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Journal of Muscle Research and Cell Motility

, Volume 36, Issue 6, pp 463–477 | Cite as

In vitro and in vivo single myosin step-sizes in striated muscle

  • Thomas P. Burghardt
  • Xiaojing Sun
  • Yihua Wang
  • Katalin Ajtai
Research Article

Abstract

Myosin in muscle transduces ATP free energy into the mechanical work of moving actin. It has a motor domain transducer containing ATP and actin binding sites, and, mechanical elements coupling motor impulse to the myosin filament backbone providing transduction/mechanical-coupling. The mechanical coupler is a lever-arm stabilized by bound essential and regulatory light chains. The lever-arm rotates cyclically to impel bound filamentous actin. Linear actin displacement due to lever-arm rotation is the myosin step-size. A high-throughput quantum dot labeled actin in vitro motility assay (Qdot assay) measures motor step-size in the context of an ensemble of actomyosin interactions. The ensemble context imposes a constant velocity constraint for myosins interacting with one actin filament. In a cardiac myosin producing multiple step-sizes, a “second characterization” is step-frequency that adjusts longer step-size to lower frequency maintaining a linear actin velocity identical to that from a shorter step-size and higher frequency actomyosin cycle. The step-frequency characteristic involves and integrates myosin enzyme kinetics, mechanical strain, and other ensemble affected characteristics. The high-throughput Qdot assay suits a new paradigm calling for wide surveillance of the vast number of disease or aging relevant myosin isoforms that contrasts with the alternative model calling for exhaustive research on a tiny subset myosin forms. The zebrafish embryo assay (Z assay) performs single myosin step-size and step-frequency assaying in vivo combining single myosin mechanical and whole muscle physiological characterizations in one model organism. The Qdot and Z assays cover “bottom-up” and “top-down” assaying of myosin characteristics.

Keywords

In vivo single myosin imaging Cardiac myosin step-frequency Cardiac myosin step-size High throughput Qdot assay Second characterization Skeletal muscle myosin mechanics 

Notes

Acknowledgments

This work was supported by National Institutes of Health grant R01AR049277.

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Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Thomas P. Burghardt
    • 1
    • 2
  • Xiaojing Sun
    • 1
  • Yihua Wang
    • 1
  • Katalin Ajtai
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyMayo Clinic RochesterRochesterUSA
  2. 2.Department of Physiology and Biomedical EngineeringMayo Clinic RochesterRochesterUSA

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