Molecular Interactions of Actin: Actin Structure and Actin-Binding Proteins

Editors C.G. dos Remedios, D.D. Thomas

Last modified: November 1, 1999
NB Please notify Cris if you want to change your entry on this site. His email address is crisdos@anatomy.usyd.edu.au

ACTIN STRUCTURE

Chapter 2
Fumio Oosawa (completed)
Historical overview: Actin assembly and interactions
Aichi Institute  of Technology, Yagusa, Toyota, Aichi 470-03, JAPAN
Fax: +81 565 485783
Contents: Introduction; The G-F Transformation; F-actin and Bond free Energy; Dynamics of F-actin;
Activation of F-actin or the Thin Filament; Sliding of F-actin on Myosin;Pathway of Free Eneergy Conversion; The State of F-actin During Sliding; Actin and Non-muscle Cells;Future of Actin Research.

Chapter 3
Hanna Strzelecka-Golaszewska (Completed)
Role of divalent cations in actin structure
Department of Muscle Biochemistry, Nencki Insitute of Experimental Biology,
3 Pasteur Str PL-02-093 Warsaw POLAND
Phone: 4822 6686182
Fax: 4822 225342
Email: hannas@nencki.gov.pl
Contents:   Introduction; Effects of cations on the state and properties of actin;
Tightly-bound cation-dependent conformational changes in G-actin; Effects
of polymerizing salts on G-actin conformation. The monomer activation;
Cation-dependent differences in F-actin structure; Conclusions.

Chapter 4
Pierre Moens, Cris dos Remedios  (Completed)
An analysis of models of F-actin
Muscle Research Unit, Institute for Biomedical Research F13, The University of Sydney
Sydney 2006, AUSTRALIA.
Phone: +61 2 93516543
Fax +61 2 93512813
Email: crisdos@anatomy.usyd.edu.au
Contents: Functionality is my middle name; Actin-binding proteins are pulling my chains; Where is my N-terminus?; FRETting over my radii; Actin preparation; Labelling of Cys-374;  Labelling the nucleotide-binding site of unlabelled and DDPM-labelled actin; Experimental design; Actin concentration after Dowex-1 treatment; Fluorescence measurements; Theoretical calculations; Determinations of Cys-374 radial coordinate; Determination of the angle - Rintra; Determination ofthe Rintra; Localisation of the probe bound t Cys-374; The orientation factor ksquared in F-actin; Nucleotide radial coordinate; Localisation of the probe in the F-actin model; Do the results exclude any other orientation of the monomer?; Conclusions.

Chapter 5
Shin'ichi Ishiwata, J. Tadashige, I. Masui, T. Nishizaka, K. Kinosita, Jr. (Completed)
Microscopic analysis of polymerization and fragmentation of an individual actin filament
Department of Physics, School of Science and Engineering, Waseda University, Okubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, JAPAN.
Phone: +81 3 5286 3437
Fax: +81 3 32002567
Email: ishiwata@mn.waseda.ac.jp
Abstract:  The polymerization and fragmentation process of individual actin filaments can be visualized by fluorescence-imaging under a conventional fluorescence microscope to view the polymerized filaments stained with the fluorescent dye, rhodamine-phalloidin.  In this way, the polymerization process at the barbed and pointed ends of actin filaments could be analyzed in the presence of Mg2+ or Ca2+.  By measuring the rate of polymerization at various G-actin concentrations, we confirmed that the depolymerization process is inhibited by the attachment of phallotoxins (phalloidin or phallacidin) to the filaments, but that the polymerization rate itself is not affected in a major way. As a consequence, the critical concentration of polymerization is essentially zero.  Also, the fragmentation process of the filaments due to the addition of high concentrations of chaotropic salts such as KSCN and KI can be observed and stabilized by Pi.  The present study demonstrated that the polymerization and fragmentation dynamics on single actin filaments can be examined under fluorescence microscopy.  The interaction of phalloidin with actin also is briefly reviewed.

Chapter 6
T. Oda, K. Makino, I. Yamashita, K. Namba, Y. Maeda (completed revised)
The helical parameters of F-actin  precisely determined from X-ray  fiber  diffraction of well-oriented sols
International Institute for Advanced Research (IIAR), Central Research Laboratories (CRL), Matsushita Electric Industrial Co. Ltd., 3-4, Hikaridai, Seika, Kyoto, 619-0237, JAPAN.
Phone: +81-774-98-2543
Fax: +81-774-98-2575
Email: toda@crl.mei.co.jp, ymaeda@crl.mei.co.jp
Abstract:   A long-range aim of our research is to obtain a high resolution structure of F-actin filament using X-ray fiber diffraction. For this purpose, preparation of well-oriented F-actin filament sols is of essential importance, because the filament orientation strongly influences resolution and quality of diffraction data, as a result, the validity of F-actin structural model. In the present work, factors governing the filament orientation were systematically studied. Key factors are the filament length and the ionic strength of solvents, whereas the pH of sols and the kinds of anions and mono-valent cations are less sensitive. Now, Taking these factors into consideration, well-oriented F-actin sols can be reproducibly prepared by the following procedure: concentration of the filaments using a low speed centrifugation and application of a strong magnetic field of 13.5 Tesla. An angular distribution of the filaments is slightly less than 2o.  From diffraction patterns of the oriented sols, the helical parameters of F-actin filament have been determined at high precision: the pitch of one-start helix is 59.8Å; the helical symmetry is 67 subunits 31 turns of the one-start helix.

 Chapter 7
Katsuzo Wakabayashi, Yutaka Ueno, Yasunobu Sugimoto, Yasunori Takezawa  (Completed)
Structural changes in F-actin structure seen by X-ray
Division of Biophysical Diffraction
Engineering, Osaka University, Toyonaka, Osaka, 560 JAPAN
Phone: 81 6 8506515
Fax: 81 6 8506557
Email: waka@bpe.es.osaka-u.ac.jp

Chapter 8
Joanna K. Krueger, Jill Trewhella  (Completed)
Neutron scattering studies of muscle protein structures and their interactions
Chemistry, Science and Technology Division, Los Alamos National Laboratories
Mail Stop G-758 Gp CST-4, Los Alamos NM87545 USA
Phone: +1 505 6672031
Fax: +1 505 6670110
Email: jtrewhella@lanl.gov
ABSTRACT: Small-angle neutron scattering with contrast variation has contributed key insights into the conformational transitions and interactions underlying muscle contraction and its regulation.  Small-angle scattering from proteins in solution provides information on their overall shapes, sizes, and relative domain or subunit movements.  The techniques of contrast variation with neutron scattering allow one to probe the conformations of components within complex assemblies.  The scattering measurements, when combined with high-resolution structural information from crystallography or NMR, have proven quite powerful in mapping the dynamic interactions in a number of muscle-related systems.  In this chapter we present a brief description of the principals underlying solution scattering with contrast variation and the information that can be obtained.  We then review some of the neutron scattering studies, and the mechanistic information that has been derived from these studies, for a number of muscle proteins complexes.  The material covered includes studies of actin polymerization, actin/tropomyosin complexes, regulation by troponinC/troponinI, myosin S1 structure, actin/myosin S1 interactions, and calmodulin/myosin light chain kinase regulation.

 Chapter 9
Edward H. Egelman, Albina Orlova (Completed)
Two conformations of G-actin related to two conformations of F-actin
Department of Cell Biology and Neuroanatomy, University of Minnesota Medical
School, 321 Church St SE Minneapolis MN55455 USA
Phone: +1 612 6260121
Fax:  +1 612 6248118
Email: egelman@egel2.med.umn.edu
Abstract:  Four X-ray crystal structures of G-actin have now been solved, and an atomic model exists for the orientation of the actin subunit in the helical filament. However, electron microscopic studies have suggested that the actin filament can exist in a number of different conformational states. Since the dynamical properties of actin filaments are likely to be important in a range of processes, from muscle contraction to the control of cell form, we have been interested in interpreting the different conformational states of F-actin seen at low resolution in terms of molecular models. We show that two different G-actin structures, an open and a closed state, can be used to model quite well the opening of the nucleotide-binding cleft that is seen in yeast actin following ATP hydrolysis and the release of phosphate.

Chapter 10
Lisa Belmont, David Drubin (Completed)
Molecular genetics of yeast actin
401 Barker Hall, Department of Molecular and Cell Biology, University of California,
Berkeley CA  94720-3202 USA
Phone: +1 510-6423692
Fax: +1 510-6426420
Email: drubin@uclink4.berkeley.edu
Email: belmont@uclink4.berkeley.edu
Abstract: The yeast, Saccharomyces cerevisiae, has proven to be a powerful model organism in which to study actin function and structure-function relationships.  This yeast has a single actin gene, a feature that greatly simplifies molecular-genetic and biochemical analysis of actin. Yeast actin is 88% identical to mammalian actin and its biochemical properties are similar to all other actins studied (Nefsky and Bretscher, 1992).  The ease of molecular genetics in yeast has allowed generation of numerous site-specific actin mutants.  Furthermore, the non-lethal actin mutants have been expressed in yeast as the sole source of actin (Shortle et al., 1984), allowing elucidation of biological importance via phenotypic analysis, and purification and biochemical characterization of mutant actins.  The ability to biochemically characterize mutant actins expressed in yeast has been especially useful since functional actin has not been expressed and purified successfully from bacteria. In this chapter, features of the published yeast actin mutants are summarized.  These various mutants have provided insights into the mechanisms of polymerization and depolymerization, binding sites of various actin-binding proteins and drugs, regulation of actin by nucleotide, and the in vivo roles of actin in yeast.
 

ACTIN-BINDING PROTEINS

Chapter 12
Amy McGough, Brian Pope, Alan Weeds (Completed)
Cofilin structure and function
Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX77030. U.S.A.
Alan Weeds and Brian Pope are at the MRC-LMB, Cambridge, England.
Phone: +1 713 7986989
Fax: +1 713 7969438
Email: amcgough@bcm.tmc.edu
Email:  agw@mrc-lmb.cam.ac.uk
Email: bjp@mrc-lmb.cam.ac.uk
Abstract: Actin polymerization and cytoskeletal reorganisation play an essentialrole in cell locomotion and many forms of motility, including phagocytosisand cytokinesis.  The rate of assembly of actin filaments in vitro is virtually diffusion controlled, but depolymerization rates are too slow to regenerate the monomer pool required for cells to advance at rates of up to 30 um/min.  Actin Depolymerizing Factors (ADF/cofilin) are ideal candidates to aid in this process.  They are localized together with actin in motile regions of cells and are essential for cell viability in yeast, C. elegans, Drosophila, and Dictyostelium.  Here we describe the structure and properties of these proteins and highlight recent work on their interactions with actin.

Chapter 13
R.J. Sheedy, Frank M. Clarke (Completed)
Predicting Interaction Sites Between Glycolytic Enzymes and Cytoskeletal Proteins Employing the Concepts of the Molecular Recognition Theory
Faculty of Science and Technology, School of Life Sciences, Nathan Qld 4111 AUSTRALIA
Phone: +61 7 38757554
Fax: +61 7 38641534
Email: f.clark@sct.gu.edu.au
Abstract:  In 1984 Blalock and Smith focussed attention on certain complementary hydropathic relationships between amino acids based on the genetic code. Amino acids specified on one strand of DNA were found to be hydropathically complementary to those encoded by the opposite strand of the DNA in the same reading frame. Consequently peptide sequences derived from the noncoding strand of DNA, or RNA that is complementary to mRNA, will have an inverted pattern of hydropathy relative to the pattern of amino acids derived from the coding nucleic acid strand. These complementary peptides have been found more often than not to specifically bind to the partner peptide specified by the coding strand.  This is the basis of the Molecular Recognition Theory (MRT) as proposed by Blalock (see Blalock, 1995) which hypothesises "that complementary nucleotide sequences specify peptides or proteins that interact through complementary shapes or structures resulting from their inverted periodicity of hydrophobic and hydrophilic amino acids". A substantial and growing literature attests to the interaction of peptides and proteins encoded by complementary nucleic acid sequences. While the physical and chemical basis of the interactions of complementary pairs of peptides is yet to be satisfactorily explained, consideration of the increasing number of observations of the phenomenon warrants that it be taken into account when investigating the molecular basis of protein-protein interactions, as it may provide the basis for predicting interacting sites between proteins and provide an understanding of the basis of their evolution.  It could be envisaged that nucleic acid and peptide complementarity were responsible for the first tenuous interactions of proteins early in the history of protein evolution. Macromolecular organisation within cells is a fundamental requirement for cell function. The evolution of viable cells required not only the evolution of functional proteins, but also, or even more so, the evolution of functional systems of interacting proteins. It is proposed that the Molecular Recognition Theory may explain the essential non-randomness of the evolution of organised protein systems and assist in elucidating the molecular basis of interacting proteins systems by predicting protein-protein interaction sites.  In this work we establish the validity of applying the Molecular Recognition Theory to identify an established actin/actin binding protein interaction site. We then apply the theory to predict an interaction site between the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin and analyse the involvement of the predicted site in the interaction between the two proteins.

Chapter  14
Murat Kekic, Cris dos Remedios (Completed)
Interactions between actin and ABPs
Muscle Research Unit, Institute for Biomedical Research F13, The University of Sydney
Sydney 2006, AUSTRALIA.
Phone: +61 2 93513266
Fax +61 2 93512813
Email: rat@anatomy.usyd.edu.au
Abstract: Actin is one of the most conserved in the cytoskeleton and is virtually unbiquitous. It is plays a critical role in many cellular functions such as cell division, replication and signal transduction. As a major component of the cytoskeleton, it is essential for force transduction during cell motility. However, actin is not alone in performing these functions. Indeed, there are more than 60 actin binding proteins (ABPs) which play a vital role in actin function. It is beyond the scope of this chapter to summarise all of these proteins/peptides and in fact, the function of many of these proteins is not clearly known. ABPs are defined as either end-binding, filament-depolymerising, monomer-binding, filament-severing, cross-linking, stabilising, or motor proteins. Much work has been done on the regulatory/modulatory effects of ABPs. A number of them are widely distributed in eukaryotic systems (Pollard & Cooper, 1986). Recent evidence highlights the importance of many ABPs. For example: (1) cofilin plays a vital role in depolymerising actin filaments (Lappalainen & Drubin, 1997). Local concentrations of cofilin are mediated by cofilin kinases and phosphatases and this reaction regulates actin filament dynamics (Arber et al., 1998); (2) profilin is a powerful regulator of actin dynamics and also participates in signal transduction to the actin cytoskeleton (see review by Schlüter et al., 1997); (3) gelsolin is a severing and capping protein (seeBurtnick et al. In this volume). Recently, the gelsolin-binding site on filamentous-actin (F-actin) was determined (McGough et al., 1998). Large conformational changes seem to be needed in both actin and gelsolin for filament severing to occur. The focus of these and many other studies has concentrated on the action of one ABP on actin. The current dogma suggests that in the regulation/modulation of actin, the binding of one ABP displaces another, e.g. gelsolin and DNase I. Little or no evidence is available to suggest that the binding of one ABP to actin increases the affinity of a second ABP. In this chapter we will examine the latest literature on ABPs and introduce the concept that one ABP can allosterically modulate the affinity of a second ABP.

Chapter 15
Paul A. Janmey, Jagesh V. Shah, Jay X. Tang, Thomas P. Stossel (Completed)
Actin filament networks
Experimental Medicine Division, Brigham & Women's Hospital LRMC #302,
221 Longwood Ave., Boston MA02115, U.S.A.
Phone: +1 617 2780382
Fax:  +1 617 7342248
Email: janmey@calvin.bwh.harvard.edu
Contents: Introduction; Viscoelastic characterization of actin networks; Elasticity of F-actin; Actin-binding proteins that alter network formation; - crosslinking proteins, Actin-severing and capping proteins; Comparison with theories for semiflexible polymers; Interaction with other polymers; Actin and microtubules; Actin and intermediate filaments; actin and nonn-cytoplasmic biopolymers; Roles for network formation independent of mechanical strength; Actin netwroks in vivo; Conclusion.

Chapter 16
Leslie Burtnick, Robert C. Robinson, Senyon Choe (Completed)
Structure and function of gelsolin
The University of British Columbia, Department of Chemistry, 2036 Main Hall,
Vancouver BC, CANADA V6T  1Z1
Phone: +1 604 8224767
Fax: +1 604 8222847
Email: burtnick@unixg.ubc.ca
Abstract: The Ca2+-free form of plasma gelsolin consists of six structurally similar domains, S1 through S6, that pack together to yield a compact globular shape in which the actin-binding sequences are not sufficiently exposed to enable interaction with actin [Burtnick LD, Koepf EK, Grimes JM, Jones EY, Stuart DI, McLaughlin PJ, Robinson RC (1997) Cell 90:661-670]. Domain S1 continues directly into S2, which is separated from S3 by a 20-residue polypeptide linker sequence. The central b-sheet of S1 lies against that of S3 to yield a continuous 10-stranded sheet. This arrangement is repeated in the second half of gelsolin, domains S4 ? S6. The two halves of the molecule are linked by an extended 50-residue polypeptide. Aside from this covalent tether, the two halves are held together in Ca2+-free gelsolin through contacts that involve S2 with S6 and with a short helical segment that follows S6 at the C-terminus of the protein. We propose that binding Ca2+ releases the non-covalent connections that join the N and C-terminal halves of gelsolin, exposing the F-actin binding site on S2 and additional sites that bind Ca2+. After initial contact has been made between S2 and the side of an actin filament, further shifting of the domains relative to each other permits S1 to locate and dock with its interaction site on actin. Simultaneously, the second half of the gelsolin molecule, connected by the 50-residue tether to the first, could bind to actin units across the filament from the first half. The result would be severing of  F-actin and capping of the freshly exposed barbed end. Such a sequence of events would require significant readjustment in the positions of the domains relative to each other. This could be accommodated by pivoting at the interdomain joints and by use of slack in the linker strands between the domains.

Chapter 17
Thomas Beck, Pierre-Alain Delley, and Michael N. Hall (completed)
How extracellular stimuli regulate the cytoskeleton
Biozentrum, University of Basel
Klingelbergstrasse 70
CH4056 Basel, Switzerland
Phone:  (+41 61) 267 2162
Fax:  (+41 61) 267 2149
Email: hall@ubaclu.unibas.ch
Abstract:  The actin cytoskeleton is remodelled in response to several different types of extracellular signals and for various purposes.  Here we review selected examples of such signals and the pathways by which these signals impinge on the actin cytoskeleton to elicit changes in cell shape or movement.  In yeast, mating pheromones, nutrients, or environmental stress controls polarization of the actin cytoskeleton and polarized cell growth. In Drosophila, developmental signals control furrow formation and dorsal closure.  In mammalian cells, growth factors control neurite outgrowth and cell migration.  Also in mammalian cells, invasive pathogens subvert the host actin cytoskeleton to enter cells.  In all cases, the signaling pathways activated by these varied extracellular signals involve Rho-type GTPases.
 

Chapter  18
Laura Machesky, Robin C. May  (completed)
ARPs, actin-like proteins
MRC-LMCB, University College London
Gower St., London, WC1E-6BT, UK
Phone: +44 171 3807248
Fax: +44 171 3807805
Email: l.m.machesky@bham.ac.uk
Abstract: The Arps (Actin-Related Proteins) constitute a recently discovered family of proteins related to actin in equence and probably 3D structure. Interestingly, many of the Arps appear to function as members of multi-protien complexes.  Arp1 functions in a 20-S complex with at least 7 different proteins which is required in cytoplasmic dynein-mediated organelle motility along microtubules.  Arp2 and Arp3 function in an 8.5-S 7-protein complex that can initiate actin polymerization, crosslink and cap actin filaments.  Arp7 and Arp9 are components of the 2000-kDa SWI/SNF complex which is thought to regulate chromatin structure to alleviate transcriptional repression.  The functions of other members of the Arp family (up to 10 family members in budding yeast) are less well characterized, but it seems that these proteins have diverse functions and may in some cases only be related to actin in sequence and structure rather than in function.  The Hsc70 family and some sugar kinases also show a significant structural relationship to actin, but are not included as Arps because they do not share significant primary sequence homology.
 
 

Molecular Interactions of Actin:

Myosin interaction, Motility Assays and Ca-Regulatory Proteins

ACTIN-MYOSIN INTERACTION

Chapter 2
Ewa Prochniewicz, David D. Thomas  and Osha Roopnarine (Completion date 1999)
Structural changes in actin and myosin due to their strong and weak interactions
Department of Biochemistry, University of Minnesota Medical School,
Minneapolis, MN 55455, USA
Phone: +1 612 6250957
Fax: +1 612 6240632
Email: ddt@ddt.biochem.umn.edu

Chapter 3
Christopher L. Berger (Completion date:  1999)
FRET: applications to myosin and acto-myosin complexes
Department of Molecular Physiology and Biophysics and Biochemistry, University of
Vermont, D-2-1 Given Building, Burlington VT05405-0068, USA.
Phone: +1 802 6560832
Fax: +1 802 6560747
Email: berger@salus.med.uvm.edu

Chapter 4
Emil Reisler, Tim Doyle (completed)
Insights into actomyosin interactions from actin mutants
Department of Chemistry & Biochemistry
UCLA, 405 Hilgard Avenue, Los Angleles CA90024-1570, USA
Phone: +1 310 8252668
Fax: +1 310 2067286
Email: reisler@ewald.mbi.ucla.edu
Abstract: The actomyosin cross-bridge cycle involves a number of steps, with both weakly and strongly bound complexes.  Crystallographic modeling, chemical modification and antibody studies have identified many of the residues involved in actomyosin interactions within the cycle.  This review discusses the use of actin mutants from both yeast and slime mold to characterize the role of specific residues on the actin molecule in different steps of the cross-bridge cycle.  Actin from these organisms shows a high degree of sequence identity with skeletal a-actin, which is reflected in their similar affinities, activity and in vitro motility, with skeletal myosin. This and the ease of genetic manipulation of these organisms and the purification of the mutated proteins provide powerful tools to investigate the interaction of actin with myosin. Three pairs of acidic residues on subdomain 1 of actin (D2/E4, D24/D25, E99/E100 in yeast) are shown to play important roles in the weak binding interactions with myosin.  An additional pair of charged residues on the N-terminus of actin facilitates the progress of cross-bridges through the cycle, by favouring the isomerization between the weakly and strongly bound states.  However, it appears that the exact location of these acidic residues on subdomain 1 is not critical to the actomyosin function, but rather overall charge density of subdomain 1 of actin affects the actomyosin interactions and function.  An I341A yeast actin was used to confirm the function of hydrophobic residues in helix 338-348 in the actomyosin rigor binding state, and the transition between weakly and strongly bound actomyosin states.  Results of the reviewed studies provide the initial mutational map of the cross-bridge cycle step-specific actomyosin interface.

Chapter 5
Patrick Chaussepied, Juliette Van Dijk  (Completed)
Role of charge in actomyosin interactions
CRBM du CNRS Biochimie, INSERM BP 5051, Route de Mende, 34033
Montpellier Cedex, FRANCE
Phone: +33 67613334
Fax: +33 67521559
Email: patrick@xerxes.crbm.cnrs-mop.fr
Contents: Introduction; Structure of the actomyosin interface; Dynamics of the actomyosin complex; Role of the ionic interactions; Studies of the ionic interactions by chemical cross-linking experiments;
Regulation of the cross-linking sites by nucleotide analogues; A new model for the actomyosin interface during the ATPase cycle; Conclusions.

Chapter 6
Hideo Asukagawa, Reiko Ohkura,  Kazuo Sutoh (Completed)
Mutagenesis of myosin and the actin-myosin interface
Department of Life Sciences, University of Tokyo at Komaba
Meguro-ku Tokyo 153, JAPAN
Phone: +81-3 54546751
Fax: +81-3 54546751
Email: cksutoh@komaba.ecc.u-tokyo.ac.jp
Abstract:  The sequence of the 50-20 kDa junction of the heavy chain of Dictyostelium myosin is ASRAKKG (residues 618-624). The basic residues in this junction (R620, K622 and K623) were replaced with alanine residues one by one(alanine-scanning mutagenesis) to examine their roles in motor function.  Ca- and Mg-ATPase activities of the mutant myosins were similar to those of the wild-type myosin, indicating that these mutations did not affect the gross conformation of myosin. Actin-activated ATPase activities of the mutants were, however, much lower at all actin concentrations examined than those of the wild-type myosin. It is very likely that the lower actin-activated ATPase activities arose from a drammatic increase in the Km values (an apparent affinityof actin and myosin during ATP hydrolysis), rather than from a decrease in Vmax values (the ATPase activity in the presence of infinite actin). Although sliding of actin filaments on these mutant myosins was much slower than that on the wild-type myosin, it reached a similar velocity at higher concentrations of myosin. These in vitro studies suggest that the observed defect of the mutant myosin mainly arose from the weaker interaction of actin and myosin during hydrolysis. When these mutant myosins were expresed in Dictyostelium myosin-null cells, they fully complemented the myosin-specific defects, indicating that these mutants were functional in them, possibly because concentrations of actin in Dictyostelium were high enough to overcome the weak affinity. These in vitro and in vivo  studies have shown that the basic residues in the 50-20 kDa junction play a crucial role in maintaining the actin-myosin accociation through ionic interactions during ATP hydrolysis, actin-myosin sliding and force generation.

 Chapter 7
Osha Roopnarine (Completion date 1999)
Familial hypertrophic cardiomyopathic mutations that affect the actin-myosin
interaction
Department of Biochemistry, University of Minnesota Medical School,
Minneapolis, MN 55455, USA
Phone: +1 612 6250113
Fax: +1 612 6240632
Email: osha@ddt.biochem.umn.edu
Anstract:
 
 

MOLTILITY-BASED ASSAYS OF ACTOMYOSIN

Chapter 9
Yoshi Ishii, Akihiko Ishijima, Toshhio Yanagida (Completed)
Coupling between chemical and mechanical events and conformation of single protein molecules
Single Molecule Processes Project, ICORP, JST, 2-4-14 Senba-Higashi, Mino, Osaka 562, JAPAN
Phone: +81 6 8575421
Fax: +81 6 8575421
Email: ishii@yanagida.jst.go.jp, yanagida@bpe.es.osaka-u.ac.jp
Abstract: Muscle contraction is achieved by a cyclic interaction of myosin and actin using the energy liberated by hydrolysis of ATP. Research has focused on the question how chemical energy is converted to the mechanical energy. Recent developments of single molecule imaging and manipulation techniques have allowed us to directly test how chemical and mechanical events are coupled. Using a single molecule imaging technique and fluorescently labeled ATP, the hydrolysis of ATP can be directly visualized. With laser trap or microneedle methods, the displacement and the forced exerted by single motor proteins can be measured with an accuracy of one nm and pN, respectively. Simultaneous measurements of single molecule ATPase and displacement show that mechanical events are not always tightly coupled to chemical events. These results indicate that the energy liberated from the ATPase reaction is stored in the actomyosin molecules for later use. To elucidate the molecular mechanism of this coupling, it is important to know the structural changes that take place within the molecules. We have developed a single molecule spectroscopy technique to monitor the dynamic behavior of proteins. These studies show a slow conformational transition between metastable states. These slow conformational transitions may explain energy storage within actomyosin.

Chapter 10
Marissa A. LaMadrid, P. Bryant Chase,  Albert M. Gordon (completed)
Motility assays of Ca regulation of actin filaments
Department of Physiology and Biophysics, University of Washington, Box 357290
Seattle, WA98195-7290, USA
Phone: +1 206 5430834
Fax: +1 206 6850619
Email: amg@u.washington.edu
Abstract:  The in vitro motility assay was utilized in an attempt to understand how calcium regulates muscle contraction by measuring the thin filament length dependence of the speed vs. pCa. No length dependence in speed of moving portions was observed for 1 mm to > 12 mm long filaments, but the shortest regulated filaments were more likely to stop moving. The results were analyzed in the context of  whether drag is significant or not. In both analyses, the data suggest that [Ca2+] is possibly controlling the strong binding time, in addition to controlling the number of motors that bind.
 
 

Ca REGULATION of THIN FILAMENTS

Chapter 12
Michael A. Geeves,  Sherwin S. Lehrer (completed)
Cooperativity in the calcium regulation of muscle contraction
Department of Muscle Research, Boston Biomedical Research Institute,
20 Staniford St., Boston MA02114, USA
Phone: +1 617 9120381
Fax: +1 617-523-6649
Email: Lehrer@BBRI.org
Email: geeves@mpi-dortmund.mpg.de
Abstract: We recently reviewed the evidence that the muscle thin filament operates as a classical Monod-Wyman-Changeux cooperative/allosteric system while interacting with myosin heads. We showed that much of the available biochemical in vitro data can be explained if actin is the catalyst (enzyme) which accelerates the loss of Pi from the myosin-ADP-Pi substrate.  Tropomyosin (Tm) is the regulatory component, myosin complexed with ADP is the activating ligand and troponin (Tn) in the absence or presence of Ca2+ is the allosteric inhibitor or activator, respectively. The myosin product complex "turns-on" the activity by shifting the equilibrium from the T (Closed) to the R-state (Open), facilitated by Tn in the presence of Ca2+. Thus, the ATPase activity of the system is mostly off in the absence of myosin heads even in the presence of Ca2+. We introduced the concept of the apparent cooperative unit size, n, the number of actin subunits turned-on by the strong binding of a myosin head and indicated that n  can vary depending upon the strength of the end-to-end interactions and flexibility of Tm. We pointed out that in the absence of Ca2+, there is significant population of another state, Blocked, which is also involved in the regulation of the myosin-actin interaction. In the previous review we discussed the essential role of Tm in providing the cooperativity. In this review we will focus on the role of Ca2+ and extend our discussion to a consideration of the various interactions among the several components which are involved in the regulation of force generation in a muscle fiber.

Chapter 13
Roger Craig, William Lehman (completed)
The ultrastructural basis of thin filament regulation
Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Ave.,
North Worcester MA016555, USA
Phone: +1 617 8562262
Fax: +1 617 8566361
Email: rcraig@umassmed.ummed.edu
Abstract: Actin filaments in muscle perform two crucial functions in contraction:  they undergo cyclic interaction with myosin crossbridges to generate filament sliding and hence contraction, and in most muscle they also regulate contraction, by controlling crossbridge interaction with actin.  In this chapter we review the contributions of electron microscopy and image processing to our understanding of the molecular mechanism of regulation in striated and smooth muscles, and we relate the results to models derived from x-ray diffraction and other approaches.  We discuss the historical development of ideas and the techniques that were successful in providing our current model. We first review our current understanding of striated muscle actin filament structure and the mode of binding of the regulatory proteins, tropomyosin and troponin.  We then describe the essence of the original "steric-blocking" model of regulation as proposed in the 1970's, the subsequent support that it received, the challenges leveled at it on the basis of conflicting structural and kinetic data, and how the original model was revised. Next we provide a state-of-the-art, near-atomic level structural model of the striated muscle thin filament in the switched OFF (low Ca2+), switched ON (high Ca2+), and potentiated (myosin-bound) states, based on recent negative staining, cryo-EM, image processing and x-ray diffraction data.  This model shows how tropomyosin physically blocks myosin-binding amino-acid clusters on actin in the OFF state and how these masked sites become available for myosin binding, by movement of tropomyosin, when thin filaments are activated.  Recent criticisms of these models are described and these criticisms evaluated.  We conclude that the steric-blocking model continues to provide a powerful explanation of the mechanism of regulation, and that the criticisms do not agree with the relevant facts. We also describe briefly how structural studies of thin filaments reconstituted using an internal deletion tropomyosin have helped advance our knowledge of regulation, and we provide new data describing the structure of native thin filaments actively interacting with myosin filaments during sliding. Finally we describe our current knowledge of smooth muscle thin filament structure, and in particular the molecular organization of the actin-binding proteins, caldesmon and calponin on the thin filament and their effects on the organization of tropomyosin.  Both caldesmon and calponin can inhibit smooth muscle actomyosin ATPase in vitro and hence are potential modulators of actin-myosin interaction, in addition to the primary regulatory switch which in smooth muscle involves phosphorylation of the regulatory light chains.  Based on structural data, we conclude that if caldesmon or calponin do modulate smooth muscle contraction in vivo, they must do so by mechanisms distinct from that used by the troponin-tropomyosin system in striated muscle.

Chapter 14
Danuta Szczesna, James D. Potter,  (completed)
The role of troponins in the Ca-regulation of muscle contraction
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine,
POB 016189 Miami FL33101-6189, USA
Phone: +1 305-243-5874
Fax:  +1 305 243-6233
Email: jpotter@chroma.med.miami.edu

Chapter 15
Masao Miki (Completed)
Structural changes between regulatory proteins and actin
Department of Applied Chemistry and Biotechnology, Fukui University, Bunkyo 3-chome,
9-1 Fukui-shi, 910, Japan
Phone: + 81 776 278786
Fax: + 81 776 278747
Email: masao@acbio.acbio.fukui-u.ac.jp
Abstract:  In this chapter, we review our recent FRET measurements and other related papers, and then introduce a new model for the Ca2+-mediated regulation of tropomyosin-troponin in skeletal muscle. More than 30 years ago, S. Ebashi and his colleagues showed that skeletal muscle contraction is regulated by tropomyosin and troponin on actin filaments in response to a change in Ca2+ concentrations. However, the detailed molecular mechanism of this regulatory process is still not well understood. A quarter century ago, the steric blocking model was proposed by H. Huxley in which tropomyosin sterically block the myosin binding site on actin during inhibitory state. Although there is no categorical proof for the model, it has been widely accepted. Fluorescence resonance energy transfer (FRET) measurements have been used to determine the spatial relationships between regulatory proteins and actin in order to test the notion of tropomyosin movement on actin filaments in response to a change in Ca2+ concentrations as predicted by the steric blocking theory. FRET measurements did not detect any significant movement of tropomyosin on the reconstituted thin filament. On the other hand, FRET measurements suggest that during inhibition, TnI moves toward the outer domain of actin. FRET measurements also indicated that the extent of this movement correlates well with pCa-dependence of tension development, and that the time scale of this movement is rapid enough to allow this movement to be directly involved in regulation of muscle contraction. These data can be incorporated into a regulation model in which, during inhibition, TnI cross-links the outer domain of actin with tropomyosin which covers seven actin-inner domains along the long-pitch helix. When the two neighboring TnIs along the long-pitch helix cross-link two actin monomers, they may cause a considerable distortion of the actin helix and/or a significant immobilization of internal motion of the outer domain of actin monomers which are located between two neighboring cross-linkings along the long-pitch helix. The distortion and/or immobilization of actin filaments may disturb the proper actin-myosin interaction during ATPase cycle.

Chapter 16
Takeyuki Wakabayashi (Completion date ?, 1999)
Ultrastructural analysis of regulation using mutants
Department of Physics, Faculty of Science, University of Tokyo,
Hongo 7-3-1, Bunkyo-ku, Tokyo 113, JAPAN
Phone: +81 776 278747
Fax: +81 3 8149717
Email: Wakabayashi@phys.s.u-tokyo.ac.jp

Chapter 17
Larry Tobacman (Completion date ?, 1999)
Regulation of contractility in cardiac muscle
Department of Internal Medicine, University of Iowa Hospitals and Clinics,
200 Hawkins Dr., Iowa City, IA52242, USA
Phone: +1 319 3563703
Fax: +1 319 3567893
Email: larry-tobacman@uiowa.edu

This page is maintained by Mo Buksh
Department of Anatomy and Histology
The University of Sydney
Sydney, 2009, Australia
mobuksh@anatomy.usyd.edu.au