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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 ionic strength dependence of M5HMM in vitro motility. Normalized filament velocity (mean ±SEM, n > 10 filaments) as a function of added potassium chloride for M5HMM expressed with LC1sa (•) and calmodulin (▴) (M5HMM loading concentration of 10–25 μg/ml). Normalized filament velocity as a function of added potassium chloride for smooth muscle myosin II (♦) from Harris and Warshaw (1992) for comparison (loading concentration of 100 μg/ml). Note the reduction in actin filament velocity at potassium chloride concentrations <125 mM for both M5HMM and myosin II. Also note the plateau at physiological ionic strengths and greater (between 125 mM and 325 mM potassium chloride) for M5HMM. Velocities were normalized to the velocity at 25 mM potassium chloride. A similar dependence of actin filament velocity was observed regardless of light chain content.
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fig3: The ionic strength dependence of M5HMM in vitro motility. Normalized filament velocity (mean ±SEM, n > 10 filaments) as a function of added potassium chloride for M5HMM expressed with LC1sa (•) and calmodulin (▴) (M5HMM loading concentration of 10–25 μg/ml). Normalized filament velocity as a function of added potassium chloride for smooth muscle myosin II (♦) from Harris and Warshaw (1992) for comparison (loading concentration of 100 μg/ml). Note the reduction in actin filament velocity at potassium chloride concentrations <125 mM for both M5HMM and myosin II. Also note the plateau at physiological ionic strengths and greater (between 125 mM and 325 mM potassium chloride) for M5HMM. Velocities were normalized to the velocity at 25 mM potassium chloride. A similar dependence of actin filament velocity was observed regardless of light chain content.

Mentions: To determine if light chain composition and/or surface attachment of M5HMM is associated with functional alterations, each myosin construct was analyzed for its ability to move actin in the in vitro motility assay over a range of ionic strengths. The results from these experiments (Table I and Fig. 3) show that M5HMM, containing only calmodulin, translocated actin at slightly higher rates than that of M5HMM containing LC1sa. This result was obtained regardless of surface attachment strategy (see Materials and methods; Table I) and at ionic strengths between 25 and 525 mM potassium chloride in the absence of methylcellulose (Fig. 3). The velocity difference is surprising, since both the unitary displacement and displacement duration were independent of light chain content (see below; Table I), suggesting that the slower filament velocity for the M5HMM is not due to a change in the working stroke size or ADP release rate, respectively (see below).


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

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

The ionic strength dependence of M5HMM in vitro motility. Normalized filament velocity (mean ±SEM, n > 10 filaments) as a function of added potassium chloride for M5HMM expressed with LC1sa (•) and calmodulin (▴) (M5HMM loading concentration of 10–25 μg/ml). Normalized filament velocity as a function of added potassium chloride for smooth muscle myosin II (♦) from Harris and Warshaw (1992) for comparison (loading concentration of 100 μg/ml). Note the reduction in actin filament velocity at potassium chloride concentrations <125 mM for both M5HMM and myosin II. Also note the plateau at physiological ionic strengths and greater (between 125 mM and 325 mM potassium chloride) for M5HMM. Velocities were normalized to the velocity at 25 mM potassium chloride. A similar dependence of actin filament velocity was observed regardless of light chain content.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: The ionic strength dependence of M5HMM in vitro motility. Normalized filament velocity (mean ±SEM, n > 10 filaments) as a function of added potassium chloride for M5HMM expressed with LC1sa (•) and calmodulin (▴) (M5HMM loading concentration of 10–25 μg/ml). Normalized filament velocity as a function of added potassium chloride for smooth muscle myosin II (♦) from Harris and Warshaw (1992) for comparison (loading concentration of 100 μg/ml). Note the reduction in actin filament velocity at potassium chloride concentrations <125 mM for both M5HMM and myosin II. Also note the plateau at physiological ionic strengths and greater (between 125 mM and 325 mM potassium chloride) for M5HMM. Velocities were normalized to the velocity at 25 mM potassium chloride. A similar dependence of actin filament velocity was observed regardless of light chain content.
Mentions: To determine if light chain composition and/or surface attachment of M5HMM is associated with functional alterations, each myosin construct was analyzed for its ability to move actin in the in vitro motility assay over a range of ionic strengths. The results from these experiments (Table I and Fig. 3) show that M5HMM, containing only calmodulin, translocated actin at slightly higher rates than that of M5HMM containing LC1sa. This result was obtained regardless of surface attachment strategy (see Materials and methods; Table I) and at ionic strengths between 25 and 525 mM potassium chloride in the absence of methylcellulose (Fig. 3). The velocity difference is surprising, since both the unitary displacement and displacement duration were independent of light chain content (see below; Table I), suggesting that the slower filament velocity for the M5HMM is not due to a change in the working stroke size or ADP release rate, respectively (see below).

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