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Title: Electron tomography of muscle cross- bridge by regulatory light chain labelling with APEX2
Authors: Mufeeda, Changaramvally Madathummal
Keywords: Science::Biological sciences
Issue Date: 2019
Source: Mufeeda, C. M. (2019). Electron tomography of muscle cross- bridge by regulatory light chain labelling with APEX2. Doctoral thesis, Nanyang Technological University, Singapore.
Abstract: Muscle contraction results from the cyclic interaction of myosin and actin by coupling energy of adenosine triphosphate (ATP) hydrolysis. During muscle contraction myosin and actin interact with each other through a structure called “cross-bridge”. Despite intense structural studies, the organisation of myosin cross-bridges on myosin filaments is not well understood. Myosin regulatory light chain (RLC) is one of the prominent proteins present at the lever arm domain of cross-bridges. Phosphorylation of RLC modulates cellular functions including muscle contraction. Moreover, RLC mutation is associated with cardiomyopathy. To understand the role of RLC in muscle contraction, high-resolution structure in its sarcomeric environment is required. Currently available high-resolution structure of myosin is inadequate to understand the role of RLC. A novel electron microscopy (EM) labelling technique based on APEX2, an engineered variant of soybean ascorbate peroxidase (APEX) protein is a promising technique to resolve the structure of specific protein in its native environment. In this study, the visualisation of the muscle cross-bridge organisation using APEX2 was attained. APEX2 provides the contrast EM by oxidation of di-aminobenzidine (DAB) substrate. For that purpose, fully functional RLCAPEX2 fusion protein was exchanged into muscle fibre. RLC-APEX2 exchanged muscle prepared in the relaxed state showed good ultrastructural preservation and good EM contrast upon chemical fixation. The presence of RLC-APEX2 in myosin cross-bridges permitted us to visualise structural features by electron tomography (ET) in relaxed state. Our standardised APEX2 based EM labelling protocol could be a promising tool for the labelling of other sarcomeric proteins in diseased states, thus permitting the direct visualisation and ultrastructural organisation of muscle proteins by ET.
Fulltext Permission: open
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