Single myosin mechanics: PicoNewton forces and nanometer steps

Department of Biochemistry, Beckman Center, Stanford University School of Medicine, California 94305.
Nature (Impact Factor: 41.46). 04/1994; 368(6467):113-9. DOI: 10.1038/368113a0
Source: PubMed


A new in vitro assay using a feedback enhanced laser trap system allows direct measurement of force and displacement that results from the interaction of a single myosin molecule with a single suspended actin filament. Discrete stepwise movements averaging 11 nm were seen under conditions of low load, and single force transients averaging 3-4 pN were measured under isometric conditions. The magnitudes of the single forces and displacements are consistent with predictions of the conventional swinging-crossbridge model of muscle contraction.

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    • "experiments presented here, the stretch force was applied using the three-bead optical trap assay (Finer et al. 1994) operating in the force-clamp mode (Takagi et al. 2006). "
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    ABSTRACT: During eccentric contraction, muscle is lengthening so that the actin-myosin cross-bridges bear a load that exceeds the force they generate during isometric contraction. Using the optical trap technique, we simulated eccentric contraction at the single molecule level and investigated the effect of load on the skeletal actomyosin lifetime at different ATP concentrations. The range of the loads was up to 17 pN above the isometric level. We found that the frequency distribution of the lifetime of the actin-bound state of the myosin molecule was biphasic: it quickly rose and then decreased slowly. The rate of the slow phase of this distribution increased with both the load and the ATP concentration. The fast phase accelerated sharply with the load, but it was independent of ATP concentration. The presence of the fast phase demonstrates that some transition(s) in the actomyosin complex occur before the myosin head becomes able to bind ATP and detach from actin. Its high sensitivity to the load indicates that the transition is load-dependent.
    Biophysics of Structure and Mechanism 06/2015; 44(6). DOI:10.1007/s00249-015-1048-3 · 2.22 Impact Factor
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    • "Optical tweezers have been used to probe the cytoskeleton, study cell motility and have enabled the targeted delivery of nanoparticles into a specified region of the interior of an individual living cell (so-called optical injection) [5]. Optical tweezers were used to resolve the step-like motions of motor proteins such as kinesin [6] and myosin [7] and for demonstrating the ability of such motors to advance for hundreds of steps [8]. However, the broader development of optical-tweezer-based techniques and related advances into the smaller research budgets and day-to-day applications are hindered by their cost that is largely dictated by use of expensive and cumbersome vibronic lasers in typical setups. "
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    ABSTRACT: Many applications of high-power laser diodes demand tight focusing. This is often not possible due to the multimode nature of semiconductor laser radiation possessing beam propagation parameter M2 values in double-digits. We propose a method of ‘interference’ superfocusing of high-M2 diode laser beams with a technique developed for the generation of Bessel beams based on the employment of an axicon fabricated on the tip of a 100 μm diameter optical fiber with high-precision direct laser writing. Using axicons with apex angle 1400 and rounded tip area as small as ~10 μm diameter, we demonstrate 2-4 μm diameter focused laser ‘needle’ beams with approximately 20 μm propagation length generated from multimode diode laser with beam propagation parameter M2=18 and emission wavelength of 960 nm. This is a few-fold reduction compared to the minimal focal spot size of ~11 μm that could be achieved if focused by an ‘ideal’ lens of unity numerical aperture. The same technique using a 1600 axicon allowed us to demonstrate few-μm-wide laser ‘needle’ beams with nearly 100 μm propagation length with which to demonstrate optical trapping of 5-6 μm rat blood red cells in a water-heparin solution. Our results indicate the good potential of superfocused diode laser beams for applications relating to optical trapping and manipulation of microscopic objects including living biological objects with aspirations towards subsequent novel lab-on-chip configurations
    Proceedings of SPIE - The International Society for Optical Engineering 03/2015; 9343:93430Q. DOI:10.1117/12.2078957 · 0.20 Impact Factor
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    • "The two parameters  r and stiff,2 k are not independently identifiable from the analyzed data. Estimated values of  r range from 4 to 20 nm [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46]. Here we fix XB stroke size at 10 nm, as there is some consensus regarding this value [40] [41] [42] [43] [44] [45] [46]. "
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    ABSTRACT: Despite extensive study over the past six decades the coupling of chemical reaction and mechanical processes in muscle dynamics is not well understood. We lack a theoretical description of how chemical processes (metabolite binding, ATP hydrolysis) influence and are influenced by mechanical processes (deformation and force generation). To address this need, a mathematical model of the muscle cross-bridge (XB) cycle based on Huxley’s sliding filament theory is developed that explicitly accounts for the chemical transformation events and the influence of strain on state transitions. The model is identified based on elastic and viscous moduli data from mouse and rat myocardial strips over a range of perturbation frequencies, and MgATP and inorganic phosphate (Pi) concentrations. Simulations of the identified model reproduce the observed effects of MgATP and MgADP on the rate of force development. Furthermore, simulations reveal that the rate of force re-development measured in slack-restretch experiments is not directly proportional to the rate of XB cycling. For these experiments, the model predicts that the observed increase in the rate of force generation with increased Pi concentration is due to inhibition of cycle turnover by Pi. Finally, the model captures the observed phenomena of force yielding suggesting that it is a result of rapid detachment of stretched attached myosin heads.
    Journal of Molecular and Cellular Cardiology 02/2015; DOI:10.1016/j.yjmcc.2015.02.006 · 4.66 Impact Factor
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