Thin filament-associated regulatory proteins control actomyosin interactions in a variety of muscle and non-muscle contractile systems. In vertebrate striated muscle, the regulatory protein complex of tropomyosin and troponin linked to actin in the thin filaments causes relaxation by blocking strong myosin-crossbridge binding onto actin in the absence of Ca2+. In smooth muscle, thin filament-associated proteins (tropomyosin, caldesmon and possibly calponin) may function in conjunction with or in addition to the well-known Ca2+-calmodulin- dependent myosin phosphorylation process to modulate actomyosin ATPase and consequently tension generation and/or maintenance. In non-muscle systems, caldesmon linked to actin may, in concert with myosin phosphorylation, also act to regulate actomyosin-dependent cytoplasmic motility. The caldesmon-tropomyosin complex, like troponin-tropomyosin, inhibits actomyosin ATPase at low intracellular Ca2+-concentration, while tropomyosin itself potentiates ATPase. We will investigate the molecular mechanisms by which thin filament-linked proteins influence actomyosin by studying The structural interactions of the proteins on thin filaments from different types of muscles and other cells. Electron microscopy (including cryomicroscopy), computer-assisted image analysis and three- dimensional reconstruction will be used to determine thin filament structure and to evaluate changes in the structural arrangement of thin filament components in """"""""on-"""""""" and in """"""""off-states"""""""". Reconstruction of troponin- and caidesmon-based native thin filaments as well as reconstruction of synthetic filaments reconstituted from the components of these systems will be carried out to determine the impact of troponin, caldesmon and calponin on tropomyosin position, two-domain actin structure, and on actomyosin-binding. Reconstructions of high precision will be fitted to the atomic map of F-actin to detail specific atomic contacts between regulatory proteins and functional domains on F-actin. Our own published reconstructions and those of others demonstrate the feasibility of these goals. We anticipate that our continued structural studies will lead to an elucidation of the molecular mechanism of troponin action in skeletal muscle and contribute towards an understanding of the role and mechanism of caldesmon and calponin in the fine tuning of the contractile response in smooth muscle. A broad understanding of the molecular mechanisms involved in the regulation of contractility in healthy tissue may aid in future evaluation of defects occurring in some disease procosses. 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 conditions such as hypertension and asthma. Moreover, the general significance of our goals is underscored by the possibility that caldesmon linked to non-muscle microfilaments may have a role in controlling cytoplasmic motile processes such as cytokinesis, and therefore may be involved in regulating cell division in normal and cancer cells.
Showing the most recent 10 out of 23 publications
