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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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Compliance-corrected step measurements. The experimental configuration for the oscillation experiments (described in Materials and methods). An example trace for M5HMM at 100 μM ATP is also shown (bottom right). Note that when myosin binds the sinusoidal oscillation of the trapped bead is clipped. The difference between clipped levels (dashed lines) provides a measure of the compliance-corrected displacement. The histogram of step increments indicates a displacement of ∼23 nm (bottom left).
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fig6: Compliance-corrected step measurements. The experimental configuration for the oscillation experiments (described in Materials and methods). An example trace for M5HMM at 100 μM ATP is also shown (bottom right). Note that when myosin binds the sinusoidal oscillation of the trapped bead is clipped. The difference between clipped levels (dashed lines) provides a measure of the compliance-corrected displacement. The histogram of step increments indicates a displacement of ∼23 nm (bottom left).

Mentions: To control for any effect of compliance within the experimental setup on our estimate of the working stroke, we pulled any compliant linkages taught by oscillating one of the trapped beads with a ∼300-nm sinusoidal displacement (Mehta et al., 1999). In these experiments, it was necessary to use sufficient myosin concentrations to assure staircase events (1 μg/ml). Representative data are shown in Fig. 6. In the absence of an attached M5HMM molecule, the bead followed the sinusoidal driving function. Upon attachment of an M5HMM molecule, clipping of the bead oscillation was observed. As other M5HMM molecules bind successively to the actin filament and undergo their working strokes, the tether length becomes progressively shorter, and the difference between the clipped levels was recorded as a measure of the true M5HMM working stroke. These data give a broad step size distribution that centers at 23 nm, thus indicating an ∼15% compliance in our system. This compliance estimate is consistent with our previous estimates (Dupuis et al., 1997; Tyska et al., 1999). In summary, the difference between murine M5HMM and the tissue-purified chicken myosin V step size cannot be accounted for by an underestimate due to surface attachment, light chain content, or system compliance.


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

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

Compliance-corrected step measurements. The experimental configuration for the oscillation experiments (described in Materials and methods). An example trace for M5HMM at 100 μM ATP is also shown (bottom right). Note that when myosin binds the sinusoidal oscillation of the trapped bead is clipped. The difference between clipped levels (dashed lines) provides a measure of the compliance-corrected displacement. The histogram of step increments indicates a displacement of ∼23 nm (bottom left).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Compliance-corrected step measurements. The experimental configuration for the oscillation experiments (described in Materials and methods). An example trace for M5HMM at 100 μM ATP is also shown (bottom right). Note that when myosin binds the sinusoidal oscillation of the trapped bead is clipped. The difference between clipped levels (dashed lines) provides a measure of the compliance-corrected displacement. The histogram of step increments indicates a displacement of ∼23 nm (bottom left).
Mentions: To control for any effect of compliance within the experimental setup on our estimate of the working stroke, we pulled any compliant linkages taught by oscillating one of the trapped beads with a ∼300-nm sinusoidal displacement (Mehta et al., 1999). In these experiments, it was necessary to use sufficient myosin concentrations to assure staircase events (1 μg/ml). Representative data are shown in Fig. 6. In the absence of an attached M5HMM molecule, the bead followed the sinusoidal driving function. Upon attachment of an M5HMM molecule, clipping of the bead oscillation was observed. As other M5HMM molecules bind successively to the actin filament and undergo their working strokes, the tether length becomes progressively shorter, and the difference between the clipped levels was recorded as a measure of the true M5HMM working stroke. These data give a broad step size distribution that centers at 23 nm, thus indicating an ∼15% compliance in our system. This compliance estimate is consistent with our previous estimates (Dupuis et al., 1997; Tyska et al., 1999). In summary, the difference between murine M5HMM and the tissue-purified chicken myosin V step size cannot be accounted for by an underestimate due to surface attachment, light chain content, or system compliance.

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