.3 Actin-based Motors 44818.3.1 Myosins 44818.3.2 Phylogenetic analysis 45118.4 Cellular Roles of Motors 45118.4.1 Cell Division 45118.4.2 Cell Polarity and Morphogenesis 45618.4.3 Cytoplasmic Streaming 45718.4.4 Microtubule Dynamics and Organization 45818.4.5 Intercellular Transport 45918.4.6 Other functions 45918.5 Regulation of Motors 46018.5.1 Calcium/Calmodulin 46018.5.2 Protein Phosphorylation 46118.6 Concluding Remarks 461

References 462

Part 4 Motors in Disease

19 Myosin Myopathies 47319.1 Introduction: Inherited Myosin Myopathy 47319.2 Cardiac Myosin Heavy Chains 47519.2.1 MyHC Structure and Function 47519.2.2 Cardiac Muscle Regulation and Disease 47719.3 Cardiac MyHC Myopathy 47819.3.1 Functional Characterization of MyHC Motor Domain Mutations 47819.3.2 Transgenic Models of Myosin-based FHC 48019.4 MyHC Interacting Proteins and FHC 48219.4.1 The Essential and Regulatory Light chains 48219.4.2 Myosin Light Chain-based FHC 48419.4.3 Myosin Binding Protein C-Based FHC 48519.4.5 Titin-based Familial Hypertrophic Cardiomyopathy 48719.5 Myosin-based Myopathies in Skeletal Muscle 48819.6 Conclusions 489

References 490

XVContents

20 The Role of Dynein in Disease 49720.1 Dynein Functional and Structural Classes 49720.2 Diseases Associated with Axonemal Defects 49820.3 Role of a Cytoplasmic Dynein Light Chain in Retinitis Pigmentosa 50020.4 Role of Cytoplasmic Dynein in the Smooth Brain Disease

Lissencephaly 501References 506

21 Molecular Motors in Sensory Defects 51121.1 Introduction 51121.2 Development of the Visual and Auditory Sensory Systems 51121.3 Visual Impairment 51321.4 Hearing Impairment 51421.5 Myosins Involved In Sensory Defects 51521.5.1 Myosin VIIA 51621.5.1.1 Structure, function, and expression of myosin VIIA 51621.5.1.2 Shaker 1 Mice and Other Models 51821.5.1.3 Usher Syndrome Type 1B 51921.5.1.4 DFNB2 and DFNA11 52021.5.2 Myosin VI 52021.5.2.1 Structure, function, and expression of myosin VI 52021.5.2.2 Snell’s waltzer mice and other models 52221.5.2.3 DFNA22 52421.5.3 Myosin XVA 52521.5.3.1 Structure, function, and expression of myosin XVA 52521.5.3.2 Shaker 2 52621.5.3.3 DFNB3 52721.5.4 MYH9 52821.5.4.1 Structure, function, and expression of MYH9 52821.5.4.2 DFNA17 52821.5.5 Myosin IIIA 52921.5.5.1 Structure, function, and expression of myosin IIIA 52921.5.5.2 Myosin IIIA mutants 53121.5.5.3 DFNB30 53121.6 Concluding Remarks 532

References 533

XVI Contents

Part 5 Beyond Biological Applications

22 Systematized Engineering of Biomotor-powered Hybrid Devices 54122.1 Introduction 54122.2 The Core Technologies 54322.2.1 Nanoscale Directed Assembly 54322.2.2 Molecular Energy Transduction 54622.2.3 Control Mechanisms 54922.2.4 Multimedia Device Construction 55222.2.5 Engineering Issues 55422.3 The Core Technologies as a Whole 556

References 557

23 Synthetic Molecular Motors 55923.1 Introduction 55923.2 Translational Synthetic Molecular Motors 55923.3 Synthetic Rotary Molecular Motors 56423.4 Chemically Driven Unidirectional Molecular Motor 56723.5 Light-driven Unidirectional Molecular Motors 56823.6 Conclusion and Prospects 575

References 575

Index 579

XVIIContents

Also of Interest

W. Ehrfeld, V. Hessel, H. Löwe

Microreactors – New Technologyfor Modern Chemistry (2000)ISBN 3-527-29590-9

S. P. Nunes and K.-V. Peinemann (Eds.)

Membrane Technology – the Chemical Industry (2001)SBN 3-527-28485-0

J. G. Sanchez Marcano and Th. T. Tsotsis

Catalytic Membranes and Membrane Reactors (2002)ISBN 3-527-30277-8

H. Schmidt-Traub (Ed.)

Chromatographic Separation –Fine Chemicals and Pharmaceutical Agents (planned 2003)

List of Contributors

XIXList of Contributors

Karen B. AvrahamDepartment of Human Geneticsand Molecular MedicineSackler School of MedicineTel Aviv UniversityTel Aviv 69978Israel

Richard BerryThe Clarendon LaboratoryUniversity of OxfordParks RoadOxford OX1 3PUUK

Richard A. van DeldenDepartment of Organic and MolecularInorganic ChemistryStratingh InstituteUniversity of GroningenNijenborgh 49747 AG GroningenThe Netherlands

Sharyn A. EndowDuke University Medical CenterDepartment of MicrobiologyP. O. Box 3020Durham, NC 27710USA

Ben L. FeringaDepartment of Organic and MolecularInorganic ChemistryStratingh InstituteUniversity of GroningenNijenborgh 49747 AG GroningenThe Netherlands

Janice A. FischerSection of Molecular Celland Developmental BiologyInstitute for Cellular and MolecularBiologyThe University of Texas at AustinAustin, TX 78712USA

Leah T. HaimoDepartment of BiologyUnversity of CaliforniaRiverside, CA 92521USA

William O. HancockDepartment of BioengineeringPennsylvania State University218 Hallowell BuildingUniversity Park, PA 16802USA

XX List of Contributors

Nobutaka HirokawaDepartment of Cell Biologyand AnatomyGraduate School of MedicineUniversity of TokyoHongo 7-3-1, Bunkyo-kuTokyo 113-0033Japan

Jonathon HowardMax Planck Instituteof Molecular Cell Biology and GeneticsPfotenhauerstrasse 10801307 DresdenGermany

Yoshiharu IshiiSingle Molecule Processes ProjectICORP, JST2-4-14 Senba-higashi, MinoOsaka 562-0035Japan

Michele C. KiekeDepartment of Genetics,Cell Biology and DevelopmentUniversity of Minnesota6-160 Jackson Hall321 Church Street SEMinneapolis, MN55455USA

Stephen M. KingDepartment of BiochemistryUniversity of ConnecticutHealth Center263 Farmington AvenueFarmington, CT 06030-3305USA

John P. KonhilasDepartment of Molecular, Cellular,and Developmental BiologyUniversity of Colorado at BoulderCampus Box 347Boulder, CO 80309-0347USA

Nataliya KorzhevaThe Public Health Research InstituteInternational Center for Public HealthAlex Goldfarb’s Laboratory225 Warren StreetNewark, NJ 07103-3535USA

Nagatoshi KoumuraDepartment of Organic and MolecularInorganic ChemistryStratingh InstituteUniversity of GroningenNijenborgh 49747 AG GroningenThe Netherlands

Leslie A. LeinwandDepartment of Molecular,Cellular, and Developmental BiologyUniversity of Colorado at BoulderCampus Box 347Boulder, CO 80309-0347USA

Mikhail K. LevinDepartment of BiochemistryRobert Wood Johnson Medical SchoolPiscataway, New JerseyNJ 08854USA

XXIList of Contributors

E. MandelkowMax-Planck-Unitfor Structural Molecular BiologyNotkestrasse 8522607 HamburgGermany

A. MarxMax-Planck-Unitfor Structural Molecular BiologyNotkestrasse 8522607 HamburgGermany

A. MogilnerCenter for Genetics and DevelopmentDepartment of MathematicsUniversity of California at DavisDavis, CA 95616USA

Carlo D. MontemagnoDepartment of BioengineeringUniversity of California Los AngelesLos Angeles, CA 90023USA

Arkady MustaevThe Public Health Research InstituteInternational Center for Public HealthAlex Goldfarb’s Laboratory225 Warren StreetNewark, NJ 07103-3535USA

Hiroyuki NojiInstitute of Industrial ScienceUniversity of Tokyo4-6-1, Komaba Meguro-kuTokyo 153-8505Japan

George OsterDepartment of Molecularand Cell Biology201 Wellman HallUniversity of CaliforniaBerkley, CA 94720-3112USA

Smita S. PatelDepartment of BiochemistryRobert Wood Johnson Medical SchoolPiscataway, New JerseyNJ 08854USA

A. S. N. ReddyDepartment of Biology and Programin Cell and Molecular BiologyColorado State UniversityFort Collins, Colorado 80523USA

J. M. ScholeyCenter for Genetics and DevelopmentSection of Molecular and CellularBiologyUniversity of California at DavisDavis, CA 95616USA

Y.-H. SongMax-Planck-Unit for StructuralMolecular BiologyNotkestrasse 8522607 HamburgGermany

Jacob J. SchmidtDepartment of BioengineeringUniversity of California Los AngelesLos Angeles, CA 90023USA

XXII List of Contributors

James A. SpudichDepartment of Biochemistryand Department of DevelopmentalBiologyStanford University School of MedicineStanford, CA 94305USA

Chin-Yin TaiDepartment of Cell BiologyUniversity of Massachusetts MedicalSchool377 Plantation St.Worcester, MA 01605USA

Reiko TakemuraDepartment of Cell Biologyand AnatomyGraduate School of MedicineUniversity of TokyoHongo 7-3-1, Bunkyo-kuTokyo 113-0033Japan

Margaret A. TitusDepartment of Genetics,Cell Biology and DevelopmentUniversity of Minnesota6-160 Jackson Hall321 Church Street SEMinneapolis, MN55455USA

Richard B. ValleeDepartment of PathologyColumbia University Collegeof Physicians and SurgeonsP & S 15-409630 W. 168th St.New York, NY 10033USA

Kristen J. VerheyUniversity of MichiganMedical SchoolDept. of Cell and Dev. Biology1335 Catharine St.Ann Arbor, MI 48109-0616USA

Hongyun WangUniversity of CaliforniaSanta Cruz1156 High StreetSanta Cruz, CA 95064USA

Matthijs K. J. ter WielDepartment of Organicand Molecular Inorganic ChemistryStratingh InstituteUniversity of GroningenNijenborgh 49747 AG GroningenThe Netherlands

Toshio YanagidaSingle Molecule Processes ProjectICORP, JST2-4-14 Senba-higashi, MinoOsaka 562-0035Japan

Part 1Basic Principles of Motor Design

1The Myosin Superfamily: An Overview

Michele C. Kieke and Margaret A. Titus

1.1An Introduction to the Myosin Superfamily

Actin filaments and the myosin motors associated with them play important rolesin many dynamic biological processes. The classic example of actin filaments andmyosin at work is during skeletal muscle contraction. But the functions of actinand myosin extend to many other cellular events, such as motility, adhesion, endo-cytosis, cytoplasmic streaming, neuron growth, structural maintenance and polar-ization. Like molecular cars on an actin track, myosins transport organelles andother cellular components, such as mRNA. Myosins can also aid in the formationor maintenance of an organized actin-based structures (such as the stereocilia ofhair cells), and play roles in intracellular signal transduction pathways (seeBaker and Titus 1998, Mermall et al. 1998, Sokac and Bement 2000, Tuxworthand Titus 2000, for reviews summarizing myosin functions).

Myosins are molecular motor proteins that use the energy from adenosine tri-phosphate (ATP) hydrolysis to generate force for directed movement along actinfilaments (see Chapter 11 by Spudich and Chapter 13 by Ishii and Yanagida). Myo-sins are composed of one to two heavy chains, and one or more light chains. Theheavy chain consists of several major domains and can include various other sub-domains or protein motifs (Fig. 1.1). The relatively conserved N-terminal motor or‘head’ domain has binding sites for both ATP and F-actin. A short region joiningthe head and neck (termed the ‘converter domain’) is believed to be responsible forproducing the force required for movement. The neck domain contains one to sixlight chain binding regions termed IQ motifs, repeats of approximately 23 to 30residues containing the sequence IQXXXRGXXXRK (Bähler and Rhoads, 2002).The divergent C-terminal globular ‘tail’ has been implicated in binding cargoand targeting the myosin to its proper location in the cell (Karcher et al., 2002).Some myosins also feature a coiled-coil domain that promotes heavy chain dimer-ization.

The founding member of the myosin family, filament-forming class II musclemyosin, was discovered nearly a century ago, and its role in muscle contraction

4 1.1 An Introduction to the Myosin Superfamily

SH3

Myosin Structural features*

Motor Neck Tail

M1

M2

M3

M4

M5

M6

M7

M8

M9

M10

M11

M12

M13

M14

M15

M16

M17

M18

+ + +1-6

C1-2

SH3

x 5

Cx 5

x 3

x 4

x 3

C PHx 3

x 6

C C

Cx 6

+ + +

+ +

chitin synthase domain

Zn+2

GPA

PDZ

polybasic region

C

C

C

SH3

C SH3

ANKx 8

Pro

+ + + = positively-charged region

C = coiled-coil regionPH = pleckstrin homology domain

SH3 = Src homology 3 domain

Zn+2 = zinc-binding domain

GPA = Gly, Pro, Ala-rich region

= PEST site

* not drawn to scale

= FERM domain

= MyTH4 domain

= IQ motif

= N-terminal extension

= protein kinase domain

= rho-GAP domain

ANK = ankyrin repeatKey

Pro = proline-rich region

has been studied extensively (Geeves and Holmes, 1999, Huxley, 2000). A combi-nation of biochemical and molecular approaches has led to the identification ofover 20 different myosin classes (Berg et al., 2001). Because of the extensiveamount of knowledge acquired regarding the properties of myosin II it is referredto as ‘conventional’ myosin; all other types of myosin are referred to as‘unconventional’.

The first unconventional myosin, myosin I, was described in 1973 by Pollard andKorn (Pollard and Korn, 1973a, 1973b). They isolated a protein with enzymaticproperties similar to myosin II (i. e. it exhibited actin-activated Mg�2-ATPase andATP-sensitive binding to actin) from the common freshwater amoeba Acantha-moeba castellanii that had a lower molecular weight than muscle myosin II (125 ver-sus 200 kD) and did not form filaments. In addition, it was determined that myo-sin I had one head rather than two. Careful analysis of this unusual molecule re-vealed that it was indeed a bona fide myosin (Korn, 1991). This work provided thefirst insights into the potential diversity and functions of the myosin superfamily.

Myosin superfamily members are grouped into different classes based on phylo-genetic analysis of motor domains (Fig. 1.2) (Berg et al., 2001, Cheney and Moose-ker, 1992, Goodson and Spudich, 1993,). Each class is designated by a Roman nu-meral, largely in the order of their discovery (note that we will refer to myosinsusing Arabic numbers for simplicity). A total of 18 classes have been officiallydesignated, but there are at least six novel myosins that have yet to be classified.Myosins have been found in a variety of organisms but no one class is universallyexpressed in all phyla. The yeasts Saccharomyces cerevisiae and Schizosaccharomycespombe have a total of five myosin genes from three classes (M1, M2, M5). Thesemyosins are shared by higher organisms, ranging from Caenorhabditis elegans(C. elegans) to mammals. The human genome includes about 40 myosin genesfrom 12 classes (Berg et al., 2001). M8, M11, and M13 are only found in plants(see Chapter 18 by Reddy; Reddy and Day, 2001), and M14 myosins are found inparasites such as Toxoplasma gondii and Plasmodium falciparum (Berg et al., 2001).A unique class of myosins (as yet undesignated) has been found in the ciliatedprotozoan Tetrahymena (Garcés and Gavin, 1998, Williams et al., 2000), suggestingthat these organisms have a distinctive set of myosins.

Cells typically express multiple myosins � the expression of at least a dozen myo-sins in a single cell type has been described (Bement et al., 1994). This includesseveral different classes of myosin, as well as two or more isoforms of several classes.Myosins from the same class can have isoform-specific roles, such as M5 isoforms inmammalian cells and yeast (Reck-Peterson et al., 2000), or they can have functionallyoverlapping roles, such as the M1s in Dictyostelium discoideum and Saccharomyces(Geli and Riezman, 1996, Goodson et al., 1996, Jung et al., 1996, Novak et al., 1995).

51 The Myosin Superfamily: An Overview

� Figure 1.1. Domain structure schematic for characterized myosin genes. Schematic illustratingthe variety of known structural motifs found in myosin genes. A general box diagram is given foreach class, although individual members of the same class may vary depending on the organismand/or particular isoform.

6 1.1 An Introduction to the Myosin Superfamily

Figure 1.2. Unrooted myosin superfamily phy-logenetic tree. Phylogenetic tree from Berg et al.,2001, constructed using myosin motor domainsequences. Species names are listed in theAbbreviations table, and some gene nameshave been shortened to save space. Sequences

predicted in full or in part from genomic clonesare indicated by an asterisk. Figure and legendtext reprinted from Molecular Biology of the Cell(2001, 12: 780�794), with permission from theAmerican Society for Cell Biology.

Abbreviations:Ac: Acanthamoeba castellaniAcl: Acetabularia cliftoniiAn: Aspergillus nidulansAt: Arabidopsis thalianaCe: Caenorhabditis elegansDd: Dictyostelium discoideumDm: Drosophila melanogaster

Hs: Homo sapiensLp: Limulus polyphemusMm: Mus musculusPf: Plasmodium falciparumRn: Rattus norvegicusSc: Saccharomyces cerevesiaeSp: Schizosaccharomyces pombe

Tg: Toxoplasma gondiiTt: Tetrahymena thermophila

CSM: Chitin synthase myosinHMWM: High molecular weight myosinMysPDZ: PDZ myosin