Thin filament-associated actin-binding proteins have dual function, controlling actomyosin-based contractility and cytoskeletal assembly in a variety of muscle and non-muscle systems. In striated muscles, the regulatory protein complex, troponin-tropomyosin, linked to thin filaments, controls contractility by sterically blocking and unblocking myosin-crossbridge binding on actin in response to changing Ca/2+ levels. In smooth muscle, thin filament-associated caldesmon and calponin may function similarly to modulate actomyosin-based motility and/or the state of cytoskeletal assembly. To accomplish our goal to determine the regulatory mechanisms by which these proteins function in striated and smooth muscles, it is crucial to assess their structural interactions on isolated and reconstituted thin filaments and their respective responses to Ca/2+, myosin-crossbridge binding and phosphorylation. For a fuller picture of filament function, it is also essential to understand the structural interactions of muscle and related non-muscle actin- cytoskeletal proteins State of the art electron microscopy, computer- assisted image analysis and three-dimensional reconstruction will be used to determine the arrangement of thin filament components on F-actin and to evaluate their position and influence on actin domains. Reconstruction will be fitted to the atomic map of F-actin to detail contacts with specific amino acid clusters on actin monomers. Our own published reconstructions and those of our colleagues demonstrate the feasibility of these goals. Our continued structural studies on troponin-tropomyosin regulated filaments will lead to an elucidation of the molecular mechanism of relaxation and activation in skeletal and cardiac muscle. Our studies to smooth muscle filament swill contribute to understanding the fine tuning of the smooth muscle contractile response and the construction of its cytoskeleton. A general understanding of the molecular mechanisms involved in the regulation of contractility in healthy muscle tissue will aid in our evaluation of defects occurring in disease. The control of smooth muscle contractility, for example, is of great importance in the regulation of vascular tone and pulmonary airway resistance, determinants in a number of disease processes such as hypertension and asthma. The wider significance of our goals is underscore by possible participation of caldesmon, calponin and proteins with consensus calponin homology-domains in such diverse cellular processes as cytokinesis, exocytosis, cortical cytoskeleton modeling and signal transduction modulation, i.e. processes that are essential to all normal cells and that can become aberrant in malignancy.
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