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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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Stepwise movements in the rising phase of the displacements. (A) The rising phase of the displacement record plotted on an expanded timescale. (B) Representative traces of stepwise movements in the rising phase at high needle stiffness (2 kHz bandwidth). Some backward steps were observed as indicated by arrows. (C) Stepwise movements in the rising phase at low needle stiffness (0.01–<0.1 pN/nm) from Kitamura et al.29. (D) The rising phase of the displacement that took place so rapidly that the steps were unclear. (E) Histogram of the pairwise distance for all the data points of stepwise movements in the rising phase at high needle stiffness (number of rising phases=80). (F) Power spectrum of the histogram of pairwise distance shown in (E). An obvious peak was observed at 0.18 nm−1 corresponding to the spatial periodicity of 5.6 nm in the histogram. (G) Histogram of the number of steps per displacement. The steps were counted by eye. Steps of ∼5.5×N nm were counted as N steps. White and gray bars indicate the results obtained at low and high needle stiffness, respectively.
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f6-1_1: Stepwise movements in the rising phase of the displacements. (A) The rising phase of the displacement record plotted on an expanded timescale. (B) Representative traces of stepwise movements in the rising phase at high needle stiffness (2 kHz bandwidth). Some backward steps were observed as indicated by arrows. (C) Stepwise movements in the rising phase at low needle stiffness (0.01–<0.1 pN/nm) from Kitamura et al.29. (D) The rising phase of the displacement that took place so rapidly that the steps were unclear. (E) Histogram of the pairwise distance for all the data points of stepwise movements in the rising phase at high needle stiffness (number of rising phases=80). (F) Power spectrum of the histogram of pairwise distance shown in (E). An obvious peak was observed at 0.18 nm−1 corresponding to the spatial periodicity of 5.6 nm in the histogram. (G) Histogram of the number of steps per displacement. The steps were counted by eye. Steps of ∼5.5×N nm were counted as N steps. White and gray bars indicate the results obtained at low and high needle stiffness, respectively.

Mentions: Figures 6A and B show the rising phases of the displacements at high needle stiffness on an expanded time scale. Displacements were not abrupt but took place in a stepwise fashion as observed previously at low needle stiffness (Fig. 6C). Most steps occurred in the forward direction but a small number were also recorded in the backward direction (indicated by arrows in Fig. 6B). Such stepwise motion was observed in the rising phase of approximately 30% of the total number of displacements observed (80 out of 274 displacements in 20 independent experiments for high needle stiffness). The probability such stepwise motion was observed was similar for low needle stiffness (66 out of 190 displacements)29. Clear steps were not observed for all displacements. In one group, the stiffness during the attachment did not increase sufficiently (<0.5 pN/nm. r.m.s. fluctuations of a needle >3 nm), so that the signal to noise ratio was not high enough to resolve the steps (65 displacements). The possibility exists that the actin filament may not have been rigidly fixed to the actin bundle on the glass surface. In another group (129 displacements), the stiffness was high enough to resolve the steps during the plateau after generating displacements. However, the displacements probably took place so rapidly that the steps in a displacement could not be reliably identified (Fig. 6D). Reliable identification of the start positions of displacements required that the myosin head attached to the actin for >3 ms and the stiffness was large29. Since the mean dwell time between the 1st and 2nd steps in displacements was approximately 5 ms, the fraction of displacements with the high-stiffness period of >3 ms before developing displacements was calculated to be <45%. Thus, first steps may have been missed in >50% of displacements, even if the stiffness increased. Furthermore, if the 2nd and 3rd steps took place within 3 ms, we could observe no steps in many displacements. Therefore, it is reasonable that we could observe no steps in many displacements (Supplement Table 1).


Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro
Stepwise movements in the rising phase of the displacements. (A) The rising phase of the displacement record plotted on an expanded timescale. (B) Representative traces of stepwise movements in the rising phase at high needle stiffness (2 kHz bandwidth). Some backward steps were observed as indicated by arrows. (C) Stepwise movements in the rising phase at low needle stiffness (0.01–<0.1 pN/nm) from Kitamura et al.29. (D) The rising phase of the displacement that took place so rapidly that the steps were unclear. (E) Histogram of the pairwise distance for all the data points of stepwise movements in the rising phase at high needle stiffness (number of rising phases=80). (F) Power spectrum of the histogram of pairwise distance shown in (E). An obvious peak was observed at 0.18 nm−1 corresponding to the spatial periodicity of 5.6 nm in the histogram. (G) Histogram of the number of steps per displacement. The steps were counted by eye. Steps of ∼5.5×N nm were counted as N steps. White and gray bars indicate the results obtained at low and high needle stiffness, respectively.
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f6-1_1: Stepwise movements in the rising phase of the displacements. (A) The rising phase of the displacement record plotted on an expanded timescale. (B) Representative traces of stepwise movements in the rising phase at high needle stiffness (2 kHz bandwidth). Some backward steps were observed as indicated by arrows. (C) Stepwise movements in the rising phase at low needle stiffness (0.01–<0.1 pN/nm) from Kitamura et al.29. (D) The rising phase of the displacement that took place so rapidly that the steps were unclear. (E) Histogram of the pairwise distance for all the data points of stepwise movements in the rising phase at high needle stiffness (number of rising phases=80). (F) Power spectrum of the histogram of pairwise distance shown in (E). An obvious peak was observed at 0.18 nm−1 corresponding to the spatial periodicity of 5.6 nm in the histogram. (G) Histogram of the number of steps per displacement. The steps were counted by eye. Steps of ∼5.5×N nm were counted as N steps. White and gray bars indicate the results obtained at low and high needle stiffness, respectively.
Mentions: Figures 6A and B show the rising phases of the displacements at high needle stiffness on an expanded time scale. Displacements were not abrupt but took place in a stepwise fashion as observed previously at low needle stiffness (Fig. 6C). Most steps occurred in the forward direction but a small number were also recorded in the backward direction (indicated by arrows in Fig. 6B). Such stepwise motion was observed in the rising phase of approximately 30% of the total number of displacements observed (80 out of 274 displacements in 20 independent experiments for high needle stiffness). The probability such stepwise motion was observed was similar for low needle stiffness (66 out of 190 displacements)29. Clear steps were not observed for all displacements. In one group, the stiffness during the attachment did not increase sufficiently (<0.5 pN/nm. r.m.s. fluctuations of a needle >3 nm), so that the signal to noise ratio was not high enough to resolve the steps (65 displacements). The possibility exists that the actin filament may not have been rigidly fixed to the actin bundle on the glass surface. In another group (129 displacements), the stiffness was high enough to resolve the steps during the plateau after generating displacements. However, the displacements probably took place so rapidly that the steps in a displacement could not be reliably identified (Fig. 6D). Reliable identification of the start positions of displacements required that the myosin head attached to the actin for >3 ms and the stiffness was large29. Since the mean dwell time between the 1st and 2nd steps in displacements was approximately 5 ms, the fraction of displacements with the high-stiffness period of >3 ms before developing displacements was calculated to be <45%. Thus, first steps may have been missed in >50% of displacements, even if the stiffness increased. Furthermore, if the 2nd and 3rd steps took place within 3 ms, we could observe no steps in many displacements. Therefore, it is reasonable that we could observe no steps in many displacements (Supplement Table 1).

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 (&lt;1 pN). Here, we measured the steps over a wide range of forces up to 4 pN. The size of the steps (&sim;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 (&gt;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