Mario Dolfi

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.

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.

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.

Mechanics of myosin function in white muscle fibres of the dogfish, Scyliorhinus canicula

Key points  •  Muscle force and shortening are generated by a structural change called the working stroke in myosin motor proteins that cross‐link the myosin and actin filaments in muscle. •  Precise values for two key parameters of the myosin motor – its mechanical stiffness and the size of the working stroke at low load – were previously only available from one type of muscle in one species, fast twitch muscles of the frog, so it was not clear how generally applicable these values were. •  We show that in dogfish fast muscle the low‐load working stroke is the same as in frog muscle, but the myosin motor stiffness is smaller. •  The results provide new insights into how the molecular properties of myosin motors in different muscle types and species may be adapted for different muscle functions.

A Kinetic Model that Explains the Transient and Steady State Responses to Mechanical and Chemical Steps Applied to Ca++ Activated Skinned Fibers from Skeletal Muscle

Force and shortening in muscle are generated by ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament. In vitro and in situ studies suggest that the working stroke is associated with the release of phosphate (Pi). We used nanometer-microsecond mechanics on skinned muscle fibers from rabbit psoas (2.4 µm sarcomere length, 12 °C) to record the velocity transient following a force step and found that the early rapid shortening, which represents the mechanical manifestation of the working stroke, is not affected by the increase in [Pi], while the subsequent transition to the steady shortening velocity is accelerated and the steady power at high loads is reduced. A new chemo-mechanical model has been proposed that reproduces the transient and steady state responses by assuming that biochemical and mechanical steps are not tightly coupled: (i) the release of the hydrolysis products (Pi and ADP) from the catalytic site of the 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 a second actin monomer (the next monomer away from the center of the sarcomere) before terminating the biochemical cycle. This model explains the efficient action of muscle molecular motors working as an ensemble. Here we demonstrate the model ability to fit the force transients elicited by jumps in either [ATP] (Goldman et al., Nature 300, 701-705, 1982; Dantzig et al., J. Physiol. 432, 639-680, 1991) or [Pi] (Dantzig et al., J. Physiol. 451, 247-278, 1992; Homsher et al., Biophys. J. 72, 1780-1791, 1997). Supported by MIUR-PRIN and ECRF-2012 (Italy).

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).