The purpose of this project will be to examine the structural organization of smooth muscle and to determine how this organization changes as contraction takes place. This study will provide important new information about how the contractile apparatus is arranged in smooth muscle, how it is integrated into the cytoplasmic matrix, and how forces are transmitted within smooth muscle cells and between the cells that make up the whole tissue. Contractile proteins and various proteins associated with dense bodies, which are the attachment sites for actin filaments in smooth muscle, will be identified, studied biochemically, and labeled with fluorescent antibodies. The way these proteins are organized into structural elements in the cell and their relationship to one another and the dense body structures will then be studied in several ways. The protein composition of isolated dense body structures and the filaments associated with them will be examined biochemically and antibodies will be made to these proteins. The phase contrast and fluorescence microscope along with digital imaging and image processing techniques will then be used to examine the location and movement of specific structures labeled with antibodies in skinned isolated smooth muscle cells during contraction. To do this, digital images, collected at fixed time intervals during shortening, will be analyzed quantitatively to provide specific information about the rate and direction of movement of structures in contracting cells and how various elements move with respect to one another. Both whole cell and localized contraction will be examined in this way. The experiments will provide an overall picture of the smooth muscle cell during contraction and specific information about the relationship of various structures to one another. These results will provide new information about the organization of smooth muscle that can be used as a knowledge base for understanding healthy and diseased smooth muscle and its relationship to other muscle and tissue types.
