Muscle contraction is a central feature of animal life. It results from interactions between myosin heads on the thick filaments and actin subunits on the thin filaments. While great progress has been made in understanding the molecular basis of contraction and its regulation, key questions remain. These include the structural basis of regulation of myosin and actin filaments, and the molecular basis of actin-myosin interaction. To elucidate these mechanisms it is crucial to determine the structures of the filaments in relaxed and activated states and to capture dynamic molecular changes. We do this using state-of-the-art cryo-EM and new millisecond time-resolved techniques, combined with 3D image reconstruction. Using these approaches: (1) We will determine the structural basis of calcium- and phosphorylation-based regulation of striated muscle myosin filaments by defining the molecular interactions occurring in isolated myosin molecules and filaments. (2) We will define the structural basis of thin filament regulation of striated muscle by analyzing troponin, tropomyosin and actin interactions at high and low Ca 2+, the dynamics of tropomyosin movement, structural alterations in myopathies, and the organization of the thin filament template protein, nebulin. (3) We will define the binding sites of myosin heads on actin at early stages of the actomyosin crossbridge cycle by analyzing thin filaments decorated with weakly bound myosin heads. In each study, reconstructions will be fitted to atomic resolution maps of F-actin, troponin or the myosin head, defining molecular contacts at near-atomic resolution. Our studies of myosin molecules and filaments will provide new insight into general mechanisms of thick filament regulation. Continued advances in our studies of troponin-tropomyosin regulated thin filaments will provide new information on native thin filament structure, the 3D organization of troponin, and the importance of tropomyosin dynamics in thin filament regulation and disease. Studies of the actomyosin complex will directly test models for the interaction of myosin heads with actin during the fundamental crossbridge cycle. Preliminary data demonstrate that all of our proposals are feasible. New insights arising from these proposals will provide a deeper understanding of the structural basis of muscle contraction and its regulation, and of actomyosin-based cell motility. Defining molecular mechanisms in healthy muscle is essential to understanding structural defects in skeletal and cardiac myopathies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR034711-13A1
Application #
6824575
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Nuckolls, Glen H
Project Start
1984-12-01
Project End
2009-04-30
Budget Start
2004-08-01
Budget End
2005-04-30
Support Year
13
Fiscal Year
2004
Total Cost
$368,550
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Lee, Kyoung Hwan; Sulbarán, Guidenn; Yang, Shixin et al. (2018) Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals. Proc Natl Acad Sci U S A 115:E1991-E2000
Mun, Ji Young; Kensler, Robert W; Harris, Samantha P et al. (2016) The cMyBP-C HCM variant L348P enhances thin filament activation through an increased shift in tropomyosin position. J Mol Cell Cardiol 91:141-7
Previs, Michael J; Mun, Ji Young; Michalek, Arthur J et al. (2016) Phosphorylation and calcium antagonistically tune myosin-binding protein C's structure and function. Proc Natl Acad Sci U S A 113:3239-44
Yang, Shixin; Woodhead, John L; Zhao, Fa-Qing et al. (2016) An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits. J Struct Biol 193:45-54
Previs, Michael J; Prosser, Benjamin L; Mun, Ji Young et al. (2015) Myosin-binding protein C corrects an intrinsic inhomogeneity in cardiac excitation-contraction coupling. Sci Adv 1:
Sulbarán, Guidenn; Alamo, Lorenzo; Pinto, Antonio et al. (2015) An invertebrate smooth muscle with striated muscle myosin filaments. Proc Natl Acad Sci U S A 112:E5660-8
Lee, Kyounghwan; Harris, Samantha P; Sadayappan, Sakthivel et al. (2015) Orientation of myosin binding protein C in the cardiac muscle sarcomere determined by domain-specific immuno-EM. J Mol Biol 427:274-86
Kirk, Jonathan A; Chakir, Khalid; Lee, Kyoung Hwan et al. (2015) Pacemaker-induced transient asynchrony suppresses heart failure progression. Sci Transl Med 7:319ra207
Mun, Ji Young; Previs, Michael J; Yu, Hope Y et al. (2014) Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism. Proc Natl Acad Sci U S A 111:2170-5
Craig, Roger; Lee, Kyoung Hwan; Mun, Ji Young et al. (2014) Structure, sarcomeric organization, and thin filament binding of cardiac myosin-binding protein-C. Pflugers Arch 466:425-31

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