Dataset Information

In vivo myosin step-size from zebrafish skeletal muscle.

ABSTRACT: Muscle myosins transduce ATP free energy into actin displacement to power contraction. In vivo, myosin side chains are modified post-translationally under native conditions, potentially impacting function. Single myosin detection provides the 'bottom-up' myosin characterization probing basic mechanisms without ambiguities inherent to ensemble observation. Macroscopic muscle physiological experimentation provides the definitive 'top-down' phenotype characterizations that are the concerns in translational medicine. In vivo single myosin detection in muscle from zebrafish embryo models for human muscle fulfils ambitions for both bottom-up and top-down experimentation. A photoactivatable green fluorescent protein (GFP)-tagged myosin light chain expressed in transgenic zebrafish skeletal muscle specifically modifies the myosin lever-arm. Strychnine induces the simultaneous contraction of the bilateral tail muscles in a live embryo, causing them to be isometric while active. Highly inclined thin illumination excites the GFP tag of single lever-arms and its super-resolution orientation is measured from an active isometric muscle over a time sequence covering many transduction cycles. Consecutive frame lever-arm angular displacement converts to step-size by its product with the estimated lever-arm length. About 17% of the active myosin steps that fall between 2 and 7 nm are implicated as powerstrokes because they are beyond displacements detected from either relaxed or ATP-depleted (rigor) muscle.

SUBMITTER: Burghardt TP

PROVIDER: S-EPMC4892436 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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In vivo myosin step-size from zebrafish skeletal muscle.

Burghardt Thomas P TP Ajtai Katalin K Sun Xiaojing X Takubo Naoko N Wang Yihua Y

Open biology 20160525 5

Muscle myosins transduce ATP free energy into actin displacement to power contraction. In vivo, myosin side chains are modified post-translationally under native conditions, potentially impacting function. Single myosin detection provides the 'bottom-up' myosin characterization probing basic mechanisms without ambiguities inherent to ensemble observation. Macroscopic muscle physiological experimentation provides the definitive 'top-down' phenotype characterizations that are the concerns in trans ...[more]

PMID: 27249818

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Project description: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.

Dataset Information

In vivo myosin step-size from zebrafish skeletal muscle.

Publications

In vivo myosin step-size from zebrafish skeletal muscle.

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