Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 Aug;79(2):962–974. doi: 10.1016/S0006-3495(00)76350-8

Characterization of single actomyosin rigor bonds: load dependence of lifetime and mechanical properties.

T Nishizaka 1, R Seo 1, H Tadakuma 1, K Kinosita Jr 1, S Ishiwata 1
PMCID: PMC1300992  PMID: 10920026

Abstract

Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling the barbed end of the actin filament with optical tweezers. For S1, the relationship between the lifetime (tau) and the externally imposed load (F) at absolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of tau(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with tau(F) for S1, suggesting that the fast component corresponds to single-headed binding and the slow component to double-headed binding. These large interaction distances, which may be a common characteristic of motor proteins, are attributed to the geometry for applying an external load. The pulling experiment has also allowed direct estimation of the number of myosin molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indicating a low torsional stiffness for HMM. From these results, we discuss the characteristics of interaction between actin and myosin, with the focus on the manner of binding of myosin.

Full Text

The Full Text of this article is available as a PDF (230.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arai Y., Yasuda R., Akashi K., Harada Y., Miyata H., Kinosita K., Jr, Itoh H. Tying a molecular knot with optical tweezers. Nature. 1999 Jun 3;399(6735):446–448. doi: 10.1038/20894. [DOI] [PubMed] [Google Scholar]
  2. Ashkin A., Schütze K., Dziedzic J. M., Euteneuer U., Schliwa M. Force generation of organelle transport measured in vivo by an infrared laser trap. Nature. 1990 Nov 22;348(6299):346–348. doi: 10.1038/348346a0. [DOI] [PubMed] [Google Scholar]
  3. Bell G. I. Models for the specific adhesion of cells to cells. Science. 1978 May 12;200(4342):618–627. doi: 10.1126/science.347575. [DOI] [PubMed] [Google Scholar]
  4. Corrie J. E., Brandmeier B. D., Ferguson R. E., Trentham D. R., Kendrick-Jones J., Hopkins S. C., van der Heide U. A., Goldman Y. E., Sabido-David C., Dale R. E. Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction. Nature. 1999 Jul 29;400(6743):425–430. doi: 10.1038/22704. [DOI] [PubMed] [Google Scholar]
  5. Erickson H. P. Reversible unfolding of fibronectin type III and immunoglobulin domains provides the structural basis for stretch and elasticity of titin and fibronectin. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10114–10118. doi: 10.1073/pnas.91.21.10114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Evans E., Ritchie K. Dynamic strength of molecular adhesion bonds. Biophys J. 1997 Apr;72(4):1541–1555. doi: 10.1016/S0006-3495(97)78802-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Finer J. T., Simmons R. M., Spudich J. A. Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature. 1994 Mar 10;368(6467):113–119. doi: 10.1038/368113a0. [DOI] [PubMed] [Google Scholar]
  8. Florin E. L., Moy V. T., Gaub H. E. Adhesion forces between individual ligand-receptor pairs. Science. 1994 Apr 15;264(5157):415–417. doi: 10.1126/science.8153628. [DOI] [PubMed] [Google Scholar]
  9. Fritz J., Katopodis A. G., Kolbinger F., Anselmetti D. Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy. Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12283–12288. doi: 10.1073/pnas.95.21.12283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Funatsu T., Harada Y., Tokunaga M., Saito K., Yanagida T. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature. 1995 Apr 6;374(6522):555–559. doi: 10.1038/374555a0. [DOI] [PubMed] [Google Scholar]
  11. Goldman Y. E., Brenner B. Special topic: molecular mechanism of muscle contraction. General introduction. Annu Rev Physiol. 1987;49:629–636. doi: 10.1146/annurev.ph.49.030187.003213. [DOI] [PubMed] [Google Scholar]
  12. Goldman Y. E. Kinetics of the actomyosin ATPase in muscle fibers. Annu Rev Physiol. 1987;49:637–654. doi: 10.1146/annurev.ph.49.030187.003225. [DOI] [PubMed] [Google Scholar]
  13. Grubmüller H., Heymann B., Tavan P. Ligand binding: molecular mechanics calculation of the streptavidin-biotin rupture force. Science. 1996 Feb 16;271(5251):997–999. doi: 10.1126/science.271.5251.997. [DOI] [PubMed] [Google Scholar]
  14. Harada Y., Noguchi A., Kishino A., Yanagida T. Sliding movement of single actin filaments on one-headed myosin filaments. Nature. 1987 Apr 23;326(6115):805–808. doi: 10.1038/326805a0. [DOI] [PubMed] [Google Scholar]
  15. Harada Y., Sakurada K., Aoki T., Thomas D. D., Yanagida T. Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay. J Mol Biol. 1990 Nov 5;216(1):49–68. doi: 10.1016/S0022-2836(05)80060-9. [DOI] [PubMed] [Google Scholar]
  16. Howard J., Hudspeth A. J., Vale R. D. Movement of microtubules by single kinesin molecules. Nature. 1989 Nov 9;342(6246):154–158. doi: 10.1038/342154a0. [DOI] [PubMed] [Google Scholar]
  17. Hunt A. J., Gittes F., Howard J. The force exerted by a single kinesin molecule against a viscous load. Biophys J. 1994 Aug;67(2):766–781. doi: 10.1016/S0006-3495(94)80537-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hunt A. J., Howard J. Kinesin swivels to permit microtubule movement in any direction. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11653–11657. doi: 10.1073/pnas.90.24.11653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huxley A. F., Simmons R. M. Proposed mechanism of force generation in striated muscle. Nature. 1971 Oct 22;233(5321):533–538. doi: 10.1038/233533a0. [DOI] [PubMed] [Google Scholar]
  20. Ishijima A., Harada Y., Kojima H., Funatsu T., Higuchi H., Yanagida T. Single-molecule analysis of the actomyosin motor using nano-manipulation. Biochem Biophys Res Commun. 1994 Mar 15;199(2):1057–1063. doi: 10.1006/bbrc.1994.1336. [DOI] [PubMed] [Google Scholar]
  21. Ishijima A., Kojima H., Funatsu T., Tokunaga M., Higuchi H., Tanaka H., Yanagida T. Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin. Cell. 1998 Jan 23;92(2):161–171. doi: 10.1016/s0092-8674(00)80911-3. [DOI] [PubMed] [Google Scholar]
  22. Ishijima A., Kojima H., Higuchi H., Harada Y., Funatsu T., Yanagida T. Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle: unitary steps and forces. Biophys J. 1996 Jan;70(1):383–400. doi: 10.1016/S0006-3495(96)79582-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ishiwata S., Kinosita K., Jr, Yoshimura H., Ikegami A. Optical anisotropy decay studies of the dynamic structure of myosin filaments. Adv Exp Med Biol. 1988;226:267–276. [PubMed] [Google Scholar]
  24. Ishiwata S., Kinosita K., Jr, Yoshimura H., Ikegami A. Rotational motions of myosin heads in myofibril studied by phosphorescence anisotropy decay measurements. J Biol Chem. 1987 Jun 15;262(17):8314–8317. [PubMed] [Google Scholar]
  25. Izrailev S., Stepaniants S., Balsera M., Oono Y., Schulten K. Molecular dynamics study of unbinding of the avidin-biotin complex. Biophys J. 1997 Apr;72(4):1568–1581. doi: 10.1016/S0006-3495(97)78804-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kamimura S., Takahashi K. Direct measurement of the force of microtubule sliding in flagella. Nature. 1981 Oct 15;293(5833):566–568. doi: 10.1038/293566a0. [DOI] [PubMed] [Google Scholar]
  27. Kinosita K., Jr, Itoh H., Ishiwata S., Hirano K., Nishizaka T., Hayakawa T. Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium. J Cell Biol. 1991 Oct;115(1):67–73. doi: 10.1083/jcb.115.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kinosita K., Jr, Yasuda R., Noji H., Ishiwata S., Yoshida M. F1-ATPase: a rotary motor made of a single molecule. Cell. 1998 Apr 3;93(1):21–24. doi: 10.1016/s0092-8674(00)81142-3. [DOI] [PubMed] [Google Scholar]
  29. Kishino A., Yanagida T. Force measurements by micromanipulation of a single actin filament by glass needles. Nature. 1988 Jul 7;334(6177):74–76. doi: 10.1038/334074a0. [DOI] [PubMed] [Google Scholar]
  30. Kitamura K., Tokunaga M., Iwane A. H., Yanagida T. A single myosin head moves along an actin filament with regular steps of 5.3 nanometres. Nature. 1999 Jan 14;397(6715):129–134. doi: 10.1038/16403. [DOI] [PubMed] [Google Scholar]
  31. Kondo H., Ishiwata S. Uni-directional growth of F-actin. J Biochem. 1976 Jan;79(1):159–171. doi: 10.1093/oxfordjournals.jbchem.a131043. [DOI] [PubMed] [Google Scholar]
  32. Kuo S. C., Ramanathan K., Sorg B. Single kinesin molecules stressed with optical tweezers. Biophys J. 1995 Apr;68(4 Suppl):74S–74S. [PMC free article] [PubMed] [Google Scholar]
  33. Kurokawa H., Fujii W., Ohmi K., Sakurai T., Nonomura Y. Simple and rapid purification of brevin. Biochem Biophys Res Commun. 1990 Apr 30;168(2):451–457. doi: 10.1016/0006-291x(90)92342-w. [DOI] [PubMed] [Google Scholar]
  34. Marston S. B. The rates of formation and dissociation of actin-myosin complexes. Effects of solvent, temperature, nucleotide binding and head-head interactions. Biochem J. 1982 May 1;203(2):453–460. doi: 10.1042/bj2030453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mehta A. D., Rock R. S., Rief M., Spudich J. A., Mooseker M. S., Cheney R. E. Myosin-V is a processive actin-based motor. Nature. 1999 Aug 5;400(6744):590–593. doi: 10.1038/23072. [DOI] [PubMed] [Google Scholar]
  36. Merkel R., Nassoy P., Leung A., Ritchie K., Evans E. Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature. 1999 Jan 7;397(6714):50–53. doi: 10.1038/16219. [DOI] [PubMed] [Google Scholar]
  37. Miyata H., Hakozaki H., Yoshikawa H., Suzuki N., Kinosita K., Jr, Nishizaka T., Ishiwata S. Stepwise motion of an actin filament over a small number of heavy meromyosin molecules is revealed in an in vitro motility assay. J Biochem. 1994 Apr;115(4):644–647. doi: 10.1093/oxfordjournals.jbchem.a124389. [DOI] [PubMed] [Google Scholar]
  38. Miyata H., Yoshikawa H., Hakozaki H., Suzuki N., Furuno T., Ikegami A., Kinosita K., Jr, Nishizaka T., Ishiwata S. Mechanical measurements of single actomyosin motor force. Biophys J. 1995 Apr;68(4 Suppl):286S–290S. [PMC free article] [PubMed] [Google Scholar]
  39. Molloy J. E., Burns J. E., Kendrick-Jones J., Tregear R. T., White D. C. Movement and force produced by a single myosin head. Nature. 1995 Nov 9;378(6553):209–212. doi: 10.1038/378209a0. [DOI] [PubMed] [Google Scholar]
  40. Moy V. T., Florin E. L., Gaub H. E. Intermolecular forces and energies between ligands and receptors. Science. 1994 Oct 14;266(5183):257–259. doi: 10.1126/science.7939660. [DOI] [PubMed] [Google Scholar]
  41. Nakajima H., Kunioka Y., Nakano K., Shimizu K., Seto M., Ando T. Scanning force microscopy of the interaction events between a single molecule of heavy meromyosin and actin. Biochem Biophys Res Commun. 1997 May 8;234(1):178–182. doi: 10.1006/bbrc.1997.6612. [DOI] [PubMed] [Google Scholar]
  42. Nishizaka T., Miyata H., Yoshikawa H., Ishiwata S., Kinosita K., Jr Mechanical properties of single protein motor of muscle studied by optical tweezers. Biophys J. 1995 Apr;68(4 Suppl):75S–75S. [PMC free article] [PubMed] [Google Scholar]
  43. Nishizaka T., Miyata H., Yoshikawa H., Ishiwata S., Kinosita K., Jr Unbinding force of a single motor molecule of muscle measured using optical tweezers. Nature. 1995 Sep 21;377(6546):251–254. doi: 10.1038/377251a0. [DOI] [PubMed] [Google Scholar]
  44. Nishizaka T., Shi Q., Sheetz M. P. Position-dependent linkages of fibronectin- integrin-cytoskeleton. Proc Natl Acad Sci U S A. 2000 Jan 18;97(2):692–697. doi: 10.1073/pnas.97.2.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nishizaka T., Yagi T., Tanaka Y., Ishiwata S. Right-handed rotation of an actin filament in an in vitro motile system. Nature. 1993 Jan 21;361(6409):269–271. doi: 10.1038/361269a0. [DOI] [PubMed] [Google Scholar]
  46. Noji H., Yasuda R., Yoshida M., Kinosita K., Jr Direct observation of the rotation of F1-ATPase. Nature. 1997 Mar 20;386(6622):299–302. doi: 10.1038/386299a0. [DOI] [PubMed] [Google Scholar]
  47. Rayment I., Holden H. M., Whittaker M., Yohn C. B., Lorenz M., Holmes K. C., Milligan R. A. Structure of the actin-myosin complex and its implications for muscle contraction. Science. 1993 Jul 2;261(5117):58–65. doi: 10.1126/science.8316858. [DOI] [PubMed] [Google Scholar]
  48. Rayment I., Rypniewski W. R., Schmidt-Bäse K., Smith R., Tomchick D. R., Benning M. M., Winkelmann D. A., Wesenberg G., Holden H. M. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science. 1993 Jul 2;261(5117):50–58. doi: 10.1126/science.8316857. [DOI] [PubMed] [Google Scholar]
  49. Sase I., Miyata H., Corrie J. E., Craik J. S., Kinosita K., Jr Real time imaging of single fluorophores on moving actin with an epifluorescence microscope. Biophys J. 1995 Aug;69(2):323–328. doi: 10.1016/S0006-3495(95)79937-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sase I., Miyata H., Ishiwata S., Kinosita K., Jr Axial rotation of sliding actin filaments revealed by single-fluorophore imaging. Proc Natl Acad Sci U S A. 1997 May 27;94(11):5646–5650. doi: 10.1073/pnas.94.11.5646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sase I., Okinaga T., Hoshi M., Feigenson G. W., Kinosita K., Jr Regulatory mechanisms of the acrosome reaction revealed by multiview microscopy of single starfish sperm. J Cell Biol. 1995 Nov;131(4):963–973. doi: 10.1083/jcb.131.4.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sellers J. R., Kachar B. Polarity and velocity of sliding filaments: control of direction by actin and of speed by myosin. Science. 1990 Jul 27;249(4967):406–408. doi: 10.1126/science.2377894. [DOI] [PubMed] [Google Scholar]
  53. Suzuki N., Miyata H., Ishiwata S., Kinosita K., Jr Preparation of bead-tailed actin filaments: estimation of the torque produced by the sliding force in an in vitro motility assay. Biophys J. 1996 Jan;70(1):401–408. doi: 10.1016/S0006-3495(96)79583-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Svoboda K., Schmidt C. F., Schnapp B. J., Block S. M. Direct observation of kinesin stepping by optical trapping interferometry. Nature. 1993 Oct 21;365(6448):721–727. doi: 10.1038/365721a0. [DOI] [PubMed] [Google Scholar]
  55. Taylor K. A., Schmitz H., Reedy M. C., Goldman Y. E., Franzini-Armstrong C., Sasaki H., Tregear R. T., Poole K., Lucaveche C., Edwards R. J. Tomographic 3D reconstruction of quick-frozen, Ca2+-activated contracting insect flight muscle. Cell. 1999 Nov 12;99(4):421–431. doi: 10.1016/s0092-8674(00)81528-7. [DOI] [PubMed] [Google Scholar]
  56. Toyoshima Y. Y., Kron S. J., McNally E. M., Niebling K. R., Toyoshima C., Spudich J. A. Myosin subfragment-1 is sufficient to move actin filaments in vitro. Nature. 1987 Aug 6;328(6130):536–539. doi: 10.1038/328536a0. [DOI] [PubMed] [Google Scholar]
  57. Toyoshima Y. Y., Toyoshima C., Spudich J. A. Bidirectional movement of actin filaments along tracks of myosin heads. Nature. 1989 Sep 14;341(6238):154–156. doi: 10.1038/341154a0. [DOI] [PubMed] [Google Scholar]
  58. Tsuda Y., Yasutake H., Ishijima A., Yanagida T. Torsional rigidity of single actin filaments and actin-actin bond breaking force under torsion measured directly by in vitro micromanipulation. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12937–12942. doi: 10.1073/pnas.93.23.12937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Vale R. D., Funatsu T., Pierce D. W., Romberg L., Harada Y., Yanagida T. Direct observation of single kinesin molecules moving along microtubules. Nature. 1996 Apr 4;380(6573):451–453. doi: 10.1038/380451a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yamada A., Ishii N., Takahashi K. Direction and speed of actin filaments moving along thick filaments isolated from molluscan smooth muscle. J Biochem. 1990 Sep;108(3):341–343. doi: 10.1093/oxfordjournals.jbchem.a123203. [DOI] [PubMed] [Google Scholar]
  61. Yanagida T., Nakase M., Nishiyama K., Oosawa F. Direct observation of motion of single F-actin filaments in the presence of myosin. Nature. 1984 Jan 5;307(5946):58–60. doi: 10.1038/307058a0. [DOI] [PubMed] [Google Scholar]
  62. Yasuda R., Noji H., Kinosita K., Jr, Yoshida M. F1-ATPase is a highly efficient molecular motor that rotates with discrete 120 degree steps. Cell. 1998 Jun 26;93(7):1117–1124. doi: 10.1016/s0092-8674(00)81456-7. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES