Ultrafast force-clamp spectroscopy is a single molecule technique based on laser tweezers with sub-millisecond and sub-nanometer resolution. The technique has been successfully applied to investigate the rapid conformational changes that occur when a myosin II motor from skeletal muscle interacts with an actin filament. Here, we share data on the kinetics of such interaction and experimental records collected under different forces [1]. The data can be valuable for researchers interested in the mechanosensitive properties of myosin II, both from an experimental and modeling point of view. The data is related to the research article "ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke" [2].

In vitro motility assay (IVMA) experiments were performed to analyze the movement of actin filaments sliding on a pavement of myosin molecules at different [ATP] and [ADP]. In standard experimental conditions at [ATP] = 2 mM, about 80% of the actin filaments move in unloaded conditions with a constant velocity. However, a fraction of at least 20% static actin filaments is always present. The accepted explanation is the occurrence of damaged "rigor"-like myosin heads that do not undergo the normal ATP-dependent cycling motion. However, in a series of IVMA experiments performed at different [ATP] we observed that the mobility of actin filaments increased with lowering [ATP]. We investigated the influence of [ATP] on the number of mobile actin filaments. IVMA experiments were performed at controlled nucleotide concentrations and the percentage of mobile filaments accurately determined by specific operator-guided software. The value of ΔG ATP involved was determined. Results showed that the number of mobile actin filaments sliding on type 2B heavy meromyosin isoform (2B HMM) increased at very low [ATP] accompanied by less negative ΔG ATP values. Similar results were obtained by increasing [ADP]. Performing experiments at the same [ATP] with different myosin types, we found a higher number of mobile actin filaments on slow type 1 HMM with respect to type 2B HMM while the highest number of mobile actin filaments was found on single-head myosin (S1 fraction). We also found that [ATP] did not influence the percentage of mobile actin filaments sliding on S1. Our results reveal novel aspects of actomyosin interaction.

Muscle wasting is due to an excessive activation of degradative pathways. It occurs in different pathologies such as cancer, AIDS, diabetes but inevitably it occurs in everybody’s life during aging. Sarcopenia is the degenerative loss of skeletal muscle mass and strength associated with aging. The ability to activate compensatory mechanisms in response to environmental stress and physiological changing is an important factor for survival and maintenance of cellular functions. A system that is often activated both in short and prolonged stress conditions is autophagy. Autophagy is required to clear the cell from dysfunctional organelles and altered proteins and is reported to decline during ageing. This reduction might contribute to age-related organ dysfunction and, in general, to ageing. Here we report that specific autophagy inhibition in muscle has a major impact on this tissue that ultimately affect life span of animals. Inhibition of autophagy exacerbates the aging-related features of muscle such as atrophy, mitochondrial dysfunction, oxidative stress and profound weakness. Mitochondrial dysfunction and oxidative stress directly affect acto-myosin interaction and force generation. These results demonstrate that aging-related muscle dysfunction is mainly caused by a failure of the autophagy system. It has been reported that lifestyle adaptations, such as caloric restriction and physical exercise are able to ameliorate several features during ageing process, moreover physical activity has been recently documented to play a fundamental physiological role in the regulation of autophagy in several tissues. Here we address also the role of autophagy during exercise. We reveal that autophagy is critical for the preservation of mitochondrial function during damaging muscle contraction. We establish that basal oxidative stress plays a crucial role in mitochondrial maintenance during normal physical activity. Therefore, autophagy is an adaptive response to exercise that ensures effective mitochondria-quality control during damaging physical activity.

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. Biophys. J., 70:401-408, 1996). Mechanical measurements have been carried out with a simplified version of the machine, in which the motor proteins (HMM from skeletal muscle of frog or rabbit) are randomly adsorbed on the flat tip of an etched optical fiber (diameter 4 μm), the position of which is controlled by a piezoelectric nano-positioner. In ATP-free solution the rupture force of the single actin-HMM bond (nano-positioner control) is 12.85 ± 0.35 pN. The bond lifetime under a load of 8 pN (force control) has a bi-exponential distribution and the time constant of the major, faster, component is 1 s. If the motor ensemble in rigor at slack length is exposed to a solution with 2 mM ATP, force develops up to a steady value of 50 pN with a rise time of 2 s, more than one order of magnitude larger than isometric force development in vivo. The difference is mainly accounted for by the compliance of the trap (7 nm/pN), which implies that to develop the steady force the motor ensemble drives 350 nm of actin filament sliding. Supported by Italian Institute of Technology-SEED, project MYOMAC (Genova).

The cellular basis of age-related tissue deterioration remains largely obscure. The ability to activate compensatory mechanisms in response to environmental stress is an important factor for survival and maintenance of cellular functions. Autophagy is activated both under short and prolonged stress and is required to clear the cell of dysfunctional organelles and altered proteins. We report that specific autophagy inhibition in muscle has a major impact on neuromuscular synaptic function and, consequently, on muscle strength, ultimately affecting the lifespan of animals. Inhibition of autophagy also exacerbates aging phenotypes in muscle, such as mitochondrial dysfunction, oxidative stress, and profound weakness. Mitochondrial dysfunction and oxidative stress directly affect acto-myosin interaction and force generation but show a limited effect on stability of neuromuscular synapses. These results demonstrate that age-related deterioration of synaptic structure and function is exacerbated by defective autophagy.

The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM(hVLC-1)) or E56G-mutated hVLC-1 (hVLC-1(E56G); TgM(E56G)). hVLC-1 or hVLC-1(E56G) expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM(hVLC-1) (1.67 pN/nm and 2.3 mu m/s, respectively) were significantly higher than myosin with hVLC-1(E56G) prepared from TgM(E56G) (1.25 pN/nm and 1.7 mu m/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5 mu m/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM(hVLC-1) (80.0 mmHg) were significantly higher than hearts from TgM(E56G) (66.2 mmHg) or C57/BL6 (59.3 +/- 3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1 > hVLC-1(E56G) approximate to mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations. (C) 2014 Elsevier Inc. All rights reserved.

Force plays a fundamental role in a wide array of biological processes, regulating, for example, enzymatic activity, kinetics of molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular complexes. Here, we present a novel force-clamp laser trap that is capable of applying constant loads to molecular complexes with lifetimes above ∼10 μs [Capitanio et al., Nature Methods 9, 1013-1019 (2012)]. Such capability is enhanced by a detection strategy allowing the investigation of very short interactions (∼100μs). Moreover, the high temporal and spatial resolution of the method enables us to probe sub-nanometer conformational changes with a time resolution of few tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2–1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. These results resolve the molecular mechanism underlying regulation of muscle contraction by force. Our technique could be straightforwardly applied to a wide variety of non-processive molecular motors, single domains of processive motors, protein-DNA and protein-RNA interactions, and conceivably to any short-lived protein-protein interaction, opening new avenues for investigating the effects of forces on biological processes.

An in vitro motility assay approach was used to investigate the mechanisms of the functional differences between myosin isoforms, by studying the effect of MgATP and MgADP on actin sliding velocity (Vf) of pure slow and fast rat skeletal myosin at different temperatures. The value of Vf depended on [MgATP] according to Michaelis–Menten kinetics, with an apparent constant (Km) of 54.2, 64.4 and 200 μm for the fast isoform and 18.6, 36.5 and 45.5 μm for the slow isoform at 20, 25 and 35°C, respectively. The presence of 2 mm MgADP decreased Vf and yielded an inhibition constant (Ki) of 377, 463 and 533 μm for the fast isoform at 20, 25 and 35°C, respectively, and 120 and 355 μm for the slow isoform at 25 and 35°C, respectively. The analysis of Km and Ki suggested that slow and fast isoforms differ in the kinetics limiting Vf. Moreover, the higher sensitivity of the fast myosin isoform to a drop in [MgATP] is consistent with the higher fatigability of fast fibres than slow fibres. From the Michaelis–Menten relation in the absence of MgADP, we calculated the rate of actomyosin dissociation by MgATP (k+ATP) and the rate of MgADP release (k‐ADP). We found values of k+ATP of 4.8 × 106, 6.5 × 106 and 6.6 × 106m−1 s−1 for the fast isoform and 3.3 × 106, 2.9 × 106 and 6.7 × 106m−1 s−1 for the slow isoform and values of k‐ADP of 263, 420 and 1320 s−1 for the fast isoform and 62, 107 and 306 s−1 for the slow isoform at 20, 25 and 35°C, respectively. The results suggest that k‐ADP could be the major determinant of functional differences between the fast and slow myosin isoforms at physiological temperatures.

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ∼10 μs, allows detection of interactions as brief as ∼100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2-1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.

It has been suggested that the rate of ADP release from acto-myosin and the rate of acto-myosin dissociation by ATP may play different roles to define unloaded shortening velocity of slow and fast myosins and tha their role might change with temperature (Nytray et al. 2006; Iorga et al. 2007). In this study, the in vitro motility assay (IVMA) approach was used to study the effect of MgATP and MgADP on actin sliding velocity (Vf) on slow and fast skeletal myosin isoforms at different temperatures. The velocity of actin filaments sliding on pure slow (myosin 1) and pure fast (myosin 2B) myosin isoforms from the rat was determined in a range of [MgATP] (0.01-2mM) and in the presence or absence of 2mM MgADP. Experiments were performed at 20, 25 and 35 °C. The rate constants of ADP release and ATP binding to acto-myosin were calculated. The inhibition of Vf by MgADP was greater in slow then fast isoforms and the rate constant of ADP release was higher in fast than slow isoform. The results suggest that, in slow isoforms, the maximum velocity could be limited by the rate of ADP dissociation from actomyosin. The ADP inhibitory effect decreased and the rate constant of ADP release increased in each isoform with temperature accounting for the increase in Vf. No differences were found between isoforms and among temperatures in the rate constant of ATP binding. The possibility that the rate of acto-myosin dissociation induced by ATP could play a role in defining Vf in fast isoforms will be discussed.

Table of Contents In this study we used an "in vitro motility assay" (IVMA) approach to investigate the effect of the variation of [MgATP] in determining the number and the sliding velocity (Vf) of actin filaments moved by fast skeletal myosin. Vf was studied on type 2B HMM from rats at 25C°, 100mM ionic strength and at varius [MgATP], [MgADP] and [Pi]. We designed a new experimental set-up to perform experiments at lower ionic strength and in buffers that had no interference with the ionic environment, in order to mimic physiological condition. This set-up allowed a complete and chemical speciation of the solutions opening the possibility to perform an accurate thermodynamic study. Therefore, along with kinetic measurements also quantitative thermodynamics measurements were carried obtaining the ∈″G of MgATP hydrolysistaking into account pH and [Mg2+]. We correlate the thermodynamics property of the system to Vf and to the number of sliding actin filaments which were assessed by a purpose-designed software. Preliminary results indicate: a) no straightly correlation between values of ∈″GATPand the velocity of actin filaments, b) an increase in thenumberof sliding actin filaments at low [MgATP] and no changes when the ratio [MgATP] / [HMM] was kept constant c) a decrease in the velocity of actin filaments at [Pi]=30mM. More investigations are required to confirm the unexpected results that indicate a complex role of MgATP and its metabolites in the modulation of actomyosin interaction.

Myosin II is the motor protein that drives muscle contraction through cyclical interactions with an actin filament. The working stroke produced by a single myosin head has been previously measured in isolated myosin molecules, but the effects of the high loads acting on the myosin molecule during muscle contraction could not be investigated. In fact, current single molecule techniques apply force with a delay of few milliseconds after actin-myosin binding, when the working stroke of skeletal muscle myosin has already been completed. Here, we developed a novel single molecule technique in which the delay between myosin binding and force application is abolished. This method is capable of resolving the development of the myosin working stroke under different loads with a very high time resolution and detecting events as short as 100 μs. We found that under loads in the range 1 to 10 pN myosin can follow two distinct pathways in its interaction with actin. In the first pathway myosin detaches from actin before producing any movement (weak binding state); these events are very fast (240 ± 23 μs), their duration does not depend on ATP concentration, and is not significantly affected by force. In the second pathway myosin steps and remains bound to actin for a longer time (strong binding state). At low forces (|F| < 2 pN) the lifetime of this second population of events linearly decreases with ATP concentration in the range 5-50 μM. At higher forces this relation becomes non-linear due to premature unbinding of myosin from actin. The working stroke is produced in two steps and its mean amplitude is found to be smaller at increasing loads and vanishes at the isometric force (5.7 ± 0.6 pN).

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by skeletal muscle wasting and atrophy. Recent evidence suggests that the impaired skeletal muscle performance in DMID is not solely dependent on a loss of contractile muscle mass. In this study the myosin motor function of mdx and control (wildtype, WT) mice was compared using pure myosin isoforms in an "in vitro motility assay" (IVMA). Actin sliding velocity (Vf) on myosin 2B extracted from single muscle fibers of gastrocnemius muscles was significantly lower in mdx mice (3.48 +/- 0.13 mu m/s, n = 18) than in WT mice (4.02 +/- 0.19 mu m/s, n = 10). No difference in Vf was found between myosin 1 extracted from soleus muscles of mdx (0.84 +/- 0.04 mu m/s, n = 13) and of WT (0.89 +/- 0.04 mu m/s, n = 10). The results suggest that the dystrophic process alters myosin molecular function, and this contributes to the functional impairment in dystrophic muscles. Muscle Nerve 40: 249-256, 2009