l-arginine and l-lysine supplementation to NaCl tenderizes porcine meat by promoting myosin extraction and actomyosin dissociation.

This study investigated the individual and combined effects of l-arginine, l-lysine, and NaCl on the ultrastructure of porcine myofibrils to uncover the mechanism underlying meat tenderization. Arg or Lys alone shortened A-bands and damaged M-lines, while NaCl alone destroyed M- and Z-lines. Overall, Arg and Lys cooperated with NaCl to destroy the myofibrillar ultrastructure. Moreover, these two amino acids conjoined with NaCl to increase myosin solubility, actin band intensity, and the protein concentration of the actomyosin supernatant. However, they decreased the turbidity and particle size of both myosin and actomyosin solutions, and the remaining activities of Ca2+- and Mg2+-ATPase. The current results revealed that Arg/Lys combined with NaCl to extract myosin and dissociate actomyosin, thereby aggravating the destruction of the myofibrillar ultrastructure. The present results provide a good explanation for the previous phenomenon that Arg and Lys cooperated with NaCl to improve meat tenderness.

Inferring scale-dependent non-equilibrium activity using carbon nanotubes.

In living systems, irreversible, yet stochastic, molecular interactions form multiscale structures (such as cytoskeletal networks), which mediate processes (such as cytokinesis and cellular motility) in a close relationship between the structure and function. However, owing to a lack of methods to quantify non-equilibrium activity, their dynamics remain poorly characterized. Here, by measuring the time-reversal asymmetry encoded in the conformational dynamics of filamentous single-walled carbon nanotubes embedded in the actomyosin network of Xenopus egg extract, we characterize the multiscale dynamics of non-equilibrium activity encoded in bending-mode amplitudes. Our method is sensitive to distinct perturbations to the actomyosin network and the concentration ratio of adenosine triphosphate to adenosine diphosphate. Thus, our method can dissect the functional coupling of microscopic dynamics to the emergence of larger scale non-equilibrium activity. We relate the spatiotemporal scales of non-equilibrium activity to the key physical parameters of a semiflexible filament embedded in a non-equilibrium viscoelastic environment. Our analysis provides a general tool to characterize steady-state non-equilibrium activity in high-dimensional spaces.

L-histidine inhibits the heat-induced gelation of actomyosin in a low ionic strength solution.

The heat-induced gelation of actomyosin plays a key role in meat processing. Our previous study showed that L-histidine could affect the characteristics of a heat-induced gel of myosin on a low ionic strength. To apply the specific effect of L-histidine to meat processing, the heat-induced gel properties of actomyosin in the presence of L-histidine were investigated. Actomyosin in a low ionic strength solution containing L-histidine did not form a gel upon heating. The dynamic rheological properties of actomyosin in low ionic strength solutions were distinct depending on the presence or absence of L-histidine. Electron microscopy showed that, heated at 50°C, actomyosin in a low ionic strength solution containing L-histidine remained a filamentous structure. The surface hydrophobicity of actomyosin was stable up to 50°C in a low ionic strength solution containing L-histidine. In conclusion, L-histidine might suppress the aggregation of actomyosin and inhibit heat-induced gelation in a low ionic strength solution.

Actomyosin Complex.

Formation of cross-bridges between actin and myosin occurs ubiquitously in eukaryotic cells and mediates muscle contraction, intracellular cargo transport, and cytoskeletal remodeling. Myosin motors repeatedly bind to and dissociate from actin filaments in a cycle that transduces the chemical energy from ATP hydrolysis into mechanical force generation. While the general layout of surface elements within the actin-binding interface is conserved among myosin classes, sequence divergence within these motifs alters the specific contacts involved in the actomyosin interaction as well as the kinetics of mechanochemical cycle phases. Additionally, diverse lever arm structures influence the motility and force production of myosin molecules during their actin interactions. The structural differences generated by myosin's molecular evolution have fine-tuned the kinetics of its isoforms and adapted them for their individual cellular roles. In this chapter, we will characterize the structural and biochemical basis of the actin-myosin interaction and explain its relationship with myosin's cellular roles, with emphasis on the structural variation among myosin isoforms that enables their functional specialization. We will also discuss the impact of accessory proteins, such as the troponin-tropomyosin complex and myosin-binding protein C, on the formation and regulation of actomyosin cross-bridges.

A generalized Flory-Stockmayer kinetic theory of connectivity percolation and rigidity percolation of cytoskeletal networks.

Actin networks are essential for living cells to move, reproduce, and sense their environments. The dynamic and rheological behavior of actin networks is modulated by actin-binding proteins such as α-actinin, Arp2/3, and myosin. There is experimental evidence that actin-binding proteins modulate the cooperation of myosin motors by connecting the actin network. In this work, we present an analytical mean field model, using the Flory-Stockmayer theory of gelation, to understand how different actin-binding proteins change the connectivity of the actin filaments as the networks are formed. We follow the kinetics of the networks and estimate the concentrations of actin-binding proteins that are needed to reach connectivity percolation as well as to reach rigidity percolation. We find that Arp2/3 increases the actomyosin connectivity in the network in a non-monotonic way. We also describe how changing the connectivity of actomyosin networks modulates the ability of motors to exert forces, leading to three possible phases of the networks with distinctive dynamical characteristics: a sol phase, a gel phase, and an active phase. Thus, changes in the concentration and activity of actin-binding proteins in cells lead to a phase transition of the actin network, allowing the cells to perform active contraction and change their rheological properties.

Evaluation of ultrasound-assisted L-histidine marination on beef M. semitendinosus: Insight into meat quality and actomyosin properties.

This paper aimed to evaluate the effects of ultrasound-assisted L-histidine marination (UMH) on meat quality and actomyosin properties of beef M. semitendinosus. Our results found that UMH treatment effectively avoided excessive liquid withdrawal, and disrupted myofibril integrity by modifying the water distribution and weakening connection of actin-myosin with increased muscle pH. The ultrasound-treated sample provided more opportunity for the filtration of L-histidine to intervene the isoelectric point and conformation of muscle protein. The activated caspase-3 and changes of ATPase activity in UMH-treated meat accelerated the postmortem ageing, and L-histidine might competitively inhibit the actin-myosin binding by the imidazole group. UMH decreased the surface hydrophobicity by shielding hydrophobic area and unfolding the actomyosin structure. In addition, the increased actomyosin solubility with smaller particle size enhanced the SH content for better cross-linking of myosin tail, and formation of heat-set gelling protein structure. Therefore, UMH treatment manifested the potential to improve beef quality.

ATP allosterically stabilizes integrin-linked kinase for efficient force generation.

SignificanceThe pseudokinase integrin-linked kinase (ILK) is a central component of focal adhesions, cytoplasmic multiprotein complexes that integrate and transduce biochemical and mechanical signals from the extracellular environment into the cell and vice versa. However, the precise molecular functions, particularly the mechanosensory properties of ILK and the significance of retained adenosine triphosphate (ATP) binding, are still unclear. Combining molecular-dynamics simulations with cell biology, we establish a role for ATP binding to pseudokinases. We find that ATP promotes the structural stability of ILK, allosterically influences the interaction between ILK and its binding partner parvin at adhesions, and enhances the mechanoresistance of this complex. On the cellular level, ATP binding facilitates efficient traction force buildup, focal adhesion stabilization, and efficient cell migration.

The Local Environment of Loop Switch 1 Modulates the Rate of ATP-Induced Dissociation of Human Cardiac Actomyosin.

Two isoforms of human cardiac myosin, alpha and beta, share significant sequence similarities but show different kinetics. The alpha isoform is a faster motor; it spends less time being strongly bound to actin during the actomyosin cycle. With alpha isoform, actomyosin dissociates faster upon ATP binding, and the affinity of ADP to actomyosin is weaker. One can suggest that the isoform-specific actomyosin kinetics is regulated at the nucleotide binding site of human cardiac myosin. Myosin is a P-loop ATPase; the nucleotide-binding site consists of P-loop and loops switch 1 and 2. All three loops position MgATP for successful hydrolysis. Loops sequence is conserved in both myosin isoforms, and we hypothesize that the isoform-specific structural element near the active site regulates the rate of nucleotide binding and release. Previously we ran molecular dynamics simulations and found that loop S291-E317 near loop switch 1 is more compact and exhibits larger fluctuations of the position of amino acid residues in beta isoform than in alpha. In alpha isoform, the loop forms a salt bridge with loop switch 1, the bridge is not present in beta isoform. Two isoleucines I303 and I313 of loop S291-E317 are replaced with valines in alpha isoform. We introduced a double mutation I303V:I313V in beta isoform background and studied how the mutation affects the rate of ATP binding and ADP dissociation from actomyosin. We found that ATP-induced actomyosin dissociation occurs faster in the mutant, but the rate of ADP release remains the same as in the wild-type beta isoform. Due to the proximity of loop S291-E317 and loop switch 1, a faster rate of ATP-induced actomyosin dissociation indicates that loop S291-E317 affects structural dynamics of loop switch 1, and that loop switch 1 controls ATP binding to the active site. A similar rate of ADP dissociation from actomyosin in the mutant and wild-type myosin constructs indicates that loop switch 1 does not control ADP release from actomyosin.

Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms.

Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2-0.5 s-1) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction.

High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex.

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8 Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.

Unraveling a Force-Generating Allosteric Pathway of Actomyosin Communication Associated with ADP and Pi Release.

The actomyosin system generates mechanical work with the execution of the power stroke, an ATP-driven, two-step rotational swing of the myosin-neck that occurs post ATP hydrolysis during the transition from weakly to strongly actin-bound myosin states concomitant with Pi release and prior to ADP dissociation. The activating role of actin on product release and force generation is well documented; however, the communication paths associated with weak-to-strong transitions are poorly characterized. With the aid of mutant analyses based on kinetic investigations and simulations, we identified the W-helix as an important hub coupling the structural changes of switch elements during ATP hydrolysis to temporally controlled interactions with actin that are passed to the central transducer and converter. Disturbing the W-helix/transducer pathway increased actin-activated ATP turnover and reduced motor performance as a consequence of prolonged duration of the strongly actin-attached states. Actin-triggered Pi release was accelerated, while ADP release considerably decelerated, both limiting maximum ATPase, thus transforming myosin-2 into a high-duty-ratio motor. This kinetic signature of the mutant allowed us to define the fractional occupancies of intermediate states during the ATPase cycle providing evidence that myosin populates a cleft-closure state of strong actin interaction during the weak-to-strong transition with bound hydrolysis products before accomplishing the power stroke.

On the mechanical response of the actomyosin cortex during cell indentations.

A mechanical model is presented to analyze the mechanics and dynamics of the cell cortex during indentation. We investigate the impact of active contraction on the cross-linked actin network for different probe sizes and indentation rates. The essential molecular mechanisms of filament stretching, cross-linking and motor activity, are represented by an active and viscous mechanical continuum. The filaments behave as worm-like chains linked either by passive rigid linkers or by myosin motors. In the first example, the effects of probe size and loading rate are evaluated using the model for an idealized rounded cell shape in which properties are based on the results of parallel-plate rheometry available in the literature. Extreme cases of probe size and indentation rate are taken into account. Afterward, AFM experiments were done by engaging smooth muscle cells with both sharp and spherical probes. By inverse analysis with finite element software, our simulations mimicking the experimental conditions show the model is capable of fitting the AFM data. The results provide spatiotemporal dependence on the size and rate of the mechanical stimuli. The model captures the general features of the cell response. It characterizes the actomyosin cortex as an active solid at short timescales and as a fluid at longer timescales by showing (1) higher levels of contraction in the zones of high curvature; (2) larger indentation forces as the probe size increases; and (3) increase in the apparent modulus with the indentation depth but no dependence on the rate of the mechanical stimuli. The methodology presented in this work can be used to address and predict microstructural dependence on the force generation of living cells, which can contribute to understanding the broad spectrum of results in cell experiments.

The Structure of Acto-Myosin.

After several decades studying different acto-myosin complexes at lower and intermediate resolution - limited by the electron microscope instrumentation available then - recent advances in imaging technology have been crucial for obtaining a number of excellent high-resolution 3D reconstructions from cryo electron microscopy. The resolution level reached now is about 3-4 Å, which allows unambiguous model building of filamentous actin on its own as well as that of actin filaments decorated with strongly bound myosin variants. The interface between actin and the myosin motor domain can now be described in detail, and the function of parts of the interface (such as, e.g., the cardiomyopathy loop) can be understood in a mechanistical way. Most recently, reconstructions of actin filaments decorated with different myosins, which show a strongly bound acto-myosin complex also in the presence of the nucleotide ADP, have become available. The comparison of these structures with the nucleotide-free Rigor state provide the first mechanistic description of force sensing. An open question is still the initial interaction of the motor domain of myosin with the actin filament. Such weakly interacting states have so far not been the subject of microscopical studies, even though high-resolution structures would be needed to shed light on the initial steps of phosphate release and power stroke initiation.

Physicochemical properties of tilapia (Oreochromis niloticus) actomyosin subjected to high intensity ultrasound in low NaCl concentrations.

Effects of high intensity ultrasound (HIU) on physicochemical properties of tilapia (Oreochromis niloticus) actomyosin in low NaCl concentrations were investigated. The protein content extracted in low NaCl concentrations (0.1-0.3 M NaCl) increased with increasing HIU intensity up to 20.62 W/cm2 (p < 0.05). The effect of HIU on actomyosin extractability in high NaCl concentrations (0.6 and 1.2 M NaCl) was less obvious. Ca2+-ATPase activity and total sulfhydryl (SH) group content decreased in both 0.2 and 0.6 M NaCl. HIU showed more pronounced effect on oxidation of the SH groups in 0.6 M NaCl, while the reactive SH content at 0.2 M NaCl increased after a prolonged exposure to HIU, suggesting conformational changes induced by HIU. Surface hydrophobicity of actomyosin in 0.6 M NaCl increased with increasing ultrasonic intensity and exposure time to a higher degree than that in 0.2 M NaCl. A greater absolute value of the zeta potential of actomyosin subjected to HIU were also observed. The HIU treatments decreased the turbidity of actomyosin incubated at 40 and 60 °C. A drastic increase in the solubility of myosin heavy chain (MHC) and actin with 0.2 M NaCl were evident when HIU treatments were applied, but degradation of MHC occurred in both 0.2 and 0.6 M NaCl. Based on particle size and microstructure, actomyosin in 0.6 M NaCl underwent more disruption by HIU than that in 0.2 M NaCl. HIU induced protein unfolding and protein dissociation, enabling better extraction in a lower NaCl concentration.

An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells.

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.

Actomyosin controls planarity and folding of epithelia in response to compression.

Throughout embryonic development and adult life, epithelia are subjected to compressive deformations. While these have been shown to trigger mechanosensitive responses such as cell extrusion and differentiation, which span tens of minutes, little is known about how epithelia adapt to compression over shorter timescales. Here, using suspended epithelia, we uncover the immediate response of epithelial tissues to the application of in-plane compressive strains (5-80%). We show that fast compression induces tissue buckling followed by actomyosin-dependent tissue flattening that erases the buckle within tens of seconds, in both mono- and multi-layered epithelia. Strikingly, we identify a well-defined limit to this response, so that stable folds form in the tissue when compressive strains exceed a 'buckling threshold' of ~35%. A combination of experiment and modelling shows that this behaviour is orchestrated by adaptation of the actomyosin cytoskeleton as it re-establishes tissue tension following compression. Thus, tissue pre-tension allows epithelia to both buffer against deformation and sets their ability to form and retain folds during morphogenesis.

Atomistic Models from Orientation and Distance Constraints Using EPR of a Bifunctional Spin Label.

We have used high-resolution orientation and distance measurements derived from electron paramagnetic resonance of a bifunctional spin label (BSL) to build and refine atomistic models of protein structure. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin's catalytic domain while myosin is in complex with actin. Constraints for orientation of individual helices were obtained in a previous study from continuous-wave electron paramagnetic resonance of myosin labeled at specific sites with BSLs in oriented muscle fibers. In this study, new distance constraints were derived from double electron-electron resonance on myosin constructs labeled with a BSL specifically at two sites. Using these complementary constraints together, we thoroughly characterize the BSL's rigid, highly stereoselective attachment to protein α-helices, which permits accurate measurements of orientation and distance. We also leverage these measurements to derive a novel, to our knowledge, structural model for myosin-II in complex with actin and MgADP and compare our model to other recent actomyosin structures. The described approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di-Cys mutation is feasible.

Influence of hydrothermal treatment on the structural and digestive changes of actomyosin.

BACKGROUND: Heat treatment induces both structural and digestive change of meat protein. However, little has been revealed regarding the associations between structural changes and digested peptides of myofibrillar proteins. This work investigated the effects of heat treatment on the structures and in vitro digestibility of actomyosin, and the peptidomics of the digests were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Heat treatment resulted in unfolding and aggregation behavior of actomyosin according to the results of surface hydrophobicity and particle size. Formation of disulfide bonds and increase in carbonyl groups that occurred during heat treatment of actomyosin indicated the oxidation of specific residues. Unfolding behavior could elevate digestibility of actomyosin by exposing residues, based on the identification of peptides in digests of actomyosin using LC-MS/MS. However, the disulfide bond proved to reduce the action of digestive proteases, since the peptides number (increased from 56 to 86 in sample heated at 70 °C for 30 min) and peptides intensity in digests largely increased after the addition of dithiothreitol (DTT). Heating at higher temperature (100 °C) induced severer aggregation and oxidation, which resulted in lower digestibility of actomyosin than that heated at 70 °C by burying or damaging partial cleavage sites for digestive proteases. CONCLUSIONS: This work highlights the huge influence of heat treatment on the multi-scale structures of myofibrillar proteins, which largely changed the peptides composition in protein digests. © 2019 Society of Chemical Industry.

Dissecting fat-tailed fluctuations in the cytoskeleton with active micropost arrays.

The ability of animal cells to crawl, change their shape, and respond to applied force is due to their cytoskeleton: A dynamic, cross-linked network of actin protein filaments and myosin motors. How these building blocks assemble to give rise to cells' mechanics and behavior remains poorly understood. Using active micropost array detectors containing magnetic actuators, we have characterized the mechanics and fluctuations of cells' actomyosin cortex and stress fiber network in detail. Here, we find that both structures display remarkably consistent power law viscoelastic behavior along with highly intermittent fluctuations with fat-tailed distributions of amplitudes. Notably, this motion in the cortex is dominated by occasional large, step-like displacement events, with a spatial extent of several micrometers. Overall, our findings for the cortex appear contrary to the predictions of a recent active gel model, while suggesting that different actomyosin contractile units act in a highly collective and cooperative manner. We hypothesize that cells' actomyosin components robustly self-organize into marginally stable, plastic networks that give cells' their unique biomechanical properties.

Relationship between the characteristics of rigor-mortis-related actomyosin and muscle fiber types in the ordinary muscle of various fishes.

BACKGROUND: Rigor mortis occurs when muscle extension vanishes through the irresistible coupling of actin and myosin by the consumption of adenosine triphosphate as energy. To clarify the cause of the differences in the progression of rigor mortis, seven fish species were used as samples. The superprecipitation reaction and Mg2+ -ATPase activity of actomyosin in dorsal ordinary muscle were measured, and the slope of the regression line between these two variables was calculated for each fish specimen. The fiber types of the dorsal ordinary muscle in each sample fish were discriminated by the stability of actomyosin ATPase at acid and alkaline preincubations. RESULT: Positive correlations were found between Mg2+ -ATPase activity and the superprecipitation reaction of actomyosin in all 27 fish specimens. The slopes of the regression lines were different not only between fish species but also in fish specimens within the same species. The area ratios of pink muscle fibers and the IIa and/or IIb subtypes of white muscle fibers in the dorsal ordinary muscle were also different between fish species, as well as in specimens within the same fish species. A positive correlation was found between the area ratios of pink muscle fibers in dorsal ordinary muscle and the slopes of the regression line. CONCLUSION: It was suggested that the differences in characteristics of rigor-mortis-related actomyosin of fish might have been caused by the differences in the interposition ratio of muscle fiber types, especially of the pink muscle fiber type, in the dorsal ordinary muscle. © 2019 Society of Chemical Industry.