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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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Displacements and forces produced by single S1 molecules at high needle stiffness (0.1–0.6 pN/nm). (A) Representative recording of the generation of displacements by a single S1 molecule (Upper, 2 kHz bandwidth) and changes in the stiffness (Lower). The stiffness was calculated from the variance of the fluctuations of the needle. The stiffness of the needle was 0.21 pN/nm at 1 µM ATP and 20°C. (B) Needle displacement averaged over all observed events at high needle stiffness (n=274). The rising phases of the displacements were synchronized at the starting position by eye and the data at each sampling point was averaged. Mean needle displacement was 8.1 nm and when corrected to S1 displacement, 9.2 nm. (C) Histogram of displacement duration. The solid line shows a single exponential fitted to the distribution by a least squares fit. Time constant was 110 ms. (D) Histogram of forces. The forces were obtained by multiplying the stiffness of the needle by the individual needle displacements. Mean force at the plateau was 2.0 pN. Forces less than 1 pN were excluded in the histogram and from the averaging process.
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f3-1_1: Displacements and forces produced by single S1 molecules at high needle stiffness (0.1–0.6 pN/nm). (A) Representative recording of the generation of displacements by a single S1 molecule (Upper, 2 kHz bandwidth) and changes in the stiffness (Lower). The stiffness was calculated from the variance of the fluctuations of the needle. The stiffness of the needle was 0.21 pN/nm at 1 µM ATP and 20°C. (B) Needle displacement averaged over all observed events at high needle stiffness (n=274). The rising phases of the displacements were synchronized at the starting position by eye and the data at each sampling point was averaged. Mean needle displacement was 8.1 nm and when corrected to S1 displacement, 9.2 nm. (C) Histogram of displacement duration. The solid line shows a single exponential fitted to the distribution by a least squares fit. Time constant was 110 ms. (D) Histogram of forces. The forces were obtained by multiplying the stiffness of the needle by the individual needle displacements. Mean force at the plateau was 2.0 pN. Forces less than 1 pN were excluded in the histogram and from the averaging process.

Mentions: Figure 3A shows a typical time course of displacements at high needle stiffness. The S1-actin interactions could be clearly identified by an increase in stiffness calculated from the reciprocal of the variance of the fluctuations of the probe43. Thermal fluctuations occurred when S1 dissociated from the actin bundle and their amplitude was dependent on the stiffness of the probe. During S1-actin attachments, the fluctuations decreased to an r.m.s. amplitude of 1.4–2.9 nm which corresponded to a stiffness of 0.5–2 pN/nm. The highest value of stiffness during attachments (∼2 pN/nm) was as large as that of an actomyosin crossbridge in muscle44.


Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro
Displacements and forces produced by single S1 molecules at high needle stiffness (0.1–0.6 pN/nm). (A) Representative recording of the generation of displacements by a single S1 molecule (Upper, 2 kHz bandwidth) and changes in the stiffness (Lower). The stiffness was calculated from the variance of the fluctuations of the needle. The stiffness of the needle was 0.21 pN/nm at 1 µM ATP and 20°C. (B) Needle displacement averaged over all observed events at high needle stiffness (n=274). The rising phases of the displacements were synchronized at the starting position by eye and the data at each sampling point was averaged. Mean needle displacement was 8.1 nm and when corrected to S1 displacement, 9.2 nm. (C) Histogram of displacement duration. The solid line shows a single exponential fitted to the distribution by a least squares fit. Time constant was 110 ms. (D) Histogram of forces. The forces were obtained by multiplying the stiffness of the needle by the individual needle displacements. Mean force at the plateau was 2.0 pN. Forces less than 1 pN were excluded in the histogram and from the averaging process.
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Related In: Results  -  Collection

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f3-1_1: Displacements and forces produced by single S1 molecules at high needle stiffness (0.1–0.6 pN/nm). (A) Representative recording of the generation of displacements by a single S1 molecule (Upper, 2 kHz bandwidth) and changes in the stiffness (Lower). The stiffness was calculated from the variance of the fluctuations of the needle. The stiffness of the needle was 0.21 pN/nm at 1 µM ATP and 20°C. (B) Needle displacement averaged over all observed events at high needle stiffness (n=274). The rising phases of the displacements were synchronized at the starting position by eye and the data at each sampling point was averaged. Mean needle displacement was 8.1 nm and when corrected to S1 displacement, 9.2 nm. (C) Histogram of displacement duration. The solid line shows a single exponential fitted to the distribution by a least squares fit. Time constant was 110 ms. (D) Histogram of forces. The forces were obtained by multiplying the stiffness of the needle by the individual needle displacements. Mean force at the plateau was 2.0 pN. Forces less than 1 pN were excluded in the histogram and from the averaging process.
Mentions: Figure 3A shows a typical time course of displacements at high needle stiffness. The S1-actin interactions could be clearly identified by an increase in stiffness calculated from the reciprocal of the variance of the fluctuations of the probe43. Thermal fluctuations occurred when S1 dissociated from the actin bundle and their amplitude was dependent on the stiffness of the probe. During S1-actin attachments, the fluctuations decreased to an r.m.s. amplitude of 1.4–2.9 nm which corresponded to a stiffness of 0.5–2 pN/nm. The highest value of stiffness during attachments (∼2 pN/nm) was as large as that of an actomyosin crossbridge in muscle44.

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