Myosin Assembly Research Articles

Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

Cloning, molecular characterization, and expression analysis of the unc45 myosin chaperone b(unc45b)gene of grass carp (Ctenopharyngodon idellus).

Unc45 myosin chaperone b(unc45b)gene is a molecular chaperone that mediates the folding, assembly and accumulation of thick-filament myosin in the formation of sarcomere, which plays an important role in the development of striated muscle and the stability of sarcomere. In this study, the complete cDNA sequence of unc45b gene of grass carp was obtained by rapid amplification of cDNA ends (RACE), and the characteristics of the unc45b protein predicted from gene sequence was analyzed by bioinformatics methods. The differential expression pattern in tissues was also detected by quantitative real-time PCR. The results showed that the full-length of unc45b gene of grass carp is 3163bp, which contains a 60bp 5'UTR, a 298bp 3'UTR, and a 2865bp open reading frame (ORF) encoding a 934 amino acid peptide. The deduced unc45b protein exhibits a homology of 92, 86, 86% with the protein of zebrafish (Danio rerio), channel catfish (Ietalurus punctatus) and tilapia (Oreochromis niloticus) respectively, and the protein contains UCS myosin head binding domain and TPR peptide repeat domain. The protein is a hydrophilic and non-secretory protein with a molecular mass and isoeletronic point of 103,699.8 and 7.39Da. The structural elements of the protein includes α-helixes and loops, and the unc45b gene highly expresses in skeletal muscle and heart in grass carp. This study laid a foundation for further research in explaining the myofibril accumulation in crisped grass carp.

Influence of cell polarity on early development of the sea urchin embryo.

Establishment and maintenance of cell polarity is critical for normal embryonic development. Previously, it was thought that the echinoderm embryo remained relatively unpolarized until the first asymmetric division at the 16-cell stage. Here, we analyzed roles of the cell polarity regulators, the PAR complex proteins, and how their disruption in early development affects later developmental milestones. We found that PAR6, aPKC, and CDC42 localize to the apical cortex as early as the 2-cell stage and that this localization is retained through the gastrula stage. Of interest, PAR1 also colocalizes with these apical markers through the gastrula stage. Additionally, PAR1 was found to be in complex with aPKC, but not PAR6. PAR6, aPKC, and CDC42 are anchored in the cortical actin cytoskeleton by assembled myosin. Furthermore, assembled myosin was found to be necessary to maintain proper PAR6 localization through subsequent cleavage divisions. Interference with myosin assembly prevented the embryos from reaching the blastula stage, while transient disruptions of either actin or microtubules did not have this effect. These observations suggest that disruptions of the polarity in the early embryo can have a significant impact on the ability of the embryo to reach later critical stages in development.

Skip residues modulate the structural properties of the myosin rod and guide thick filament assembly

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report here that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility. Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. In addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.

Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation.

Primitive streak formation in the chick embryo involves large scale highly coordinated flows of over 100.000 cells in the epiblast. These large scale tissue flows and deformations can be correlated with specific anisotropic cell behaviours in the forming mesendoderm through a combined light-sheet microscopy and computational analysis. Relevant behaviours include apical contraction, elongation along the apical-basal axis followed by ingression as well as asynchronous directional cell intercalation of small groups of mesendoderm cells. Cell intercalation is associated with sequential, directional contraction of apical junctions, the onset, localisation and direction of which correlate strongly with the appearance of active Myosin II cables in aligned apical junctions in neighbouring cells. Use of a class specific Myosin inhibitors and gene specific knockdowns show that apical contraction and intercalation are Myosin II dependent and also reveal critical roles for Myosin I and Myosin V family members in the assembly of junctional Myosin II cables.

A mutation in the converter subdomain of Aspergillus nidulans MyoB blocks constriction of the actomyosin ring in cytokinesis

We have identified a mutant allele of the Aspergillus nidulans homologue of myosin II (myoB; AN4706), which prevents normal septum formation. This is the first reported myosin II mutation in a filamentous fungus. Strains expressing the myoBG843D allele produce mainly aberrant septa at 30°C and are completely aseptate at temperatures above 37°C. Conidium formation is greatly reduced at 30°C and progressively impaired with increasing temperature. Sequencing of the myoBG843D allele identified a point mutation predicted to result in a glycine-to-aspartate amino acid substitution at residue 843 in the myosin II converter domain. This residue is conserved in all fungal, plant, and animal myosin sequences that we have examined. The mutation does not prevent localization of the myoBG843D gene product to contractile rings, but it does block ring constriction. MyoBG843D rings at sites of abortive septation disassemble after an extended period and dissipate into the cytoplasm. During contractile ring formation, both wild type and mutant MyoB::GFP colocalize with actin – an association that begins at the pre-ring “string” stage. Down-regulation of wild-type myoB expression under control of the alcA promoter blocks septation but does not prevent actin from aggregating at putative septation sites – the actin rings, however, do not fully coalesce. Both septation and targeting of MyoB are blocked by disruption of filamentous actin using latrunculin B. We propose a model in which myosin assembly at septation sites depends upon the presence of F-actin, but assembly of the actin component of contractile rings depends upon normal levels of myosin only for the final stages of ring compaction.

UCS proteins: chaperones for myosin and co-chaperones for Hsp90.

The UCS (UNC-45/CRO1/She4p) family of proteins has emerged as chaperones that are specific for the folding, assembly and function of myosin. These proteins participate in various important myosin-dependent cellular processes that include myofibril organization and muscle functions, cell differentiation, cardiac and skeletal muscle development, cytokinesis and endocytosis. Mutations in the genes that code for UCS proteins cause serious defects in these actomyosin-based processes. Homologs of UCS proteins can be broadly divided into (1) animal UCS proteins, generally known as UNC-45 proteins, which contain an N-terminal tetratricopeptide repeat (TPR) domain in addition to the canonical UCS domain, and (2) fungal UCS proteins, which lack the TPR domain. Structurally, except for TPR domain, both sub-classes of UCS proteins comprise of several irregular armadillo (ARM) repeats that are divided into two-domain architecture: a combined central-neck domain and a C-terminal UCS domain. Structural analyses suggest that UNC-45 proteins form elongated oligomers that serve as scaffolds to recruit Hsp90 and/or Hsp70 to form a multi-protein chaperoning complex that assists myosin heads to fold and simultaneously organize them into myofibrils. Similarly, fungal UCS proteins may dimerize to promote folding of non-muscle myosins as well as determine their step size along actin filaments. These findings confirm UCS proteins as a new class of myosin-specific chaperones and co-chaperones for Hsp90. This chapter reviews the implications of the outcome of studies on these proteins in cellular processes such as muscle formation, and disease states such as myopathies and cancer.

Chaperone-mediated reversible inhibition of the sarcomeric myosin power stroke

Molecular chaperones are required for successful folding and assembly of sarcomeric myosin in skeletal and cardiac muscle. Here, we show that the chaperone UNC-45B inhibits the actin translocation function of myosin. Further, we show that Hsp90, another chaperone involved in sarcomere development, allows the myosin to resume actin translocation. These previously unknown activities may play a key role in sarcomere development, preventing untimely myosin powerstrokes from disrupting the precise alignment of the sarcomere until it has formed completely.

Regulation of structure and function of sarcomeric actin filaments in striated muscle of the nematode Caenorhabditis elegans.

The nematode Caenorhabditis elegans has been used as a valuable system to study structure and function of striated muscle. The body wall muscle of C. elegans is obliquely striated muscle with highly organized sarcomeric assembly of actin, myosin, and other accessory proteins. Genetic and molecular biological studies in C. elegans have identified a number of genes encoding structural and regulatory components for the muscle contractile apparatuses, and many of them have counterparts in mammalian cardiac and skeletal muscles or striated muscles in other invertebrates. Applicability of genetics, cell biology, and biochemistry has made C. elegans an excellent system to study mechanisms of muscle contractility and assembly and maintenance of myofibrils. This review focuses on the regulatory mechanisms of structure and function of actin filaments in the C. elegans body wall muscle. Sarcomeric actin filaments in C. elegans muscle are associated with the troponin-tropomyosin system that regulates the actin-myosin interaction. Proteins that bind to the side and ends of actin filaments support ordered assembly of thin filaments. Furthermore, regulators of actin dynamics play important roles in initial assembly, growth, and maintenance of sarcomeres. The knowledge acquired in C. elegans can serve as bases to understand the basic mechanisms of muscle structure and function.

S100A4 regulates the Src-tyrosine kinase dependent differentiation of Th17 cells in rheumatoid arthritis

Objectives: To evaluate the role of S100A4, a calcium-binding regulator of nonmuscle myosin assembly, for T-cell responses in rheumatoid arthritis. Methods: Arthritis was induced in the methylated bovine serum albumin (mBSA)-immunized mice lacking the entire S100A4 protein (S100A4KO) and in wild-type counterparts treated with short hairpin ribonucleic acid (shRNA)-lentiviral constructs targeting S100A4 (S100A4-shRNA). The severity of arthritis was evaluated morphologically. T-cell subsets were characterized by the expression of master transcription factors, and functionally by proliferation activity and cytokine production. The activity of the Scr-kinases Fyn and Lck was assessed by the autophosphorylation of C-terminal thyrosine and by the phosphorylation of the CD5 cytodomain. The interaction between S100A4 and the CD5 cytodomain was analysed by nuclear magnetic resonance spectrophotometry. Results: S100A4-deficient mice (S100A4KO and S100A4-shRNA) had significantly alleviated morphological signs of arthritis and joint damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice accumulated Foxp3+ Treg cells, while the number of RORγt+ and (pTyr705)STAT3+ cells was reduced. S100A4-deficient mice had a limited formation of Th17-cells with low retinoic acid orphan receptor gamma t (RORγt) mRNA and IL17 production in T-cell cultures. S100A4-deficient mice had a low expression and activity of T-cell receptor (TCR) inhibitor CD5 and low (pTyr705)STAT3 (signal transducer and activator of transcription 3), which led to increased (pTyr352)ZAP-70 (theta-chain associated protein kinase of 70kDa), lymphocyte proliferation and production of IL2. In vitro experiments showed that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards the CD5 cytodomain. Spectrometry demonstrates an interaction between the CD5 cytodomain and EF2-binding sites of S100A4. Conclusion: The present study demonstrates that S100A4 plays an important part in the pathogenesis of arthritis. It controls CD5-dependent differentiation of Th17 cells by regulating the activity of the Src-family kinases Lck and Fyn.

Mechanochemical regulation of oscillatory follicle cell dynamics in the developing Drosophila egg chamber.

During tissue elongation from stage 9 to stage 10 in Drosophila oogenesis, the egg chamber increases in length by ∼1.7-fold while increasing in volume by eightfold. During these stages, spontaneous oscillations in the contraction of cell basal surfaces develop in a subset of follicle cells. This patterned activity is required for elongation of the egg chamber; however, the mechanisms generating the spatiotemporal pattern have been unclear. Here we use a combination of quantitative modeling and experimental perturbation to show that mechanochemical interactions are sufficient to generate oscillations of myosin contractile activity in the observed spatiotemporal pattern. We propose that follicle cells in the epithelial layer contract against pressure in the expanding egg chamber. As tension in the epithelial layer increases, Rho kinase signaling activates myosin assembly and contraction. The activation process is cooperative, leading to a limit cycle in the myosin dynamics. Our model produces asynchronous oscillations in follicle cell area and myosin content, consistent with experimental observations. In addition, we test the prediction that removal of the basal lamina will increase the average oscillation period. The model demonstrates that in principle, mechanochemical interactions are sufficient to drive patterning and morphogenesis, independent of patterned gene expression.

OP0176 S100a4 Regulates the Src-Thyrosine Kinase Dependent Differentiation of TH17 Cells in Rheumatoid Arthritis

BackgroundA calcium-binding protein S100A4 regulates non-muscle myosin assembly and activity and plays important part in cell motility and differentiation. The binding of S100A4 to calcium drives conformation changes and permits...

The myosin chaperone UNC45B is involved in lens development and autosomal dominant juvenile cataract

Genome-wide linkage analysis, followed by targeted deep sequencing, in a Danish multigeneration family with juvenile cataract revealed a region of chromosome 17 co-segregating with the disease trait. Affected individuals were heterozygous for two potentially protein-disrupting alleles in this region, in ACACA and UNC45B. As alterations of the UNC45B protein have been shown to affect eye development in model organisms, effort was focused on the heterozygous UNC45B missense mutation. UNC45B encodes a myosin-specific chaperone that, together with the general heat shock protein HSP90, is involved in myosin assembly. The mutation changes p.Arg805 to Trp in the UCS domain, an amino acid that is highly conserved from yeast to human. UNC45B is strongly expressed in the heart and skeletal muscle tissue, but here we show expression in human embryo eye and zebrafish lens. The zebrafish mutant steif, carrying an unc45b nonsense mutation, has smaller eyes than wild-type embryos and shows accumulation of nuclei in the lens. Injection of RNA encoding the human wild-type UNC45B protein into the steif homozygous embryo reduced the nuclei accumulation and injection of human mutant UNC45B cDNA in wild-type embryos resulted in development of a phenotype similar to the steif mutant. The p.Arg805Trp alteration in the mammalian UNC45B gene suggests that developmental cataract may be caused by a defect in non-muscle myosin assembly during maturation of the lens fiber cells.

Molecular Chaperone Mediated Inhibition of the Myosin Power Stroke may be Critical for Sarcomere Assembly

Molecular chaperones are required for successful folding and assembly of sarcomeric myosin. Here, we show that the chaperone UNC-45B stabilizes a non-translocation competent, actin-bound conformation of myosin that preserves its normal ATPase function. This novel locked actomyosin conformation may be necessary for assembly into myofibrils during development and after mechanical stress, and is fully reversible by the general chaperone Hsp90. We propose that a major function of UNC-45B is to prevent the power stroke from occurring during myosin biogenesis. In the forming sarcomere, this would prevent untimely force from being applied to thin filaments, which could disrupt their orderly assembly into the semi-crystalline sarcomere. Hsp90 could then serve both to assist in the completion of the myosin fold and to release the UNC-45B-mediated block, allowing the now fully formed myofibrils to contract.

Characterization of Three Full-length Human Nonmuscle Myosin II Paralogs

Nonmuscle myosin IIs (NM IIs) are a group of molecular motors involved in a wide variety of cellular processes including cytokinesis, migration, and control of cell morphology. There are three paralogs of the NM II heavy chain in humans (IIA, IIB, and IIC), each encoded by a separate gene. These paralogs are expressed at different levels according to cell type and have different roles and intracellular distributions in vivo. Most previous studies on NM II used tissue-purified protein or expressed fragments of the molecule, which presents potential drawbacks for characterizing individual paralogs of the intact protein in vitro. To circumvent current limitations and approach their native properties, we have successfully expressed and purified the three full-length human NM II proteins with their light chains, using the baculovirus/Sf9 system. The enzymatic and structural properties of the three paralogs were characterized. Although each NM II is capable of forming bipolar filaments, those formed by IIC tend to contain fewer constituent molecules than those of IIA and IIB. All paralogs adopt the compact conformation in the presence of ATP. Phosphorylation of the regulatory light chain leads to assembly into filaments, which bind to actin in the presence of ATP. The nature of interactions with actin filaments is shown with different paralogs exhibiting different actin binding behaviors under equivalent conditions. The data show that although NM IIA and IIB form filaments with similar properties, NM IIC forms filaments that are less well suited to roles such as tension maintenance within the cell.

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

Chaperoning myosin assembly in muscle formation and aging.

The activity and assembly of various myosin subtypes is coordinated by conserved UCS (UNC-45/CRO1/She4p) domain proteins. One founding member of the UCS family is the Caenorhabditis elegans UNC-45 protein important for the organization of striated muscle filaments. Our recent structural and biochemical results demonstrated that UNC-45 forms a protein chain with defined periodicity of myosin interaction domains. Intriguingly, the UNC-45 chain serves as docking platform for myosin molecules, which promotes ordered spacing and incorporation of myosin into contractile muscle sarcomeres. The physiological relevance of this observation was demonstrated in C. elegans by transgenic expression of UNC-45 chain formation mutants, which provokes defects in muscle structure and size. Collaborating with the molecular chaperones, Hsp70 and Hsp90, chain formation of UNC-45 links myosin folding with myofilament assembly. Here, we discuss our recent findings on the dynamic regulation of UNC-45 structure and stability in the context of muscle regeneration mechanisms that are affected in myopathic diseases and during aging.

Regulation of assembly of bipolar myosin II filaments

Myosin is a motor protein that has many different functions, including muscle contraction, cell transport, and cell adhesion. The current project focuses on non‐muscle myosin II B in Drosophila. Myosin is composed of heavy chains and light chains. There are two identical heavy chains that each contain an N‐terminal globular head region, neck region, and a C‐terminal long α‐helical tail region. There are also two types of light chains, the regulatory light chain and essential light chain. The phosphorylation state of the regulatory light chain regulates whether myosin is in an active or inactive position. In myosin's inactive position, it is believed that part of the tail region binds to the neck region. To understand the mechanism underlying myosin activation and inactivation, it is essential to pinpoint the site where the neck region binds to the tail region. We approach this question by expressing varying lengths of the tail region and assay neck region binding to these tail region fragments, thereby systematically narrowing down the location of binding on the primary structure of the tail region. To accomplish this, we must first coexpress the neck region with the light chains. So far, it has been confirmed that the heavy chain can be coexpressed with the light chains and form the desired trimeric complex. The information gained from this experiment will help us better understand the regulation of non‐muscle myosin II B.

Special Issue: Biomolecular Motors and Motor Assemblies

Since the first in vitro observations of movement driven by molecular motors by Sheetz and Spudich in 1983, 30 years of work have brought us toward a better foundation in understanding how molecular motors play roles in different organisms and how they can be used in biotechnology applications. Recent advances in this area were highlighted in a session ‘‘Biomolecular Motors and Force Generation’’ at the first US Association for Computational Mechanics Thematic Conference on Multiscale Methods and Validation in Medicine and Biology held in February 2012. This event featuring 15 oral presentations, including a keynote speech by George Oster, generated fruitful discussions among researchers in the field and led us to conceive of this special issue. A majority of biomolecular motors are ATPdependent, coupling ATP hydrolysis energy to mechanical work. These motors include cytoskeletal myosin, kinesin, and dynein, as well as nucleic acidbased translocases, ATP-dependent chaperones, and ATP synthase. Each molecular motor generates forward movement along its specific substrate (myosin on actin, kinesin and dynein on microtubule, ATPdependent chaperones on peptides, nucleic acid-based translocases on DNA or RNA, and ATP synthase on the central shaft). The motor–substrate interactions and their corresponding functions, and the interactions between individual motors are one focus of this special issue. For the interaction between myosin and actin filaments, Yasuhiro Inoue and Taiji Adachi illustrate how actin–myosin catch bonds affect actomyosin aggregate formation. Using computer simulations they found that the catch-bond behavior facilitates the assembly of myosin in a concentration dependent manner. The actin–myosin interaction thus can influence the properties of actin–myosin networks at a larger scale which in turn affect cellular morphogenesis. In muscle, the interaction between a myosin motor and an actin filament can be affected by the binding of neighboring myosins to the actin filament. This cooperative effect is not captured by classical models based on linear partial differential equations. Sam Walcott’s contribution is a new model which includes these cooperative effects and is an exciting step towards a complete multi-scale description of muscle contraction. Travis Stewart and his colleagues from the research group of Josh Baker describe an experimental study which shows that the details of actin–myosin interactions affect muscle shortening speeds not only by defining the detachment rates but also by influencing the attachment kinetics. In contrast to actomyosin’s cooperativity, kinesins often hinder each other except under high loads, and the detailed mechanism behind this load-dependent cooperativity is unclear. Uppulury et al. used a stochastic model to identify biochemical and structural parameters affecting this negative cooperativity. This contribution continues the advances made by Anatoly Kolomeisky’s and Michael Diehl’s groups in the understanding of kinesin-driven intracellular transport. The behavior of two elastically coupled motors is studied theoretically by Florian Berger et al., who derived explicit results to describe the general dynamics of pairs of biomolecular motors. These results enable the prediction of distinct behaviors of different motor species and build upon prior work by Reinhard Lipowsky and Stefan Klumpp. A second focus of this special issue is kinesin’s counterpart in microtubule-associated transport, the dynein motor. Recently solved crystal structures of dynein facilitate further analysis of dynein activities. Dynein belongs to the AAA+ protein superfamily, forming a ring-shaped structure with multiple catalytic sites. Wonmuk Hwang and Matthew Lang used a coarse-grained model to study dynein as well as ClpX and HslU, two ATP-dependent peptide translocases. They found that nucleotide-free and nucleotide occupied subunits work together to generate force while enabling a variety of motor designs. Nandini Shekar and Jun Wu review the accumulated knowledge and its Address correspondence to Henry Hess, Department of Biomedical Engineering, Columbia University, New York, NY, USA. Electronic mail: hh2374@columbia.edu, jcliao@columbia.edu Cellular and Molecular Bioengineering, Vol. 6, No. 1, March 2013 ( 2012) pp. 1–2 DOI: 10.1007/s12195-012-0268-1

Analysis of Myosin Motor Domain Interactions with its Chaperone UNC-45 at the Single Molecule Level

Myosins are actin-based motor proteins that convert chemical energy from ATP hydrolysis into mechanical work. Movement of myosin heads along actin filaments is a result of structurally complex conformational changes in the myosin motor domain induced by ATP binding and hydrolysis. UNC-45, a member of the UCS family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly of myosin into muscle thick filaments in vivo. The molecular mechanism of the myosin-UNC-45 interaction in the promoting proper folding of the myosin head domain is not known. We have devised a novel approach to elucidate the interaction of the UNC-45 chaperone with the myosin motor domain utilizing single molecule atomic force microscopy (AFM). By chemically coupling a titin I27 polyprotein to the motor domain of myosin we synthesized a chimera protein that possesses the property of a “molecular reporter”. Our new construct provides a specific attachment point and the well-characterized mechanical fingerprint of the titin octamer in the AFM measurements. Refolding experiments of the chimeric S1-I27 molecules showed that the myosin motor domain interfered with the refolding of otherwise robust I27 modules, presumably by recruiting them into a misfolded state. The presence of UNC-45 restored the folding of the titin I27 domains. We identify the canonical UCS domain of UNC-45 as the essential component of chaperone like activity. This approach enables the study the myosin-UNC-45 interactions at a single molecule level and their consequences for motor domain folding and misfolding in mechanistic detail.