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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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The dependence of actin filament velocity on the number of interacting M5HMM heads. Normalized filament velocity as a function the number of M5HMM heads available to interact with each filament at a loading concentration of 5 (▪) and 1 μg/ml (•). Note the independence of M5HMM-driven actin filament velocity on the number of interacting heads. The number of available heads was calculated via surface ATPase assays (described in Materials and methods). For comparison, normalized filament velocity as a function the number of smooth muscle myosin heads available to interact with each filament (▴; data from Harris and Warshaw, 1993) at a loading concentration of 10 μg/ml. (Bottom solid line) A least squares fit of the myosin II data to Eq. 1, revealing a 3% duty cycle for the myosin II data. (Top solid line) A least squares fit of the M5HMM data to Eq. 1, revealing a 51% duty cycle. Dashed lines represent the range of the fit determined by standard error of the estimate.
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fig4: The dependence of actin filament velocity on the number of interacting M5HMM heads. Normalized filament velocity as a function the number of M5HMM heads available to interact with each filament at a loading concentration of 5 (▪) and 1 μg/ml (•). Note the independence of M5HMM-driven actin filament velocity on the number of interacting heads. The number of available heads was calculated via surface ATPase assays (described in Materials and methods). For comparison, normalized filament velocity as a function the number of smooth muscle myosin heads available to interact with each filament (▴; data from Harris and Warshaw, 1993) at a loading concentration of 10 μg/ml. (Bottom solid line) A least squares fit of the myosin II data to Eq. 1, revealing a 3% duty cycle for the myosin II data. (Top solid line) A least squares fit of the M5HMM data to Eq. 1, revealing a 51% duty cycle. Dashed lines represent the range of the fit determined by standard error of the estimate.

Mentions: The actin filament velocity was plotted as a function of N (Fig. 4) and the best fit (f ± SE) of the data to Eq. 1 resulted in a 51 ± 10% duty cycle estimate (Fig. 4, top solid line). One characteristic of such a high duty cycle is the observation that maximum actin filament velocity was maintained when only a few heads were available to interact with the actin filament. It is worth noting that the duty cycle may in fact be >50%, but the sensitivity of the estimate is extremely low between 50 and 100% (Fig. 4); therefore, interpretation of these data with regards to processive versus nonprocessive motility should be taken with caution. Published data from smooth muscle myosin II (Harris and Warshaw, 1993) with a 3.4 ± 0.1% duty cycle are plotted for comparison (Fig. 4, bottom solid line) to demonstrate the striking difference between the M5HMM and myosin II duty cycles.


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

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

The dependence of actin filament velocity on the number of interacting M5HMM heads. Normalized filament velocity as a function the number of M5HMM heads available to interact with each filament at a loading concentration of 5 (▪) and 1 μg/ml (•). Note the independence of M5HMM-driven actin filament velocity on the number of interacting heads. The number of available heads was calculated via surface ATPase assays (described in Materials and methods). For comparison, normalized filament velocity as a function the number of smooth muscle myosin heads available to interact with each filament (▴; data from Harris and Warshaw, 1993) at a loading concentration of 10 μg/ml. (Bottom solid line) A least squares fit of the myosin II data to Eq. 1, revealing a 3% duty cycle for the myosin II data. (Top solid line) A least squares fit of the M5HMM data to Eq. 1, revealing a 51% duty cycle. Dashed lines represent the range of the fit determined by standard error of the estimate.
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Related In: Results  -  Collection

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fig4: The dependence of actin filament velocity on the number of interacting M5HMM heads. Normalized filament velocity as a function the number of M5HMM heads available to interact with each filament at a loading concentration of 5 (▪) and 1 μg/ml (•). Note the independence of M5HMM-driven actin filament velocity on the number of interacting heads. The number of available heads was calculated via surface ATPase assays (described in Materials and methods). For comparison, normalized filament velocity as a function the number of smooth muscle myosin heads available to interact with each filament (▴; data from Harris and Warshaw, 1993) at a loading concentration of 10 μg/ml. (Bottom solid line) A least squares fit of the myosin II data to Eq. 1, revealing a 3% duty cycle for the myosin II data. (Top solid line) A least squares fit of the M5HMM data to Eq. 1, revealing a 51% duty cycle. Dashed lines represent the range of the fit determined by standard error of the estimate.
Mentions: The actin filament velocity was plotted as a function of N (Fig. 4) and the best fit (f ± SE) of the data to Eq. 1 resulted in a 51 ± 10% duty cycle estimate (Fig. 4, top solid line). One characteristic of such a high duty cycle is the observation that maximum actin filament velocity was maintained when only a few heads were available to interact with the actin filament. It is worth noting that the duty cycle may in fact be >50%, but the sensitivity of the estimate is extremely low between 50 and 100% (Fig. 4); therefore, interpretation of these data with regards to processive versus nonprocessive motility should be taken with caution. Published data from smooth muscle myosin II (Harris and Warshaw, 1993) with a 3.4 ± 0.1% duty cycle are plotted for comparison (Fig. 4, bottom solid line) to demonstrate the striking difference between the M5HMM and myosin II duty cycles.

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