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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.

No MeSH data available.


Related in: MedlinePlus

(A) Schematic drawing of the experimental apparatus. The system was built on an inverted microscope. A green laser (YAG532) was used as the light source for both single molecule imaging (objective-type TIRFM) and nanometry (bright field illumination) by changing the incident angle of the laser by moving the mirror, MM. The fluorescent image of single Cy3-BDTC-S1 molecules was collected by the lower objective (Obj1), magnified by a projection lens (PL), and detected by an intensified-SIT camera (ISIT) under TIR illumination mode. The magnified image of the probe was obtained by the upper objective (Obj2) and a concave lens (L5) under bright field illumination mode. Displacement of the needle was monitored using a split-photodiode (PD). The red laser (He-Ne 633) was used for monitoring the probe position during single molecule manipulation (See text for details). ND, neutral density filters; Exp, beam expanders; λ/4, quarter-wave plates; L1&3, concave lens; L2&4, convex lens; DM1&2, dichroic mirrors; NF, notch filter; and BF, bandpass filter. (B) Imaging and nano-manipulation of single S1 molecules. A single S1 molecule, which had been biotinylated and fluorescently labeled by Cy3 at its regulatory light chain, was specifically attached to the tip of a scanning probe through a biotin-streptavidin bond and observed as a single fluorescent spot. The displacement produced when the S1 molecule was brought into contact with an actin bundle bound to a glass surface in the presence of ATP was determined by measuring the position of the needle with sub-nanometer accuracy. The S1 was rigidly attached to a fairly large scanning probe, so it was assumed to stably interact with an actin filament without diffusing away just like in muscle. (C) The schematics of the measurement geometry. A ZnO whisker crystal, whose length was 5–10 µm and radius of curvature of the tip was ∼15 nm, was attached to the tip of a very fine glass microneedle, 100 µm long and 0.3 µm in diameter. The glass needle was set perpendicular to the longitudinal axis of the actin bundle (Lower). The magnified image of the whisker + needle was projected onto the split-photodiode to measure the nanometer displacement (Upper).
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f1-1_1: (A) Schematic drawing of the experimental apparatus. The system was built on an inverted microscope. A green laser (YAG532) was used as the light source for both single molecule imaging (objective-type TIRFM) and nanometry (bright field illumination) by changing the incident angle of the laser by moving the mirror, MM. The fluorescent image of single Cy3-BDTC-S1 molecules was collected by the lower objective (Obj1), magnified by a projection lens (PL), and detected by an intensified-SIT camera (ISIT) under TIR illumination mode. The magnified image of the probe was obtained by the upper objective (Obj2) and a concave lens (L5) under bright field illumination mode. Displacement of the needle was monitored using a split-photodiode (PD). The red laser (He-Ne 633) was used for monitoring the probe position during single molecule manipulation (See text for details). ND, neutral density filters; Exp, beam expanders; λ/4, quarter-wave plates; L1&3, concave lens; L2&4, convex lens; DM1&2, dichroic mirrors; NF, notch filter; and BF, bandpass filter. (B) Imaging and nano-manipulation of single S1 molecules. A single S1 molecule, which had been biotinylated and fluorescently labeled by Cy3 at its regulatory light chain, was specifically attached to the tip of a scanning probe through a biotin-streptavidin bond and observed as a single fluorescent spot. The displacement produced when the S1 molecule was brought into contact with an actin bundle bound to a glass surface in the presence of ATP was determined by measuring the position of the needle with sub-nanometer accuracy. The S1 was rigidly attached to a fairly large scanning probe, so it was assumed to stably interact with an actin filament without diffusing away just like in muscle. (C) The schematics of the measurement geometry. A ZnO whisker crystal, whose length was 5–10 µm and radius of curvature of the tip was ∼15 nm, was attached to the tip of a very fine glass microneedle, 100 µm long and 0.3 µm in diameter. The glass needle was set perpendicular to the longitudinal axis of the actin bundle (Lower). The magnified image of the whisker + needle was projected onto the split-photodiode to measure the nanometer displacement (Upper).

Mentions: Using these assays, displacements of single myosin molecules have been measured to be 4 to 25 nm per ATP28. In order to examine the underling mechanism of movement, it is essential to investigate the process driving the displacement. However, past experiments have not resolved the rising phase of the displacements due to a poor signal to noise ratio. This is because the displacements were measured by observing the movement of actin, not myosin, and thus the compliance (1/stiffness) of the linkage between the optically trapped beads or microneedle and the actin filament damped the signal to noise ratio. To overcome this problem, we have developed a more direct method of measuring the displacements of the myosin head by using a scanning probe29 (Fig. 1). The series stiffness during acto-S1 interaction significantly increased to >1 pN/nm, compared to that obtained with optical trapping experiments (0.05–0.2 pN/nm)30,31. Resulting thermal fluctuations of the probe, namely, the noise of the measurements, was reduced from 4–9 to <2 nm r.m.s. This improvement was critical to resolve the process generating these ∼10–20 nm displacements. Furthermore, the myosin head rigidly attached to a relatively large scanning probe could steadily interact with actin without diffusing away from the actin filament as it does in muscle (H. E. Huxley, personal communication). Using this improved method, we demonstrated that the displacements did not take place abruptly but instead developed in a stepwise fashion. The size of the steps during the rising phase was 5.3 nm, which coincided with the actin monomer pitch (5.5 nm), and the number of steps per displacement was distributed in the range of one to five. Since each displacement corresponded to one biochemical cycle of ATP turnover, the result shows a step is not tightly coupled to the ATP turnover in a one to one fashion. This one-to-many coupling between the ATP turnover and the mechanical event is in accordance with a loose-coupling mechanism17,32.


Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro
(A) Schematic drawing of the experimental apparatus. The system was built on an inverted microscope. A green laser (YAG532) was used as the light source for both single molecule imaging (objective-type TIRFM) and nanometry (bright field illumination) by changing the incident angle of the laser by moving the mirror, MM. The fluorescent image of single Cy3-BDTC-S1 molecules was collected by the lower objective (Obj1), magnified by a projection lens (PL), and detected by an intensified-SIT camera (ISIT) under TIR illumination mode. The magnified image of the probe was obtained by the upper objective (Obj2) and a concave lens (L5) under bright field illumination mode. Displacement of the needle was monitored using a split-photodiode (PD). The red laser (He-Ne 633) was used for monitoring the probe position during single molecule manipulation (See text for details). ND, neutral density filters; Exp, beam expanders; λ/4, quarter-wave plates; L1&3, concave lens; L2&4, convex lens; DM1&2, dichroic mirrors; NF, notch filter; and BF, bandpass filter. (B) Imaging and nano-manipulation of single S1 molecules. A single S1 molecule, which had been biotinylated and fluorescently labeled by Cy3 at its regulatory light chain, was specifically attached to the tip of a scanning probe through a biotin-streptavidin bond and observed as a single fluorescent spot. The displacement produced when the S1 molecule was brought into contact with an actin bundle bound to a glass surface in the presence of ATP was determined by measuring the position of the needle with sub-nanometer accuracy. The S1 was rigidly attached to a fairly large scanning probe, so it was assumed to stably interact with an actin filament without diffusing away just like in muscle. (C) The schematics of the measurement geometry. A ZnO whisker crystal, whose length was 5–10 µm and radius of curvature of the tip was ∼15 nm, was attached to the tip of a very fine glass microneedle, 100 µm long and 0.3 µm in diameter. The glass needle was set perpendicular to the longitudinal axis of the actin bundle (Lower). The magnified image of the whisker + needle was projected onto the split-photodiode to measure the nanometer displacement (Upper).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036627&req=5

f1-1_1: (A) Schematic drawing of the experimental apparatus. The system was built on an inverted microscope. A green laser (YAG532) was used as the light source for both single molecule imaging (objective-type TIRFM) and nanometry (bright field illumination) by changing the incident angle of the laser by moving the mirror, MM. The fluorescent image of single Cy3-BDTC-S1 molecules was collected by the lower objective (Obj1), magnified by a projection lens (PL), and detected by an intensified-SIT camera (ISIT) under TIR illumination mode. The magnified image of the probe was obtained by the upper objective (Obj2) and a concave lens (L5) under bright field illumination mode. Displacement of the needle was monitored using a split-photodiode (PD). The red laser (He-Ne 633) was used for monitoring the probe position during single molecule manipulation (See text for details). ND, neutral density filters; Exp, beam expanders; λ/4, quarter-wave plates; L1&3, concave lens; L2&4, convex lens; DM1&2, dichroic mirrors; NF, notch filter; and BF, bandpass filter. (B) Imaging and nano-manipulation of single S1 molecules. A single S1 molecule, which had been biotinylated and fluorescently labeled by Cy3 at its regulatory light chain, was specifically attached to the tip of a scanning probe through a biotin-streptavidin bond and observed as a single fluorescent spot. The displacement produced when the S1 molecule was brought into contact with an actin bundle bound to a glass surface in the presence of ATP was determined by measuring the position of the needle with sub-nanometer accuracy. The S1 was rigidly attached to a fairly large scanning probe, so it was assumed to stably interact with an actin filament without diffusing away just like in muscle. (C) The schematics of the measurement geometry. A ZnO whisker crystal, whose length was 5–10 µm and radius of curvature of the tip was ∼15 nm, was attached to the tip of a very fine glass microneedle, 100 µm long and 0.3 µm in diameter. The glass needle was set perpendicular to the longitudinal axis of the actin bundle (Lower). The magnified image of the whisker + needle was projected onto the split-photodiode to measure the nanometer displacement (Upper).
Mentions: Using these assays, displacements of single myosin molecules have been measured to be 4 to 25 nm per ATP28. In order to examine the underling mechanism of movement, it is essential to investigate the process driving the displacement. However, past experiments have not resolved the rising phase of the displacements due to a poor signal to noise ratio. This is because the displacements were measured by observing the movement of actin, not myosin, and thus the compliance (1/stiffness) of the linkage between the optically trapped beads or microneedle and the actin filament damped the signal to noise ratio. To overcome this problem, we have developed a more direct method of measuring the displacements of the myosin head by using a scanning probe29 (Fig. 1). The series stiffness during acto-S1 interaction significantly increased to >1 pN/nm, compared to that obtained with optical trapping experiments (0.05–0.2 pN/nm)30,31. Resulting thermal fluctuations of the probe, namely, the noise of the measurements, was reduced from 4–9 to <2 nm r.m.s. This improvement was critical to resolve the process generating these ∼10–20 nm displacements. Furthermore, the myosin head rigidly attached to a relatively large scanning probe could steadily interact with actin without diffusing away from the actin filament as it does in muscle (H. E. Huxley, personal communication). Using this improved method, we demonstrated that the displacements did not take place abruptly but instead developed in a stepwise fashion. The size of the steps during the rising phase was 5.3 nm, which coincided with the actin monomer pitch (5.5 nm), and the number of steps per displacement was distributed in the range of one to five. Since each displacement corresponded to one biochemical cycle of ATP turnover, the result shows a step is not tightly coupled to the ATP turnover in a one to one fashion. This one-to-many coupling between the ATP turnover and the mechanical event is in accordance with a loose-coupling mechanism17,32.

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