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Actin Sliding Velocities are Influenced by the Driving Forces of Actin-Myosin Binding

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Abstract

Unloaded shortening speeds, V, of muscle are thought to be limited by actin-bound myosin heads that resist shortening, or V = a · d · τ −1on where τ −1on is the rate at which myosin detaches from actin and d is myosin’s step size. The a-term describes the efficiency of force transmission between myosin heads, and has been shown to become less than one at low myosin densities in a motility assay. Molecules such as inorganic phosphate (P i ), and blebbistatin inhibit both V and actin-myosin strong binding kinetics suggesting a link between V and attachment kinetics. To determine whether these small molecules slow V by increasing resistance to actin sliding or by decreasing the efficiency of force transmission, a, we determine how inhibition of V by P i and blebbistatin changes the force exerted on actin filaments during an in vitro sliding assay, measured from changes in the rate, τ −1break , at which actin filaments break. Upon addition of 30 mM P i to a low (30 μM) [ATP] motility buffer V decreased from 1.8 to 1.3 μm s−1 and τ −1break from 0.029 to 0.018 s−1. Upon addition of 50 μM blebbistatin to a low [ATP] motility buffer, V decreased from 1.0 to 0.7 μm s−1 and τ −1break from 0.059 to 0.022 s−1. These results imply that blebbistatin and P i slow V by decreasing force transmission, a, not by increasing resistive forces, implying that actin-myosin attachment kinetics influence V.

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References

  1. Amrute-Nayak, M., M. Antognozzi, T. Scholz, H. Kojima, and B. Brenner. Inorganic phosphate binds to the empty nucleotide binding pocket of conventional myosin II. J. Biol. Chem. 283:3773–3781, 2008.

    Article  Google Scholar 

  2. Baker, J. E., C. Brosseau, P. Fagnant, and D. M. Warshaw. Myosin V processivity: multiple kinetic pathways for head-to-head coordination. J. Biol. Chem. 278:28533–28539, 2003.

    Article  Google Scholar 

  3. Baker, J. E., C. Brosseau, P. B. Joel, and D. M. Warshaw. The biochemical kinetics underlying actin movement generated by one and many skeletal muscle myosin molecules. Biophys. J. 82:2134–2147, 2002.

    Article  Google Scholar 

  4. Baker, J. E., I. Brust-Mascher, S. Ramachandran, L. E. LaConte, and D. D. Thomas. A large and distinct rotation of the myosin light chain domain occurs upon muscle contraction. Proc. Natl Acad. Sci. USA. 95:2944–2949, 1998.

    Article  Google Scholar 

  5. Baker, J. E., E. W. LaConte, I. Brust-Mascher, and D. Thomas. Mechanochemical coupling in spin-labeled, active, isometric muscle. Biophys. J. 77(5):2657–2665, 1999.

    Article  Google Scholar 

  6. Cooke, R. Actomyosin interaction in striated muscle. Physiol. Rev. 77:671–697, 1997.

    Google Scholar 

  7. Cooke, R., and E. Pate. The effects of ADP and phosphate on the contraction of muscle fibers. Biophys. J. 48:789–798, 1985.

    Article  Google Scholar 

  8. Dantzig, J. A., Y. E. Goldman, N. C. Millar, J. Lacktis, and E. Homsher. Reversal of the cross-bridge force-generating transition by photogeneration of phosphate in rabbit psoas muscle fibres. J. Physiol. 451:247–278, 1992.

    Google Scholar 

  9. Debold, E. P., J. P. Schmitt, J. R. Moore, J. B. Patlak, S. E. Beck, J. G. Seidman, C. Seidman, and D. M. Warshaw. Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse α-cardiac myosin in the laser trap assay. Am. J. Physiol. Heart Circ. Physiol. 293(1):H284–H291, 2007.

    Article  Google Scholar 

  10. Doi, M., and S. F. Edwards. The Theory of Polymer Dynamics; New York, NY. Oxford University Inc. 1986.

  11. Eisenberg, E., and T. L. Hill. Muscle contraction and free energy transduction in biological systems. Science 227:999–1006, 1985.

    Article  Google Scholar 

  12. Fabiato, A., and F. Fabiato. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. (Paris) 75:463–505, 1979.

    Google Scholar 

  13. Farman, G. P., K. Tachampa, R. Mateja, O. Cazorla, A. Lacampagne, and P. P. de Tombe. Blebbistatin: use as inhibitor of muscle contraction. Pflügers Archiv Eur. J. Physiol. 4552:995–1005, 2008.

    Article  Google Scholar 

  14. Finer, J. T., R. M. Simmons, and J. A. Spudich. Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368:113–119, 1994.

    Article  Google Scholar 

  15. Goldman, Y. E. Kinetics of the actomyosin ATPase in muscle fibers. Annu. Rev. Physiol. 49:637–654, 1987.

    Article  Google Scholar 

  16. Guilford, W. H., D. E. Dupuis, G. Kennedy, J. B. Patlak, and D. M. Warshaw. Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap. Biophys. J. 72:1006–1021, 1997.

    Article  Google Scholar 

  17. Harada, Y., A. Noguchi, A. Kishino, and T. Yanagida. Sliding movement of single actin filaments on one-headed myosin filaments. Nature 326:805–808, 1987.

    Article  Google Scholar 

  18. Harris, D. E., and D. M. Warshaw. Smooth and skeletal muscle myosin both exhibit low duty cycles at zero load in vitro. J. Biol. Chem. 268:14764–14768, 1993.

    Google Scholar 

  19. Herrmann, C., J. Wray, F. Travers, and T. Barman. Research Article. Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPases. An example of an uncompetitive inhibitor. Biochemistry 31:12227–12232, 1992.

    Article  Google Scholar 

  20. Hooft, A. M., E. J. Maki, K. K. Cox, and J. E. Baker. An accelerated state of myosin-based actin motility. Biochemistry 46:3513–3520, 2007.

    Article  Google Scholar 

  21. Huxley, A. F. Muscle structure and theories of contraction. Prog. Biophys. 7:255–315, 1957.

    Google Scholar 

  22. Huxley, H. E. The mechanism of muscular contraction. Science 164:1356–1365, 1969.

    Article  Google Scholar 

  23. Jackson, D. R. J., and J. E. Baker. The energentics of allosteric regulation of ADP release from myosin heads. Phys. Chem. Chem. Phys. 11:4808–4814, 2009.

    Article  Google Scholar 

  24. Kovacs, M., J. Toth, C. Hetenyi, A. Malnasi-Csizmadia, and J. R. Sellers. Mechanism of Blebbistatin Inhibition of Myosin II. J. Biol. Chem. 279:35557–35563, 2004.

    Article  Google Scholar 

  25. Kovács, M., J. Tóth, C. Hetényi, A. Málnási-Csizmadia, and J. R. Sellers. Mechanism of blebbistatin inhibition of myosin II. J. Biol. Chem. 279(34):35557–35563, 2004.

    Article  Google Scholar 

  26. Kron, S. J., and J. A. Spudich. Fluorescent actin filaments move on myosin fixed to a glass surface. Proc. Natl Acad. Sci. USA. 83:6272–6276, 1986.

    Article  Google Scholar 

  27. Lehrer, S. S., and M. A. Geeves. The muscle thin filament as a classical cooperative/allosteric regulatory system. J. Mol. Biol. 277:1081–1089, 1998.

    Article  Google Scholar 

  28. Limouze, J., A. F. Straight, T. Mitchison, and J. R. Sellers. Specificity of blebbistatin, an inhibitor of myosin II. J. Muscle Res. Cell Motil. 25:337–341, 2004.

    Article  Google Scholar 

  29. Lymn, R. W., and E. W. Taylor. Mechanism of the actomyosin ATPase: effect of actin on the ATP hydrolysis step. Biochemistry 10:4617–4624, 1971.

    Article  Google Scholar 

  30. Molloy, J. E., J. E. Burns, J. Kendrick-Jones, R. T. Tregear, and D. C. White. Movement and force produced by a single myosin head. Nature 378:209–212, 1995.

    Article  Google Scholar 

  31. Pardee, J. D., and J. A. Spudich. Methods Enzymol. 85 Pt B:164–181, 1982.

    Google Scholar 

  32. Pate, E., and R. Cooke. A model of crossbridge action: the effects of ATP, ADP, and Pi. J. Muscle Res. Cell Motil. 10:181–196, 1989.

    Article  Google Scholar 

  33. Ramamurthy, B., C. M. Yengo, A. F. Straight, T. J. Mitchison, and H. L. Sweeney. Kinetic mechanism of blebbistatin inhibition of nonmuscle myosin IIB. Biochemistry 43:14832–14839, 2004.

    Article  Google Scholar 

  34. Reedy, M. K., K. C. Holmes, and R. T. Tregear. Induced changes in orientation of the cross-bridges of glycerinated insect flight muscle. Nature 207:1276–1280, 1965.

    Article  Google Scholar 

  35. Regnier, M., P. B. Chase, and D. A. Martyn. COntractile properties of rabbit psoas muscle fibres inhibited by beryllium fluoride. J. Muscle Res. Cell Motil. 20:425–432, 1999.

    Article  Google Scholar 

  36. Sakamoto, T., J. Limouze, C. A. Combs, A. F. Straight, and J. R. Sellers. Blebbistatin, a myosin II inhibitor. Biochemistry 44:584–588, 2005.

    Article  Google Scholar 

  37. Sellers, J. R. Myosins (2nd ed.). Ed.: Oxford University Press, Oxford UK, 1999.

    Google Scholar 

  38. Shaw, M. A., E. M. Ostap, and Y. E. Goldman. Mechanism of inhibition of skeletal muscle actomyosin by N-benzyl-p-toluenesulfonamide. Biochemistry 42:6128–6135, 2003.

    Article  Google Scholar 

  39. Spudich, J. A. How molecular motors work.Nature 372:515–518, 1994.

    Google Scholar 

  40. Takagi, Y., E. E. Homsher, Y. E. Goldman, and H. Shuman. Force generation in single conventional actomyosin complexes under high dynamic load. Biophys. J. 90:1295–1307, 2006.

    Article  Google Scholar 

  41. Tsuda, Y., H. Yasutake, A. Ishijima, and T. Yanagida. Torsional rigidity of single actin filaments and actin–actin bond breaking force under torsion measured directly by in vitro micromanipulation. Proc. Natl Acad. Sci. USA. 93:12937–12942, 1996.

    Google Scholar 

  42. Uyeda, T. Q., S. J. Kron, and J. A. Spudich. Myosin step size: Estimation from slow sliding movement of actin over low densities of heavy meromyosin. J. Mol. Biol. 214:699–710, 1990.

    Article  Google Scholar 

  43. Warshaw, D. M., J. M. Desrosiers, S. S. Work, and K. M. Trybus. Smooth muscle myosin cross-bridge interactions modulate actin filament sliding velocity in vitro. J. Cell Biol. 111:453–463, 1990.

    Article  Google Scholar 

  44. Warshaw, D. M., J. M. Desrosiers, S. S. Work, and K. M. Trybus. Effects of MgATP, MgADP, and Pi on actin movement by smooth muscle myosin. J. Biol. Chem. 266:24339–24343, 1991.

    Google Scholar 

  45. Zhao, L., E. Pate, A. J. Baker, and R. Cooke. The myosin catalytic domain does not rotate during the working power stroke. Biophys. J. 69:994–999, 1995.

    Article  Google Scholar 

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Acknowledgments

We thank Kevin Facemyer for his helpful suggestions and Olivia John for purifying proteins. This study was funded by NIH 1R01HL090938.

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  1. Department of Biochemistry and Molecular Biology, University of Nevada Reno School of Medicine, 1664 N. Virginia St. Howard Building, MS 330, Reno, NV, 89557, USA

    Travis J. Stewart, Del Ray Jackson Jr., Ryan D. Smith, Steven F. Shannon, Christine R. Cremo & Josh E. Baker

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Correspondence to Josh E. Baker.

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Associate Editor Frank C.-P. Yin oversaw the review of this article.

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Stewart, T.J., Jackson, D.R., Smith, R.D. et al. Actin Sliding Velocities are Influenced by the Driving Forces of Actin-Myosin Binding. Cel. Mol. Bioeng. 6, 26–37 (2013). https://doi.org/10.1007/s12195-013-0274-y

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