In Vivo Orientation of Single Myosins in a Zebrafish Embryo

Cardiac and skeletal myosin is highly organized in the muscle lattice where it powers contraction by transducing ATP free energy into the mechanical work of moving actin in a mechanism known as transduction/mechanical coupling. While muscle myosin can move actin in vitro, its in vivo environment is crowded and constrained by the fiber lattice. In vivo, myosin side chains are modified during- and post-translation by mutation, phosphorylation, deamidation, and oxidation under normal, diseased, or aging conditions and all potentially impacting transduction/mechanical coupling. Single myosin detection provides highly prized "bottom-up" quantitative characterization of myosin that tests hypotheses without the ambiguities inherent in ensemble derived observations. The marriage of in vivo and single myosin detection to study human cardiac or skeletal muscle contraction in zebrafish embryo models is a multi-scaled technology for basic and translational research. It allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism levels of phenotype with confidence that all interactions and modifications are appropriately contributing their impact to myosin conformation. In vivo single myosin lever-arm orientation was observed at super-resolution using a photoactivatable GFP (PAGFP) tagged myosin light chain expressed in zebrafish skeletal muscle. Imaging was aided by an innovative microfluidic design for embryo confinement. Tag specificity was demonstrated by the simultaneous observation of 2-photon fluorescence emission and second harmonic generation (SHG) from myosin. Single molecule detection used highly inclined and laminated optical sheet (HILO) illumination and was verified by quantized photoactivation or photobleaching. Single molecule emission patterns from relaxed muscle indicated a highly orientationally confined lever-arm orientation. Results demonstrate detection of single myosin orientation in vivo. The zebrafish muscle system serves as an in vivo model for human disease and aging effects on myosin. Research supported by NIH R01AR049277 and R01HL095572.