Myofibril structure of insect flight muscle (IFM) is being studied by coordinated use of electron microscopy, X-ray diffraction, quantitative microscopy and biochemical analysis, and emphasis on the structure and behavior of myosin crossbridges. Crossbridges are the primary movers in muscle contraction and most non-muscle motility, and their highly ordered lattice arrangement in IFM permits averaging methods to extract crossbridge structure indifferent states which can be related to different phases of their ratchet-like cycle. X-ray and optical diffraction criteria will guide our search for better fixation of actin and crossbridges by modifying glutaraldehyde with tannic acid, diamines, and polyethylene-glycol triazine. Quick-freeze and freeze-substitution methods will be optimized for thin- sectioning of stable states like rigor and relaxed before attempting the study of dynamic structural transitions. 3-D computer reconstructions from 10-25 nm sections, based on multiple tilt views and on oblique cross sections, will be completed for rigor and applied to a group of necleotide-induced states, to establish definitively the variation in size, shape and domain alignment of crossbridges. Computer modelling will explore how EM structure of actin and crossbridges of rigor can be reconciled with X-ray intensity modelling. EM-mechanical correlation studies of single fibers will characterize two new crossbridge configurations encountered in glycol-AMPPNP- induced progression of states, and will explore rigor induction and tension to examine the length of the power-stroke, strength of bridge attachment, and effects of crossbridge """"""""slippage"""""""". We expect to finish coordinated collaborative studies of crossbridge orientation by EM/X-ray and spin-labelling, and antibody localization of troponin within the crossbridge lattice. IN a different approach, quantitative microscopy (EM and interference), buoyant density banding in Percoll, and X-ray diffraction measurement of lattice spacing will be coordinated in studies of dry mass content of myofilaments and fibrils, to produce mass distribution profiles of insect and rabbit sarcomeres, ascertain absolute protein conon in sarcomere cross- bands, and to resolve an anomalous discrepancy between filament mass and whole fibril mass.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
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Molecular Cytology Study Section (CTY)
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Duke University
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Fee, Lanette; Lin, Weili; Qiu, Feng et al. (2017) Myosin II sequences for Lethocerus indicus. J Muscle Res Cell Motil 38:193-200
Hu, Zhongjun; Taylor, Dianne W; Edwards, Robert J et al. (2017) Coupling between myosin head conformation and the thick filament backbone structure. J Struct Biol 200:334-342
Hu, Zhongjun; Taylor, Dianne W; Reedy, Michael K et al. (2016) Structure of myosin filaments from relaxed Lethocerus flight muscle by cryo-EM at 6 Å resolution. Sci Adv 2:e1600058
Arakelian, Claudia; Warrington, Anthony; Winkler, Hanspeter et al. (2015) Myosin S2 origins track evolution of strong binding on actin by azimuthal rolling of motor domain. Biophys J 108:1495-1502
Wu, Shenping; Liu, Jun; Reedy, Mary C et al. (2012) Structural changes in isometrically contracting insect flight muscle trapped following a mechanical perturbation. PLoS One 7:e39422
Perz-Edwards, Robert J; Reedy, Michael K (2011) Electron microscopy and x-ray diffraction evidence for two Z-band structural states. Biophys J 101:709-17
Perz-Edwards, Robert J; Irving, Thomas C; Baumann, Bruce A J et al. (2011) X-ray diffraction evidence for myosin-troponin connections and tropomyosin movement during stretch activation of insect flight muscle. Proc Natl Acad Sci U S A 108:120-5
Wu, Shenping; Liu, Jun; Reedy, Mary C et al. (2010) Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions. PLoS One 5:
Wu, Shenping; Liu, Jun; Reedy, Mary C et al. (2009) Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle. J Struct Biol 168:485-502
Bekyarova, T I; Reedy, M C; Baumann, B A J et al. (2008) Reverse actin sliding triggers strong myosin binding that moves tropomyosin. Proc Natl Acad Sci U S A 105:10372-7

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