In vivo myosin step-size from zebrafish skeletal muscle

• Published in: Open biology Vol. 6; no. 5; p. 160075
• Main Authors: Burghardt, Thomas P., Ajtai, Katalin, Sun, Xiaojing, Takubo, Naoko, Wang, Yihua
• Format: Journal Article
• Language: English
• Published: England The Royal Society 01.05.2016
• Subjects: Actins - metabolism; Adenosine Triphosphate - metabolism; Animals; Animals, Genetically Modified; Binding Sites; Green Fluorescent Proteins - metabolism; Highly Inclined Thin Illumination; Humans; Muscle, Skeletal - drug effects; Muscle, Skeletal - embryology; Muscle, Skeletal - metabolism; Myosin Light Chains - chemistry; Myosin Light Chains - genetics; Myosin Light Chains - metabolism; Single Myosin Detection; single myosin detection in vivo; Strychnine - pharmacology; Strychnine Induced Contraction; Transgenic Zebrafish Skeletal Muscle; Zebrafish - embryology; Zebrafish - genetics; Zebrafish Skeletal Myosin Powerstroke; Zebrafish Skeletal Myosin Step-Size; transgenic zebrafish skeletal muscle; zebrafish skeletal myosin step-size; zebrafish skeletal myosin powerstroke; strychnine induced contraction; single myosin detection in vivo; highly inclined thin illumination
• ISSN: 2046-2441; 2046-2441
• DOI: 10.1098/rsob.160075

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.