Molecular Motor Myosin Research Articles

Myopalladin promotes muscle growth through modulation of the serum response factor pathway.

BackgroundMyopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe.MethodsConstitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro.ResultsMKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway.ConclusionsMyopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.

Stiffness of Cargo-Motor Linkage Tunes Myosin VI Motility and Response to Load.

We examine the effect of cargo-motor linkage stiffness on the mechanobiological properties of the molecular motor myosin VI. We use the programmability of DNA nanostructures to modulate cargo-motor linkage stiffness and combine it with high-precision optical trapping measurements to measure the effect of linkage stiffness on the motile properties of myosin VI. Our results reveal that a stiff cargo-motor linkage leads to shorter step sizes and load-induced anchoring of myosin VI, while a flexible linkage results in longer steps with frequent detachments from the actin filament under load. Our findings suggest a novel regulatory mechanism for tuning the dual cellular roles of the anchor and transporter ascribed to myosin VI.

Large and reversible myosin-dependent forces in rigidity sensing.

Cells sense the rigidity of their environment through localized pinching, which occurs when myosin molecular motors generate contractions within actin filaments anchoring the cell to its surroundings. We present high-resolution experiments performed on these elementary contractile units in cells. Our experimental results challenge the current understanding of molecular motor force generation. Surprisingly, bipolar myosin filaments generate much larger forces per motor than measured in single molecule experiments. Further, contraction to a fixed distance, followed by relaxation at the same rate, is observed over a wide range of matrix rigidities. Lastly, step-wise displacements of the matrix contacts are apparent during both contraction and relaxation. Building upon a generic two-state model of molecular motor collections, we interpret these unexpected observations as spontaneously emerging features of a collective motor behavior. Our approach explains why, in the cellular context, collections of resilient and slow motors contract in a stepwise fashion while collections of weak and fast motors do not. We thus rationalize the specificity of motor contractions implied in rigidity sensing compared to previous in vitro observations.

Cytoskeletal stiffening in synthetic hydrogels

Although common in biology, controlled stiffening of hydrogels in vitro is difficult to achieve; the required stimuli are commonly large and/or the stiffening amplitudes small. Here, we describe the hierarchical mechanics of ultra-responsive hybrid hydrogels composed of two synthetic networks, one semi-flexible and stress-responsive, the other flexible and thermoresponsive. Heating collapses the flexible network, which generates internal stress that causes the hybrid gel to stiffen up to 50 times its original modulus; an effect that is instantaneous and fully reversible. The average generated forces amount to ~1 pN per network fibre, which are similar to values found for stiffening resulting from myosin molecular motors in actin. The excellent control, reversible nature and large response gives access to many biological and bio-like applications, including tissue engineering with truly dynamic mechanics and life-like matter.

Dynamical mechanism of stepping of the molecular motor myosin V along actin filament and simulation in an actual system

On the base of the random motion of myosin, the structural asymmetry of the actin filament, and the interaction between the motor and the track, the present study proposes a physical mechanism for the processive stepping of the two-headed molecular motor myosin V along the actin filament. The detachment from actin filament and forward swing of the trailing head, which are caused by increased entropy and the release of the bending energy in the neck region, are the initial steps of the stepping process. The detached head then rotates and randomly moves around the still attached head. A step is completed after the detached head detects the next binding site. In the mechanism, the direction of myosin V movement was determined on the basis of the configuration of the actomyosin system. The role of ATP is the energy provider for the local random motion. The applicability of the dynamic mechanism was evaluated on a simulation system that preserved most of the characteristics of the actomyosin system.

Assessing the Contribution of Active and Passive Stresses in C. elegans Elongation.

The role of the actomyosin network is investigated in the elongation of C. elegans during embryonic morphogenesis. We present a model of active elongating matter that combines prestress and passive stress in nonlinear elasticity. Using this model we revisit recently published data from laser ablation experiments to account for why cells under contraction can lead to an opening fracture. By taking into account the specific embryo geometry, we obtain quantitative predictions for the contractile forces exerted by the molecular motors myosin II for an elongation up to 70% of the initial length. This study demonstrates the importance of active processes in embryonic morphogenesis and the interplay between geometry and nonlinear mechanics during morphological events. In particular, it outlines the role of each connected layer of the epidermis compressed by an apical extracellular matrix that distributes the stresses during elongation.

Common and Specific Functions of Nonmuscle Myosin II Paralogs in Cells.

Various forms of cell motility critically depend on pushing, pulling, and resistance forces generated by the actin cytoskeleton. Whereas pushing forces largely depend on actin polymerization, pulling forces responsible for cell contractility and resistance forces maintaining the cell shape require interaction of actin filaments with the multivalent molecular motor myosin II. In contrast to muscle-specific myosin II paralogs, nonmuscle myosin II (NMII) functions in virtually all mammalian cells, where it executes numerous mechanical tasks. NMII is expressed in mammalian cells as a tissue-specific combination of three paralogs, NMIIA, NMIIB, and NMIIC. Despite overall similarity, these paralogs differ in their molecular properties, which allow them to play both unique and common roles. Importantly, the three paralogs can also cooperate with each other by mixing and matching their unique capabilities. Through specialization and cooperation, NMII paralogs together execute a great variety of tasks in many different cell types. Here, we focus on mammalian NMII paralogs and review novel aspects of their kinetics, regulation, and functions in cells from the perspective of how distinct features of the three myosin II paralogs adapt them to perform specialized and joint tasks in the cells.

Molecular to organismal chirality is induced by the conserved myosin 1D.

The emergence of asymmetry from an initially symmetrical state is a universal transition in nature. Living organisms show asymmetries at the molecular, cellular, tissular, and organismal level. However, whether and how multilevel asymmetries are related remains unclear. In this study, we show that Drosophila myosin 1D (Myo1D) and myosin 1C (Myo1C) are sufficient to generate de novo directional twisting of cells, single organs, or the whole body in opposite directions. Directionality lies in the myosins' motor domain and is swappable between Myo1D and Myo1C. In addition, Myo1D drives gliding of actin filaments in circular, counterclockwise paths in vitro. Altogether, our results reveal the molecular motor Myo1D as a chiral determinant that is sufficient to break symmetry at all biological scales through chiral interaction with the actin cytoskeleton.

A single myosin sets chirality at all scales

Developmental Biology When viewed externally, most organisms appear symmetric between the left and right sides. However, many organs are left-right asymmetric. Whether macroscopic asymmetries are directly related to molecular-level chirality remains an open question. Working in Drosophila , Lebreton et al. found that the conserved molecular motor myosin 1D induced stereotyped chirality at all biological scales—from F-actin turning in vitro to the organ level and even organismal behavior. Thus, a single conserved myosin can generate de novo nano-to-macroscopic changes in form and direction through chiral interaction with the actin cytoskeleton. Science , this issue p. [949][1] [1]: /lookup/doi/10.1126/science.aat8642

Modeling the Axon as an Active Partner with the Growth Cone in Axonal Elongation

Forces generated by the growth cone are vital for the proper development of the axon and thus brain function. Although recent experiments show that forces are generated along the axon, it is unknown whether the axon plays a direct role in controlling growth cone advance. Here, we use analytic and finite element modeling of microtubule dynamics and the activity of the molecular motors myosin and dynein to investigate mechanical force balance along the length of the axon and its effects on axonal outgrowth. Our modeling indicates that the paradoxical effects of stabilizing microtubules and the consequences of microtubule disassembly on axonal outgrowth can be explained by changes in the passive and active mechanical properties of axons. Our findings suggest that a full understanding of growth cone motility requires a consideration of the mechanical contributions of the axon. Our study not only has potential applications during neurodevelopment but might also help identify strategies to manipulate and promote axonal regrowth to treat neurodegeneration.

A myosin II nanomachine mimicking the striated muscle

The contraction of striated muscle (skeletal and cardiac muscle) is generated by ATP-dependent interactions between the molecular motor myosin II and the actin filament. The myosin motors are mechanically coupled along the thick filament in a geometry not achievable by single-molecule experiments. Here we show that a synthetic one-dimensional nanomachine, comprising fewer than ten myosin II dimers purified from rabbit psoas, performs isometric and isotonic contractions at 2 mM ATP, delivering a maximum power of 5 aW. The results are explained with a kinetic model fitted to the performance of mammalian skeletal muscle, showing that the condition for the motor coordination that maximises the efficiency in striated muscle is a minimum of 32 myosin heads sharing a common mechanical ground. The nanomachine offers a powerful tool for investigating muscle contractile-protein physiology, pathology and pharmacology without the potentially disturbing effects of the cytoskeletal—and regulatory—protein environment.

Repetitive stretching of giant liposomes utilizing the nematic alignment of confined actin

Giant liposomes encapsulating cytoskeletons have been constructed to further understand the mechanisms of cell movement and develop cell-sized chemical machineries. Innovative studies demonstrating liposomal movements using microtubules and the molecular motors kinesin/dynein have been reported. However, no one has succeeded in generating repetitive motions controlled by external stimuli. Here we show that if the actin concentration in liposomes is comparable to that of cytoplasm of living cells, the liposomes can be deformed into spindle shapes by encapsulating only actin filaments, even without the molecular motor myosin. Furthermore, their shapes can be changed reversibly between spindle and sphere shapes by adjusting osmotic pressure or by light irradiation of fluorescent-labeled actin. In the latter case, the repetitive shape changes are accompanied with stretching and shrinking of filopodia- or acrosome projection-like extensions. Our results indicate that filamentous polymer of variable length like actin filament is a potential material for the reproduction of cell-like movement.

Classifying Cardiac Actin Mutations Associated With Hypertrophic Cardiomyopathy.

Mutations in the cardiac actin gene (ACTC1) are associated with the development of hypertrophic cardiomyopathy (HCM). To date, 12 different ACTC1 mutations have been discovered in patients with HCM. Given the high degree of sequence conservation of actin proteins and the range of protein–protein interactions actin participates in, mutations in cardiac actin leading to HCM are particularly interesting. Here, we suggest the classification of ACTC1 mutations based on the location of the resulting amino acid change in actin into three main groups: (1) those affecting only the binding site of the myosin molecular motor, termed M-class mutations, (2) those affecting only the binding site of the tropomyosin (Tm) regulatory protein, designated T-class mutations, and (3) those affecting both the myosin- and Tm-binding sites, called MT-class mutations. To understand the precise pathogenesis of cardiac actin mutations and develop treatments specific to the molecular cause of disease, we need to integrate rapidly growing structural information with studies of regulated actomyosin systems.

Estimation of actomyosin active force maintained by tropomyosin and troponin complex under vertical forces in the in vitro motility assay system.

The interaction between actin filaments and myosin molecular motors is a power source of a variety of cellular functions including cell division, cell motility, and muscular contraction. In vitro motility assay examines actin filaments interacting with myosin molecules that are adhered to a substrate (e.g., glass surface). This assay has been the standard method of studying the molecular mechanisms of contraction under an optical microscope. While the force generation has been measured through an optically trapped bead to which an actin filament is attached, a force vector vertical to the glass surface has been largely ignored with the in vitro motility assay. The vertical vector is created by the gap (distance) between the trapped bead and the glass surface. In this report, we propose a method to estimate the angle between the actin filament and the glass surface by optically determining the gap size. This determination requires a motorized stage in a standard epi-fluorescence microscope equipped with optical tweezers. This facile method is applied to force measurements using both pure actin filaments, and thin filaments reconstituted from actin, tropomyosin and troponin. We find that the angle-corrected force per unit filament length in the active condition (pCa = 5.0) decreases as the angle between the filament and the glass surface increases; i.e. as the force in the vertical direction increases. At the same time, we demonstrate that the force on reconstituted thin filaments is approximately 1.5 times larger than that on pure actin filaments. The range of angles we tested was between 11° and 36° with the estimated measurement error less than 6°. These results suggest the ability of cytoplasmic tropomyosin isoforms maintaining actomyosin active force to stabilize cytoskeletal architecture.

Dissecting the Molecular Mechanism for Familial Cardiomyopathies

The heart needs to generate sufficient force and power to pump blood to the body and to adjust its power output in response to stresses. Mutations of contractile proteins, such as cardiac troponin T, can lead to familial cardiomyopathies, the leading cause of sudden cardiac death in people under the age of 30. Two common forms of cardiomyopathy, hypertrophic (HCM) and dilated (DCM) are characterized by thickening or thinning of the ventricular wall, respectively. However, how changes in contractile proteins at the molecular level lead to the disease phenotype is not well understood. To address this problem, we examined two point mutations in human cardiac troponin T (TnT), R92Q and ΔK210, which cause hypertrophic and dilated cardiomyopathy, respectively, in human patients. We have reconstituted the calcium-based regulation of actomyosin in vitro using tissue purified and recombinant cardiac proteins. Similar to previous studies, in vitro motility assays which reconstitute the calcium-dependent interactions between myosin and regulated thin filaments revealed that R92Q is shifted towards submaximal calcium activation while ΔK210 is shifted towards supermaximal calcium activation. We dissected the molecular mechanism of these differential changes using a combination of fluorescence equilibrium titrations and stopped-flow experiments, and show that these effects are due to alterations in the free energies of the biochemical transitions that regulate muscle activation. In particular, we see that these mutations differentially affect calcium-dependent positioning of tropomyosin along the thin filament. We propose that these changes affect the regulation of force production by the molecular motor myosin, leading to alterations in mechanosensing by heart tissue.

The Giant Protein Titin Regulates the Length of the Striated Muscle Thick Filament-Titin Rules

The contractile machinery of heart and skeletal muscles that powers life has as its most essential component the thick filament, comprised of the molecular motor myosin. The thick filament is of a precisely controlled length, defining thereby the force level that muscles generate and how this force varies with muscle length. It has been speculated that the mechanism by which the length of the thick filament is so exquisitely controlled involves the giant protein titin, but no conclusive support for or against exists. Here we show studies on a mouse model in which we deleted two of titin's C-zone super-repeats, located in the A-band region of the sarcomere. Structural studies using immunoelectron microscopy and super-resolution optical microscopy in both cardiac and skeletal muscles reveal a thick filament length that is reduced by ∼170 nm; functional studies reveal reduced force generation and a dilated cardiomyopathy (DCM) phenotype. Our studies show for the first time the important role of titin in regulating thick filament length, with each of titin's C-zone repeats being responsible for a quantal 43 nm thick filament length. We conclude that thick filament length regulation is titin-based and that this mechanism is crucial for maintaining muscle health.

Mechanical parameters of the molecular motor myosin II determined in permeabilised fibres from slow and fast skeletal muscles of the rabbit.

The different performance of slow and fast muscles is mainly attributed to diversity of the myosin heavy chain (MHC) isoform expressed within them. In this study fast sarcomere-level mechanics has been applied to Ca2+ -activated single permeabilised fibres isolated from soleus (containing the slow myosin isoform) and psoas (containing the fast myosin isoform) muscles of rabbit for a comparative definition of the mechano-kinetics of force generation by slow and fast myosin isoforms in situ. The stiffness and the force of the slow myosin isoform are three times smaller than those of the fast isoform, suggesting that the stiffness of the myosin motor is a determinant of the isoform-dependent functional diversity between skeletal muscles. These results open the question of the mechanism that can reconcile the reduced performance of the slow MHC with the higher efficiency of the slow muscle. The skeletal muscle exhibits large functional differences depending on the myosin heavy chain (MHC) isoform expressed in its molecular motor, myosin II. The differences in the mechanical features of force generation by myosin isoforms were investigated in situ by using fast sarcomere-level mechanical methods in permeabilised fibres (sarcomere length 2.4μm, temperature 12°C, 4% dextran T-500) from slow (soleus, containing the MHC-1 isoform) and fast (psoas, containing the MHC-2X isoform) skeletal muscle of the rabbit. The stiffness of the half-sarcomere was determined at the plateau of Ca2+ -activated isometric contractions and in rigor and analysed with a model that accounted for the filament compliance to estimate the stiffness of the myosin motor (ε). ε was 0.56±0.04 and 1.70±0.37pNnm-1 for the slow and fast isoform, respectively, while the average strain per attached motor (s0 ) was similar (∼3.3nm) in both isoforms. Consequently the force per motor (F0 =εs0 ) was three times smaller in the slow isoform than in the fast isoform (1.89±0.43 versus 5.35±1.51pN). The fraction of actin-attached motors responsible for maximum isometric force at saturating Ca2+ (T0,4.5 ) was 0.47±0.09 in soleus fibres, 70% larger than that in psoas fibres (0.29±0.08), so that F0 in slow fibres was decreased by only 53%. The lower stiffness and force of the slow myosin isoform open the question of the molecular basis of the higher efficiency of slow muscle with respect to fast muscle.

Long-range coupling between ATP-binding and lever-arm regions in myosin via dielectric allostery.

A protein molecule is a dielectric substance, so the binding of a ligand is expected to induce dielectric response in the protein molecule, considering that ligands are charged or polar in general. We previously reported that binding of adenosine triphosphate (ATP) to molecular motor myosin actually induces such a dielectric response in myosin due to the net negative charge of ATP. By this dielectric response, referred to as "dielectric allostery," spatially separated two regions in myosin, the ATP-binding region and the actin-binding region, are allosterically coupled. In this study, from the statistically stringent analyses of the extensive molecular dynamics simulation data obtained in the ATP-free and the ATP-bound states, we show that there exists the dielectric allostery that transmits the signal of ATP binding toward the distant lever-arm region. The ATP-binding-induced electrostatic potential change observed on the surface of the main domain induced a movement of the converter subdomain from which the lever arm extends. The dielectric response was found to be caused by an underlying large-scale concerted rearrangement of the electrostatic bond network, in which highly conserved charged/polar residues are involved. Our study suggests the importance of the dielectric property for molecular machines in exerting their function.

The giant protein titin regulates the length of the striated muscle thick filament

The contractile machinery of heart and skeletal muscles has as an essential component the thick filament, comprised of the molecular motor myosin. The thick filament is of a precisely controlled length, defining thereby the force level that muscles generate and how this force varies with muscle length. It has been speculated that the mechanism by which thick filament length is controlled involves the giant protein titin, but no conclusive support for this hypothesis exists. Here we show that in a mouse model in which we deleted two of titin’s C-zone super-repeats, thick filament length is reduced in cardiac and skeletal muscles. In addition, functional studies reveal reduced force generation and a dilated cardiomyopathy (DCM) phenotype. Thus, regulation of thick filament length depends on titin and is critical for maintaining muscle health.

Myosin isoform expressed in metastatic prostate cancer stimulates cell invasion

During metastasis, tumor cells migrate out of their original tissue to invade other organs. Secretion of exosomes and metalloproteases is essential for extracellular matrix remodeling, enabling migration through tissue barriers. Metastatic prostate cancer is differentiated by expression of the rare isoform A of the molecular motor myosin IC, however the function of this isoform remained unknown. Here we show that it contributes causatively to the invasive motility of prostate cancer cells. We found that the isoform associates with metalloprotease-containing exosomes and stimulates their secretion. While the data show that myosin IC is involved in prostate cancer cell migration, migration outside extracellular matrix in vitro proves little affected specifically by isoform A. Nevertheless, this isoform stimulates invasion through extracellular matrix, pointing to a critical role in secretion. Both the secretion and invasion depend on the integrity of the motor and lipid-binding domains of the protein. Our results demonstrate how myosin IC isoform A is likely to function in metastasis, driving secretion of exosomes that enable invasion of prostate cancer cells across extracellular matrix barriers. The new data identify a molecule suitable for a mechanistically grounded development into a marker and target for prognosis, detection, and treatment of invasive prostate cancer.