Luca Melli

The working stroke of the myosin II motor in muscle is not tightly coupled to release of orthophosphate from its active site.

•  Force and shortening in muscle are caused by ATP‐driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half‐sarcomere. Crystallographic studies indicate that the working stroke consists in an interdomain movement of the myosin motor associated with the release of inorganic phosphate (Pi). •  Here the coupling of the working stroke with the release of Pi is studied in situ using fast half‐sarcomere mechanics on skinned fibres from rabbit psoas. •  The isotonic velocity transient following stepwise force reductions superimposed on isometric contraction measures the mechanical manifestation of the working stroke and its rate of regeneration. •  The results indicate that the release of Pi from the catalytic site of an actin‐attached myosin motor can occur at any stage of the working stroke, and a myosin motor uses two consecutive actin monomers to maximize the power during shortening.

The contributions of filaments and cross-bridges to sarcomere compliance in skeletal muscle

Muscle contraction is driven at the molecular level by a structural working stroke in the head domain of the myosin cross‐bridge linking the thick and thin filaments. Crystallographic models suggest that the working stroke corresponds to a relative movement of 11 nm between the attachments of the head domain to the thin and thick filaments. The molecular mechanism of force generation depends on the relationship between cross‐bridge force and movement, which is determined by cross‐bridge and filament compliances. Here we measured the compliance of the cross‐bridges and of the thin and thick filaments by combining mechanical and X‐ray diffraction experiments. The results show that cross‐bridge compliance is relatively low and fully accounted for by the elasticity of the myosin head, suggesting that the myosin cross‐bridge generates isometric force by a small sub‐step of the 11 nm stroke that drives filament sliding at low load.

An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

•  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.

PicoNewton-Millisecond Force Steps Reveal the Transition Kinetics and Mechanism of the Double-Stranded DNA Elongation

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 k(B)T, accounting for the stability of the basic conformation of DNA, and explains why ds-DNA can remain in equilibrium as it overstretches.

Effects of myosin heavy chain (MHC) plasticity induced by HMGCoA-reductase inhibition on skeletal muscle functions

The rate‐limiting step of cholesterol biosynthetic pathway is catalyzed by 3‐hydroxy‐3‐methylglu‐taryl 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. FASEB J. 25, 4037–4047 (2011). www.fasebj.org

Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics

Under a tension of ∼65 pN, double-stranded DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the λ-phage DNA with different melting degrees and temperatures (10–25°C). The shortening transient following a 20–35 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses owing to reannealing of melted segments. The lengthening transients following a 2–35 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is one-third of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the interstrand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition.

Force and number of myosin motors during muscle shortening and the coupling with the release of the ATP hydrolysis products

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.

A myosin II nanomachine mimicking the striated muscle

The contraction of striated muscle (skeletal and cardiac muscle) is generated by ATP-dependent interactions between the molecular motor myosin II and the actin filament. The myosin motors are mechanically coupled along the thick filament in a geometry not achievable by single-molecule experiments. Here we show that a synthetic one-dimensional nanomachine, comprising fewer than ten myosin II dimers purified from rabbit psoas, performs isometric and isotonic contractions at 2 mM ATP, delivering a maximum power of 5 aW. The results are explained with a kinetic model fitted to the performance of mammalian skeletal muscle, showing that the condition for the motor coordination that maximises the efficiency in striated muscle is a minimum of 32 myosin heads sharing a common mechanical ground. The nanomachine offers a powerful tool for investigating muscle contractile-protein physiology, pathology and pharmacology without the potentially disturbing effects of the cytoskeletal—and regulatory—protein environment.There is interest in mimicking striated muscle for a range of applications including nanomachines. Here, the authors report on synthetic 1D nanomachines which are used to study an ensemble of myosin motors interacting with an actin filament with potential to create assays of muscle related diseases

Fast Force Clamp in Optical Tweezers: A Tool to Study the Kinetics of Molecular Reactions

A dual-laser optical tweezers has been developed to study the mechanics of motor proteins or DNA filaments. A bead attached to one end of the specimen is trapped in the confocal point of the two lasers, while the other end is connected to a three-dimensional piezo-stage. The instrument can be operated under computer control either as a length clamp, applying length steps or ramps, or as a force clamp, applying abrupt changes in load of fixed magnitude and direction. The dynamic range of the instrument (0.5–75,000 nm in length and 0.5–200 pN in force) and the speed of the force feedback permit recording the kinetics of molecular and intermolecular phenomena such as the overstretching transition in double-stranded DNA (ds-DNA) or the generation of force and shortening by an ensemble of myosin motors pulling on an actin filament. We demonstrate the performance of the system in recording for the first time the transient kinetics of the ds-DNA overstretching transition, which allows the determination of the underlying reaction parameters, such as rate constants and distance to the transitions state.

Half-Sarcomere Mechanics and Energetics Indicate that Myosin Motors Slip Between Two Consecutive Actin Monomers during their Working Stroke

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).