The long term goal of this research project is to understand the molecular mechanism of force production through 3-D visualization of myosin molecular motors in situ in muscle. The research focuses on the structure of the large waterbug Lethocerus sp. because its filament lattice is the best ordered of all known muscles types thereby making it an excellent candidate for 3-D imaging as well as facilitating the trapping of many myosin motors into similar states. Lethocerus, like many insects, utilize a stretch activation mechanism to operate their flight muscles. Stretch activation also occurs in vertebrate striated and cardiac muscle, where in the case of cardiac muscle, it is an important part of the rhythmic contractions. Specimen preparation emphasizes rapid freezing and freeze substitution which traps molecular motions with millisecond time resolution. The structure of isolated Lethocerus thick filament in the relaxed state will be investigated using electron cryomicroscopy. Myosin motors during isometric contraction itself and following mechanical perturbations such as quick stretch and release will be trapped by fast freezing and imaged in 3-D with the specific aim of obtaining a higher resolution structure. Rigor fibers swollen in low ionic strength buffers enhance the visibility of the 1-helical coiled-coil domain that links the myosin head to the thick filament backbone.
The specific aim of this study will be to derive structural rules that define the position on the thick filament from which force producing myosin heads must originate and thereby test a new model for the weak-to-strong binding transi- tion in myosin. Specimens with enhanced numbers of weak binding myosin heads will be trapped by rapid freezing to further improve their characterization. Particular emphasis will be place on myosin heads binding to troponin, which may be important players in the stretch activation mechanism of muscle contraction. Improvements to increase structure homogeneity are pro- posed so that higher resolution images of active molecular motors can be obtained to better de- fine structural intermediates in the weak-to-strong transition and in force production itself. We will utilize electron tomography to obtain 3-D images of individual muscle motors and use multivariate data analysis to identify groups having similar structure for subsequent averaging to improve the signal-to-noise ratio and resolution. Continued refinements of our unique tomographic methods are proposed in order to increase resolution, improve the reconstructions and the rate at which 3-D maps can be produced. Atomic models based on the crystal structures of actin and myosin will be quantitatively fit within the envelope of the reconstruction and used to make pre- dictions of domain orientations, actin binding affinity and progress through the working stroke.

Public Health Relevance

One of the characteristics of cardiac muscle is its response to a stretch, which generally causes a delayed increase in tension. The molecular basis of stretch activation is unknown. Stretch activation is critical to flight by certain genera of insects making them an excellent model system with which to investigate this phenomenon.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-IMST-J (03))
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Flicker, Paula F
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Florida State University
Schools of Arts and Sciences
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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
Rusu, Mara; Hu, Zhongjun; Taylor, Kenneth A et al. (2017) Structure of isolated Z-disks from honeybee flight muscle. J Muscle Res Cell Motil 38:241-250
Hu, Guiqing; Taylor, Dianne W; Liu, Jun et al. (2017) Identification of interfaces involved in weak interactions with application to F-actin-aldolase rafts. J Struct Biol :
Banerjee, Chaity; Hu, Zhongjun; Huang, Zhong et al. (2017) The structure of the actin-smooth muscle myosin motor domain complex in the rigor state. J Struct Biol 200:325-333
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 108:1495-502
Winkler, Hanspeter; Taylor, Kenneth A (2013) Marker-free dual-axis tilt series alignment. J Struct Biol 182:117-24
Winkler, Hanspeter; Wu, Shenping; Taylor, Kenneth A (2013) Electron tomography of paracrystalline 2D arrays. Methods Mol Biol 955:427-60
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
Luther, Pradeep K; Winkler, Hanspeter; Taylor, Kenneth et al. (2011) Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle. Proc Natl Acad Sci U S A 108:11423-8

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