Myosins

PROTEINS AND CELL REGULATION

Volume 7

Series Editors: Professor Anne RidleyLudwig Institute for Cancer Researchand Department of Biochemistry and Molecular BiologyUniversity College LondonLondonUnited Kingdom

Professor Jon FramptonProfessor of Stem Cell BiologyInstitute for Biomedical Research,Birmingham University Medical School,Division of Immunity and InfectionBirminghamUnited Kingdom

Aims and Scope

Our knowledge of the ways in which a cell communicates with its environment and how it responds toinformation received has reached a level of almost bewildering complexity. The large diagrams of cells tobe found on the walls of many a biologist’s office are usually adorned with parallel and interconnectingpathways linking the multitude of components and suggest a clear logic and understanding of the roleplayed by each protein. Of course this two-dimensional, albeit often colourful representation takes noaccount of the three-dimensional structure of a cell, the nature of the external and internal milieu, thedynamics of changes in protein levels and interactions, or the variations between cells in different tissues.

Each book in this series, entitled “Proteins and Cell Regulation”, will seek to explore specific proteinfamilies or categories of proteins from the viewpoint of the general and specific functions they provideand their involvement in the dynamic behaviour of a cell. Content will range from basic proteinstructure and function to consideration of cell type-specific features and the consequences of disease-associated changes and potential therapeutic intervention. So that the books represent the most up-to-dateunderstanding, contributors will be prominent researchers in each particular area. Although aimed atgraduate, postgraduate and principal investigators, the books will also be of use to science and medicalundergraduates and to those wishing to understand basic cellular processes and develop novel therapeuticinterventions for specific diseases.

Myosins

A Superfamily of Molecular Motors

Edited by

Lynne M. ColuccioBoston Biomedical Research Institute, Watertown, MA, U.S.A.

A C.I.P. Catalogue record for this book is available from the Library of Congress.

ISBN 978-1-4020-6516-3 (HB)ISBN 978-1-4020-6519-4 (e-book)

Published by Springer,P.O. Box 17, 3300 AA Dordrecht, The Netherlands.

www.springer.com

Printed on acid-free paper

Legend to cover figure: The cover illustration shows molecules of different myosin classes,visualized in the electron microscope by negative staining. Classes II (left) and V (right, top panel)

have two heads at one end of a coiled-coil �-helical tail. Class I myosins, Ib (right, third panel) and Ic(right, bottom panel), have one head. Class VI (right, second panel) is monomeric in these

examples, but can be dimeric. Head length varies between 14 nm and 32 nm depending on the numberof light chains, which varies between one and six (myosin 1b, myosin V). The resolution in the

pictures is 2 nm and the magnification is different in each case. Courtesy of Drs. Chun Feng Song,Matthew Walker and John Trinick, Leeds University, UK.

All Rights Reserved© 2008 Springer

No part of this work may be reproduced, stored in a retrieval system, or transmitted inany form or by any means, electronic, mechanical, photocopying, microfilming, recording

or otherwise, without written permission from the Publisher, with the exceptionof any material supplied specifically for the purpose of being entered

and executed on a computer system, for exclusive use by the purchaser of the work.

PREFACE

Few would have predicted 20 years ago that myosins constitute a superfamilywith at least two-dozen classes and that these molecular motors are involvedin a multitude of intracellular activities including cell division, cell movement,intracellular transport and signal transduction. Application of state-of-the-artcellular and molecular biological, structural biological, genetic, biochemical andbiophysical techniques has provided and continues to provide critical informationregarding the structure–function relationship; and the cellular roles of variousmyosins in organisms as diverse as protozoa, yeast, plants and higher animals. Theassociation of myosins with diseases including neurological disorders, immuno-deficiencies, cardiomyopathies, hearing and vision loss testify to the importanceof understanding the biochemical properties and cellular roles of myosins. The16 chapters in this volume summarize the tremendous progress made in studyingmembers of the myosin superfamily in recent years and offer critical insight intowhat future research will yield. I would like to express my sincere gratitude to theauthors of this volume. It was a pleasure to work with each of you and I thank youfor the considerable efforts in making this international endeavor possible. I alsothank John Trinick from the University of Leeds, UK, for the elegant montageof images of single molecules of myosins on the cover, which beautifully showsthe structures of some of these amazing molecules. The able assistance of MarliesVlot from Springer and Indumadhi Srinivasan from Integra Software Services forproduction of the book is greatly appreciated.

September 2007 Lynne ColuccioWatertown, MA

v

CONTENTS

List of Contributors ix

Colour Plates xiii

1. The Structural and Functional Diversity of the Myosin Familyof Actin-Based Molecular Motors 1Mark S. Mooseker and Bernardo J. Foth

2. Myosin Structure 35Kenneth C. Holmes

3. The Myosin Family: Biochemical and Kinetic Properties 55Mohammed El-Mezgueldi and Clive R. Bagshaw

4. Myosin I 95Lynne M. Coluccio

5. Myosin II: Sarcomeric Myosins, the Motors of Contraction in Cardiacand Skeletal Muscles 125Carlo Reggiani and Roberto Bottinelli

6. Smooth-Muscle Myosin II 171Christine R. Cremo and David J. Hartshorne

7. Non-Muscle Myosin II 223Mary Anne Conti, Sachiyo Kawamoto, and Robert S. Adelstein

8. Class III Myosins 265Andréa Dosé, Jennifer Lin-Jones, and Beth Burnside

9. Myosin V 289James R. Sellers and Lois S. Weisman

10. Myosin VI: A Multifunctional Motor Protein 325Folma Buss and John Kendrick-Jones

11. Myosin VII 353Aziz El-Amraoui, Amel Bahloul, and Christine Petit

vii

viii CONTENTS

12. Plant Myosins VIII, XI, and XIII 375Keiichi Yamamoto

13. Class IX Myosins 391Martin Bähler

14. Myosin X 403Melinda M. Divito and Richard E. Cheney

15. Myosin Class XIV and Other Myosins in Protists 421Karine Frénal, Bernardo J. Foth, and Dominique Soldati

16. Myosin XVa 441Erich T. Boger, Gregory I. Frolenkov, Thomas B. Friedman,and Inna A. Belyantseva

Index 469

LIST OF CONTRIBUTORS

Robert S. Adelstein National Heart, Lung, and Blood Institute, NationalInstitutes of Health, 10 Center Dr MSC 1762, Building 10, Room 8N202,Bethesda, MD 20892-1762 USA/ Tel. 001 301 496 1865/ Fax. 001 301 402 1542/adelster@mail.nih.gov

Clive R. Bagshaw Department of Biochemistry, University of Leicester,Leicester LE1 9HN, UK. / Tel. +44 (0) 116 229 7048/ Fax. +44 (0) 116 229 7018/crb5@le.ac.uk

Amel Bahloul Unité de Génétique des Déficits Sensoriels, INSERM UMRS 587,Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris cedex 15, France /Tel. 33 1 4568 88 89/Fax. 33 1 40 61 34 42/ abahloul@pasteur.fr

Martin Bähler Institut für Allgemeine Zoologie und Genetik, Westfälische-Wilhelms Universität, Schlossplatz 5, 48149 Münster, Germany/ Tel. +49 251 83238 74/ Fax. +49 251 83 247 23/ baehler@uni-muenster.de

Inna A. Belyantseva Section on Human Genetics, Laboratory of MolecularGenetics, National Institute on Deafness and Other Communication Disorders,National Institutes of Health, Rockville, MD 20850/ Tel. 001 301 435 8204/ Fax.001 301 402 7580/ belyants@nidcd.nih.gov

Erich T. Boger Section on Human Genetics, Laboratory of Molecular Genetics,National Institute on Deafness and Other Communication Disorders, NationalInstitutes of Health, Rockville, MD 20850/ Tel. 001 301 435 8110/ Fax. 001 301402 7580/ bogere@nidcd.nih.gov

Roberto Bottinelli Department of Experimental Medicine, Human PhysiologyUnit, University of Pavia, Via Forlanini 6, I-27100 Pavia, Italy/ Tel. +39 0382987257/ Fax. +39 0382 987257 or 987664/ roberto.bottinelli@unipv.it

Beth Burnside U. C. Berkeley, Department of Molecular and Cell Biology, 391LSA 3200, Berkeley, CA 94720-3200 USA / Tel. 001 510 642 2439/ Fax. 001510-643-6791/ burnside@socrates.berkeley.edu

Folma Buss Cambridge Institute for Medical Research, University of Cambridge,Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK/ Tel. +44(0)1223 763225/ Fax. +44 (0)1223 762640/ fb1@mole.bio.cam.ac.uk

ix

x LIST OF CONTRIBUTORS

Richard E. Cheney Department of Cell and Molecular Physiology, Universityof North Carolina, Chapel Hill, NC 27599-7545 USA / Tel. 001 919 966 0331/Fax. 001 919 966 6927/ richard_cheney@med.unc.edu

Lynne M. Coluccio Boston Biomedical Research Institute, 64 Grove St.,Watertown, MA 02472 USA/ Tel. 001 617 658 7784/ Fax. 001 617 972 1761/coluccio@bbri.org

Mary Anne Conti National Heart, Lung, and Blood Institute, National Institutesof Health, 10 Center Dr MSC 1762, Building 10, Room 8N202, Bethesda,MD 20892-1762 USA/ Tel. 001 301 496 1912/ Fax. 001 301 402 1542/contim@mail.nih.gov

Christine R. Cremo Department of Biochemistry and Molecular Biology,University of Nevada School of Medicine, Reno NV 89557-0014/ Tel. 001 775784 7033/ Fax. 001 775 784 1419/ cremo@unr.edu

Melinda M. DiVito Department of Cell and Molecular Physiology, Universityof North Carolina, Chapel Hill, NC, 27599-7545 USA/ Tel. 001 919 966 1190/Fax. 001 919 966 6927/ melinda_divito@med.unc.edu

Andréa Dosé Department of Molecular and Cell Biology, University ofCalifornia Berkeley, 391 Life Sciences Addition 3200, Berkeley, CA, 94720-3200/Tel. 001 510 642 2439/ Fax. 001 510 643 6791/ acdose@socrates.berkeley.edu

Aziz El-Amraoui Unité de Génétique des Déficits Sensoriels, INSERM UMRS587, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris cedex 15, France /Tel. 331 45 68 88 92/ Fax. 33 1 40 61 34 42/ elaz@pasteur.fr

Mohammed El-Mezgueldi Department of Biochemistry, University of Leicester,Leicester LE1 9HN, UK/ Tel. +44 (0) 116 229 7102/ Fax. +44 (0) 116 229 7018/mem16@le.ac.uk

Bernardo J. Foth School of Biological Sciences, Nanyang TechnologicalUniversity, 60 Nanyang Drive, Singapore 637551/ Tel. +65 6316 2927/ Fax. +656791 3856/ bfoth@ntu.edu.sg

Karine Frénal Department of Microbiology and Molecular Medicine, Facultyof Medicine, University of Geneva, CMU, 1 Rue Michel-Servet, CH-1211Geneva 4 Switzerland/ Tel. +41 22 379 5656 / Fax. + 41 22 379 5702/Karine.Frenal@medecine.unige.ch

Thomas B. Friedman Section on Human Genetics, Laboratory of MolecularGenetics, National Institute on Deafness and Other Communication Disorders,National Institutes of Health, Rockville, MD 20850 USA /Tel. 001 301 4967882/Fax. 001 301 402 7580/ friedman@nidcd.nih.gov

LIST OF CONTRIBUTORS xi

Gregory I. Frolenkov Department of Physiology, University of Kentucky,Lexington, KY 40536/ Tel. 001 859 323 8729 / Fax. 001 859 323 1070/Gregory.Frolenkov@uky.edu

David J. Hartshorne Muscle Biology Group, Department of NutritionalSciences, University of Arizona, Tucson, AZ 85721-0038/Tel. 001 520 621 7239/Fax. 001 520 621 1396/ hartshor@ag.arizona.edu

Kenneth C. Holmes Department of Biophysics, Max Planck Institut fürmedizinische Forschung, Jahnstraße 29, 69120 Heidelberg, Germany/ Tel. +496221 486270/ Fax. +49 6221 486437/ holmes@mpimf-heidelberg.mpg.de

Sachiyo Kawamoto National Heart, Lung, and Blood Institute, NationalInstitutes of Health, 10 Center Dr MSC 1762, Building 10, Room 8N202,Bethesda, MD 20892-1762 USA/ Tel. 001 301 435 8034/ Fax. 001 301 402 1542/kawamots@mail.nih.gov

John Kendrick-Jones MRC Laboratory of Molecular Biology, Hills Road,Cambridge CB2 2QH, UK/ Tel. +44 (0)1223 402409/ Fax. +44 (0)1223 213556/jkj@mrc-lmb.cam.ac.uk

Jennifer Lin-Jones Department of Molecular and Cell Biology, University ofCalifornia Berkeley, 391 Life Sciences Addition 3200, Berkeley, CA, 94720-3200/Tel. 001 510 642 2439/ Fax. 001 510 643 6791/ linjones@berkeley.edu

Mark S. Mooseker Department of Molecular, Cellular and DevelopmentalBiology, 352 Kline Biology Tower, Yale University, New Haven, CT 06520-8104/Tel. 001 203 432 3468/ Fax. 001 203 432 3468/ mark.mooseker@yale.edu

Christine Petit Unité de Génétique des Déficits Sensoriels, INSERM UMRS587, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris cedex 15, France/ Tel. 331 45 68 88 90/ Fax. 33 1 40 61 34 42/ cpetit@pasteur.fr

Carlo Reggiani Department of Anatomy and Physiology, University of Padova,Via Marzolo 3, 35131 Padova, Italy/ Tel. +39 049827 5313/ Fax. +39 049 8275301/ carlo.reggiani@unipd.it

James R. Sellers NIH NHLBI, Lab. of Molecular Cardiology, 10 CenterDr. Bldg 10 8N117, Bethesda, MD 20892-1760 USA/ Tel. 001 301 402 1542/Fax. 001 301 402 1542/ sellersj@nhlbi.nih.gov

Dominique Soldati Department of Microbiology and Molecular Medicine,Faculty of Medicine, University of Geneva, CMU, 1 Rue Michel-Servet,CH-1211 Geneva 4, Switzerland/ Tel. +41 22 379 5672/ Fax. +41 22 379 5702/Dominique.Soldati-Favre@medecine.unige.ch

xii LIST OF CONTRIBUTORS

Lois S. Weisman Department of Cell and Developmental Biology. University ofMichigan, 210 Washtenaw Ave., Rm. 6437, Ann Arbor, MI 48109-2216, USA/Tel. 001 734 647 2539/ Fax. 001 734 615 5493/ lweisman@umich.edu

Keiichi Yamamoto Department of Biology, Chiba University, Inage-ku,263-8522, Chiba, Japan/ Tel./Fax. +81 43 290 2809/ k_yamamoto@faculty.chiba-u.jp

COLOUR PLATES

Plate 1.

�Plate 1. Schematic representation of “traditional” myosin classes including their known organ-ismal distribution. This classification is based on phylogenetic analysis of the conserved myosinmotor domains (see, e.g., Foth et al., 2006). The three groups as indicated on the right representclasses of myosins containing MyTH4 (and FERM) domains (group A), myosins containing DIL(dilute) domains and related classes (group B), and closely related classes II and XVIII (groupC) (Foth et al., 2006). Three groups of myosins have been proposed to represent the ancestraland most ancient myosins (Richards and Cavalier-Smith, 2005): class I, the FERM/MyTH4-domaincontaining myosins (group A), and the DIL-domain containing myosins (group B). The portrayedprotein domains are primarily based on predictions by SMART (http://smart.embl-heidelberg.de/)and CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). This figure is adapted from Fothet al., 2006; copyright 2006 National Academy of Sciences, U. S. A. Species abbreviations: Ac,Acanthamoeba castellanii; Acl, Acetabularia cliftonii; Af, Aspergillus fumigatus; At, Arabidopsisthaliana; Cb, Caenorhabditis briggsae; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Cp,Cryptosporidium parvum; Dm, Drosophila melanogaster; Gg, Gallus gallus; Hs, Homo sapiens;Mm, Mus musculus; Pf, Plasmodium falciparum; Sc, Saccharomyces cerevisiae; Tb, Trypanosomabrucei; Tc, Trypanosoma cruzi; Tg, Toxoplasma gondii; Tpn, Thalassiosira pseudonana; Tt,Tetrahymena thermophila. Sequence accession numbers: Ac_HMWMI_IV: A23662; Acl_myo2_XIII:AAB53062; Af_csE_XVII: EAL93639; At_MYA1_XI: NP_173201; At_VIIIa: AAD50052;Cb_HUM4_XII: CAE70637; Ce_HUM3_VI: NP_490856; Ci_XIX: AK116916; CpMyoI_XXIV:CAD98475; Dm_10A_XV: NP_572669; Dm_29D_XX: AAF52683; Dm_NinaC_III: AAA28721;Gg_FSk_II: AAB47555; Hs_FSkE_II: P11055; Hs_IB: NP_036355; Hs_IC (Ibeta): CAA67131;Hs_IE: NP_004989; Hs_MyoIG: EAL23748; Hs_IXa: AAD49195; Hs_MYO5C_V: AAF78783;Hs_Myr8_XVI: NP_055826; Hs_X: AAF37875; Hs_XV: AAF05903; Mm_shaker_VII: NP_032689;Mm_XVIIIa: CAI24424; PfMyoF_XXII: CAD52416; PfMyoK_VI: AAN35999; Sc_Myo2_Va:P19524; Tb_Myo1_IB: AAZ10929; Tc_Myo2_XXI: EAN87803; TgMyoA_XIVa: AAC47724;TgMyoC_XIVb: AAL30895; TgMyoG_XXIII: ABA01553; TgMyoH_XIVc: TgTwinScan_6293(http://v3-0.toxodb.org/); Tpn_135766_XXII: 135766 (http://genome.jgi-psf.org/); TtMyo1_XIVd:AAC24454. Protein domain accession numbers (SMART, COG, and Pfam databases): ankyrin:SM00248; ATS1 = alpha-tubulin suppressor: COG5184; C1 = protein kinase C conserved region 1:SM00109; chitin synthase: PF03142; Cyt-b5 = Cytochrome b5-like heme/steroid binding: PF00173;DIL = dilute: PF01843; FERM (Band 4.1): SM00295; FYVE: SM00064; myosin tail homology 1 =MyTH1: PF06017; myosin tail homology 4 = MyTH4: SM00139; N-terminal SH3-like: PF02736; PDZ:SM00228; PH = pleckstrin homology: SM00233; RA = Ras association: SM00314; RhoGAP = GTPase-activator protein for Rho-like GTPases: SM00324; SH3 = Src homology 3: SM00326; UBA = ubiquitinassociated: SM00165; WD40: SM00320; WW: SM00456 (See Figure 1.1, p. 4.)

Plate 2. The post-rigor structure of the myosin motor domain (Rayment et al., 1993a). The structure ofthe myosin cross-bridge shown as a ribbon diagram in the orientation it would take if bound to actin andviewed from the pointed (–) end of the actin filament. The N-terminus is shown green and the nucleotidebinding P-loop and adjoining helix are shown yellow; the upper 50K is red; the lower 50K domain isgrey. Note the cleft separating the upper and lower 50K domains. The lower 50K domain appears tobe the primary actin-binding site. The N-terminal boundary of the upper 50K domain comprises thedisordered loop 1 (between the points marked A and B). The upper and lower 50K domains are alsoconnected by a disordered loop (loop 2 between C and D). The C-terminal long helix (dark blue) carriestwo calmodulin-like light chains and joins onto the thick filament. The relay helix and converter domainare shown. In this conformation of the cross-bridge (post-rigor state) the lever arm is in the post-powerstroke position or “DOWN”, as in the rigor state. The colouring corresponds with sub-domain bound-aries. For clarity, the proximal end of the relay helix is shown light blue although it is actually part ofthe lower 50K domain. The distal end (beyond the kink) is firmly attached to the converter domain. Inthe post-rigor structure the relay helix is straight (no kink). This figure is from Geeves and Holmes, 2005(SeeFigure 2.7, p. 43.)

Plate 3. Shown is the ATP-binding site: the P-loop, switch 1 (SW1) switch 2 (SW2) and the relay helixand converter domain for a, the near-rigor state and b, the pre-power stroke state. Note the rotation ofthe converter and lever arm accompanying the movement of switch 2 (See Figure 2.8, p. 44.)

Plate 4. The converter regions of myosin VI and myosin V. The converter subdomain of the myosinmotor immediately follows the SH1 helix and precedes the lever arm. The myosin VI converter (green)is extended by insert 2 (dark purple) plus a calmodulin (CaM; light purple; with Ca2+ ions as red balls),which function to redirect the lever arm helix (beginning at green star; Lys-809). For comparison, theSH1 helix and converter of myosin V (red) are overlaid (from Park et al., 2007)(See Figure 2.13, p. 51.)

iP.PDA.M

PTA.M

PDA.M.AiP.PDA.M

PTA.M

M.A)b(M.A+PDA.M.A)a(

Plate 5. Pie chart to illustrate the duty ratios of (a) fast skeletal myosin contracting under unloadedconditions (b) mammalian single headed myosin V based on the data of De La Cruz et al. at 7.5 �M actin(De La Cruz et al., 1999). The double-headed myosin V has a duty ratio of >0�99 due to the alternatingactivity of the heads. The overall time (360�) represents the time for one ATPase cycle (1/kcat) whilethe black and blue sectors represent the relative time attached to actin (1% in skeletal myosin II and67% in myosin V)(See Colour Plate 5) (See Figure 3.1, p. 57.)

xilehHS=2hctiwS=1hctiwS=poolP=

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76

5

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21

mret-N

1pool

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mret-C

tfelcrenni

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ATP

Plate 6. Schematic representation of a myosin motor domain to show the major features referred to inthe text. Subdomains are colored as follows: green, N-terminal subdomain; red, upper 50K subdomain;pink, lower 50K subdomain; blue, 20K C-terminal subdomain which leads into the rod region of theintact myosin II molecule. The light chains bound to the lever arm region are omitted for simplicity.The figure is from C. R. Bagshaw (2007) Myosin mechanochemistry. Structure 15(5): 511–2 ©ElsevierLtd (See Figure 3.2, p. 73.)

Plate 7. Localization of class I myosins. A schematic depicting intracellular structures and/or functionswith which class I myosins are associated. 1, microvilli from intestinal brush border; 2, intracellularorganelles; 3, stereocilia on hair cells of inner ear; 4, cell cortex; 5, pseudopods; 6, exocytosis; 7,endocytosis; 8, phagocytosis; and 9, nucleus, where it is associated with the transcription machinery.Modified from the original drawing in Mermal et al. (Mermall et al., 1998) (See Figure 4.3, p. 108.)

Plate 8. Structure of myosin II and subfragments. Myosin II can be proteolysed to form distinctfragments. Light meromyosin (LMM) is the C-terminal two thirds of the coiled-coil tail (rod) andsubfragment 2 (S2) is the remaining one-third with its N-terminus at the head-tail junction. Heavymeromyosin (HMM) contains the S2 portion of the tail plus both subfragment 1 (S1) head fragments.Each S1 heavy chain can be digested by trypsin at 2 loop regions to form fragments of 25, 50 and 23kDa. The color for each region is matched in the atomic structure of S1, shown below. The regulatorydomain contains a portion of the 23-kDa fragment of the heavy chain and the essential and regulatorylight chains. The motor domain forms the remainder of the molecule (See Figure 6.1, p. 173.)

Plate 9. A cartoon illustrating the domain organization of myosin VI with the motor domain, the 53amino-acid insert (the reverse gear), the IQ motif that binds calmodulin and the tail domains labeled 3to 6. The sequence of the central charged region 4 is shown above the cartoon illustrating the repeatingpattern of positively (yellow) and negatively (green) charged amino acids. Below is shown the keyfeatures of the C-terminal targeting domain with the sequences of the large insert in myosin VI from(a) human brain and (b) human intestine and the conserved sequence of the small insert. The positionsof the phosphorylated T residues in the TINT sequence (labeled with P) and the binding sites forGIPC/optineurin, PIP2 and Dab2 are shown (See Figure 10.1, p. 328.)

Plate 10. The possible roles of myosin VI in endocytosis. The left immunofluorescent image showsthe localization of expressed GFP-tagged myosin VI in normal rat kidney fibroblasts and enlarged onthe right its colocalization with clathrin in vesicles in a merged image. In the model at step (1) myosinVI may initially bind to PIP2 and Dab2 bound to the cytoplasmic tail of a receptor at the plasmamembrane and by moving the complex may cluster the receptors together to initiate the formation of aclathrin-coated pit. At step (2) myosin VI may promote pit formation by pulling the plasma membraneinto the cell and then aid in the pinching off of the membrane to form a clathrin-coated vesicle. At step(3) myosin VI may move the newly formed clathrin-coated vesicle into the cell where it is uncoated andthen at step (4) transport the vesicle through the cortical actin filament network for its eventual deliveryto its final destination, possibly a recycling endosome from where it can subsequently be recycled backto the plasma membrane (step 5) (See Figure 10.2, p. 337.)

Plate 11. Myosin VI is present at the Golgi complex and involved in exocytosis. In the model, proteinssynthesized in the endoplasmic reticulum (ER) are transported to the Golgi complex, where they undergopost-translational modification and are sorted for delivery to their specific intracellular destinations. Atthe TGN myosin VI is involved in the constitutive secretion pathway. At step (1) it could sort proteinsinto different subdomains for delivery to distinct cellular compartments and could also be involved invesicle formation and budding rather like its proposed role in endocytosis at the plasma membrane. Atstep (2) myosin VI could then be involved in short distance transport of the vesicles ultimately destinedfor delivery to the plasma membrane. In polarized epithelial cells myosin VI is required for the sortingand transport of new membrane proteins with specific sorting signals to the basolateral domain [step(3)]. A complex of myosin VI with the adaptor protein optineurin and the regulatory protein Rab8 ispresent in recycling endosomes (RE), which are the sorting station for basolateral transport in polarizedepithelial cells (See Figure 10.3, p. 339.)

MOTOR DOMAIN

ENDOCYTOSIS

CELL DIVISION

CELL MOTILITY

SECRETION

CELL SHAPEMEMBRANEDYNAMICS

deafness

secretorydiarrhea

Cardiomyopathy

Neurodegeneration

cancer

Plate 12. The roles of myosin VI in the cell and its possible involvement in a number ofdiseases/disorders. The motor domain may be involved in transporting vesicles or protein complexeseither as an array of monomers or dimers to specific cellular destinations and may also have anchoringfunctions to tether vesicles to actin filaments or cross-link actin filaments to membranes. The C-terminaltargeting domain via specific adaptor proteins such as Dab2, GIPC, SAP97 and optineurin is involvedin the cellular processes outlined and either the absence, mutation or over-expression of myosin VI mayplay a role in the diseases/disorders shown (See Figure 10.4, p. 345.)

Plate 13. (A) Myosin VIIa in the sensory cells of the mouse auditory organ (hair cells). The right panelis a schematic representation of the mammalian auditory epithelium, known as the organ of Corti. Theinner (IHC) and outer (OHC) hair cells are flanked by various types of supporting cells that do notexpress myosin VIIa. (B) Diagrams of developing (E18 to P5) and mature (P15) auditory hair bundles inthe mouse. The upper panel illustrates the organization of the hair bundle, a group of large, specializedmicrovilli – known as stereocilia– in which mechanoelectrical transduction of sound stimuli takes place.The height of stereocilia rows rises in a staircase pattern towards the kinocilium, the genuine cilium. Inmammals, the kinocilium disappears in mature auditory hair cells. Lower panels: At E18, the stereociliathat form the growing hair bundle are held together by various side-links spreading along their entirelength. At P5, one single apical link, the tip link (TL), believed to gate the mechanoelectrical transductionchannel, and different lateral-links connect the stereocilia. From the tip down to the base of stereocilia,these links are the horizontal top links (HL), shaft links (SL), and ankle links (AL). Subsequently,the ankle links and shaft links disappear, as shown at P15, and are replaced by top connectors thatare maintained in adulthood (adapted from Goodyear et al., 2005). (C) Hair bundle disorganizationin a shaker-1 (Myo7a4626SB� newborn mouse. The kinocilium is colored in yellow. Scanning electronmicroscopy (source: Gaelle Lefèvre). (D) Myosin VIIa and other USH proteins in the hair bundle. Inthe hair-cell body apical region, myosin VIIa is likely to be required for the transfer of most USHproteins up to the stereocilia. In the developing stereocilia, cadherin 23, protocadherin 15, usherin andVlgr1, make up links that are intracellularly attached to the scaffold proteins harmonin or whirlin. Thesescaffold proteins are directly or indirectly (via myosin VIIa) connected to the actin filament core of thestereocilia. Dysfunction or absence of any of the USH proteins leads to disruption of the hair bundlecohesiveness (See Figure 11.3, p. 362.)

Plate 14. Myosin VIIa in retinal pigment epithelium and photoreceptor cells. Middle panel: Schematicrepresentation of the retinal cell layers. Light gets through several cell layers before it reaches thephotoreceptors (rods and cones). The apical microvilli of the retinal pigment epithelium (RPE) cellssurround the tip of the photoreceptor outer segment (OS), which is where phototransduction takes place.Approximately 10% of the outer segments are replaced each day, with new discs being formed proxi-mally and others shed distally by the RPE cells. The synaptic terminals, containing the ribbons, connectthe photoreceptors with horizontal cells, bipolar cells and ganglion cells. Abbreviations: ONL, outernuclear layer; INL, inner nuclear layer, GCL, ganglion cells layer. Left panel: Schematic representationof the apical region of a REP cell. Melanosomes (M) display fast, bidirectional microtubule-dependentlong-range movements in the cell body, driven by kinesin/dynein motor proteins (1), At the cellperiphery, cargos can switch from transport along microtubules to transport along actin microfilaments,and conversely from actin filaments to microtubules (e.g., phagosomes). The myosin VIIa/MyRIP/rab27atripartite complex ensures this switch (green arrow) for melanosomes (2), enabling the retention and/orlocal movement of the melanosomes along RPE microvilli (3), Myosin VIIa also plays a role in thetransfer of phagosomes from the microvilli to the cell body (4; orange arrow). Right panel: Schematicrepresentation of a photoreceptor connecting cilium and its calycal process. The proteins en route tothe photoreceptor outer segment, e.g., rhodopsin, transducins…etc, are believed to dock at the levelof the basal bodies (BB) to be transported distally via the connecting cilium (CC). At the tip of thephotoreceptor inner segment (IS), myosin VIIa may be involved in the transfer of opsin (green arrow)to the connecting cilium and its transport to the outer segment (See Figure 11.4, p. 366.)

Plate 15. Schematic models showing the structures of the three classes of plant myosins (See Figure12.1, p. 377.)

A B C

10 μm

Plate 16. Myo10 localizes to the tips of filopodia. (A) Immunostaining of endogenous Myo10. (B)Phalloidin staining of actin. (C) Overlay of panels A and B. In this HeLa cell, Myo10 is present atthe tips of filopodia (arrowheads). Endogenous Myo10 generally localizes to the tips of filopodia aswell as other regions of dynamic actin such as membrane ruffles, the leading edge of lamellipodia, andphagocytic cups (See Figure 14.2, p. 409.)

Plate 17. Scheme of a Toxoplasma gondii tachyzoite with a detailed model of the “glideosome”, themolecular machinery promoting gliding motility. Abbreviations: GAP45 and GAP50, gliding associatedprotein 45 kDa and 50 kDa, respectively; IMC, Inner Membrane Complex; IMP, Inner MembraneParticle, MyoA, myosin A; MLC or MTIP, Myosin light chain or Myosin tail-interacting protein (SeeFigure 15.2, p. 424.)

Plate 18. Endogenous myosin XVa andwhirlin protein expression in the organ ofCorti and vestibular hair-cell stereocilia. Inall images the actin core of stereocilia wascounterstained with rhodamine-phalloidin(red). (A-C) Development of myosin XVaimmunoreactivity (green) in the E18.5mouse organ of Corti hair cells (opticalplane of the stereocilia level). (A) MyosinXVa immunoreactivity was detected at thetips of longer stereocilia of one row of innerhair cells (IHCs) and three rows of outerhair cells (OHCs) at the basal turn of thecochlea. Note that at E18.5 immature stere-ocilia still cover most of the apical surfaceof IHCs (arrow) and OHCs (arrowhead) andare about the same length as the longerstereocilia, but have practically no myosinXVa-specific signal at their tips. As part ofthe normal process of hair-cell differenti-ation, these underdeveloped stereocilia areresorbed from the apical surface of maturehair cells. (B) In the middle turn of theE18.5 mouse cochlea, more mature IHCshave a low amount of myosin XVa at thetips of stereocilia (arrow), while immaturebundles of OHCs have barely visiblemyosin XVa immunoreactivity at the tips

(arrowhead). (C) Immature stereocilia of both IHCs (arrow) and OHCs (arrowheads) of the apical turnof the cochlea are not clearly visible when stained with rhodamine-phalloidin and do not possess myosinXVa immunoreactivity. (D-F ) Localization of myosin XVa at the tips of stereocilia of mouse vestibularhair cells. (D) Myosin XVa immunoreactivity (green) at the tips of hair-cell stereocilia of ampulla fromE14.5 mouse, when the gradation in height of stereocilia is just becoming evident. (E) Myosin XVa(green) at the tips of stereocilia of hair cells from adult mouse ampulla. (F ) Myosin XVa at the tips ofsaccular hair-cell stereocilia of adult mouse. (G) Localization of myosin XVa at the tips of stereociliaof three rows of OHCs and one row of IHCs of P5 mouse organ of Corti (green). (H) Myosin XVa atthe tips of OHC stereocilia of adult guinea pig organ of Corti (green). (I) IHC stereocilia of adult ratorgan of Corti were splayed by pressing on the coverslip to visualize myosin XVa at the tip of eachstereocilium. Arrow points to a stereocilium that was enlarged in J–L. (J ) Myosin XVa staining isconcentrated at the tip (green), while no detectable staining of myosin XVa is evident on the side of astereocilium. (K) Rhodamine-phalloidin staining of the F-actin core of the same stereocilium. F-actin isnot detected at the tip where myosin XVa is concentrated. (L) Double staining reveals partial overlap ofmyosin XVa signal (green, J ) with F-actin core of stereocilia (red, K). (M-N ) Localization of whirlin(green) in organ of Corti hair-cell stereocilia of adult (M) and postnatal day 7 (N ) wild type mouse.Scale bars 5 �m (See Figure 16.4, p. 453.)

Plate 19. Helios gene-gun mediated trans-fection of vestibular and cochlear sensoryepithelia explants with expression vectorscontaining GFP-Myo15a or DsRed/GFP-Whrn. In all images, the actin core of stere-ocilia was counterstained with rhodamine-phalloidin (red). (A) Saccular hair celltransfected with GFP-Myo15a shows greenfluorescent signal at the tips of stereociliaconfirming the localization of endogenousmyosin XVa. (B-C) Distention of stere-ocilia tips 96 hours after transfectionwith GFP-Myo15a. (B) GFP-myosin XVa(green) at the tips of stereocilia of a trans-fected hair cell of a P8 utricular explant.Stereocilia bundles of two other nontrans-fected hair cells are depicted in the sameimage. (C) Nomarski image of the samearea of the utricular explant. Arrows pointto the distended stereocilia tips filled withGFP-myosin XVa. Arrowheads point to thestereocilia of nontransfected hair cells. (D)GFP-myosin XVa at the tips of stereociliaof transfected OHC 8 hrs after transfection.(E) GFP−myosin-XVa[C592Y], and (F )

GFP-myosin-XVa[R167A, G388A] are concentrated in the cell bodies and are not targeted to stereociliatips in vestibular hair cells of wild-type mouse. (G-I) Simultaneous transfection of GFP-Myo15a andDsRed-Whrn into the vestibular hair cell of a wild-type mouse. GFP-Myo15a (G), DsRed-Whrn (H) andmerged image (I). Note that the fluorescence of GFP-myosin-XVa and DsRed-whirlin are proportionalto each other and increases with stereocilia length. (J ) GFP-whirlin is concentrated in the cell bodyand is not targeted to stereocilia tips in vestibular hair cell of Myo15ash2 homozygous mouse. (K-N )Stereocilia of Myo15ash2 vestibular (K) and auditory (L-N ) hair cells do not elongate after transfectionwith GFP-Myo15a[-PDZL], lacking the C-terminal PDZ ligand (green). In panel K, staining with anti-whirlin antibody is shown in blue, GFP-Myo15a[-PDZL] is in green. (O-Q)An auditory hair cell of aMyo15ash2 mouse transfected with GFP-Myo15a and neighbouring non-transfected control cells (48 hpost-transfection). (O), A merge of rhodamine-phalloidin staining (P) with the GFP-myosin XVa image(Q). (R) Restoration of the staircase shape of a stereocilia bundle of a Myo15ash2 vestibular hair cell 67h after transfection with GFP-Myo15a. (S) Accumulation of GFP-whirlin at the tips of IHC stereocilia.Transfected and neighboring non-transfected P5 rat auditory hair cells 21 hours post-transfection. (T )Vestibular hair cell of P3 wild-type mouse 65 hours post-transfection with GFP-Whrn. (U ) Restorationof the staircase shape of a stereocilia bundle in a Whrnwi vestibular hair cell 48 h after transfectionwith GFP-Whrn. (V -X) Exogenous GFP-myosin-XVa (green, V ) recruits endogenous whirlin stainedwith anti-whirlin antibody (blue, W ) to stereocilia tips of a Myo15ash2 vestibular hair cell transfectedwith GFP-Myo15a (X, merged image). There is no anti-whirlin immunoreactivity in the stereocilia ofneighbouring non-transfected hair cells. Sensory explants were harvested at P2–P5 and transfected thenext day. Scale bars, 5 �m (See Figure 16.5, p. 454.)