Our research investigates the roles that various cytoskeletal proteins play in the structure and function of nonmuscle cells. The intestinal epithelial brush bo rder chosen as a model system for these studies contains one of the most stable and extensively organized actin-based cytoskeletons found in nonmuscle cells. We isolate brush borders from intestinal epithelial cells with their cytoskeletons intact and in quantities sufficient for structural and biochemical analysis. We use a wide variety of morphological, biochemical, immunological, and molecular techniques to analyze various brush-border proteins. The strength of the brush border as an experimental system is that it allows us to relate what we learn from our analyses of individual proteins to their organization and function in the brush-border cytoskeleton. We also are able to investigate the roles o f the various proteins in establishing the brush border during development.

Recently, we have focused our interest on a cellular isoform of the muscle protein titin that we discovered in the brush border. Titins are the largest ribosome -translated peptides found to date. In muscle, titin plays a key role in establishing and maintaining the integrity of the contractile sarcomere unit through in teraction with the myosin thick filaments. Minititins found in invertebrates also appear to play a regulatory role in muscle contraction through an intrinsic ki nase activity. Using immunofluorescence microscopy with an antibody that we raised against cellular titin, we have found cellular titin colocalized with myosin filaments in the brush border and in the stress fibers and cleavage furrows in all other cells that we have investigated. Our biochemical investigations of proteins purified from brush borders have revealed that cellular titin has the capability of organizing myosin filaments in vitro into arrays that with electron microscopy appear similar to those found in vivo. This finding indicates that cellular titin may play a key organizational role in important cytoskeletal assemblies in all cells. We also have evidence that cellular titin may play a crucial role in assembling sarcomeres during muscle differentiation. Currently, we are using biochemical and molecular approaches to determine roles of a putative cellular titin kinase activity. One approach is to confirm kinase activity and find the in vivo substrate. A second approach involves the molecular cloning of cellular titin. The size of titin makes this a monumental task. Nevertheless, sequence analysis of certain regions of the titin sequence would elucidate important aspect s of the kinase domain and myosin-binding domains. Cloning these regions would make it possible to investigate cellular titin functional domains through bacterial expression of fusion proteins and generating dominant negative mutations in transfected cells. O ur generation and screening of expression cDNA libraries has yielded two possible cellular titin clones, which now are being characterized. This molecular chara cterization will continue to constitute an important thrust in our multifaceted approach to the study of cytoskeletal structure and function in the future.