Abstract
Recent advances in single-molecule labeling and detection techniques allow high-resolution imaging of the motion of single molecules. Molecular motors are biological machines that convert chemical energy into mechanical work. Myosin Va (MyoVa) is a well-characterized processive molecular motor, essential for cargo transport in living organisms. Quantum dots (Qdots) are fluorescent semiconductor nanocrystals that are extremely useful for single-molecule studies in biological sciences. High-resolution video microscopy and single-particle tracking of a Qdot-labeled MyoVa motor molecule allow the detection of individual steps in vitro and in live cells.
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References
Krementsov, D. N., Krementsova, E. B., and Trybus, K. M. (2004) Myosin V: regulation by calcium, calmodulin, and the tail domain. J. Cell Biol. 164, 877–886.
Cronan, J. E., Jr. (1990) Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins. J. Biol. Chem. 265, 10327–10333.
Warshaw, D. M., Kennedy, G. G., Work, S. S., Krementsova, E. B., Beck, S., and Trybus, K. M. (2005) Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity. Biophys. J. 88, L30–32.
Hodges, A. R., Krementsova, E. B., and Trybus, K. M. (2007) Engineering the processive run length of Myosin V. J. Biol. Chem. 282, 27192–27197.
Ali, M. Y., Lu, H., Bookwalter, C. S., Warshaw, D. M., and Trybus, K. M. (2008) Myosin V and Kinesin act as tethers to enhance each others’ processivity. Proc. Natl. Acad. Sci. USA 105, 4691–4696.
Baker, J. E., Krementsova, E. B., Kennedy, G. G., Armstrong, A., Trybus, K. M., and Warshaw, D. M. (2004) Myosin V processivity: multiple kinetic pathways for head-to-head coordination. Proc. Natl. Acad. Sci. USA 101, 5542–5546.
Pardee, J. D., and Spudich, J. A. (1982) Purification of muscle actin. Methods Cell Biol. 24, 271–289.
Warshaw, D. M., Desrosiers, J. M., Work, S. S., and Trybus, K. M. (1990) Smooth muscle myosin cross-bridge interactions modulate actin filament sliding velocity in vitro. J. Cell Biol. 111, 453–463.
Hermanson, G. T. (2008) Bioconjugate techniques, 2nd ed., Elsevier Academic Press, Amsterdam; Boston.
Liu, J., Taylor, D. W., Krementsova, E. B., Trybus, K. M., and Taylor, K. A. (2006) Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography. Nature 442, 208–211.
Wang, F., Thirumurugan, K., Stafford, W. F., Hammer, J. A., 3rd, Knight, P. J., and Sellers, J. R. (2004) Regulated conformation of myosin V. J. Biol. Chem. 279, 2333–2336.
Ali, M. Y., Krementsova, E. B., Kennedy, G. G., Mahaffy, R., Pollard, T. D., Trybus, K. M., and Warshaw, D. M. (2007) Myosin Va maneuvers through actin intersections and diffuses along microtubules. Proc. Natl. Acad. Sci. USA 104, 4332–4336.
Yildiz, A., Forkey, J. N., McKinney, S. A., Ha, T., Goldman, Y. E., and Selvin, P. R. (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065.
Sage, D., Neumann, F. R., Hediger, F., Gasser, S. M., and Unser, M. (2005) Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamic. IEEE Trans. Image Process 14, 1372–1383.
Kerssemakers, J. W., Munteanu, E. L., Laan, L., Noetzel, T. L., Janson, M. E., and Dogterom, M. (2006) Assembly dynamics of microtubules at molecular resolution. Nature 442, 709–712.
Okada, C. Y., and Rechsteiner, M. (1982) Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell 29, 33–41.
Acknowledgments
The authors thank Guy Kennedy for optomechanical support, Samantha Previs for technical expertise, and Kathleen Trybus for providing MyoVa protein. This work was supported by funds from the National Institutes of Health HL007944 (to S.R.N.) and HL059408, GM094229, and HL085489 (to D.M.W.).
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Nelson, S.R., Ali, M.Y., Warshaw, D.M. (2011). Quantum Dot Labeling Strategies to Characterize Single-Molecular Motors. In: Mashanov, G., Batters, C. (eds) Single Molecule Enzymology. Methods in Molecular Biology, vol 778. Humana Press. https://doi.org/10.1007/978-1-61779-261-8_8
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DOI: https://doi.org/10.1007/978-1-61779-261-8_8
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