The long-term objective of this research is to gain a greater understanding of the structure and function of actin-based cytoskeletons. This project is designed to investigate the roles of T-protein, a huge protein that recently has been discovered as a component of the extensively characterized intestinal epithelial cell brush border cytoskeleton. Based on polypeptide molecular weight, molecular morphology, and immunocrossreactivity, brush border T-protein appears to be a nonmuscle isoform of the muscle protein titin. Muscle titin is a key structural component associated with the myosin thick filaments in sarcomeres. Likewise, T-protein is co-localized with myosin in brush borders, and T-protein and brush border myosin assemble into supramolecular structures in vitro. The specific aims of this proposal are to: 1) determine the existence and structural organization of T-protein in different domains of the intestinal epithelial cell brush border cytoskeleton and in cytoskeletal structures, including stress fibers, in other types of cells; 2) investigate the role of T- protein in organizing brush border cytoskeletal assembly during embryonic development of intestinal epithelial cells; and 3) characterize interactions between T-protein and other cytoskeletal components, including myosin. Results of these studies should elucidate the importance of nonmuscle titin in governing the organization of cytoskeletons and their ability to produce force for critical cellular processes. %%% The most obvious form of movement in biological systems is muscular motility. In muscle tissue, and particularly in skeletal muscle, contractile behavior results in gross movements of organs, such as the bending of an elbow or the turn of a head. These gross movements are the composite result of intracellular movements of protein complexes with respect to one another. The major intracellular contractile proteins in muscle are actin and myosin, which form oligomeric filaments that slide past each other in an ATP-fueled controlled reaction that results in cellular shortening. Whole muscles shorten (contract) as a composite result of the cells within the muscle shortening. However, cellular and intracellular movement is not limited to muscle tissue; virtually all types of eukaryotic cells display various actin / myosin based motility behaviors, ranging from cellular crawling to the separation of daughter cells after mitosis. While the muscle system is quite well understood in molecular detail, the extent to which these non- muscle actin /myosin based motility systems resemble muscle, in terms of molecular organization, is unclear. The Principal Investigator has now identified a non-muscle version of a major muscle protein which plays an important role in muscle actomyosin based motility. The experiments which will be performed in this project will elucidate the role played by the T-protein in non- muscle motility, and will thereby contribute greatly to our general understanding of cellular movement.
