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.

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

“…The (β,α) data (open blue squares) refers to lever-arm orientation. The rigidity of the S1/GFP coordination for skeletal and cardiac isoforms was also demonstrated (Sun et al 2014). …”

“…Images were 1 sec exposures recorded over 1 min (60 exposures in a record). We identified single molecule events with quantized activation over background intensity or quantized photobleaching back to background from the points of light appearing in the image over time (Sun et al 2014). …”

“…Single relaxed skeletal myosin lever-arms in live zebrafish embryos were imaged and analyzed with orientation super-resolution for the first time using a photoactivatable tag on a myosin light chain expressed in the embryo (Sun et al 2014). Tag specificity was demonstrated by the simultaneous observation of 2-P fluorescence and SHG signals from the muscle ( Fig 7 ).…”

“…Single myosin orientation super-resolution measurements from the photoactivated RLC-PAGFP tagged myosin lever-arms in active isometric muscle were made just as described for relaxed muscle (Sun et al 2014). Fig 9 panel c shows dipole (open black triangles) and lever-arm orientation (open blue squares) in the active isometric muscle.…”

“…These disease linked mutants spanned the RLC structure and indicated position dependent change to lever-arm functionality. We adapted this single molecule technology to in vivo experimentation in zebrafish embryos (Sun et al 2014). …”