Abstract
Recent research efforts from several groups have addressed the question of whether the amplitude of myosin's unitary step size is proportional to the length of the neck region. Unconventional myosin V, which has an extended neck region with 6IQ motifs, provides a natural template by which to test the lever arm model via mutational analysis. The most stringent test requires that a series of single-headed molecules from the same myosin class be analyzed. Here we characterized the unitary mechanics of three single-headed fragments of myosin V expressed in the baculovirus/insect cell system. Each construct consisted of the motor domain (MD) and a variable number of IQ motifs (MD2IQ, MD4IQ and MD6IQ) that bind calmodulin, followed by an epitope tag so that the molecule can be attached to the nitrocellulose surface via an antibody. The results show a correlation between the unitary step size and the number of IQ motifs, confirming that the myosin neck region acts as a lever. The step size of MD2IQ is twice that observed from single-headed subfragments of class II myosins with the same neck length. Our results are discussed in relation to data obtained concurrently from other laboratories with similar constructs.
Similar content being viewed by others
References
Anson M, Geeves MA, Kurzawa SE and Manstein DJ (1996) Myosin motors with artificial lever arms. EMBO J 15: 6069–6074.
Burgess S, Walker M, Wang F, Sellers JR, White HD, Knight PJ and Trinick J (2002) The prepower stroke conformation of myosin V. J Cell Biol 159: 983–991.
Dominguez R, Freyzon Y, Trybus KM and Cohen C (1998) Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain:visualization of the pre-power stroke state. Cell 94: 559–571.
Finer JT, Mehta AD and Spudich JA (1995) Characterization of single actin-myosin interactions. Biophys J 68: 291S–296S.
Forkey JN, Quinlan ME, Shaw MA, Corrie JE and Goldman YE (2003) Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization. Nature 422: 399–404.
Guilford WH, Dupuis DE, Kennedy G, Wu J, Patlak JB and Warshaw DM (1997) Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap. Biophys J 72: 1006–1021.
Houdusse A, Kalabokis VN, Himmel D, Szent-Gyorgyi AG and Cohen C (1999) Atomic structure of scallop myosin subfragment S1 complexed with MgADP:a novel conformation of the myosin head. Cell 97: 459–470.
Houdusse A, Szent-Gyorgyi AG and Cohen C (2000) Three conformational states of scallop myosin S1. Proc Natl Acad Sci USA 97: 11238–11243.
Howard J (2001) Mechanics of Motor Proteins and the Cytoskeleton. Sinauer Associates, Inc., Sunderland, MA. p. 145.
Howard J and Spudich JA (1996) Is the lever arm of myosin a molecular elastic element? Proc Natl Acad Sci USA 93: 4462–4464.
Huxley HE (1990) Sliding laments and molecular motile systems. J Biol Chem 265: 8347–8350.
Kad NM, Rovner AS, Fagnant PM, Joel PB, Kennedy G, Patlak JB, Warshaw D and Trybus KM (2003) A mutant heterodimeric myosin with one inactive head generates maximal displacement. J Cell Biol, in press.
Kohler D, Ruff C, Meyhofer E and Bahler M (2003) Different degrees of lever arm rotation control myosin step size. J. Cell Biol. 161: 237–241.
Kollmar M, Durrwang U, Kliche W, Manstein DJ and Kull FJ (2002) Crystal structure of the motor domain of a class-I myosin. EMBO J 21: 2517–2525.
Lowey S, Waller GS and Bandman E (1991) Neonatal and adult myosin heavy chains form homodimers during avian skeletal muscle development. J Cell Biol 113: 303–310.
Mehta A (2001) Myosin learns to walk. J Cell Sci 114: 1981–1998.
Mehta AD, Rock RS, Rief M, Spudich JA, Mooseker MS and CheneyRE (1999) Myosin-V is a processive actin-based motor. Nature 400: 590–593.
Mercer JA, Seperack PK, Strobel MC, Copeland NG and Jenkins NA (1991) Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 349: 709–713.
Mermall V, Post PL and Mooseker MS (1998) Unconventional myosins in cell movement, membrane trasac, and signal transduction. Science 279: 527–533.
Molloy JE, Burns JE, Kendrick-Jones J, Tregear RT and White DC (1995) Movement and force produced by a single myosin head. Nature 378: 209–212.
Moore LA, Arrizubieta MJ, Tidyman WE, Herman LA and Bandman E (1992) Analysis of the chicken fast myosin heavy chain family. Localization of isoform-specific antibody epitopes and regions of divergence. J Mol Biol 225: 1143–1151.
Moore JR, Krementsova EB, Trybus KM and Warshaw DM (2001) Myosin V exhibits a high duty cycle and large unitary displacement. J Cell Biol 155: 625–635.
Moore JR, Krementsova E, Trybus KM and Warshaw DM (2002) Molecular mechanics of expressed-monomeric myosin V fragments with different neck lengths. Biophys J 82 (1): 409a.
O'Reilly DR, Miller LK and Luckow VA (1992) Baculovirus Expression Vectors. A Laboratory Manual. W. H. Freeman and Co., New York.
Purcell TJ, Morris C, Spudich JA and Sweeney HL (2002) Role of the lever arm in the processive stepping of myosin V. Proc Natl Acad Sci USA 99: 14159–14164.
Rock RS, Rice SE, Wells AL, Purcell TJ, Spudich JA and Sweeney HL (2001) Myosin VI is a processive motor with a large step size. Proc Natl Acad Sci USA 98: 13655–13659.
Ruff C, Furch M, Brenner B, Manstein DJ and Meyhofer E (2001) Single-molecule tracking of myosins with genetically engineered amplier domains. Nat Struct Biol 8: 226–229.
Sakamoto T, Wang F, Schmitz S, Xu Y, Molloy JE, Veigel C and Sellers JR (2003) Neck length and processivity of myosin V. J Biol Chem 278: 29201–29207.
Schott DH, Collins RN and Bretscher A (2002) Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length. J Cell Biol 156: 35–39.
Shih WM, Gryczynski Z, Lakowicz JR and Spudich JA (2000) A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin. Cell 102: 683–694.
Tanaka H, Homma K, Iwane AH, Katayama E, Ikebe R, Saito J, Yanagida T and Ikebe M (2002) The motor domain determines the large step of myosin-V. Nature 415: 192–195.
Trybus KM (1994) Regulation of expressed truncated smooth muscle myosins. Role of the essential light chain and tail length. J Biol Chem 269: 20819–20822.
Trybus KM, Freyzon Y, Faust LZ and Sweeney HL (1997) Spare the rod, spoil the regulation:necessity for a myosin rod. Proc Natl Acad Sci USA 94: 48–52.
Trybus KM, Krementsova E and Freyzon Y (1999) Kinetic characterization of a monomeric unconventional myosin V construct. J Biol Chem 274: 27448–27456.
Tyska MJ and Warshaw DM (2002) The myosin power stroke. Cell Motil Cytoskeleton 51: 1–15.
Tyska MJ, Dupuis DE, Guilford WH, Patlak JB, Waller GS, Trybus KM, Warshaw DM and Lowey S (1999) Two heads of myosin are better than one for generating force and motion. Proc Natl Acad Sci USA 96: 4402–4407.
Uyeda TQ, Abramson PD and Spudich JA (1996) The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc Natl Acad Sci USA 93: 4459–4464.
Veigel C, Bartoo ML, White DC, Sparrow JC and Molloy JE (1998) The stiffiness of rabbit skeletal actomyosin cross-bridges determined with an optical tweezers. Biophys J 75: 1424–1438.
Veigel C, Wang F, Bartoo ML, Sellers JR and Molloy JE (2002) The gated gait of the processive molecular motor, myosin V. Nat Cell Biol 4: 59–65.
Walker ML, Burgess SA, Sellers JR, Wang F, Hammer JA, III, Trinick J and Knight PJ (2000) Two-headed binding of a processive myosin to F-actin. Nature 405: 804–807.
Warshaw DM, Desrosiers JM, Work SS and Trybus KM (1990) Smooth muscle myosin cross-bridge interactions modulate actin lament sliding velocity in vitro. J Cell Biol 111: 453–463.
Warshaw DM, Guilford WH, Freyzon Y, Krementsova E, Palmiter KA, Tyska MJ, Baker JE and Trybus KM (2000) The light chain binding domain of expressed smooth muscle heavy meromyosin acts as a mechanical lever. J Biol Chem 275: 37167–37172.
Wells JA and Yount RG (1979) Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment. Proc Natl Acad Sci USA 76: 4966–4970.
Work SS and Warshaw DM (1992) Computer-assisted tracking of actin lament motility. Anal Biochem 202: 275–285.
Yanagida T, Kitamura K, Tanaka H, Hikikoshi IA and Esaki S (2000) Single molecule analysis of the actomyosin motor. Curr Opin Cell Biol 12: 20–25.
Yengo CM, De la Cruz EM, Safer D, Ostap EM and Sweeney HL (2002) Kinetic characterization of the weak binding states of myosin V. Biochemistry 41: 8508–8517.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Moore, J.R., Krementsova, E.B., Trybus, K.M. et al. Does the myosin V neck region act as a lever?. J Muscle Res Cell Motil 25, 29–35 (2004). https://doi.org/10.1023/B:JURE.0000021394.48560.71
Issue Date:
DOI: https://doi.org/10.1023/B:JURE.0000021394.48560.71