Exciting recent progress promises continued revelations from time-resolved structural analysis of motor molecules in muscle, to clarify individualmotor actions as well as their integrated ensemble performance in whole muscles. High paracrystalline order of glycerinated insect flight muscle (IFM)from giant waterbug Lethocerus gives distinctive X-ray diffraction (X-ray) from each physiological state, and allows unexcelled thin-section electron microscope (EM) images of myosin crossbridges. Comparing image Fouriers with native X-ray patterns measures and validates fidelity of thin-section EM. New synchrotron X-ray beamlines and detectors allow new methods. We will coordinate time-sliced synchrotron X-ray diffraction with EM 3- D reconstructions (3-Ds) from quick-frozen fibers to integrate mechanical with structural data for millisecond-timed snapshots of the myosin power stroke. 3-D analysis of crossbridge diversity in active states is based on 3-D EM tomography, which can present non-averages structures, but also allows partial or full averaging, as well as 3D correspondence analysis to classify and count different crossbridge shapes. ti Load-induced distortions of IFM high-force crossbridges in active, rigor and AMPPNP states will be mechanically synchronized by step/ramp length changes and studied by X-ray, 3D EM of cryo- and chem- fixed fibers, and fitting of crystal-structure models of myosin and actin. Force generation schemes will be modeled from these fittings. A Low-force (pre-powerstroke) active bridges, to be favored at high [Ca~+J by cooling, high [Pi],and Pi analogs (BDM, vanadate, A1F4~, BeFx), will be compared with high-force bridges using X-ray, 3D-EM and fitting of atomic models, (ft X-ray cryodiffraction of quick-frozen IFM fibers will be developed to protect against high-flux radiation damage while recording high resolution exposures, and to freeze-capture stable, evolving and maybe dynamic states. 4 Myosin's S1-S2 junction at the crossbridge origin in IFM will be probed by low-salt lattice swelling and high-salt loosening of the thick filament backbone. A Thick filament and A-band structure in relaxed IFM will be collaboratively modeled from our X-ray data at 2?, 20? and 40? (normal flight temperature in vivo), using our supporting EM studies to generate model geometry.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AR014317-36
Application #
7477901
Study Section
Special Emphasis Panel (NSS)
Program Officer
Nuckolls, Glen H
Project Start
1979-05-01
Project End
2011-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
36
Fiscal Year
2008
Total Cost
$517,672
Indirect Cost
Name
Duke University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
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
Szakmary, Akos; Reedy, Mary; Qi, Hongying et al. (2009) The Yb protein defines a novel organelle and regulates male germline stem cell self-renewal in Drosophila melanogaster. J Cell Biol 185:613-27
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
Pyhtila, Brook; Zheng, Tianli; Lager, Patrick J et al. (2008) Signal sequence- and translation-independent mRNA localization to the endoplasmic reticulum. RNA 14:445-53
Littlefield, Kimberly P; Ward, Andrew B; Chappie, Joshua S et al. (2008) Similarities and differences between frozen-hydrated, rigor acto-S1 complexes of insect flight and chicken skeletal muscles. J Mol Biol 381:519-28

Showing the most recent 10 out of 35 publications