Thin filament-associated actin-binding proteins control both actomyosin-based muscle contraction and cytoskeletal formation. To elucidate the mechanisms required for muscle thin filaments to function, it is crucial to determine the structural interactions of the regulatory proteins involved. As a means of achieving our objective to understand the physiology of cardiac, skeletal and smooth muscle control systems, we will examine the architecture of muscle thin filaments at a fundamental structural level and characterize the changing interactions of thin filament-linked proteins that regulate muscle activity. We will use state-of- the-art electron microscopy and electron tomography coupled with image analysis and 3D reconstruction to establish the macromolecular structure of actin-binding proteins on thin filament actin. Using these techniques: (1) We aim to determine the structural basis of troponin-tropomyosin regulation of cardiac and skeletal muscle activity by analyzing interactions of tropomyosin and troponin on thin filaments, which are governed by Ca2+binding to troponin and myosin-crossbridge binding on actin. To accomplish this goal, (A) we will test our newly proposed atomic model for troponin-tropomyosin localization on thin filaments by generating single particle and electron tomographic reconstructions;(B) we will test the hypothesis that mobile domains of troponin-I latch onto actin to constrain tropomyosin in the inhibitory "blocking" state characteristic of relaxed muscle;(C) we will test both the hypothesis that tropomyosin assumes the contours of the F-actin helix as a relatively stiff coiled coiled-coil and the alternative view that tropomyosin is flexible. (2) We will test the hypothesis that mutant cardiac troponin and different tropomyosin variants perturb muscle regulation by causing an imbalance in tropomyosin's position that alters the regulatory state of thin filaments. (3) We will assess the regulatory role of thin filament-linked caldesmon and calponin in defining tropomyosin position in vascular and visceral muscle. (4) We will determine the structure of nebulin bound to actin to complete our map of thin filaments. In each study, reconstructions fitted to the atomic resolution maps of F-actin will demarcate molecular contacts of binding proteins with actin at near atomic resolution ("hybrid crystallography"). Lay summary: Studies on troponin-tropomyosin regulated filaments, with particular attention devoted to normal and mutant proteins derived from cardiac muscle, will lead to an elucidation of the molecular regulatory mechanisms governing cardiac contraction, which is essential for tracing cardiovascular disease processes. Studies on smooth muscle filaments will aid in understanding the fine-tuning of smooth muscle contraction thus revealing key controls for vascular tone and pulmonary airway resistance, determinants in, e.g., hypertension and asthma. Actin filaments and associated proteins are major participates in diverse cellular systems, underscoring the broad significance of the proposed work. Our goal is to elucidate the control mechanisms that regulate cardiovascular and skeletal muscle activity. We will examine structural changes at a molecular level that are orchestrated by regulatory proteins and which control muscle shortening and force production. Understanding the underlying molecular physiology governing contraction and relaxation in heart muscle and blood vessels is key to deciphering cardiovascular disease processes, controlling blood pressure and identifying novel targets for drug development.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL036153-22
Application #
8197634
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Gao, Yunling
Project Start
1986-09-30
Project End
2012-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
22
Fiscal Year
2012
Total Cost
$402,188
Indirect Cost
$154,688
Name
Boston University
Department
Physiology
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Gu, Changkyu; Chang, Joann; Shchedrina, Valentina A et al. (2014) Regulation of dynamin oligomerization in cells: the role of dynamin-actin interactions and its GTPase activity. Traffic 15:819-38
Orzechowski, Marek; Moore, Jeffrey R; Fischer, Stefan et al. (2014) Tropomyosin movement on F-actin during muscle activation explained by energy landscapes. Arch Biochem Biophys 545:63-8
Lehman, William; Li, Xiaochuan Edward; Orzechowski, Marek et al. (2014) The structural dynamics of *-tropomyosin on F-actin shape the overlap complex between adjacent tropomyosin molecules. Arch Biochem Biophys 552-553:68-73
Li, Xiaochuan Edward; Orzechowski, Marek; Lehman, William et al. (2014) Structure and flexibility of the tropomyosin overlap junction. Biochem Biophys Res Commun 446:304-8
Yang, Shixin; Barbu-Tudoran, Lucian; Orzechowski, Marek et al. (2014) Three-dimensional organization of troponin on cardiac muscle thin filaments in the relaxed state. Biophys J 106:855-64
Orzechowski, Marek; Li, Xiaochuan Edward; Fischer, Stefan et al. (2014) An atomic model of the tropomyosin cable on F-actin. Biophys J 107:694-9
Orzechowski, Marek; Fischer, Stefan; Moore, Jeffrey R et al. (2014) Energy landscapes reveal the myopathic effects of tropomyosin mutations. Arch Biochem Biophys 564:89-99
Lehman, William; Orzechowski, Marek; Li, Xiaochuan Edward et al. (2013) Gestalt-binding of tropomyosin on actin during thin filament activation. J Muscle Res Cell Motil 34:155-63
Sousa, Duncan R; Stagg, Scott M; Stroupe, M Elizabeth (2013) Cryo-EM structures of the actin:tropomyosin filament reveal the mechanism for the transition from C- to M-state. J Mol Biol 425:4544-55
Lehman, William; Galinska-Rakoczy, Agnieszka; Hatch, Victoria et al. (2009) Structural basis for the activation of muscle contraction by troponin and tropomyosin. J Mol Biol 388:673-81