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KEY POINTS A nanomachine made of an ensemble of seven heavy-meromyosin (HMM) fragments of muscle myosin interacting with an actin filament is able to mimic the half-sarcomere generating steady force and constant-velocity shortening. To preserve Ca2+ as a free parameter, the Ca2+ -insensitive gelsolin fragment TL40 is used to attach the correctly oriented actin filament to the laser-trapped bead acting as a force transducer. The new method reveals that the performance of the nanomachine powered by myosin from frog hind-limb muscles depends on [Ca2+ ], an effect mediated by a Ca2+ -binding site in the regulatory light chain of HMM. The Ca2+ -sensitivity is class-specific because the performance of the nanomachine powered by mammalian skeletal muscle myosin is Ca2+ independent. A model simulation is able to interface the nanomachine performance with that of the muscle of origin and provides a molecular explanation of the functional diversity of muscles with different orthologue isoforms of myosin. ABSTRACT An ensemble of seven heavy-meromyosin (HMM) fragments of myosin-II purified from the hindlimb muscles of the frog (Rana esculenta) is used to drive a synthetic nanomachine that pulls an actin filament in the absence of confounding effects of other sarcomeric proteins. In the present version of the nanomachine the +end of the actin filament is attached to the laser trapped bead via the Ca2+ -insensitive gelsolin fragment TL40, making [Ca2+ ] a free parameter. Frog myosin performance in 2 mm ATP is affected by Ca2+ : in 0.1 mm Ca2+ , the isometric steady force (F0 , 15.25 pN) is increased by 50% (P = 0.004) with respect to that in Ca2+ -free solution, the maximum shortening velocity (V0 , 4.6 μm s-1 ) is reduced by 27% (P = 0.46) and the maximum power (Pmax , 7.6 aW) is increased by 21% (P = 0.17). V0 reduction is not significant for the paucity of data at low force, although it is solidified by a similar decrease (33%, P

The evidence, in both resting and active muscle, for the presence of an I-band spring element like titin that anchors the Z line to the end of the thick filament did not yet produce a proper theoretical treatment in a complete model of the half-sarcomere. The textbook model developed by A. F. Huxley and his collaborators in 1981, which provides that the half-sarcomere (hs) compliance is due to the contribution of the compliances of the thin and thick filaments and actin-attached myosin motors, predicts that at any sarcomere length (SL) the absence of attached motors results in an infinite half-sarcomere compliance, in contrast with the observations. Growing evidence for the presence of a titin-like I-band spring urges the 1981 model to be implemented to include the contribution of this element in the mechanical model of the half-sarcomere. The model described here represents a tool for the interpretation of measurements of hs stiffness at increasing SL, which is important either in relation to the mechanism of stabilisation of SL against the consequence of sarcomere inhomogeneity in active force generation, or for investigations on the role of titin as mechano-sensor in thick filament regulation. Moreover the model opens the possibility for understanding the functional differences related to the titin isoform of various muscle types and the mechanism by which mutations in titin gene lead to myopathies.

Key points Direct binding of rumenic acid to the cardiac myosin-2 motor domain increases the release rate for orthophosphate and increases the Ca2+ responsiveness of cardiac muscle at low load. Physiological cellular concentrations of rumenic acid affect the ATP turnover rates of the super-relaxed and disordered relaxed states of β-cardiac myosin, leading to a net increase in myocardial metabolic load. In Ca2+ -activated trabeculae, rumenic acid exerts a direct inhibitory effect on the force-generating mechanism without affecting the number of force-generating motors. In the presence of saturating actin concentrations rumenic acid binds to the β-cardiac myosin-2 motor domain with an EC50 of 200 nM. Molecular docking studies provide information about the binding site, the mode of binding, and associated allosteric communication pathways. Free rumenic acid may exceed thresholds in cardiomyocytes above which contractile efficiency is reduced and interference with small molecule therapeutics, targeting cardiac myosin, occurs. Abstract Based on experiments using purified myosin motor domains, reconstituted actomyosin complexes and rat heart ventricular trabeculae, we demonstrate direct binding of rumenic acid, the cis-delta-9-trans-delta-11 isomer of conjugated linoleic acid, to an allosteric site located in motor domain of mammalian cardiac myosin-2 isoforms. In the case of porcine β-cardiac myosin, the EC50 for rumenic acid varies from 10.5 μM in the absence of actin to 200 nM in the presence of saturating concentrations of actin. Saturating concentrations of rumenic acid increase the maximum turnover of basal and actin-activated ATPase activity of β-cardiac myosin approximately 2-fold but decrease the force output per motor by 23% during isometric contraction. The increase in ATP turnover is linked to an acceleration of the release of the hydrolysis product orthophosphate. In the presence of 5 μM rumenic acid, the difference in the rate of ATP turnover by the super-relaxed and disordered relaxed states of cardiac myosin increases from 4-fold to 20-fold. The equilibrium between the two functional myosin states is not affected by rumenic acid. Calcium responsiveness is increased under zero-load conditions but unchanged under load. Molecular docking studies provide information about the rumenic acid binding site, the mode of binding, and associated allosteric communication pathways. They show how the isoform-specific replacement of residues in the binding cleft induces a different mode of rumenic acid binding in the case of non-muscle myosin-2C and blocks binding to skeletal muscle and smooth muscle myosin-2 isoforms.

The emergent properties of the array arrangement of the molecular motor myosin II in the sarcomere of the striated muscle, generation of steady force and shortening, can be studied in vitro with a synthetic nanomachine, made by an ensemble of eight HMM myosin fragments from rabbit psoas muscle carried on a piezoelectric nanopositioner and brought to interact with a properly oriented actin filament attached via gelsolin (a Ca2+-regulated actin binding protein) to a bead trapped by a Dual Laser Optical Tweezers. The application of the original version of the nanomachine to investigation of the Ca2+-dependent regulation mechanisms of the other sarcomeric (regulatory or cytoskeleton) proteins, adding them on at a time, was anyway prevented by the impossibility to preserve Ca2+ as a free parameter. Here the nanomachine is implemented by assembling the bead-attached actin filament with the Ca2+-insensitive gelsolin fragment TL40. The performance of the nanomachine is determined either in the absence or in the presence of 0.1 mM Ca2+ (the concentration required for BTA preparation with gelsolin). The nanomachine exhibits a maximum power output of 5.4 aW, independently of [Ca2+], opening the possibility for future studies of the Ca2+-dependent function/dysfunction of regulatory and cytoskeletal proteins.

α-catenin is a crucial protein at cell junctions that provides connection between the actin cytoskeleton and the cell membrane. At adherens junctions (AJs), α-catenin forms heterodimers with β-catenin that are believed to resist force on F-actin. Outside AJs, α-catenin forms homodimers that directly connect the cell membrane to the actin cytoskeleton, but their mechanosensitive properties are inherently unknown.Surprisingly, by using ultra-fast laser tweezers we found that a single α-β-catenin heterodimer does not resist force but instead slips along F-actin in the direction of force. Conversely, the action of 5 to 10 α-β-catenin heterodimers together with force applied toward F-actin pointed end engaged a molecular switch in α-catenin, which unfolded and strongly bound F-actin as a cooperative catch bond. Similarly, an α-catenin homodimer formed an asymmetric catch bond with F-actin triggered by protein unfolding under force. Our data suggest that α-catenin clustering together with intracellular tension engage a fluid-to-solid phase transition at the membrane-cytoskeleton interface.

In a contracting muscle, myosin cross-bridges extending from thick filaments pull the interdigitating thin (actin-containing) filaments during cyclical ATP-driven interactions toward the center of the sarcomere, the structural unit of striated muscle. Cross-bridge attachments in the sarcomere have been reported to exhibit a similar stiffness under both positive and negative forces. However, in vitro measurements on filaments with a sparse complement of heads detected a decrease of the cross-bridge stiffness at negative forces attributed to the buckling of the subfragment 2 tail portion. Here, we review some old and new data that confirm that cross-bridge stiffness is nearly linear in the muscle filament lattice. The implications of high myosin stiffness at positive and negative strains are considered in muscle fibers and in nonmuscle intracellular cargo transport.

Key points Fast sarcomere-level mechanics in intact trabeculae, which allows the definition of the mechano-kinetic properties of cardiac myosin in situ, is a fundamental tool not only for understanding the molecular mechanisms of heart performance and regulation, but also for investigating the mechanisms of the cardiomyopathy-causing mutations in the myosin and testing small molecules for therapeutic interventions. The approach has been applied to measure the stiffness and force of the myosin motor and the fraction of motors attached during isometric twitches of electrically paced trabeculae under different extracellular Ca2+ concentrations. Although the average force of the cardiac myosin motor (∼6 pN) is similar to that of the fast myosin isoform of skeletal muscle, the stiffness (1.07 pN nm-1 ) is 2- to 3-fold smaller. The increase in the twitch force developed in the presence of larger extracellular Ca2+ concentrations is fully accounted for by a proportional increase in the number of attached motors. Abstract The mechano-kinetic properties of the cardiac myosin were studied in situ, in trabeculae dissected from the right ventricle of the rat heart, by measuring the stiffness of the half-sarcomere both at the twitch force peak (Tp ) of an electrically paced intact trabecula at different extracellular Ca2+ concentrations ([Ca2+ ]o ), and in the same trabecula after skinning and induction of rigor. Taking into account the contribution of filament compliance to half-sarcomere compliance and the lattice geometry, we found that the stiffness of the cardiac myosin motor is 1.07 ± 0.09 pN nm-1 , which is slightly larger than that of the slow myosin isoform of skeletal muscle (0.6-0.8 pN nm-1 ) and 2- to 3-fold smaller than that of the fast skeletal muscle isoform. The increase in Tp from 61 ± 4 kPa to 93 ± 9 kPa, induced by raising [Ca2+ ]o from 1 to 2.5 mm at sarcomere length ∼2.2 μm, is accompanied by an increase of the half-sarcomere stiffness that is explained by an increase of the fraction of actin-attached motors from 0.08 ± 0.01 to 0.12 ± 0.02, proportional to Tp . Consequently, each myosin motor bears an average force of 6.14 ± 0.52 pN independently of Tp and [Ca2+ ]o . The application of fast sarcomere-level mechanics to intact trabeculae to define the mechano-kinetic properties of the cardiac myosin in situ represents a powerful tool for investigating cardiomyopathy-causing mutations in the myosin motor and testing specific therapeutic interventions.

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 are investigated in situ by using fast sarcomere-level mechanical methods in permeabilised fibres (sarcomere length 2.4 μm, temperature 12°C, 4% dextran T-500) from the slow (soleus, containing the MHC-1 isoform) and the 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 accounts for the filament compliance to estimate the stiffness of the myosin motor (e). e is 0.56 ± 0.04 pN nm−1 and 1.70 ± 0.37 pN nm−1 for the slow and fast isoform respectively, while the average strain per attached motor (s0) is similar (≈ 3.3 nm) in both isoforms. Consequently the force per motor (F0 = e ⋅ s0) is three times smaller in the slow isoform than in the fast isoform (1.89 ± 0.43 pN versus 5.35 ± 1.51 pN). The fraction of actin-attached motors responsible for maximum isometric force at saturating Ca2+ (T0,4.5) is 0.47 ± 0.09 in soleus fibres, 70% larger than that in psoas fibres (0.29 ± 0.08), so that F0 in slow fibres is 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. This article is protected by copyright. All rights reserved

Regulation of genomic activity occurs through the manipulation of DNA by competent mechanoenzymes. Force-clamp optical tweezers that allow the structural dynamics of the DNA molecule to be measured were used here to investigate the kinetics of mechanically-driven strand reannealing. When the force on the torsionally unconstrained λ-phage DNA is decreased stepwise from above to below the overstretching transition, reannealing occurs via discrete shortening steps separated by exponentially distributed time intervals. Kinetic analysis reveals a transition barrier 0.58 nm along the reaction coordinate and an average reannealing-step size of ∼750 bp, consistent with the average bp interval separating segments of more than 10 consecutive AT bases. In an AT-rich DNA construct, in which the distance between segments of more than 10 consecutive AT is reduced to ∼210 bps, the reannealing step reduces accordingly without changes in the position of the transition barrier. Thus, the transition barrier for reannealing is determined by the presence of segments of more than 10 consecutive AT bps independent of changes in sequence composition, while the length of the reannealing strand changes according to the distance between poly-AT segments at least 10 bps long.

The function as a collective motor of skeletal muscle myosin II is studied in vitro with a synthetic bio-machine, consisting of an ensemble of myosin motors interacting with a single actin filament attached with the correct polarity to a bead trapped in the focus of a Dual Laser Optical Tweezers (DLOT, Bianco et al. Biophys. J.101:866-874, 2011). The motor ensemble provides the condition for cyclic interactions with the actin filament, allowing the development of steady force and filament sliding. The mechanical outputs of the machine are measured by means of the DLOT, which acts as a force transducer (range 0.5-200 pN and resolution ∼0.3 pN), and a piezoelectric nano-positioner carrying the support for the myosin motors, which acts as a length transducer (range 1-75.000 nm and resolution ∼1 nm). Here is reported the performance of a first version of the machine, consisting of an ensemble of myosin motors purified from frog skeletal muscle randomly adsorbed on the surface of a chemically etched single-mode optical fibre with diameter 4 µm. Isometric and isotonic contractions are reproduced by the motor ensemble in solution with physiological [ATP] (2 mM) and temperature 21 °C. Following a drop in force from the maximum isometric value (F0) to a lower value (F), the actin filament slides at a constant velocity (V) which is larger the smaller the force, as expected from the in vivo force-velocity relation. Up to five F-V points for each interaction can be determined, allowing the definition of the maximum power at F ∼0.3 F0 (V ∼2 µm/s) and demonstrating the unequalled ability of the synthetic machine to define the power of native and engineered myosin II motors from striated muscle. Supported by IIT-SEED, Genova and ECRF, 2015 (Italy).