In vitro and in vivo single myosin step-sizes in striated muscle
- 235 Downloads
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.
KeywordsIn vivo single myosin imaging Cardiac myosin step-frequency Cardiac myosin step-size High throughput Qdot assay Second characterization Skeletal muscle myosin mechanics
This work was supported by National Institutes of Health grant R01AR049277.
- Burns CG, Milan DJ, Grande EJ, Rottbauer W, MacRae CA, Fishman MC (2005) High-throughput assay for small molecules that modulate zebrafish embryonic heart rate. Nat Chem Biol 1:263–264. http://www.nature.com/nchembio/journal/v1/n5/suppinfo/nchembio732_S1.html
- Howe K et al. (2013) The zebrafish reference genome sequence and its relationship to the human genome Nature 496:498–503 doi: 10.1038/nature12111. http://www.nature.com/nature/journal/vaop/ncurrent/abs/nature12111.html#supplementary-information
- Hurlstone AFL et al. (2003) The Wnt/[beta]-catenin pathway regulates cardiac valve formation. Nature 425:633–637. http://www.nature.com/nature/journal/v425/n6958/suppinfo/nature02028_S1.html
- Miller Mark S, Farman Gerrie P, Braddock Joan M, Soto-Adames Felipe N, Irving Thomas C, Vigoreaux Jim O, Maughan David W (2011) Regulatory light chain phosphorylation and n-terminal extension increase cross-bridge binding and power output in drosophila at in vivo myofilament lattice spacing. Biophys J 100:1737–1746. doi: 10.1016/j.bpj.2011.02.028 PubMedCentralCrossRefPubMedGoogle Scholar
- Wang L, Muthu P, Szczesna-Cordary D, Kawai M (2013a) Characterizations of myosin essential light chain’s N-terminal truncation mutant Δ43 in transgenic mouse papillary muscles by using tension transients in response to sinusoidal length alterations. J Muscle Res Cell Motil 34:93–105CrossRefPubMedGoogle Scholar