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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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Load dependent properties of the 5.5-nm steps. (A) The definition of the direction of the forces. When the S1 was pulled by the needle against its proceeding direction, determined by the direction of major displacement as shown in Fig. 4, the force was defined as positive. (B) Force-velocity curve of individual S1 molecules. The velocity was obtained by dividing the step size, 5.5 nm, by the dwell time (Filled circles). Bars indicate the standard deviations for 10–30 steps. Open circles indicate the velocity corrected by the anisotropy of the stepping direction. The solid line shows the Hill’s curve fitted to the corrected velocity. (C) Load dependence of the stepping anisotropy. (D) Histogram of work done by each 5.5-nm step at low (white bars) and at high needle stiffness (gray bars). Average work done per 5.5-nm step at higher needle stiffness was 1.8 kBT.
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f7-1_1: Load dependent properties of the 5.5-nm steps. (A) The definition of the direction of the forces. When the S1 was pulled by the needle against its proceeding direction, determined by the direction of major displacement as shown in Fig. 4, the force was defined as positive. (B) Force-velocity curve of individual S1 molecules. The velocity was obtained by dividing the step size, 5.5 nm, by the dwell time (Filled circles). Bars indicate the standard deviations for 10–30 steps. Open circles indicate the velocity corrected by the anisotropy of the stepping direction. The solid line shows the Hill’s curve fitted to the corrected velocity. (C) Load dependence of the stepping anisotropy. (D) Histogram of work done by each 5.5-nm step at low (white bars) and at high needle stiffness (gray bars). Average work done per 5.5-nm step at higher needle stiffness was 1.8 kBT.

Mentions: Though high needle stiffness leads to higher forces, the data at high needle stiffness included the dwell times over all force levels. To quantitatively compare the mechanical properties of individual actomyosin motors with those of muscle, their force-velocity curve was investigated. As mentioned above, displacements started at various force levels due to thermal fluctuations of a needle and thus steps also took place at various force levels. Force levels were measured at the dwell times of the steps and the direction of the force was defined as depicted in Figure 7A. The equilibrium position of the needle was taken as the zero level of force, and the force was defined as positive when the S1 pulled the needle. When S1 was pushed by the needle, the force was defined as negative. The velocity was obtained by dividing the step size (∼5.5 nm) by the dwell time, and the forces were obtained from the recorded force levels.


Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro
Load dependent properties of the 5.5-nm steps. (A) The definition of the direction of the forces. When the S1 was pulled by the needle against its proceeding direction, determined by the direction of major displacement as shown in Fig. 4, the force was defined as positive. (B) Force-velocity curve of individual S1 molecules. The velocity was obtained by dividing the step size, 5.5 nm, by the dwell time (Filled circles). Bars indicate the standard deviations for 10–30 steps. Open circles indicate the velocity corrected by the anisotropy of the stepping direction. The solid line shows the Hill’s curve fitted to the corrected velocity. (C) Load dependence of the stepping anisotropy. (D) Histogram of work done by each 5.5-nm step at low (white bars) and at high needle stiffness (gray bars). Average work done per 5.5-nm step at higher needle stiffness was 1.8 kBT.
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Related In: Results  -  Collection

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f7-1_1: Load dependent properties of the 5.5-nm steps. (A) The definition of the direction of the forces. When the S1 was pulled by the needle against its proceeding direction, determined by the direction of major displacement as shown in Fig. 4, the force was defined as positive. (B) Force-velocity curve of individual S1 molecules. The velocity was obtained by dividing the step size, 5.5 nm, by the dwell time (Filled circles). Bars indicate the standard deviations for 10–30 steps. Open circles indicate the velocity corrected by the anisotropy of the stepping direction. The solid line shows the Hill’s curve fitted to the corrected velocity. (C) Load dependence of the stepping anisotropy. (D) Histogram of work done by each 5.5-nm step at low (white bars) and at high needle stiffness (gray bars). Average work done per 5.5-nm step at higher needle stiffness was 1.8 kBT.
Mentions: Though high needle stiffness leads to higher forces, the data at high needle stiffness included the dwell times over all force levels. To quantitatively compare the mechanical properties of individual actomyosin motors with those of muscle, their force-velocity curve was investigated. As mentioned above, displacements started at various force levels due to thermal fluctuations of a needle and thus steps also took place at various force levels. Force levels were measured at the dwell times of the steps and the direction of the force was defined as depicted in Figure 7A. The equilibrium position of the needle was taken as the zero level of force, and the force was defined as positive when the S1 pulled the needle. When S1 was pushed by the needle, the force was defined as negative. The velocity was obtained by dividing the step size (∼5.5 nm) by the dwell time, and the forces were obtained from the recorded force levels.

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