Limits...
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.

Show MeSH

Related in: MedlinePlus

Laser trap data time series of single M5HMM molecules. (A) Representative time series for M5HMM at a flow cell loading of 1 μg/ml. For clarity, a 3-s portion of the total ∼140-s time series is shown. The corresponding MV histogram for this 3-s time series is shown with the baseline population indicated by a “B.” The event populations are numbered consecutively and are separated from the baseline by an increase in mean displacement and a reduction in system variance. (B) Representative 220-s time series for M5HMM at a flow cell loading of 0.1 μg/ml. The corresponding MV histogram for the entire time series is also shown. Note that the variance of the second event population is between baseline and the first event population. (C) To illustrate event size and duration more clearly, a 15-s portion of the time series in B is also shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198872&req=5

fig5: Laser trap data time series of single M5HMM molecules. (A) Representative time series for M5HMM at a flow cell loading of 1 μg/ml. For clarity, a 3-s portion of the total ∼140-s time series is shown. The corresponding MV histogram for this 3-s time series is shown with the baseline population indicated by a “B.” The event populations are numbered consecutively and are separated from the baseline by an increase in mean displacement and a reduction in system variance. (B) Representative 220-s time series for M5HMM at a flow cell loading of 0.1 μg/ml. The corresponding MV histogram for the entire time series is also shown. Note that the variance of the second event population is between baseline and the first event population. (C) To illustrate event size and duration more clearly, a 15-s portion of the time series in B is also shown.

Mentions: To observe M5HMM displacements at high resolution (<1 nm), we used an optical trapping assay (Dupuis et al., 1997; Guilford et al., 1997). Fig. 5 shows displacement data for M5HMM obtained with a flow cell loading of 1 μg/ml (Fig. 5 A) and 0.1 μg/ml (Fig. 5, B and C). At the higher surface density, displacement traces with frequent staircase events were observed where the actin filament was pulled for several 20-nm step increments (Fig. 5 A). These step increments were discernible as individual displacement event populations offset by ∼20 nm in the mean variance (MV) histogram (Fig. 5 A) and at a lower bead position variance compared with baseline, which is characteristic of a stiffer system because of myosin's attachment to actin. Interestingly, the step size within the staircases appears to decrease with distance from trap center, suggesting that load may influence the size of the working stroke.


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

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

Laser trap data time series of single M5HMM molecules. (A) Representative time series for M5HMM at a flow cell loading of 1 μg/ml. For clarity, a 3-s portion of the total ∼140-s time series is shown. The corresponding MV histogram for this 3-s time series is shown with the baseline population indicated by a “B.” The event populations are numbered consecutively and are separated from the baseline by an increase in mean displacement and a reduction in system variance. (B) Representative 220-s time series for M5HMM at a flow cell loading of 0.1 μg/ml. The corresponding MV histogram for the entire time series is also shown. Note that the variance of the second event population is between baseline and the first event population. (C) To illustrate event size and duration more clearly, a 15-s portion of the time series in B is also shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Laser trap data time series of single M5HMM molecules. (A) Representative time series for M5HMM at a flow cell loading of 1 μg/ml. For clarity, a 3-s portion of the total ∼140-s time series is shown. The corresponding MV histogram for this 3-s time series is shown with the baseline population indicated by a “B.” The event populations are numbered consecutively and are separated from the baseline by an increase in mean displacement and a reduction in system variance. (B) Representative 220-s time series for M5HMM at a flow cell loading of 0.1 μg/ml. The corresponding MV histogram for the entire time series is also shown. Note that the variance of the second event population is between baseline and the first event population. (C) To illustrate event size and duration more clearly, a 15-s portion of the time series in B is also shown.
Mentions: To observe M5HMM displacements at high resolution (<1 nm), we used an optical trapping assay (Dupuis et al., 1997; Guilford et al., 1997). Fig. 5 shows displacement data for M5HMM obtained with a flow cell loading of 1 μg/ml (Fig. 5 A) and 0.1 μg/ml (Fig. 5, B and C). At the higher surface density, displacement traces with frequent staircase events were observed where the actin filament was pulled for several 20-nm step increments (Fig. 5 A). These step increments were discernible as individual displacement event populations offset by ∼20 nm in the mean variance (MV) histogram (Fig. 5 A) and at a lower bead position variance compared with baseline, which is characteristic of a stiffer system because of myosin's attachment to actin. Interestingly, the step size within the staircases appears to decrease with distance from trap center, suggesting that load may influence the size of the working stroke.

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