Motor proteins drive motion in living systems. Myosin motors adsorbed on a surface propel actin filaments by hydrolyzing ATP. This makes them interesting systems for applications in nanotechnology, e.g. as sensors, for transporting molecular cargo or driving other forms of molecular motion. However, their effective functioning requires the proper combination of materials with adequate surface chemistry and hydrophobic properties. Here, we investigate a set of materials systems used as substrates and analyze their compatibility with the actomyosin system. As a reference, we used glass slides coated with trimethylchlorosilane (TMCS) where coating is performed in liquid phase, since this is a commonly used approach. We then explored an alternative vapor phase deposition method to coat glass slides with various silane compounds: in addition to TMCS, we also used perfluoro-octyltrichlorosilane (FOTCS) and perfluoro-dodecyltrichlorosilane (FDDTCS). In vitro motility assays (IVMAs), where surface-adsorbed myosin motor fragments propel actin filaments, were then used to measure the sliding velocity on the different surfaces. Filaments propelled on FOTCS-functionalized surfaces by chemical vapor deposition exhibited the highest average sliding velocity (3.9 +/- 1.2 mu m/s; mean +/- SD) and retained a high fraction of motile actin filaments (87%), comparable to TMCS-functionalized surfaces (3.3 +/- 0.4 mu m/s, 90% motile). In addition, we also used a UV-curable polymer as active substrate material, which we have successfully treated to either promote or inhibit motor adsorption and therefore motility. We have evaluated the hydrophobic characteristics and the roughness of the different functionalized surfaces. In addition, we patterned microchannels with physical and chemical contrast, to confine the motor adsorption and consequently motion of the myosin- driven actin filaments to the patterned microchannel bottoms. This gas-phase deposition technique uses just a low cost commercial oven and offers a promising method for tailoring the surface properties of various materials, paving the way for standardizing and advancing the application of myosin-propelled actin filaments in nanotechnology and microdevices.

The release of the ATP hydrolysis product, orthophosphate (Pi), from the myosin active site, together with force-generating structural changes, is central to actomyosin energy transduction, but the temporal order of these events remains unclear. A range of data, interpreted using simple kinetic schemes (that do not account for varying cross-bridge strains) suggests that force generation is closely associated with the attachment of the myosin head to actin, preceding Pi-release. However, the addition of a branched pathway to the kinetic scheme is needed to account for the lower sensitivity of the isometric ATP-turnover rate to Pi compared with that of force. In contrast, a branched pathway does not appear necessary if the data are analyzed using a mechanokinetic model that incorporates the myosin strain distribution. Here, we corroborated this idea using a model in which Pi-release from the active site precedes the force-generating power-stroke. We explain the effect based on two components underlying the reduction in isometric force with increased [Pi]. The larger component arises from pre-power-stroke cross-bridges with high large elastic strain, whereas the smaller component results from cross-bridges attaching with low elastic strain. Because only the latter myosin heads undergo ATPase cycles, force exhibits greater Pi-sensitivity than ATPase activity. Changes in model parameter values that minimize the width of the cross-bridge strain distribution do not eliminate the difference in Pi-sensitivity between isometric force and ATPase. Such changes, including reduced actin affinity in a pre-power-stroke state, also lead to a proportional reduction in isometric force and in the number of attached cross-bridges with increased [Pi]. In conclusion, our data suggest that a mechanokinetic model explains the combined changes in isometric force, ATPase activity, and the number of attached cross-bridges with varied [Pi] more directly than apparently simpler kinetic schemes. A central feature of these results is the explicit demonstration of two components of isometric force with different physiological roles.

Myosin II is a molecular motor that converts chemical energy derived from ATP hydrolysis into mechanical work. Myosin II isoforms are responsible for muscle contraction and a range of cell functions relying on the development of force and motion. When the motor attaches to actin, ATP is hydrolyzed and inorganic phosphate (Pi) and ADP are released from its active site. These reactions are coordinated with changes in the structure of myosin, promoting the so-called "power stroke" that causes the sliding of actin filaments. The general features of the myosin-actin interactions are well accepted, but there are critical issues that remain poorly understood, mostly due to technological limitations. In recent years, there has been a significant advance in structural, biochemical, and mechanical methods that have advanced the field considerably. New modeling approaches have also allowed researchers to understand actomyosin interactions at different levels of analysis. This paper reviews recent studies looking into the interaction between myosin II and actin filaments, which leads to power stroke and force generation. It reviews studies conducted with single myosin molecules, myosins working in filaments, muscle sarcomeres, myofibrils, and fibers. It also reviews the mathematical models that have been used to understand the mechanics of myosin II in approaches focusing on single molecules to ensembles. Finally, it includes brief sections on translational aspects, how changes in the myosin motor by mutations and/or posttranslational modifications may cause detrimental effects in diseases and aging, among other conditions, and how myosin II has become an emerging drug target.

Introduction Small molecular compounds that affect the force, and motion-generating actin-myosin interaction in the heart have emerged as alternatives to treat or alleviate symptoms in severe debilitating conditions, such as cardiomyopathies and heart failure. Omecamtiv mecarbil (OM) is such a compound developed to enhance cardiac contraction. In addition to potential therapeutic use, its effects may help to elucidate myosin energy transduction mechanisms in health and disease and add insights into how the molecular properties govern contraction of large myosin ensembles in cardiac cells. Despite intense studies, the effects of OM are still incompletely understood.Methods Here we take an in silico approach to elucidate the issue. First, we modify a model, previously used in studies of skeletal muscle, with molecular parameter values for human ventricular beta-myosin to make it useful for studies of both myosin mutations and drugs. Repeated tests lead to at a set of parameter values that allow faithful reproduction of range of functional variables of cardiac myocytes. We then apply the model to studies of OM.Results and discussion The results suggest that major effects of OM such as large reduction of the maximum velocity with more limited effects on maximum isometric force and slowed actin-activated ATPase can be accounted for by two key molecular effects. These encompass a reduced difference in binding free energy between the pre- and post-power-stroke states and greatly increased activation energy for the lever arm swing during the power-stroke. Better quantitative agreement, e.g., isometric force minimally changed from the control value by OM is achieved by additional changes in model parameter values previously suggested by studies of isolated proteins.

The relative timing of the force-generating power stroke and release of the ATP-hydrolysis product orthophosphate (Pi) in actomyosin energy transduction is debated. It may be explored by studying the tension response to sudden changes in [Pi] during isometric muscle contraction (Pi-transients; rate constant k_Pi) and by the rate of redevelopment of isometric force (k_tr) after a period of unloaded shortening at varied [Pi]. Most studies of these types are interpreted using simple kinetic schemes that ignore the range of elastic strains of actin-attached myosin cross-bridges. We found that the only simple scheme which accounts for the experimental findings of single exponential Pi-transients with k_Pi approximate to ktr has force-generation coincident with actin-myosin attachment. This characteristics could compromise the high power output of muscle. We therefore turned to a mechanokinetic model, allowing consideration of the varying elastic cross-bridge strains. Our model assumes Pi-release between cross-bridge attachment and the force-generating power stroke. However, power strokes only occur if cross-bridges attach in a pre-power-stroke state with zero or negative elastic strain (counteracting shortening). The model suggests two components of the Pi-transients. One is attributed to slow cross-bridge detachment from the pre-power-stroke state at positive elastic strain upon Pi-binding. The other is due to Pi-induced shifts in equilibrium with rapid power stroke reversal. The slow component dominates for all parameter values tested but the fast component is ubiquitous, predicting a biphasic Pi-transient in disagreement with experiments. Strikingly, however, the mechanokinetic model gives different predictions than apparently similar simple kinetic schemes and we do not rule out the existence of parameter values leading to a negligible fast component. We also show that the assumption of secondary Pi-binding sites on myosin outside the active site removes the fast component albeit without predicting that ktr approximate to kPi. Additional studies are required to finally corroborate that k_tr approximate to k_Pi in experiments but also to further develop mechanokinetic models combined with multistep Pi-release.

Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac beta-myosin heavy chain (beta-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current similar to 4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

In the in vitro motility assay (IVMA), actin filaments are observed while propelled by surface-adsorbed myosin motor fragments such as heavy meromyosin (HMM). In addition to fundamental studies, the IVMA is the basis for a range of lab-on-a-chip applications, e.g. transport of cargoes in nanofabricated channels in nanoseparation/biosensing or the solution of combinatorial mathematical problems in network-based biocomputation. In these applications, prolonged myosin function is critical as is the potential to repeatedly exchange experimental solutions without functional deterioration. We here elucidate key factors of importance in these regards. Our findings support a hypothesis that early deterioration in the IVMA is primarily due to oxygen entrance into in vitro motility assay flow cells. In the presence of a typically used oxygen scavenger mixture (glucose oxidase, glucose, and catalase), this leads to pH reduction by a glucose oxidase-catalyzed reaction between glucose and oxygen but also contributes to functional deterioration by other mechanisms. Our studies further demonstrate challenges associated with evaporation and loss of actin filaments with time. However, over 8 h at 21-26 degrees C, there is no significant surface desorption or denaturation of HMM if solutions are exchanged manually every 30 min. We arrive at an optimized protocol with repeated exchange of carefully degassed assay solution of 45 mM ionic strength, at 30 min intervals. This is sufficient to maintain the high-quality function in an IVMA over 8 h at 21-26 degrees C, provided that fresh actin filaments are re-supplied in connection with each assay solution exchange. Finally, we demonstrate adaptation to a microfluidic platform and identify challenges that remain to be solved for real lab-on-a-chip applications.

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (k(cat)). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

Calcium binding to troponin, with subsequent displacement of its linked tropomyosin molecule on the thin filament surface, cooperates with myosin binding to actin in the contractile regulation of striated muscle. The intertwined role of these systems is studied in the present issue of JGP by Ishii et al. (https://doi.org/10.1085/jgp.202313414). A particularly interesting feature of the paper, except for studying both skeletal and cardiac muscle proteins, is that the experiments unlike most other similar studies are performed at physiological temperature (35-40(degrees)C).