EXCEED THE SPACE PROVIDED. Thin filament-associated actin-binding proteins control both actomyosin-based contractilityand cytoskeletal formation in muscle and non-muscle cells. To elucidate these mechanisms, it is crucial to determine the structural interactionsof the proteins involved. We address several interrelated questions fundamental to understanding muscle thin filament function: (1) What is the architecture of the thin filament in skeletal, cardiac and smooth muscles? (2) What are the changing structural interactionsof thin filament-linked proteinsthat regulate muscle activity? (3) How do these proteins interact with actin to form the muscle cytoskeleton? We use state-of-the-art electron microscopy, image analysis and 3-D reconstruction to establishthe macromolecular structureof components bound on thin filament actin. Using these techniques: (A) We aim to determine the structural basis of troponin-tropomyosinregulation of skeletal and cardiac muscle activity by analyzing interactions of tropomyosin and troponin on thin filaments and the effects of Ca2 and myosin-crossbridgebinding. (B) To help understandthe organization of the cortical actin cytoskeletonof muscle cells, we aim to determine the structural interactions of dystrophin and utrophin with filamentous F-actin by examining the binding of their distinctcalponin homology (CH)-domains. (C) We aim to assess the structural role of thin filament- associated caldesmon as a possible modulator of actomyosin-based motility and cytoskeletal assembly in smooth muscle. (D) Studies of the structureof nebulin bound to actinwill be part of our continuing investigationof the functional design of thin filaments. In each study, reconstructionswill be fitted to the atomic resolution maps of F-actin to define molecular contacts of binding proteins with actin. Further, such 'hybrid crystallography' will be used to fit newly solved crystal structures of troponin, tropomyosin, dystrophin and utrophin domains within EM density maps to attain near atomic resolution. Our ongoing studies on troponin-trepomyosin regulated filaments will lead to an elucidation of the molecular mechanism of relaxation and activation in skeletal and cardiac muscle. Our successfully initiated structural studies on utrophin and dystrophinwill establish their unique features as cytoskeletal elements, informationapplicable to designing genetic therapies for muscular dystrophy.Studies on smooth muscle filaments will contributeto understanding the fine-tuning of the smooth muscle contractile response. Such modulation affects vascular tone and pulmonary airway resistance, determinants in, e.g., hypertension and asthma. The wider significance of our goals is underscored by the role of actin and associated proteins indiverse and vital cellular mechanisms that can become aberrant in malignancy. PERFORMANCE SITE ========================================Section End===========================================

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL036153-15
Application #
6819255
Study Section
Special Emphasis Panel (ZRG1-SSS-B (01))
Program Officer
Rabadan-Diehl, Cristina
Project Start
1986-09-30
Project End
2007-11-30
Budget Start
2004-12-01
Budget End
2005-11-30
Support Year
15
Fiscal Year
2005
Total Cost
$323,000
Indirect Cost
Name
Boston University
Department
Physiology
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Lehman, William; Li, Xiaochuan; Kiani, Farooq A et al. (2018) Precise Binding of Tropomyosin on Actin Involves Sequence-Dependent Variance in Coiled-Coil Twisting. Biophys J 115:1082-1092
Farman, Gerrie P; Rynkiewicz, Michael J; Orzechowski, Marek et al. (2018) HCM and DCM cardiomyopathy-linked ?-tropomyosin mutations influence off-state stability and crossbridge interaction on thin filaments. Arch Biochem Biophys 647:84-92
Rynkiewicz, Michael J; Fischer, Stefan; Lehman, William (2016) The propensity for tropomyosin twisting in the presence and absence of F-actin. Arch Biochem Biophys 609:51-58
Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel et al. (2015) Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back. Mol Biosyst 11:2180-9
Begonja, Antonija Jurak; Pluthero, Fred G; Suphamungmee, Worawit et al. (2015) FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets. Blood 126:80-8
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; Suphamungmee, Worawit; Janco, Miro et al. (2012) The flexibility of two tropomyosin mutants, D175N and E180G, that cause hypertrophic cardiomyopathy. Biochem Biophys Res Commun 424:493-6
Suphamungmee, Worawit; Nakamura, Fumihiko; Hartwig, John H et al. (2012) Electron microscopy and 3D reconstruction reveals filamin Ig domain binding to F-actin. J Mol Biol 424:248-56
East, Daniel A; Sousa, Duncan; Martin, Stephen R et al. (2011) Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind co-operatively to actin and regulate myosin. Biochem J 438:265-73
Moore, Jeffrey R; Li, Xiaochuan; Nirody, Jasmine et al. (2011) Structural implications of conserved aspartate residues located in tropomyosin's coiled-coil core. Bioarchitecture 1:250-255

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