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Myosin V exhibits a high duty cycle and large unitary displacement.

Moore JR, Krementsova EB, Trybus KM, Warshaw DM - J. Cell Biol. (2001)

Bottom Line: The 20-nm unitary step represents the myosin V working stroke and is independent of the mode of M5(HMM) attachment to the motility surface or light chain content.The large M5(HMM) working stroke is consistent with the myosin V neck acting as a mechanical lever.The second step is characterized by an increased displacement variance, suggesting a model for how the two heads of myosin V function in processive motion.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA.

ABSTRACT
Myosin V is a double-headed unconventional myosin that has been implicated in organelle transport. To perform this role, myosin V may have a high duty cycle. To test this hypothesis and understand the properties of this molecule at the molecular level, we used the laser trap and in vitro motility assay to characterize the mechanics of heavy meromyosin-like fragments of myosin V (M5(HMM)) expressed in the Baculovirus system. The relationship between actin filament velocity and the number of interacting M5(HMM) molecules indicates a duty cycle of > or =50%. This high duty cycle would allow actin filament translocation and thus organelle transport by a few M5(HMM) molecules. Single molecule displacement data showed predominantly single step events of 20 nm and an occasional second step to 37 nm. The 20-nm unitary step represents the myosin V working stroke and is independent of the mode of M5(HMM) attachment to the motility surface or light chain content. The large M5(HMM) working stroke is consistent with the myosin V neck acting as a mechanical lever. The second step is characterized by an increased displacement variance, suggesting a model for how the two heads of myosin V function in processive motion.

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Double step events. (A) Representative “double step” event for smooth muscle myosin II taken at higher myosin surface densities where presumably more than one molecule is available to interact with the actin filament. Note that the second step is characterized by a similar duration and lower variance than the first step. (B) Representative “double step” event for myosin V, which gives rise to the intermediate variance 37-nm steps (summarized in Table III). Note the higher variance and longer duration of the second step. “B” denotes the baseline population, and event populations are numbered to highlight at the respective mean and variance for each particular population.
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fig8: Double step events. (A) Representative “double step” event for smooth muscle myosin II taken at higher myosin surface densities where presumably more than one molecule is available to interact with the actin filament. Note that the second step is characterized by a similar duration and lower variance than the first step. (B) Representative “double step” event for myosin V, which gives rise to the intermediate variance 37-nm steps (summarized in Table III). Note the higher variance and longer duration of the second step. “B” denotes the baseline population, and event populations are numbered to highlight at the respective mean and variance for each particular population.

Mentions: At comparable flow cell loadings, myosin II molecules generate primarily unitary displacements. However, with increased density (2–5 μg/ml) applied to the coverslip surface, myosin II can also produce staircases with step increments of ∼10 nm (Table III; see Fig. 8 A), which presumably reflect the interaction of multiple myosin molecules with actin (see below). Based on system geometry and the surface ATPase assays, we estimate that at 1 μg/ml there are on average 1.7 M5HMM molecules on the surface of the bead platform that can interact with the actin filament. This surface density coupled to the high duty cycle of M5HMM increases the probability that the staircase events observed at the 1 μg/ml loading (Fig. 5 A) result from the interaction of more than one M5HMM molecule with actin.


Myosin V exhibits a high duty cycle and large unitary displacement.

Moore JR, Krementsova EB, Trybus KM, Warshaw DM - J. Cell Biol. (2001)

Double step events. (A) Representative “double step” event for smooth muscle myosin II taken at higher myosin surface densities where presumably more than one molecule is available to interact with the actin filament. Note that the second step is characterized by a similar duration and lower variance than the first step. (B) Representative “double step” event for myosin V, which gives rise to the intermediate variance 37-nm steps (summarized in Table III). Note the higher variance and longer duration of the second step. “B” denotes the baseline population, and event populations are numbered to highlight at the respective mean and variance for each particular population.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198872&req=5

fig8: Double step events. (A) Representative “double step” event for smooth muscle myosin II taken at higher myosin surface densities where presumably more than one molecule is available to interact with the actin filament. Note that the second step is characterized by a similar duration and lower variance than the first step. (B) Representative “double step” event for myosin V, which gives rise to the intermediate variance 37-nm steps (summarized in Table III). Note the higher variance and longer duration of the second step. “B” denotes the baseline population, and event populations are numbered to highlight at the respective mean and variance for each particular population.
Mentions: At comparable flow cell loadings, myosin II molecules generate primarily unitary displacements. However, with increased density (2–5 μg/ml) applied to the coverslip surface, myosin II can also produce staircases with step increments of ∼10 nm (Table III; see Fig. 8 A), which presumably reflect the interaction of multiple myosin molecules with actin (see below). Based on system geometry and the surface ATPase assays, we estimate that at 1 μg/ml there are on average 1.7 M5HMM molecules on the surface of the bead platform that can interact with the actin filament. This surface density coupled to the high duty cycle of M5HMM increases the probability that the staircase events observed at the 1 μg/ml loading (Fig. 5 A) result from the interaction of more than one M5HMM molecule with actin.

Bottom Line: The 20-nm unitary step represents the myosin V working stroke and is independent of the mode of M5(HMM) attachment to the motility surface or light chain content.The large M5(HMM) working stroke is consistent with the myosin V neck acting as a mechanical lever.The second step is characterized by an increased displacement variance, suggesting a model for how the two heads of myosin V function in processive motion.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA.

ABSTRACT
Myosin V is a double-headed unconventional myosin that has been implicated in organelle transport. To perform this role, myosin V may have a high duty cycle. To test this hypothesis and understand the properties of this molecule at the molecular level, we used the laser trap and in vitro motility assay to characterize the mechanics of heavy meromyosin-like fragments of myosin V (M5(HMM)) expressed in the Baculovirus system. The relationship between actin filament velocity and the number of interacting M5(HMM) molecules indicates a duty cycle of > or =50%. This high duty cycle would allow actin filament translocation and thus organelle transport by a few M5(HMM) molecules. Single molecule displacement data showed predominantly single step events of 20 nm and an occasional second step to 37 nm. The 20-nm unitary step represents the myosin V working stroke and is independent of the mode of M5(HMM) attachment to the motility surface or light chain content. The large M5(HMM) working stroke is consistent with the myosin V neck acting as a mechanical lever. The second step is characterized by an increased displacement variance, suggesting a model for how the two heads of myosin V function in processive motion.

Show MeSH
Related in: MedlinePlus