title: The Qdot-labeled actin super-resolution motility assay measures low-duty cycle muscle myosin step size.
abstract: Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescently labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdots were imaged in time with total internal reflection fluorescence microscopy and then spatially localized to 1-3 nm using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full-length β-cardiac myosin (MYH7). The average Qdot-actin velocity matches measurements with rhodamine-phalloidin-labeled actin. The sHMM Qdot-actin velocity histogram contains low-velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm and larger compliance compared to that of sHMM depending on the MYH7 surface concentration. Low-duty cycle skeletal and cardiac myosin present challenges for a single-molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions, and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space and then characterized using super-resolution. The approach provides a quick, quantitative, and inexpensive step size measurement for low-duty cycle muscle myosin.
authors: Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota 55905, USA.
authors: Yihua
authors: Y
authors: Wang
authors: Katalin
authors: K
authors: Ajtai
authors: Thomas P
authors: TP
authors: Burghardt
mesh_terms: D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000465:Algorithms; D000818:Animals; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D008747:Methylcellulose / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000032:analysis / Q000378:metabolism*; D015879:Myosin Subfragments / Q000032:analysis / Q000378:metabolism*; D010590:Phalloidine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D045663:Quantum Dots; D011817:Rabbits; D012235:Rhodamines / Q000032:analysis* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D013552:Swine
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D012235:Rhodamines; C504731:rhodamine-phalloidin; D010590:Phalloidine; D008747:Methylcellulose; D018995:Myosin Heavy Chains
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