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Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin-actin interactions increases with the increase in shortening velocity. We used fast half-sarcomere mechanics on skinned muscle fibres to determine the relation between shortening velocity and the number and strain of myosin motors and the effect of orthophosphate concentration. A model simulation of the myosin-actin reaction explains the results assuming that orthophosphate and then ADP are released with rates that increase as the motor progresses through the working stroke. The ADP release rate further increases by one order of magnitude with the rise of negative strain in the final motor conformation. These results provide the molecular explanation of the relation between the rate of energy liberation and shortening velocity during muscle contraction. The chemo-mechanical cycle of the myosin II--actin reaction in situ has been investigated in Ca(2+)-activated skinned fibres from rabbit psoas, by determining the number and strain (s) of myosin motors interacting during steady shortening at different velocities (V) and the effect of raising inorganic phosphate (Pi) concentration. It was found that in control conditions (no added Pi ), shortening at V ≤ 350 nm s(-1) per half-sarcomere, corresponding to force (T) greater than half the isometric force (T0 ), decreases the number of myosin motors in proportion to the reduction of T, so that s remains practically constant and similar to the T0 value independent of V. At higher V the number of motors decreases less than in proportion to T, so that s progressively decreases. Raising Pi concentration by 10 mM, which reduces T0 and the number of motors by 40-50%, does not influence the dependence on V of number and strain. A model simulation of the myosin-actin reaction in which the structural transitions responsible for the myosin working stroke and the release of the hydrolysis products are orthogonal explains the results assuming that Pi and then ADP are released with rates that increase as the motor progresses through the working stroke. The rate of ADP release from the conformation at the end of the working stroke is also strain-sensitive, further increasing by one order of magnitude within a few nanometres of negative strain. These results provide the molecular explanation of the relation between the rate of energy liberation and the load during muscle contraction.

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Nonmuscle myosin 2 (NM-2) powers cell motility and tissue morphogenesis by assembling into bipolar filaments that interact with actin. Although the enzymatic properties of purified NM-2 motor fragments have been determined, the emergent properties of filament ensembles are unknown. Using single myosin filament in vitro motility assays, we report fundamental differences in filaments formed of different NM-2 motors. Filaments consisting of NM2-B moved processively along actin, while under identical conditions, NM2-A filaments did not. By more closely mimicking the physiological milieu, either by increasing solution viscosity or by copolymerization with NM2-B, NM2-A containing filaments moved processively. Our data demonstrate that both the kinetic and mechanical properties of these two myosins, in addition to the stochiometry of NM-2 subunits, can tune filament mechanical output. We propose altering NM-2 filament composition is a general cellular strategy for tailoring force production of filaments to specific functions such as maintaining tension or remodeling actin.

Force generation in the muscle sarcomere is driven by the head domain of the myosin molecule extending from the thick filament to form cross-bridges with the actin-containing thin filament. Following attachment, a structural working stroke in the head pulls the thin filament towards the centre of the sarcomere, producing, under unloaded conditions, a filament sliding of ∼ 11 nm. The mechanism of force generation by the myosin head depends on the relationship between cross-bridge force and movement, which is determined by compliances of the cross-bridge (C(cb)) and filaments. By measuring the force dependence of the spacing of the high-order myosin- and actin-based X-ray reflections from sartorius muscles of Rana esculenta we find a combined filament compliance (Cf) of 13.1 ± 1.2 nm MPa(-1), close to recent estimates from single fibre mechanics (12.8 ± 0.5 nm MPa(-1)). C(cb) calculated using these estimates is 0.37 ± 0.12 nm pN(-1), a value fully accounted for by the compliance of the myosin head domain, 0.38 ± 0.06 nm pN(-1), obtained from the intensity changes of the 14.5 nm myosin-based X-ray reflection in response to 3 kHz oscillations imposed on single muscle fibres in rigor. Thus, a significant contribution to C(cb) from the myosin tail that joins the head to the thick filament is excluded. The low C(cb) value indicates that the myosin head generates isometric force by a small sub-step of the 11 nm stroke that drives filament sliding at low load. The implications of these results for the mechanism of force generation by myosins have general relevance for cardiac and non-muscle myosins as well as for skeletal muscle.

Double stranded DNA pulled to ∼65 pN undergoes an overstretching transition from the basic conformation (B-form) to a 1.7 times longer conformation that represents a fundamental stage in the structural transitions during DNA recombination, replication and repair. By using a dual laser optical tweezers with a fast force feedback (2 ms rise time), we recorded the length transient following force steps imposed on the λ-phage DNA with different degrees of melting and at different temperatures (10-25°C). The rate-force relations obtained from the lengthening transient under our rapid force clamp following 2-35 pN force pull to the overstretching force shows that the whole 70% extension is a two state reaction from the B-form to an S-form that precedes and is independent of melting. The shortening transient following 20-35 pN force drop from the overstretching force is made by stepwise shortenings and pauses due to S-B transitions and reannealing of the melted segments. The temperature dependence of the lengthening transient shows that the entropic contribution to the reaction at room temperature is only 1/3 of the entropy change expected from thermal melting. The structural parameters that describe the distances from the B and the S state to the transition state, do not depend on temperature. Accordingly the total elongation of a single two-state unit, and therefore q, the index of cooperativity (22bp) of the reaction, do not depend on temperature, suggesting that it arises from structural factors, such as the nucleic acid sequence. Supported by IIT-SEED (Genova) and Ente Cassa di Risparmio di Firenze.

Skeletal muscle shortens faster against a lower load. This force-velocity relationship is the fundamental determinant of muscle performance in vivo and is due to ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half-sarcomere. Crystallographic studies suggest that the working stroke is associated with the release of phosphate (Pi) and consists of 70 deg tilting of a light-chain domain that connects the catalytic domain of the myosin motor to the myosin tail and filament. However, the coupling of the working stroke with Pi release is still an unsolved question. Using nanometre-microsecond mechanics on skinned muscle fibres, we impose stepwise drops in force on an otherwise isometric contraction and record the isotonic velocity transient, to measure the mechanical manifestation of the working stroke of myosin motors and the rate of its regeneration in relation to the half-sarcomere load and [Pi]. We show that the rate constant of the working stroke is unaffected by [Pi], while the subsequent transition to steady velocity shortening is accelerated. We propose a new chemo-mechanical model that reproduces the transient and steady state responses by assuming that: (i) the release of Pi from the catalytic site of a myosin motor can occur at any stage of the working stroke, and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to the next actin monomer during filament sliding. This model explains the efficient action of muscle molecular motors working as an ensemble in the half-sarcomere.

Key points • Force and shortening in muscle are due to the ATP-powered motor protein myosin II, polymerized in two bipolar arrays of motors that pull the two overlapping actin filaments toward the centre of the sarcomere. • The parameters of the myosin motor in situ have been best characterized for the skeletal muscle of the frog, from which single intact cells can be isolated allowing fast sarcomere level mechanics to be applied. • Up to now no reliable methods have been developed for the study of frog myosin with single molecule techniques. • In this work a new protocol for extraction and conservation of frog muscle myosin allows us to estimate the sliding velocity of actin on myosin (VF) and its modulation by pH, myosin density, temperature and substrate concentration. • By integrating in vitro and in situ parameters of frog muscle myosin we can relate kinetic and mechanical steps of the acto-myosin ATPase.

We study the kinetics of the overstretching transition in λ-phage double-stranded (ds) DNA from the basic conformation (B state) to the 1.7-times longer and partially unwound conformation (S state), using the dual-laser optical tweezers under force-clamp conditions at 25°C. The unprecedented resolution of our piezo servo-system, which can impose millisecond force steps of 0.5–2 pN, reveals the exponential character of the elongation kinetics and allows us to test the two-state nature of the B-S transition mechanism. By analyzing the load-dependence of the rate constant of the elongation, we find that the elementary elongation step is 5.85 nm, indicating a cooperativity of ∼25 basepairs. This mechanism increases the free energy for the elementary reaction to ∼94 kBT, accounting for the stability of the basic conformation of DNA, and explains why ds-DNA can remain in equilibrium as it overstretches.

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).

Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin-actin interactions increases with the increase in shortening velocity. We used fast half-sarcomere mechanics on skinned muscle fibres to determine the relation between shortening velocity and the number and strain of myosin motors and the effect of orthophosphate concentration. A model simulation of the myosin-actin reaction explains the results assuming that orthophosphate and then ADP are released with rates that increase as the motor progresses through the working stroke. The ADP release rate further increases by one order of magnitude with the rise of negative strain in the final motor conformation. These results provide the molecular explanation of the relation between the rate of energy liberation and shortening velocity during muscle contraction. The chemo-mechanical cycle of the myosin II--actin reaction in situ has been investigated in Ca(2+)-activated skinned fibres from rabbit psoas, by determining the number and strain (s) of myosin motors interacting during steady shortening at different velocities (V) and the effect of raising inorganic phosphate (Pi) concentration. It was found that in control conditions (no added Pi ), shortening at V ≤ 350 nm s(-1) per half-sarcomere, corresponding to force (T) greater than half the isometric force (T0 ), decreases the number of myosin motors in proportion to the reduction of T, so that s remains practically constant and similar to the T0 value independent of V. At higher V the number of motors decreases less than in proportion to T, so that s progressively decreases. Raising Pi concentration by 10 mM, which reduces T0 and the number of motors by 40-50%, does not influence the dependence on V of number and strain. A model simulation of the myosin-actin reaction in which the structural transitions responsible for the myosin working stroke and the release of the hydrolysis products are orthogonal explains the results assuming that Pi and then ADP are released with rates that increase as the motor progresses through the working stroke. The rate of ADP release from the conformation at the end of the working stroke is also strain-sensitive, further increasing by one order of magnitude within a few nanometres of negative strain. These results provide the molecular explanation of the relation between the rate of energy liberation and the load during muscle contraction.

During muscle contraction, the myosin motors emerging from the myosin filament in each half-sarcomere form cross-bridges with the opposing actin filament, pulling it towards the center of the sarcomere through a structural working stroke. Motors are mechanically coupled via their filament attachments, and the co-operative action of this coupled system is the basic functional unit of muscle, so determining the elastic properties of the half-sarcomere components is crucial for understanding the mechanism of this collective motor. To that end we used mechanical and X-ray diffraction measurements on single fibers and whole muscles of the frog. 4kHz length oscillations were imposed on single fibers (4°C, 2.15µm sarcomere length) to determine how the half-sarcomere compliance (Chs) is modulated by force. The results indicate the presence of an elastic element in parallel with the array of cross-bridges with a compliance of 200 nm/MPa, ca20 times larger than that attributed to the cross-bridges. X-ray diffraction from whole frog muscles allows precise measurements of the spacing of the high-order myosin- and actin-based reflections, which report changes in the strain of the two filaments. We found that, at forces >0.4 the isometric tetanic force, the filament compliance is constant and contributes 13.1±1.1 nm/MPa to Chs, from which a cross-bridge compliance of 0.35±0.13 nm/pN is calculated. This value is not significantly different from that of the myosin head alone, 0.38±0.06 nm/pN, estimated from changes in the intensity of the 14.5-nm X-ray reflection from the axial repeat of myosin heads during rapid length oscillations in rigor fibers. We conclude that cross-bridge compliance is low and fully accounted for by the compliance of the myosin head, without a significant contribution from the rod connecting the head to the filament backbone. Supported by MIUR-PRIN and FIRB (ITALY).

Skeletal muscle shortens faster against a lower load. This force–velocity relationship is the fundamental determinant of muscle performance in vivo and is due to ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half-sarcomere. Crystallographic studies suggest that the working stroke is associated with the release of phosphate (Pi) and consists of 70 deg tilting of a light-chain domain that connects the catalytic domain of the myosin motor to the myosin tail and filament. However, the coupling of the working stroke with Pi release is still an unsolved question. Using nanometre–microsecond mechanics on skinned muscle fibres, we impose stepwise drops in force on an otherwise isometric contraction and record the isotonic velocity transient, to measure the mechanical manifestation of the working stroke of myosin motors and the rate of its regeneration in relation to the half-sarcomere load and [Pi]. We show that the rate constant of the working stroke is unaffected by [Pi], while the subsequent transition to steady velocity shortening is accelerated. We propose a new chemo-mechanical model that reproduces the transient and steady state responses by assuming that: (i) the release of Pi from the catalytic site of a myosin motor can occur at any stage of the working stroke, and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to the next actin monomer during filament sliding. This model explains the efficient action of muscle molecular motors working as an ensemble in the half-sarcomere.

""The rate-limiting step of cholesterol biosynthetic. pathway is catalyzed by 3-hydroxy-3-methylglutaryl. coenzyme reductase (HGMR), whose inhibitors,. the statins, widely used in clinical practice to treat. hypercholesterolemia, often cause myopathy, and. rarely rhabdomyolysis. All studies to date are limited to. the definition of statin-induced myotoxicity omitting to. investigate whether and how HMGR inhibition influences. muscle functions. To this end, 3-mo-old male rats. (Rattus norvegicus) were treated for 3 wk with a daily. intraperitoneal injection of simvastatin (1.5 mg\\\/kg\\\/d),. and biochemical, morphological, mechanical, and functional. analysis were performed on extensor digitorum. longus (EDL) muscle. Our results show that EDL. muscles from simvastatin-treated rats exhibited reduced. HMGR activity; a 15% shift from the fastest. myosin heavy-chain (MHC) isoform IIb to the slower. IIa\\\/x; and reduced power output and unloaded shortening. velocity, by 41 and 23%, respectively, without any. change in isometric force and endurance. Moreover,. simvastatin-treated rats showed a decrease of maximum. speed reached and the latency to fall off the rotaroad. (30%). These results indicate that the molecular. mechanism of the impaired muscle function following. statin treatment could be related to the plasticity of fast. MHC isoform expression.—Trapani, L., Melli, L., Segatto,. M., Trezza, V., Campolongo, P., Jozwiak, A.,. Swiezewska, E., Pucillo, L.P., Moreno, S., Fanelli, F.,. Linari, M., Pallottini, V. Effects of myosin heavy chain. (MHC) plasticity induced by HMGCoA-reductase inhibition. on skeletal muscle functions.""

We report the progress toward the realization of a synthetic sarcomere-like machine consisting of an array of motor proteins, regularly distributed on an inorganic nano-structured surface, interacting with a single actin filament. The mechanical output of the bio-machine is measured by means of a Dual Laser Optical Tweezers system (DLOT, range 0.5-200 pN force and 1-10,000 nm displacement) under either nano-positioner control or force control (Bianco et al. Biophys. J. 101:866-874, 2011). The correct polarity of the actin filament (5-15 μm long) is controlled by attaching its barbed end to a trapped bead via gelsolin (Suzuki et al.

The coupling between chemical and mechanical steps of actomyosin ATPase cycle was studied in situ by using fast mechanical protocols in Ca2+-activated demembranated fibres from rabbit psoas under sarcomere length control (sarcomere length 2.4 μm, temperature 12°C). We determined the effects of the concentration of inorganic phosphate (Pi) on the force-velocity relation (T-V), on the stiffness-velocity relation (e-V) and on the isotonic velocity transient following a stepwise drop in force from the isometric plateau force (T0) (Piazzesi et al. J Physiol 545:145, 2002). With respect to control (no added Pi), the increase of [Pi] to 10 mM, i) reduced T0 by 50-60%, decreased the curvature of the T-V relation by 30% and increased the unloaded shortening velocity (V0) by 19%; ii) decreased the relative half-sarcomere stiffness at each shortening velocity by an extent that indicates that Pi has little effect on the force per attached myosin motor; iii) did not change the rate of early rapid shortening (phase 2) following the stepwise drop in force, while reduced its size and made the subsequent pause of shortening (phase 3) briefer. Steady state and transient mechanical responses and the known related energetics (Potma and Stienen J Physiol 496:1, 1996) are simulated with a kinetic-mechanical model of the actomyosin ATPase cycle that incorporates Huxley and Simmons mechanism of force generation. Muscle power and efficiency during isotonic shortening at high and intermediate loads can be predicted only if myosin motors at an intermediate stage of both the working stroke and product release can slip to the next Z-ward actin monomer.Supported by MIUR, Ministero della Salute and Ente Cassa di Risparmio di Firenze (Italy).

Rhodium-catalyzed CC coupling reactions, involving ring opening of strained molecules, have been studied using diphenylmethylenecyclopropanes as models. It has been established that, as rhodium takes control of the ring opening process, activated olefins can react to form open-chain unsaturated compounds. In the presence of acids, protonation of the substrate competes with olefin incorporation, and intramolecular aromatic electrophilic substitution, leading to indene derivatives, can be obtained. Aromatic substitution can also occur in a sequential process, involving ring opening, 3-butenoic acid addition, leading to linear and branched isomeric complexes, and selective formation of a benzocycloheptene derivative from the branched isomer. The regioselectivity of 3-butenoic acid insertion is contrasted with the non-regioselectivity observed with the same acid in reactions which probably involve metallacycle formation.

By using a dual laser optical tweezers with a fast force feedback, we were able to record the exponential elongation following force steps imposed on Δ-phage ds-DNA molecule and on two ∼3000 bp segments of the molecule, with either high (59%) or low (46%) CG content. The rate of elongation (r) following a 2pN step imposed on the whole molecule in the region of the overstretching transition varies with the force in a U shaped way, while its corresponding elongation varies in a reverse-U shaped way (Bianco et al., Biophys. J. 101, 866-874, 2011). The minimum of the r - force relation (rmin) and the corresponding maximum elongation (ΔLe) do not change significantly with temperature in the range 25-10°C (mean values 4.9 ± 0.2 s-1 and 3.9 ± 0.2 μm respectively) and are shifted progressively to higher forces at lower temperature. The load- and temperature-dependence of the elongation rate supports the two state (B-S) nature of the transition, yielding a cooperativity of 22 bp and revealing the absence of an enthalpic contribution to the transition free energy barrier.At room temperature the AT rich segment shows large hysteresis on relaxation, implying a predominance of melting on overstretching, while the CG rich segment does not show hysteresis. At temperature below 10°C hysteresis completely disappears for both segments. rmin and ΔLe are 3.9 ±1.4 s-1 and 0.3 ± 0.1 nm for the CG and 4.3 ± 1.3 s-1 and 0.3 ± 0.1 nm for the AT. Fitting the data with the two state reaction model shows a cooperativity of ∼ 15 and 32 for the AT and CG respectively, which correlates with the average distance between groups of more than four consecutive A or T bases.Supported by IIT-SEED (Italy).

Until now the structure, kinetics and energetics of the transition from the basic conformation of ds-DNA (B state) to the 1.7 times longer and partially unwound conformation (S state) have not been defined. The force-extension relation of the ds-DNA of λ-phage is measured here with unprecedented resolution using a dual laser optical tweezers that can impose millisecond steps in force of 0.5-2 pN via beads attached to opposite strands of the molecule (temperature 25 °C). This approach reveals the kinetics of the transition between intermediate states of ds-DNA and uncovers the load-dependence of the rate constant of the unitary reaction step. We show that (1) the elongation (ΔL) following the force step imposed on the molecule in the region of overstretching transition has an exponential time course with a rate constant (r) that depends solely on the force attained by the step (F) ; (2) r is related to the extent of elongation ΔL0 through a power equation that is linearized by double log transformation. The size of the force step does not affect the slope (0.6) of the linearized relation, but shifts its vertical position. These results indicate that r is not related to viscous resistence to elongation-untwisting of the molecule, but only to the kinetics of a two-state reaction and explain the absence of hysteresis in the force-extension relation at equilibrium.Supported by Ente Cassa di Risparmio di Firenze.

We study the kinetics of the overstretching transition in lambda-phage double-stranded (ds) DNA from the basic conformation (B state) to the 1.7-times longer and partially unwound conformation (S state). Using a dual-laser optical tweezers with unprecedented time resolution we have recently demonstrated that millisecond force steps of 0.5-2 pN applied to the dsDNA at 25 °C in the range 62-72 pN (the overstretching transition region) induce elongation responses with exponential time course, suggesting a two-state nature of the B-S transition (Bianco et al., Biophys. J. 101, 866-874, 2011). The load-dependence of the rate constant of the elongation allowed to define the elementary elongation step (∼5.8 nm) and thus the degree of cooperativity (∼25 base pairs). Here we expand the investigation determining the effect of temperature (range 10-25 °C) on the kinetics of the overstretching transition. The U-shaped relation between the rate constant of elongation and the force is progressively shifted to higher forces by the reduction in temperature, so that at 10 °C the force for the minimum rate is 70 pN, 4 pN higher than at 25 °C. Instead, both the minimum value of the rate constant and the degree of cooperativity are temperature independent, suggesting that the transition barrier between the compact and the extended state is basically entropic. All-atoms molecular dynamics simulations support these conclusions and quantitatively reproduce the enthalpic profile determined by fitting the experimental data. According to simulations, overstretched DNA is characterized by a limited amount of residual base pairing and a rather efficient hydrophobic screening of apolar regions.Supported by IIT-SEED and Ente Cassa di Risparmio di Firenze (Italy).