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25 February 2009 Structural and molecular conformation of myosin in intact muscle fibers by second harmonic generation
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Abstract
Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored with regard to intrinsic SHG in highly ordered biological samples. As shown by fractional extraction of proteins, myosin is the source of SHG signal in skeletal muscle. SHG is highly dependent on symmetries and provides selective information on the structural order and orientation of the emitting proteins and the dynamics of myosin molecules responsible for the mechano-chemical transduction during contraction. We characterise the polarization-dependence of SHG intensity in three different physiological states: resting, rigor and isometric tetanic contraction in a sarcomere length range between 2.0 μm and 4.0 μm. The orientation of motor domains of the myosin molecules is dependent on their physiological states and modulate the SHG signal. We can discriminate the orientation of the emitting dipoles in four different molecular conformations of myosin heads in intact fibers during isometric contraction, in resting and rigor. We estimate the contribution of the myosin motor domain to the total second order bulk susceptibility from its molecular structure and its functional conformation. We demonstrate that SHG is sensitive to the fraction of ordered myosin heads by disrupting the order of myosin heads in rigor with an ATP analog. We estimate the fraction of myosin motors generating the isometric force in the active muscle fiber from the dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an isometric contraction.
© (2009) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, M. Linari, G. Piazzesi, V. Lombardi, and F. S. Pavone "Structural and molecular conformation of myosin in intact muscle fibers by second harmonic generation", Proc. SPIE 7183, Multiphoton Microscopy in the Biomedical Sciences IX, 71831W (25 February 2009); https://doi.org/10.1117/12.809721
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