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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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Schematic representation comparing the sequences of the expressed myosin V constructs to wild-type myosin V. We constructed two COOH terminally truncated myosin V constructs, M5HMM and M5HMM(tag), where amino acids 1099–1853 were removed. Note that removal of the residues, which include the tail region of the molecule, results in a M5HMM protein that resembles the proteolytic HMM fragment of vertebrate smooth and skeletal muscle. M5HMM and M5HMM(tag) are identical except that M5HMM(tag) contains the S2.2 epitope for specific attachment to the motility surface (described in Materials and methods).
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fig1: Schematic representation comparing the sequences of the expressed myosin V constructs to wild-type myosin V. We constructed two COOH terminally truncated myosin V constructs, M5HMM and M5HMM(tag), where amino acids 1099–1853 were removed. Note that removal of the residues, which include the tail region of the molecule, results in a M5HMM protein that resembles the proteolytic HMM fragment of vertebrate smooth and skeletal muscle. M5HMM and M5HMM(tag) are identical except that M5HMM(tag) contains the S2.2 epitope for specific attachment to the motility surface (described in Materials and methods).

Mentions: A double-headed HMM-like fragment of the murine myosin V heavy chain was expressed using the Baculovirus/insect cell system (Fig. 1). The heavy chain was coexpressed with calmodulin, which was identified in the purified myosin V HMM (M5HMM) by its characteristic calcium-dependent shift in gel migration rate. This preparation contains three minor bands in the light chain region (molecular weight ∼20–30 kD), which react with anti-FLAG monoclonal antibody by Western blotting, confirming that they are derived from the COOH terminus of the myosin V heavy chain (Fig. 2) and are not essential light chains. The minor band (Fig. 2, arrowhead), which does not react with anti-FLAG antibody, is likely to be the complementary NH2-terminal piece of the heavy chain. In the mechanical assays, exogenous calmodulin was added (see Materials and methods) to the assay solution to assure full occupancy of the six IQ motifs.


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

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

Schematic representation comparing the sequences of the expressed myosin V constructs to wild-type myosin V. We constructed two COOH terminally truncated myosin V constructs, M5HMM and M5HMM(tag), where amino acids 1099–1853 were removed. Note that removal of the residues, which include the tail region of the molecule, results in a M5HMM protein that resembles the proteolytic HMM fragment of vertebrate smooth and skeletal muscle. M5HMM and M5HMM(tag) are identical except that M5HMM(tag) contains the S2.2 epitope for specific attachment to the motility surface (described in Materials and methods).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Schematic representation comparing the sequences of the expressed myosin V constructs to wild-type myosin V. We constructed two COOH terminally truncated myosin V constructs, M5HMM and M5HMM(tag), where amino acids 1099–1853 were removed. Note that removal of the residues, which include the tail region of the molecule, results in a M5HMM protein that resembles the proteolytic HMM fragment of vertebrate smooth and skeletal muscle. M5HMM and M5HMM(tag) are identical except that M5HMM(tag) contains the S2.2 epitope for specific attachment to the motility surface (described in Materials and methods).
Mentions: A double-headed HMM-like fragment of the murine myosin V heavy chain was expressed using the Baculovirus/insect cell system (Fig. 1). The heavy chain was coexpressed with calmodulin, which was identified in the purified myosin V HMM (M5HMM) by its characteristic calcium-dependent shift in gel migration rate. This preparation contains three minor bands in the light chain region (molecular weight ∼20–30 kD), which react with anti-FLAG monoclonal antibody by Western blotting, confirming that they are derived from the COOH terminus of the myosin V heavy chain (Fig. 2) and are not essential light chains. The minor band (Fig. 2, arrowhead), which does not react with anti-FLAG antibody, is likely to be the complementary NH2-terminal piece of the heavy chain. In the mechanical assays, exogenous calmodulin was added (see Materials and methods) to the assay solution to assure full occupancy of the six IQ motifs.

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