These observations have led to a postulation that ROCK is involved in myosin filament formation and regulation of filament length. Fragmented myosin filaments due to ROCK inhibition could have functional consequences on smooth muscle response to mechanical oscillations, such as those imposed by a DI. In the same study, Lan et al. The authors propose that fragmented myosin filaments could be lost from the actin filament lattice more easily than long filaments during large-amplitude DI-mimicking oscillations, leading to loss of contractility.
Both discoveries support a new phenotype of asthma in which ROCK signaling is augmented. Besides many other implications on airway smooth muscle contractility and stiffness Wang et al. Lability of filamentous myosin II in smooth muscle is crucial for proper function of the muscle. Filament lability is determined by intracellular chemical environment, contractile stimulation, and the associated signaling pathways regulating RLC phosphorylation, as well as the strains associated with changes in cell dimension.
Besides its role in regulating contractility, myosin filament lability is also indispensable in the process of length adaptation in smooth muscle. The labile nature of smooth muscle myosin filament underlies the phenomenon of myosin evanescence and contributes to cellular malleability of smooth muscle.
This malleability is required for normal function of the muscle but may be altered in a diseased state. Increased ROCK expression in asthma may increase myosin filament stability and thereby underlie the failure of DI-induced bronchodilation and bronchoprotection in asthma. Author contributions: L. Wang prepared the first draft of the manuscript including the text, references, and figures.
Chitano contributed to the text and figures. Seow made final modifications to the text, references, and figure legends, as well as all final decisions on the content of the manuscript. Schematic diagram of a myosin II molecule. SM1 and SM2 heterodimers are drawn for illustration purposes only. Controversy exists about whether in living cells they exist as homodimers Kelley et al. S1 and S2, HMM subfragments. Adapted from Chen et al.
Proposed model for myosin filament formation in vitro. Unphosphorylated myosin II exists in folded monomeric form. It can assemble to form antiparallel folded dimers and then antiparallel folded tetramers. Antiparallel straight dimers and tetramers assemble to form filaments. Bipolar filaments are formed in striated muscle and side-polar filaments are formed in smooth muscle. Adapted from Dasbiswas et al.
Proposed model for myosin filament evanescence during reversible length adaptation process. For illustration purposes, there are two and three contractile units in series in the muscle cell adapted to 0. Smooth muscle adapted to a shorter length 0. Nonmuscle myosin intermediates are not shown, as they do not participate in the adaptation process.
Dotted lines highlight a contractile unit. Adapted from Chitano et al. Sign In or Create an Account. Advanced Search. User Tools. Sign In. Skip Nav Destination Article Navigation. Review February 19 Filament evanescence of myosin II and smooth muscle function Lu Wang Lu Wang.
This Site. Google Scholar. Pasquale Chitano , Pasquale Chitano. Chun Y. Seow Correspondence to Chun Y. Seow: chun. Author and Article Information. Pasquale Chitano. This work is part of a special collection on myofilament function and disease. Received: September 30 Accepted: January 19 Online Issn: J Gen Physiol 3 : e Article history Received:. Cite Icon Cite. Henk L. Granzier served as editor.
The authors declare no competing financial interests. Search ADS. Mechanical control of cAMP signaling through integrins is mediated by the heterotrimeric Galphas protein. Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils.
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Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules. Two heads are required for phosphorylation-dependent regulation of smooth muscle myosin. A nucleation--elongation mechanism for the self-assembly of side polar sheets of smooth muscle myosin.
Role of intracellular pH in relaxation of porcine tracheal smooth muscle by respiratory gases. Ordering of myosin II filaments driven by mechanical forces: experiments and theory.
Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo. Both heads of tissue-derived smooth muscle heavy meromyosin bind to actin in the presence of ADP. Mechanism of Myofilament Sliding in Muscle Contraction. Advances in Experimental Medicine and Biology. Effect of deep inspiration on airway conductance in subjects with allergic rhinitis and allergic asthma. Isolation of a novel PDZ-containing myosin from hematopoietic supportive bone marrow stromal cell lines.
Density of myosin filaments in the rat anococcygeus muscle, at rest and in contraction. Godfraind-De Becker. Analysis of the birefringence of the smooth muscle anococcygeus of the rat, at rest and in contraction. The variation in isometric tension with sarcomere length in vertebrate muscle fibres.
Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function. Influence of calcium on myosin thick filament formation in intact airway smooth muscle. Effect of inspiratory flow rate on bronchomotor tone in normal and asthmatic subjects.
Role of the COOH-terminal nonhelical tailpiece in the assembly of a vertebrate nonmuscle myosin rod. Antibodies probe for folded monomeric myosin in relaxed and contracted smooth muscle.
Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle. Regulation of the function of mammalian myosin and its conformational change.
Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: preparation of heavy meromyosin and subfragment 1 with intact 20 dalton light chains. Correlation of enzymatic properties and conformation of smooth muscle myosin. Effects of phosphorylation of light chain residues threonine 18 and serine 19 on the properties and conformation of smooth muscle myosin.
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A comparative study. Studies on the effect of phosphorylation of the 20, Mr light chain of vertebrate smooth muscle myosin. Myosin repertoire expansion coincides with eukaryotic diversification in the Mesoproterozoic era. Conformation, filament assembly, and activity of single-headed smooth muscle myosin.
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Regulation of phosphorylation of myosin heavy chain during the chemotactic response of Dictyostelium cells. Importance of the time interval between FEV1 measurements in a methacholine provocation test.
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Assembly of smooth muscle myosin by the 38k protein, a homologue of a subunit of pre-mRNA splicing factor Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells. To adapt to the changing requirements, the cell's cytoskeleton constantly remodels through the action of myosin II motor clusters that interact with numerous actin filaments simultaneously.
Here we study the various roles of myosin II clusters in the formation and evolution of in vitro actomyosin networks as a model system for the cell's cytoskeleton.
In our experiments the motor clusters can vary in size between 14 and myosin II molecules and apply forces ranging from several to tens of piconewtons. During the initial process of network formation the motor clusters become embedded within the network structure, where they act as internal active cross-linkers. The stresses building-up in the networks lead to complex dynamics and can drive their contraction and rupture, depending on the motor concentration and cluster size.
Myosin II motors are shown to be unique motors that function as complex machines that can perform a diversity of tasks, thereby regulating the nature of the assembled network and facilitating its formation.
Ideses, A. Sonn-Segev, Y. Roichman and A. To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page. If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given. If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given.
Read more about how to correctly acknowledge RSC content. Fetching data from CrossRef. One reason is that muscle myosin is non-processive when isolated and works as a group with other muscle myosins in the highly-structured sarcomere, the minimal mechanical unit of muscle, which is composed of rigorously arranged myosin II-based thick filaments Fig. This design and the non-processivity make it difficult to directly observe the internal dynamics of the rapid and minute displacements of myosin II at the single molecular level.
Although myosin II in a thick filament forms a dimer, only the monomeric form is shown for simplicity. Arrowheads indicate myosin heads. Average occupancy, 5. Cartoon of the complex and histogram of the distance between heads on actin two-headed arrow are also shown below. Data were obtained from at least 10 independent experiments.
Error bars indicate SD. In the intermediate states of force generation, myosin heads dissociate from actin upon ATP binding to the catalytic site and undergo rapid association and dissociation weak binding 22 , 23 with actin to diffusively search landing sites. Upon binding, they take the strong binding state and generate force through a conformational change lever-arm swing.
The weak binding followed by the strong binding is critical for fully transmitting the lever-arm swing to the thick filament backbone or actin filament, because the myosin head is connected to the backbone through S2, which has an elasticity 24 that would absorb the small displacement of the lever-arm swing if the landing sites were random 1. Also, the multi-step and reversible mechanics of the lever-arm swing is important for the response of muscle to rapid mechanical perturbations Thus, to elucidate the molecular mechanism of the overall force generation cycle, it is essential to directly visualize and quantitatively measure the dynamics of elementary mechanical processes i.
To observe the motion of the heads in thick filaments in vitro , synthesis of the thick filaments is necessary; however, synthetic filaments composed of purified myosin II self-assembled in a conventional manner 26 do not mimic the symmetric bipolar filaments observed in sarcomeres and instead assemble randomly Here, to overcome the limitation of conventional approaches, we engineered thick filaments using three-dimensional DNA origami 28 , 29 , 30 and recombinant human myosin II The addressability of the DNA origami enabled the precise positioning of myosin heads in the filament 32 , resulting in clear observation of the myosin shape by high-speed atomic force microscopy HS-AFM Thus, we directly visualized a reversible two-step lever-arm swing, which provides a molecular basis for explaining the dynamic characteristics 25 of muscle contraction.
Further, we also observed rapid weak bindings of microseconds dwell time by darkfield imaging of a bifunctionally attached gold nanoparticle GNP to the myosin head. Overall, we found a biased binding mechanism based on the Brownian ratchet 1 , which fully transmits the lever-arm swing to the thick filament backbone or actin filament. We prepared thick filaments consisting of a helix-bundle DNA origami rod as a backbone Fig.
For simplicity, linkers along only one side of the backbone were designed and spaced To attach S1 of myosin IIa to the linker, a base oligonucleotide handle was attached to the end of the linker, and the complementary oligonucleotide, or antihandle, was labeled to a SNAP-tag at the C-terminus of S1 Supplementary Fig.
We observed our thick filament using AFM Fig. S1 was attached to the linker with The lever-arm swing of myosin II has been studied by in vitro motility assays 9 , spectroscopic studies 6 , 7 , and atomic structures 4 , 5 and is widely accepted to be the powerstroke of all myosins. However, the lever-arm swing of muscle myosin in the actomyosin complex has not been directly visualized during force generation, although the lever-arm swing of unconventional myosin V has been directly observed by HS-AFM 34 and there are spectroscopic and force measurements that point directly to tilting of the myosin II lever arm.
We applied HS-AFM at hundreds of milliseconds time resolution 34 to our thick filament for direct observation. We prepared thick filaments occupied with six myosin heads and observed the translocation of the actin filaments along a thick filament in the presence of ATP Fig.
When coupled to the actin translocation, the orientation of S1 presumably the lever-arm domain of S1 relative to the actin filament changed. The orientation change frequently occurred in a two-step manner Fig. The head displacement of the first step was 3. This two-step motion of the powerstroke is consistent with previous reports that suggest muscle myosin adopts at least two different phases of the bound state with actin during force generation 16 , 35 , The changes in orientation of the lever-arm and the head displacement are schematically summarized in Fig.
Direct observation of a two-step and reversible powerstroke by high-speed AFM. Arrows indicate the sliding direction. Lower cartoon shows the myosin shape in the images. AFM images and cartoons on the right show the head displacement.
Data were obtained from at least ten independent experiments. Next, fully utilizing the programmability of our DNA origami-based thick filament, we constructed an assay system for high-speed observation of a single myosin head in the filament. We attached a single myosin head at position 3 in Fig. Our microscopy achieved 0. In the AFM image, the profiles of the parallel lines along an actin filament and the crossing linkers show the periodicity of the intensity peaks, with the peaks of the linkers positioned between the peaks of the actin pitch Fig.
This observation suggests that the engineered thick filament forms a complex with an actin filament, maintaining a geometry similar to the myosin-labeled engineered thick filament in Fig.
Figure 3f shows a two-dimensional histogram of the trajectory of a myosin head on an image plane. Furthermore, myosin heads anisotropically bound to actin along the major axis Fig.
We frequently found a two-step displacement in the rising phase of the strong bindings Fig. To confirm this speculation, we constructed a myosin IIa mutant lacking the lever-arm domain lever-arm-less S1, Supplementary Fig.
A trajectory of lever-arm-less S1 is shown in Fig. To compare step sizes displacements from the mean position of the detached state to strong binding state between wild-type and lever-arm-less S1, we used a hidden Markov model, which has been widely used for single-molecule trajectory analysis 39 see Methods.
The obtained step size decreased from We concluded the difference, In addition, individual displacements of lever-arm-less S1 were biased in one direction, which strongly suggested a biased binding mechanism of the myosin heads to actin. Figure 4e shows the estimated geometry of the rigor complex from the binding step size and the linker angle Fig. Experimental design for high-speed observations of a myosin head in thick filaments.
A handle sequence red projecting from the linker dark blue contains three handles bound to one antihandle labeled to myosin S1 light blue and two antihandles bound to GNP orange. The head, oligonucleotides and GNP are drawn to scale.
The line profile is measured along the red and blue lines. The blue line was decided by shifting the red line in the perpendicular direction. The numbering of the linkers corresponds to Fig.
Measured values dashed line were fitted by multiple Gaussian distributions solid line. The numbering corresponds to c.
The distance axis indicates the relative distance from the position of the intensity peak black dashed line on the actin filament between linkers 3 and 4.
Linkers 4, 5, and 6 are positioned between the peaks of an actin helical pitch. Linkers 1, 2, and 3 are not shown, because they frequently detached from actin in the absence of myosin. Shadowed areas indicate the standard error. The elliptical shape is consistent with the prediction from the geometry of the complex Supplementary Fig.
The two peaks of the plot correspond to the mean position of the detached state and the binding state, respectively. Tracking the myosin head with microsecond time resolution. A histogram of the trajectory is shown side-by-side.
The binding displacement was calculated by a hidden Markov model Methods. Cyan and magenta arrowheads indicate the end points of the two transitions. Center line, median; box limits, upper and lower quartiles; whiskers, 1. CI confidential interval. The geometry was estimated by the linker angle Fig.
This hypothesis is supported by observing that the binding dwell time depended on the ATP concentration Fig. The displacement of the lever-arm swing, This is probably because the second step occurred too quickly to be observed, while in the AFM observation, the myosin and actin were adsorbed onto mica or lipid, which suppressed mechanical transitions, as previously suggested The ATP binding rate was fit to a straight line with a slope of 0.
See Methods for details. Error bars indicate standard deviations. The points were obtained by the cumulative frequency plots in Supplementary Fig. Data were obtained from at least three independent experiments for each ATP concentration.
A scattering image of GNP was used to decide the position of the myosin head. Each spike indicates a binding event of Cy3ATP to a myosin head. The time between spikes corresponds to ATP waiting time. To dissect the biased binding process, we analyzed the trajectory in detail using nonparametric Bayesian inference, which was recently applied to single-molecule trajectory analysis 41 see Methods.
This inference method is capable of detecting hidden molecular states without defining the number of states beforehand, which is unlike the hidden Markov model fitting used above.
We tested the detection accuracy of nonparametric Bayesian inference by simulated trajectories while assuming weak binding states are hidden in the detached state.
An analysis of our experimental data with lever-arm-less S1 is shown in Fig. Similar to the results of the simulated data Supplementary Fig. Based on the inferred parameters, we calculated the accessibility of the myosin head to the binding position and the dwell time of the transient binding states Fig.
Furthermore, the distance between the mean position of the detached state and the most compatible position leading to the longest dwell time was consistent with the step size This result suggests that while weakly binding to actin molecules during the detached state, a myosin head achieves biased binding at the most compatible actin molecule as a Brownian ratchet Figure 7b shows a model that summarizes the experimental results of the elementary mechanical processes for force generation in the actomyosin complex by the interaction of myosin II in our engineered thick filament.
Position-dependent weak binding detected by nonparametric Bayesian inference. The blue line indicates the trajectory of lever-arm-less S1 labeled with GNP.
Magenta and cyan plots indicate the inferred mean position of the transient binding states and detached state, respectively. Data were obtained from three independent and reproduced experiments. Representative data are shown. The calculated accessibilities access ratio are 0. These values are shown in Fig.
Errors in the values indicate standard deviations. Dwell times were fit to the cumulative distribution function of a single exponential decay. Brownian ratchet mechanism underpinned by position-dependent weak binding. The accessibility blue is plotted against the left axis, and the dwell time magenta is plotted against the right axis.
The dashed line indicates the inferred mean position of the detached state, which is the cyan plot in Fig. The dwell time is prolonged times upon the transition from weak binding to strong binding. A myosin head achieves strong biased binding from weak binding during free diffusion as a Brownian ratchet, followed by a multiple-step and reversible lever-arm swing.
In vitro single-molecule assays using optical tweezers 13 , 14 , 15 , 16 are conventionally used to measure the displacement and force of myosin heads, but they cannot observe the movement of muscle myosin heads directly. In the present work, the controllable labeling of myosins, actin binding proteins and GNPs in our DNA origami-based thick filament provided a reliable assay system for directly visualizing the mechanistic details of myosins during force generation under geometric conditions that resemble those in muscle.
Also, we found the linker angle had a significant dependency on the position Fig. This position dependency of the angles may impose different geometric constraints on myosins at different positions in our thick filament. Nonetheless, our AFM experiments show that the lever-arm angles and powerstroke sizes were independent of the position on the thick filament Supplementary Fig. Additionally, the estimated binding displacements of GNP at different positions suggest that the position dependency of the angle will have negligible effects on the behaviour of the GNP Supplementary Fig.
These theoretical models successfully explain the steady state mechanical characteristics of muscle contraction such as A. Moreover, these models remain the standard for explaining the whole mechanical characteristics of muscle contraction, but no direct experimental evidence has validated them. Our results confirm their assumptions and provide quantitative values, such as the number of tilting steps, angles of the lever-arm, the attachment and detachment kinetics of weak binding, and the spatial asymmetricity.
A complete list of all oligonucleotides sequences can be found in Supplementary Data 2. The folded DNA origami rods were purified by glycerol gradient ultracentrifugation according to Lin et al.
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