Single Myosins Research Articles

Direct visualization of human myosin II force generation using DNA origami-based thick filaments

The sarcomere, the minimal mechanical unit of muscle, is composed of myosins, which self-assemble into thick filaments that interact with actin-based thin filaments in a highly-structured lattice. This complex imposes a geometric restriction on myosin in force generation. However, how single myosins generate force within the restriction remains elusive and conventional synthetic filaments do not recapitulate the symmetric bipolar filaments in sarcomeres. Here we engineered thick filaments using DNA origami that incorporate human muscle myosin to directly visualize the motion of the heads during force generation in a restricted space. We found that when the head diffuses, it weakly interacts with actin filaments and then strongly binds preferentially to the forward region as a Brownian ratchet. Upon strong binding, the two-step lever-arm swing dominantly halts at the first step and occasionally reverses direction. Our results illustrate the usefulness of our DNA origami-based assay system to dissect the mechanistic details of motor proteins.

Single cardiac ventricular myosins are autonomous motors.

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored in vitro by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin in vivo down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded in vitro single myosin assay indicates quantitative complementarity with the in vivo mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule.

Coordinated force generation of skeletal myosins in myofilaments through motor coupling

In contrast to processive molecular motors, skeletal myosins form a large motor ensemble for contraction of muscles against high loads. Despite numerous information on the molecular properties of skeletal myosin, its ensemble effects on collective force generation have not been rigorously clarified. Here we show 4 nm stepwise actin displacements generated by synthetic myofilaments beyond a load of 30 pN, implying that steps cannot be driven exclusively by single myosins, but potentially by coordinated force generations among multiple myosins. The simulation model shows that stepwise actin displacements are primarily caused by coordinated force generation among myosin molecules. Moreover, the probability of coordinated force generation can be enhanced against high loads by utilizing three factors: strain-dependent kinetics between force-generating states; multiple power stroke steps; and high ATP concentrations. Compared with other molecular motors, our findings reveal how the properties of skeletal myosin are tuned to perform cooperative force generation for efficient muscle contraction.

Mechanism of Cooperative Force Generations between Skeletal Myosins

To understand the molecular mechanism of cooperative force generation between skeletal myosin molecules, we measured forces generated by myosin-rod cofilaments, in which approximately 17 myosin molecules interact with single actins at the mixing ratio used in this study. Combined with results from the computational model, three factors are important for synchronization of power strokes between myosin motors. First, strain-dependent kinetics are necessary to couple mechanochemical cycles between myosins. Second, multiple power stroke states further enhance a chance of power stroke synchronization. Finally, the physiological ATP concentration is another important factor to enhance a chance of power stroke synchronization, since the strain-dependent transitions accompanied by the first or second power stroke are the rate limiting steps at higher ATP concentrations. Consequently, our computational model predicts that most of steps were generated by synchronous executions of power strokes between several myosin motors at 1 mM ATP, while they are generated primarily by single myosins. Thus, ensemble average curves of steps obtained from our model were distinctively different between 1 mM and 10 μM ATP. These results were consistent with experimental results, supporting our conclusions.

Local heat activation of single myosins based on optical trapping of gold nanoparticles.

Myosin is a mechano-enzyme that hydrolyzes ATP in order to move unidirectionally along actin filaments. Here we show by single molecule imaging that myosin V motion can be activated by local heat. We constructed a dark-field microscopy that included optical tweezers to monitor 80 nm gold nanoparticles (GNP) bound to single myosin V molecules with nanometer and submillisecond accuracy. We observed 34 nm processive steps along actin filaments like those seen when using 200 nm polystyrene beads (PB) but dwell times (ATPase activity) that were 4.5 times faster. Further, by using DNA nanotechnology (DNA origami) and myosin V as a nanometric thermometer, the temperature gradient surrounding optically trapped GNP could be estimated with nanometer accuracy. We propose our single molecule measurement system should advance quantitative analysis of the thermal control of biological and artificial systems like nanoscale thermal ratchet motors.

Intermolecular Cooperativity of Skeletal Myosins Enhances Force Output in Myofilaments

Muscle contraction is powered by the cyclic interaction of skeletal myosin molecules with actin filaments. Recent experiments suggest cooperative actions between myosin molecules, when part of an ensemble. In order to gain more insight into the mechanism of intermolecular cooperativity between myosins in a myofilament, displacements of actin generated by ∼17 interacting myosins embedded in myosin-rod cofilament were measured by optical tweezers. Results showed stepwise displacements of actin (3-6 nm) under high loads of ∼30 pN at every 1-2 ms. Dwell times gradually increased with increasing loads, which is distinctively different from highly load-dependent characteristics of dwell time observed in single myosins, implying that the number of force generating myosins increases with increasing loads. These results suggest that stepwise displacements may be generated by synchronous force generations of myosin molecules. In order to elucidate potential mechanisms of synchronous actions between myosins, we developed the simulation model, consisting of 17 myosins arranged in series with six transition states defined during actomyosin mechanochemical cycles. We have two types of model, implemented with either one or two power strokes. Interestingly, two power stroke model generates stepwise movements of actin at loads up to 30 pN, but one power stroke model generates forces up to 20 pN. These differences in force output are attributed to modulation of number of synchronous power stroke motors. The numbers of synchronous power stroke motors increase with increasing loads from 1.5 to 3 molecules in two power stroke model on average, while they are virtually the same in one power stroke model. Combined with results from both in-vitro and in-silico experiments, multiple power stroke states associated with strain-dependent kinetics are essential for synchronous force generations between myosins, that is a key feature for force enhancement of myosin ensembles.

In Vivo Orientation of Single Myosin Lever Arms in Zebrafish Skeletal Muscle

Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo.

In Vivo Orientation of Single Myosins in a Zebrafish Embryo

Cardiac and skeletal myosin is highly organized in the muscle lattice where it powers contraction by transducing ATP free energy into the mechanical work of moving actin in a mechanism known as transduction/mechanical coupling. While muscle myosin can move actin in vitro, its in vivo environment is crowded and constrained by the fiber lattice. In vivo, myosin side chains are modified during- and post-translation by mutation, phosphorylation, deamidation, and oxidation under normal, diseased, or aging conditions and all potentially impacting transduction/mechanical coupling. Single myosin detection provides highly prized bottom-up quantitative characterization of myosin that tests hypotheses without the ambiguities inherent in ensemble derived observations. The marriage of in vivo and single myosin detection to study human cardiac or skeletal muscle contraction in zebrafish embryo models is a multi-scaled technology for basic and translational research. It allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism levels of phenotype with confidence that all interactions and modifications are appropriately contributing their impact to myosin conformation. In vivo single myosin lever-arm orientation was observed at super-resolution using a photoactivatable GFP (PAGFP) tagged myosin light chain expressed in zebrafish skeletal muscle. Imaging was aided by an innovative microfluidic design for embryo confinement. Tag specificity was demonstrated by the simultaneous observation of 2-photon fluorescence emission and second harmonic generation (SHG) from myosin. Single molecule detection used highly inclined and laminated optical sheet (HILO) illumination and was verified by quantized photoactivation or photobleaching. Single molecule emission patterns from relaxed muscle indicated a highly orientationally confined lever-arm orientation. Results demonstrate detection of single myosin orientation in vivo. The zebrafish muscle system serves as an in vivo model for human disease and aging effects on myosin. Research supported by NIH R01AR049277 and R01HL095572.

2SDA-06 骨格筋ミオシン分子複合体の力発生に特化したミオシン1分子の特性とダイナミクス(2SDA 新学術領域「少数性生物学-個と多数の狭間が織りなす生命現象の探究-」共催,少数個分子の協同が生み出す生命機能のメカニズム,シンポジウム,日本生物物理学会第51回年会(2013年度))

2SDA-06 骨格筋ミオシン分子複合体の力発生に特化したミオシン1分子の特性とダイナミクス(2SDA 新学術領域「少数性生物学-個と多数の狭間が織りなす生命現象の探究-」共催,少数個分子の協同が生み出す生命機能のメカニズム,シンポジウム,日本生物物理学会第51回年会(2013年度))

Non-Linear Elasticity of Skeletal Myosins is Essential for Collective Force Generation in Muscle

Muscle contraction occurs through the rotation of the myosin heads pulling actin filaments past myosin filaments. In order to achieve contractions efficiently, it is crucial for myosins to generate force collectively and to minimize the interference between motors. In the previous studies, it was suggested that the elasticity of myosin is linear so that myosins generate the large drag force opposing to muscle contraction if negatively strained. However, none of the previous studies has investigated the elasticity of single myosin explicitly in the negative strain region. Therefore, we investigated the elasticity of single skeletal myosins in both the positive and negative strain directions. In the absence of ATP, single myosins embedded in synthetic myosin-rod filaments were tightly bound to actin filaments and were stretched and shortened repeatedly by oscillating the two trapped beads on the both ends of acting filament manipulated by optical tweezers. The elasticity of myosins was characterized by obtaining the force-displacement curves, where forces on myosin heads were estimated by measuring the displacement of two beads, and displacements of myosin heads were obtained by tracking the position of quantum dots attached to the actin filament. We found that the elasticity is non-linear, in which stiffness is high (∼2.8 pN/nm) and low (∼0.02 pN/nm) in the positive and negative strain regions. The non-linear elasticity ensures high force generation with a small stretching of the elastic portion, and minimizing the drag force of negatively-strained myosins. Furthermore, the estimates of the actin sliding distance by the non-linear elasticity model were more consistent with experimental results observed in 20μM ATP than those by the linear elasticity model. Therefore, the non-linear elasticity might be an essential mechanical property of single myosin designed for the collective force generation in muscle.

Non-linear Elasticity of Single Skeletal Myosins Designed for Efficient Muscle Contractions

Non-linear Elasticity of Single Skeletal Myosins Designed for Efficient Muscle Contractions

Nonlinear Elasticity and an 8-nm Working Stroke of Single Myosin Molecules in Myofilaments

Using optical trapping and fluorescence imaging techniques, we measured the step size and stiffness of single skeletal myosins interacting with actin filaments and arranged on myosin-rod cofilaments that approximate myosin mechanics during muscle contraction. Stiffness is dramatically lower for negatively compared to positively strained myosins, consistent with buckling of myosin's subfragment 2 rod domain. Low stiffness minimizes drag of negatively strained myosins during contraction at loaded conditions. Myosin's elastic portion is stretched during active force generation, reducing apparent step size with increasing load, even though the working stroke is approximately constant at about 8 nanometers. Taking account of the nonlinear nature of myosin elasticity is essential to relate myosin's internal structural changes to physiological force generation and filament sliding.

Measuring Single Myosins at Work

Measuring Single Myosins at Work

3P-132 骨格筋ミオシンのステップサイズは負荷に伴い減少する(分子モーター(3),第46回日本生物物理学会年会)

3P-132 骨格筋ミオシンのステップサイズは負荷に伴い減少する(分子モーター(3),第46回日本生物物理学会年会)