Abstract
Throughout the cell, motor proteins work together to drive numerous molecular processes and functions. For example, ensembles of myosin motors collectively transport vesicles and organelles, maintain membrane homeostasis, and drive muscle contraction. Studying these motors in groups has become increasingly important with work demonstrating the emergence of ensemble behavior distinct from individual motor behavior. One powerful technique that has been used in the last decade is DNA nanotechnology, which provides precise control over spacing and organization of patterned motor proteins. Until recently, however, most studies combining DNA nanostructures and molecular motors have been confined to discrete DNA structures with limited attachment points for motor proteins. In this chapter, we describe a new approach for making synthetic motor filaments using DNA nanotubes. We present methods for preparing myosin VI-labeled nanotubes and testing these nanotubes using a general in vitro motility setup. Overall, these nanotubes can easily be used to study other large ensembles of molecular motors, such as muscle myosin or ciliary dynein, both proteins that work in large motor ensembles to drive key cellular functions.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Baker JE, Brosseau C, Joel PB, Warshaw DM (2002) The biochemical kinetics underlying actin movement generated by one and many skeletal muscle myosin molecules. Biophys J 82(4):2134–2147. https://doi.org/10.1016/S0006-3495(02)75560-4
Derr ND, Goodman BS, Jungmann R, Leschziner AE, Shih WM, Reck-Peterson SL (2012) Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. Science 338(6107):662–665. https://doi.org/10.1126/science.1226734
Hariadi RF, Cale M, Sivaramakrishnan S (2014) Myosin lever arm directs collective motion on cellular actin network. Proc Natl Acad Sci U S A 111(11):4091–4096. https://doi.org/10.1073/pnas.1315923111
Hariadi RF, Sommese RF, Sivaramakrishnan S (2015) Tuning myosin-driven sorting on cellular actin networks. eLife 4. doi:https://doi.org/10.7554/eLife.05472
Walcott S, Warshaw DM, Debold EP (2012) Mechanical coupling between myosin molecules causes differences between ensemble and single-molecule measurements. Biophys J 103(3):501–510. https://doi.org/10.1016/j.bpj.2012.06.031
Hariadi RF, Sommese RF, Adhikari AS, Taylor RE, Sutton S, Spudich JA, Sivaramakrishnan S (2015) Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments. Nat Nanotechnol 10(8):696–700. https://doi.org/10.1038/nnano.2015.132
Yin P, Hariadi RF, Sahu S, Choi HM, Park SH, Labean TH, Reif JH (2008) Programming DNA tube circumferences. Science 321(5890):824–826. https://doi.org/10.1126/science.1157312
Heuser T, Raytchev M, Krell J, Porter ME, Nicastro D (2009) The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella. J Cell Biol 187(6):921–933. https://doi.org/10.1083/jcb.200908067
Huxley HE (1969) The mechanism of muscular contraction. Science 164(3886):1356–1365
Kron SJ, Toyoshima YY, Uyeda TQ, Spudich JA (1991) Assays for actin sliding movement over myosin-coated surfaces. Methods Enzymol 196:399–416
Nakao K, Minobe W, Roden R, Bristow MR, Leinwand LA (1997) Myosin heavy chain gene expression in human heart failure. J Clin Invest 100(9):2362–2370. https://doi.org/10.1172/JCI119776
Weith A, Sadayappan S, Gulick J, Previs MJ, Vanburen P, Robbins J, Warshaw DM (2012) Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain. J Mol Cell Cardiol 52(1):219–227. https://doi.org/10.1016/j.yjmcc.2011.09.019
Acknowledgments
This work was supported by the American Heart Association Scientist Development Grant (13SDG14270009) and the NIH (1DP2 CA186752-01 and 1-R01-GM-105646-01-A1) to SS. RFS is a Life Sciences Research Foundation postdoctoral fellow.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Sommese, R.F., Sivaramakrishnan, S. (2018). Engineering Synthetic Myosin Filaments Using DNA Nanotubes. In: Lavelle, C. (eds) Molecular Motors. Methods in Molecular Biology, vol 1805. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8556-2_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8556-2_5
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8554-8
Online ISBN: 978-1-4939-8556-2
eBook Packages: Springer Protocols