title: In vivo orientation of single myosin lever arms in zebrafish skeletal muscle.
abstract: Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo.
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Sun
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Ekker
authors.affiliation: School of Molecular Biosciences, Washington State University, Pullman, Washington.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Shelden
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota.
authors.forename: Naoko
authors.initials: N
authors.lastname: Takubo
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota.
authors.forename: Yihua
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota; Department of Physiology and Biomedical Engineering, Mayo Clinic Rochester, Rochester, Minnesota. Electronic address: burghardt@mayo.edu.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Burghardt
mesh_terms: D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D062105:Molecular Docking Simulation; D009126:Muscle Relaxation; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000502:physiology; D009206:Myocardium / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D015027:Zebrafish
chemical_list: D018994:Myosin Light Chains; D049452:Green Fluorescent Proteins; D009218:Myosins