Harmonic force spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules

Sung, Jongmin; Nag, Suman; Mortensen, Kim I.; Vestergaard, Christian; Sutton, Shirley; Ruppel, Kathleen M.; Flyvbjerg, Henrik; Spudich, James A.

doi:10.1038/ncomms8931

Cited by 76 publications

(119 citation statements)

References 51 publications

(104 reference statements)

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“…Forces that resist the powerstroke slow the rate of ADP release at physiological ATP concentrations. The distance to the transition state for actomyosin dissociation is 0.97 nm (36,37), such that 1 pN of load slows the rate of actin detachment from 83 s À1 to 66 s À1 . This force sensitivity is more modest than Myo1b, Myo1c, or smooth muscle myosin-II, which is likely important for cardiac myosin's role in generating sufficient power to counteract cardiac afterload.…”

Section: Myosin-ic (Gene: Myo1c)mentioning

confidence: 99%

A Perspective on the Role of Myosins as Mechanosensors

Greenberg

Arpag

Tüzel

et al. 2016

Biophysical Journal

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Cells are dynamic systems that generate and respond to forces over a range of spatial and temporal scales, spanning from single molecules to tissues. Substantial progress has been made in recent years in identifying the molecules and pathways responsible for sensing and transducing mechanical signals to short-term cellular responses and longer-term changes in gene expression, cell identity, and tissue development. In this perspective article, we focus on myosin motors, as they not only function as the primary force generators in well-studied mechanobiological processes, but also act as key mechanosensors in diverse functions including intracellular transport, signaling, cell migration, muscle contraction, and sensory perception. We discuss how the biochemical and mechanical properties of different myosin isoforms are tuned to fulfill these roles in an array of cellular processes, and we highlight the underappreciated diversity of mechanosensing properties within the myosin superfamily. In particular, we use modeling and simulations to make predictions regarding how diversity in force sensing affects the lifetime of the actomyosin bond, the myosin power output, and the ability of myosin to respond to a perturbation in force for several nonprocessive myosin isoforms.

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Section: Myosin-ic (Gene: Myo1c)mentioning

confidence: 99%

A Perspective on the Role of Myosins as Mechanosensors

Greenberg

Arpag

Tüzel

et al. 2016

Biophysical Journal

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“…The detailed protocol on how to set up the dual-beam optical trap instrument used in this study is described in Sung et al (2010). The instrumentation used in this study is described in detail elsewhere (Sung et al, 2015). …”

Section: Methodsmentioning

confidence: 99%

“…Motors were further purified by a two-step purification procedure through actin sedimentation, the first with rigor attachment of motors to actin filaments without ATP and the second with inactive motors remaining bound to actin at high ATP concentration. Detailed protocols on cloning, virus production, and protein expression and purification are found elsewhere (Sommese et al, 2013; Sung et al, 2015). …”

Section: Sample Preparations: Proteins Reagents and Buffersmentioning

confidence: 99%

“…Moreover, the sampling efficiency is improved with HFS, so we could collect enough data from an individual molecule for a range of forces to get full load-dependent kinetics curves for individual molecules. In Sung et al (2015), we demonstrated the power of HFS by directly measuring the force-dependent ADP release kinetics of human β-cardiac myosin II (Fig. 1), as an example of load-dependent transient molecular interactions.…”

Section: Introductionmentioning

confidence: 97%

“…We recently developed an approach orthogonal to current force-clamp methods, a new method that we refer to as harmonic force spectroscopy (HFS) for reasons given later (Sung et al, 2015). HFS can detect and apply forces to weak and transient molecular interactions.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

How to Measure Load-Dependent Kinetics of Individual Motor Molecules Without a Force-Clamp

Sung

Mortensen

Spudich

et al. 2017

Methods in Enzymology

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Single-molecule force spectroscopy techniques, including optical trapping, magnetic trapping, and atomic force microscopy, have provided unprecedented opportunities to understand biological processes at the smallest biological length scales. For example, they have been used to elucidate the molecular basis of muscle contraction and intracellular cargo transport along cytoskeletal filamentous proteins. Optical trapping is among the most sophisticated single-molecule techniques. With exceptionally high spatial and temporal resolutions, it has been extensively utilized to understand biological functions at the single molecule level, such as conformational changes and force-generation of individual motor proteins or force-dependent kinetics in molecular interactions. Here, we describe a new method, “Harmonic Force Spectroscopy (HFS).” With a conventional dual-beam optical trap and a simple harmonic oscillation of the sample stage, HFS can measure the load-dependent kinetics of transient molecular interactions, such as a human β-cardiac myosin II interacting with an actin filament. We demonstrate that the ADP release rate of an individual human β-cardiac myosin II molecule depends exponentially on the applied load, which provides a clue to understanding the molecular mechanism behind the force-velocity curve of a contracting cardiac muscle. The experimental protocol and the data analysis are simple, fast, and efficient. This chapter provides a practical guide to the method: basic concepts, experimental setup, step-by-step experimental protocol, theory, data analysis, and results.

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Single‐Molecule Investigation of Load‐Dependent Actomyosin Dissociation Kinetics for Cardiac and Slow Skeletal Myosin

Wang,

Nayak,

Kraft

et al. 2024

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Myosins are ATP‐powered, force‐generating motor proteins involved in cardiac and muscle contraction. The external load experienced by the myosins modulates and coordinates their function in vivo. Here, this study investigates the tension‐sensing mechanisms of rabbit native β‐cardiac myosin (βM‐II) and slow skeletal myosins (SolM‐II) that perform in different physiological settings. Using mobile optical tweezers with a square wave‐scanning mode, a range of external assisting and resisting loads from 0 to 15 pN is exerted on single myosin molecules as they interact with the actin filament. Influenced of load on specific strongly‐bound states in the cross‐bridge cycle is examined by adjusting the [ATP]. The results implies that the detachment kinetics of actomyosin ADP.Pi strongly‐bound force‐generating state are load sensitive. Low assisting load accelerates, while the resisting load hinders the actomyosin detachment, presumably, by slowing both the Pi and ADP release. However, under both high assisting and resisting load, the rate of actomyosin dissociation decelerates. The transition from actomyosin ADP.Pi to ADP state appears to occur with a higher probability for βM‐II than SolM‐II. This study interpret that dissociation of at least three strongly‐bound actomyosin states are load‐sensitive and may contribute to functional diversity among different myosins.

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Harmonic force spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules

Cited by 76 publications

References 51 publications

A Perspective on the Role of Myosins as Mechanosensors

A Perspective on the Role of Myosins as Mechanosensors

How to Measure Load-Dependent Kinetics of Individual Motor Molecules Without a Force-Clamp

Single‐Molecule Investigation of Load‐Dependent Actomyosin Dissociation Kinetics for Cardiac and Slow Skeletal Myosin

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