Smooth muscle, which lines the periphery of hollow organs, contracts to change the shape of the organ or maintains tension to fix the shape. An understanding of the regulation of smooth muscle contraction could help to form the basis for understanding a number of diseases, including hypertension in the vascular system and asthma in the airways of the lung. Smooth muscle contraction is primarily regulated by Ca2+-calmodulin dependent phosphorylation of myosin in the thick filament. Evidence is accumulating for an additional regulatory mechanism associated with the actin thin filament involving caldesmon, a long, thin, actin-binding protein, and tropomyosin. A much shorter isoform of caldesmon is also found in a wide range of non-muscle tissue including brain, liver, kidney, and platelets. Non-muscle caldesmon is thought to play a role in regulating the shape and motility of cells. However very little is known about its function. Thus the long range goal of this project is to uncover the molecular mechanisms whereby caldesmon, in concert with tropomyosin, regulates smooth muscle contraction and non-muscle motility and shape. The immediate objective is to map the arrangement of smooth muscle and non-muscle caldesmon, and tropomyosin, on the actin filament. The results of such studies will be correlated with actomyosin ATPase activity, the test tube analogue of contraction, in order to connect structure with function.
These aims will be pursued with reconstituted and native thin filaments and intact myofibrils. The project will make use of spectroscopic and cross-linking probes specifically attached to particular domains of caldesmon and tropomyosin in order to probe these regions of the proteins in their interaction with each other and other contractile proteins. In particular, spin labels, fluorescence labels, photocross-linkers, and disulfide cross-linkers will be used. Electron microscopy and the hydrodynamic techniques of viscosity and analytical ultracentrifugation will also be employed.
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