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Motility of myosin V regulated by the dissociation of single calmodulin

Abstract

Myosin V is a calmodulin-binding motor protein. The dissociation of single calmodulin molecules from individual myosin V molecules at 1 μM Ca2+ correlates with a reduction in sliding velocity in an in vitro motility assay. The dissociation of two calmodulin molecules at 5 μM Ca2+ correlates with a detachment of actin filaments from myosin V. To mimic the regulation of myosin V motility by Ca2+ in a cell, caged Ca2+ coupled with a UV flash system was used to produce Ca2+ transients. During the Ca2+ transient, myosin V goes through the functional cycle of reduced sliding velocity, actin detachment and reattachment followed by the recovery of the sliding velocity. These results indicate that myosin V motility is regulated by Ca2+ through a reduction in actin-binding affinity resulting from the dissociation of single calmodulin molecules.

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Figure 1: Effects of Ca2+ on the in vitro motility assay of myosin V.
Figure 2: Effect of Ca2+ on the dissociation of CaM from myosin V.
Figure 3: Ca2+-dependent dissociation rate of CaM from myosin V (Supplementary Methods online).
Figure 4: Reversible regulation of myosin V function with transient Ca2+ generated by UV photolysis of caged Ca2+.
Figure 5: Model for the reversible regulation of myosin V motor functions by Ca2+ in the presence of free CaM.

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Acknowledgements

We thank N. Sasaki and S. Uemura for giving important suggestions and helping in purification of myosin V, and T. Ando and J. M. West for critical reading of the manuscript. The research was supported by Grants-in-Aid for Scientific Research in Priority Areas from the Japan Ministry of Education, Culture, Sports, Science and Technology (H.H.). H.A.N. was the recipient of a postdoctoral scholarship from the Japan Society for the Promotion of Science.

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Correspondence to Hideo Higuchi.

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Supplementary information

Supplementary Video 1

The reversible attachment and movement of actin filaments by myosin–V. An in vitro motility was performed in the presence of 10 µM CaM and transient Ca2+concentrations, which was generated locally by UV photolysis of caged Ca2+ within a time frame of 4 s. The number indicates the time in seconds and tens of milliseconds. The time period of the UV flash was indicated by “UV”. Bar 10 µm. This movie is a supplement for Figure 4a. (MOV 1437 kb)

Supplementary Methods (PDF 36 kb)

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Nguyen, H., Higuchi, H. Motility of myosin V regulated by the dissociation of single calmodulin. Nat Struct Mol Biol 12, 127–132 (2005). https://doi.org/10.1038/nsmb894

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