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Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro

View Article: PubMed Central - PubMed

ABSTRACT

We have previously measured the process of displacement generation by a single head of muscle myosin (S1) using scanning probe nanometry. Given that the myosin head was rigidly attached to a fairly large scanning probe, it was assumed to stably interact with an underlying actin filament without diffusing away as would be the case in muscle. The myosin head has been shown to step back and forth stochastically along an actin filament with actin monomer repeats of 5.5 nm and to produce a net movement in the forward direction. The myosin head underwent 5 forward steps to produce a maximum displacement of 30 nm per ATP at low load (<1 pN). Here, we measured the steps over a wide range of forces up to 4 pN. The size of the steps (∼5.5 nm) did not change as the load increased whereas the number of steps per displacement and the stepping rate both decreased. The rate of the 5.5-nm steps at various force levels produced a force-velocity curve of individual actomyosin motors. The force-velocity curve from the individual myosin heads was comparable to that reported in muscle, suggesting that the fundamental mechanical properties in muscle are basically due to the intrinsic stochastic nature of individual actomyosin motors. In order to explain multiple stochastic steps, we propose a model arguing that the thermally-driven step of a myosin head is biased in the forward direction by a potential slope along the actin helical pitch resulting from steric compatibility between the binding sites of actin and a myosin head. Furthermore, computer simulations show that multiple cooperating heads undergoing stochastic steps generate a long (>60 nm) sliding distance per ATP between actin and myosin filaments, i.e., the movement is loosely coupled to the ATPase cycle as observed in muscle.

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Related in: MedlinePlus

Imaging of single Cy3-labeled S1 (Cy3-BDTC-S1) molecules captured on the tip of the scanning probe under TIRFM. (A) Fluorescence image of a Cy3-BDTC-S1 molecule captured on the tip of the probe (arrow). The S1 molecules were clearly observed as fluorescent spots. Bar=5 µm. (B and C) Typical time trajectories of the fluorescence intensity of a single (B) and double (C) Cy3-BDTC-S1 molecule captured onto the tip of the probe. Arrows indicate photo-bleaching. (D and E) Distributions of fluorescence intensities from Cy3-BDTC-S1 captured onto the tip of the probe (D) and adhering to the glass surface (E) were well fitted with Gaussian distributions centered at 230±88 and 290±76 (mean±s.d.), respectively.
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f2-1_1: Imaging of single Cy3-labeled S1 (Cy3-BDTC-S1) molecules captured on the tip of the scanning probe under TIRFM. (A) Fluorescence image of a Cy3-BDTC-S1 molecule captured on the tip of the probe (arrow). The S1 molecules were clearly observed as fluorescent spots. Bar=5 µm. (B and C) Typical time trajectories of the fluorescence intensity of a single (B) and double (C) Cy3-BDTC-S1 molecule captured onto the tip of the probe. Arrows indicate photo-bleaching. (D and E) Distributions of fluorescence intensities from Cy3-BDTC-S1 captured onto the tip of the probe (D) and adhering to the glass surface (E) were well fitted with Gaussian distributions centered at 230±88 and 290±76 (mean±s.d.), respectively.

Mentions: S1 molecules were labeled at the RLC with a fluorescent dye complex (Cy3-BDTC) in an almost one (0.95) to one molar ratio (see Methods). The number of S1 molecules captured onto the probe tip was determined from the fluorescence intensity and photobleaching behavior. The fluorescence was observed by TIRFM26,37 (Fig. 2).


Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro
Imaging of single Cy3-labeled S1 (Cy3-BDTC-S1) molecules captured on the tip of the scanning probe under TIRFM. (A) Fluorescence image of a Cy3-BDTC-S1 molecule captured on the tip of the probe (arrow). The S1 molecules were clearly observed as fluorescent spots. Bar=5 µm. (B and C) Typical time trajectories of the fluorescence intensity of a single (B) and double (C) Cy3-BDTC-S1 molecule captured onto the tip of the probe. Arrows indicate photo-bleaching. (D and E) Distributions of fluorescence intensities from Cy3-BDTC-S1 captured onto the tip of the probe (D) and adhering to the glass surface (E) were well fitted with Gaussian distributions centered at 230±88 and 290±76 (mean±s.d.), respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036627&req=5

f2-1_1: Imaging of single Cy3-labeled S1 (Cy3-BDTC-S1) molecules captured on the tip of the scanning probe under TIRFM. (A) Fluorescence image of a Cy3-BDTC-S1 molecule captured on the tip of the probe (arrow). The S1 molecules were clearly observed as fluorescent spots. Bar=5 µm. (B and C) Typical time trajectories of the fluorescence intensity of a single (B) and double (C) Cy3-BDTC-S1 molecule captured onto the tip of the probe. Arrows indicate photo-bleaching. (D and E) Distributions of fluorescence intensities from Cy3-BDTC-S1 captured onto the tip of the probe (D) and adhering to the glass surface (E) were well fitted with Gaussian distributions centered at 230±88 and 290±76 (mean±s.d.), respectively.
Mentions: S1 molecules were labeled at the RLC with a fluorescent dye complex (Cy3-BDTC) in an almost one (0.95) to one molar ratio (see Methods). The number of S1 molecules captured onto the probe tip was determined from the fluorescence intensity and photobleaching behavior. The fluorescence was observed by TIRFM26,37 (Fig. 2).

View Article: PubMed Central - PubMed

ABSTRACT

We have previously measured the process of displacement generation by a single head of muscle myosin (S1) using scanning probe nanometry. Given that the myosin head was rigidly attached to a fairly large scanning probe, it was assumed to stably interact with an underlying actin filament without diffusing away as would be the case in muscle. The myosin head has been shown to step back and forth stochastically along an actin filament with actin monomer repeats of 5.5 nm and to produce a net movement in the forward direction. The myosin head underwent 5 forward steps to produce a maximum displacement of 30 nm per ATP at low load (<1 pN). Here, we measured the steps over a wide range of forces up to 4 pN. The size of the steps (∼5.5 nm) did not change as the load increased whereas the number of steps per displacement and the stepping rate both decreased. The rate of the 5.5-nm steps at various force levels produced a force-velocity curve of individual actomyosin motors. The force-velocity curve from the individual myosin heads was comparable to that reported in muscle, suggesting that the fundamental mechanical properties in muscle are basically due to the intrinsic stochastic nature of individual actomyosin motors. In order to explain multiple stochastic steps, we propose a model arguing that the thermally-driven step of a myosin head is biased in the forward direction by a potential slope along the actin helical pitch resulting from steric compatibility between the binding sites of actin and a myosin head. Furthermore, computer simulations show that multiple cooperating heads undergoing stochastic steps generate a long (>60 nm) sliding distance per ATP between actin and myosin filaments, i.e., the movement is loosely coupled to the ATPase cycle as observed in muscle.

No MeSH data available.


Related in: MedlinePlus