Single-Molecule Analysis of Actomyosin in the Presence of Osmolyte

  • Mitsuhiro Iwaki
  • Kohji Ito
  • Keisuke Fujita


Actomyosin is a protein complex composed of myosin and actin, which is well known for being the minimal contractile unit of muscle. The chemical free energy of ATP is converted into mechanical work by the complex, and the single-molecule mechanical properties of myosin are well characterized in vitro. However, the aqueous solution environment in in vitro assay is far from that in cells, where biomolecules are crowded, which influences osmotic pressure, and processes such as folding, and association and diffusion of proteins. Here, to bridge the gap between in vitro and in-cell environment, we observed mechanical motion of actomyosin-V in the presence of the osmolyte sucrose, as a model system. Single-molecule observation of myosin-V motor domains (heads) on actin filament at varying sucrose concentration revealed modulated mechanical elementary processes suggesting increased affinity of heads with actin and more robust force generation possibly accompanied by a sliding motion of myosin head along actin.


Actomyosin Osmolyte Single-molecule observation Myosin-V Force generation mechanism 


  1. Amano K, Yoshidome T, Iwaki M, Suzuki M, Kinoshita M (2010) Entropic potential field formed for a linear-motor protein near a filament: Statistical-mechanical analyses using simple models. J Chem Phys 133:045103CrossRefGoogle Scholar
  2. Ando T, Asai H (1977) The effects of solvent viscosity on the kinetic parameters of myosin and heavy meromyosin ATPase. J Bioenerg Biomembr 9:283–288CrossRefGoogle Scholar
  3. Boersma AJ, Zuhorn IS, Poolman B (2015) A sensor for quantification of macromolecular crowding in living cells. Nature methods 12: 227-229, 221 p following 229CrossRefGoogle Scholar
  4. Cayley S, Lewis BA, Guttman HJ, Record MT Jr (1991) Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo. J Mol Biol 222:281–300CrossRefGoogle Scholar
  5. Cookson NA, Cookson SW, Tsimring LS, Hasty J (2010) Cell cycle-dependent variations in protein concentration. Nucleic Acids Res 38:2676–2681CrossRefGoogle Scholar
  6. De La Cruz EM, Wells AL, Rosenfeld SS, Ostap EM, Sweeney HL (1999) The kinetic mechanism of myosin V. Proc Natl Acad Sci USA 96:13726–13731CrossRefGoogle Scholar
  7. Dunn AR, Spudich JA (2007) Dynamics of the unbound head during myosin V processive translocation. Nat Struct Mol Biol 14:246–248CrossRefGoogle Scholar
  8. Foth BJ, Goedecke MC, Soldati D (2006) New insights into myosin evolution and classification. Proc Natl Acad Sci USA 103:3681–3686CrossRefGoogle Scholar
  9. Fujita K, Iwaki M, Iwane AH, Marcucci L, Yanagida T (2012) Switching of myosin-V motion between the lever-arm swing and brownian search-and-catch. Nature communications 3:956CrossRefGoogle Scholar
  10. Fulton AB (1982) How crowded is the cytoplasm? Cell 30:345–347CrossRefGoogle Scholar
  11. Geeves MA, Jeffries TE (1988) The effect of nucleotide upon a specific isomerization of actomyosin subfragment 1. Biochem J 256:41–46CrossRefGoogle Scholar
  12. Harada Y, Noguchi A, Kishino A, Yanagida T (1987) Sliding movement of single actin filaments on one-headed myosin filaments. Nature 326:805–808CrossRefGoogle Scholar
  13. Highsmith S, Duignan K, Cooke R, Cohen J (1996) Osmotic pressure probe of actin-myosin hydration changes during ATP hydrolysis. Biophys J 70:2830–2837CrossRefGoogle Scholar
  14. Howard J (2001) Mechanics of motor proteins and the cytoskeleton. Sinauer Associates, MAGoogle Scholar
  15. Iwaki M, Iwane AH, Shimokawa T, Cooke R, Yanagida T (2009) Brownian search-and-catch mechanism for myosin-VI steps. Nat Chem Biol 5:403–405CrossRefGoogle Scholar
  16. Iwaki M, Tanaka H, Iwane AH, Katayama E, Ikebe M, Yanagida T (2006) Cargo-binding makes a wild-type single-headed myosin-VI move processively. Biophys J 90:3643–3652CrossRefGoogle Scholar
  17. Iwaki M, Wickham SF, Ikezaki K, Yanagida T, Shih WM (2016) A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads. Nature communications 7:13715CrossRefGoogle Scholar
  18. Kitamura K, Tokunaga M, Iwane AH, Yanagida T (1999) A single myosin head moves along an actin filament with regular steps of 5.3 nanometres. Nature 397:129–134CrossRefGoogle Scholar
  19. Mehta AD, Rock RS, Rief M, Spudich JA, Mooseker MS, Cheney RE (1999) Myosin-V is a processive actin-based motor. Nature 400:590–593CrossRefGoogle Scholar
  20. Millar NC, Geeves MA (1983) The limiting rate of the ATP-mediated dissociation of actin from rabbit skeletal muscle myosin subfragment 1. FEBS Lett 160:141–148CrossRefGoogle Scholar
  21. Rief M, Rock RS, Mehta AD, Mooseker MS, Cheney RE, Spudich JA (2000) Myosin-V stepping kinetics: a molecular model for processivity. Proc Natl Acad Sci USA 97:9482–9486CrossRefGoogle Scholar
  22. Shiroguchi K, Kinosita K Jr (2007) Myosin V walks by lever action and Brownian motion. Science 316:1208–1212CrossRefGoogle Scholar
  23. Siemankowski RF, Wiseman MO, White HD (1985) ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle. Proc Natl Acad Sci USA 82:658–662CrossRefGoogle Scholar
  24. Svoboda K, Schmidt CF, Schnapp BJ, Block SM (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365:721–727CrossRefGoogle Scholar
  25. Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300:2061–2065CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Quantitative Biology Center, RIKEN/Graduate School of Frontier BiosciencesOsaka UniversityOsakaJapan
  2. 2.Graduate School of ScienceChiba UniversityChibaJapan

Personalised recommendations