Research and Professional Interests:

Our research investigates the roles that various cytoskeletal proteins play in the structure and function of nonmuscle cells. The intestinal epithelial brush border 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 of 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 interaction with the myosin thick filaments. Minititins found in invertebrates also appear to play a regulatory role in muscle contraction through an intrinsic kinase 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 aspects 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. Our generation and screening of expression cDNA libraries has yielded two possible cellular titin clones, which now are being characterized. This molecular characterization will continue to constitute an important thrust in our multifaceted approach to the study of cytoskeletal structure and function in the future.

Selected Publications:

Volosky, J., and T. Keller. 1991. 3'-Untranslated regions and multiple polyadenylation signals are conserved between chicken and human cellular myosin II transcripts. Gene Expression 1: 223-231.

Gordon, P. V., and T. Keller. 1992. Functional coupling to brush border creatine kinase imparts a selective energetic advantage to contractile ring myosin in intestinal epithelial cells. Cell Motil. Cytoskel. 23: 38-44.

Eilertsen, K., and T. Keller. 1992. Identification and characterization of two huge proteins of the brush border cytoskeleton: evidence for a cellular isoform of titin. J. Cell Biol. 119: 549-557.

Eilertsen, K., S. Kazmierski, and T. Keller. 1994. Cellular titin-myosin interaction: in vitro assembly and isoform specificity. J. Cell Biol. 126: 1201-1210.

Keller, T. 1995. Structure and function of titin and nebulin. Curr. Opinion Cell Biol. 7: 32-38.

Eilertsen, K. E., S. T. Kazmierski, and T. Keller. 1997. Interaction of alpha-actinin with cellular titin. Eur. J. Cell Biol. 74: 361-364.

Keller, T. 1997. Muscle structure: molecular bungees. Nature 387: 233-235.

Griparic, L., J. M. Volosky, and T. Keller. 1998. Cloning and expression of chicken CLIP-170 and restin isoforms. Gene 206: 195-208.

Griparic, L., and T. Keller. 1998. Identification and expression of two novel CLIP-170/restin isoforms expressed predominantly in muscle. Biochim. Biophys. Acta 1405: 35-46.

Griparic, L., and T. Keller. 1999. Differential usage of two 5’ splice sites in a complex exon generates additional protein sequence complexity in chicken CLIP-170 isoforms. Biochim. Biophys. Acta 1449: 119-124.

Keller, L. R., T. C. S. Keller, and J. H. Evans. 1999. Experimental Developmental Biology: A Laboratory Manual. Academic Press, San Diego.

Keller, T. 1999. The Cell in Health and Disease. Thompson Learning Custom Publishing, Cincinnati, Ohio.

Keller, T., K. Eilertsen, M. Higginbotham, S. Kazmierski, K.-T. Kim, and M. Velichkova. 2000. Role of titin in nonmuscle and smooth muscle cells. Adv. Exp. Med Biol. 481: 265-277.

Kim, K., and T. Keller. 2002. Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro. J. Cell Biol. 156: 101-111.

Graduate Students:

Cavnar, Peter J
Fazel, Arif
Gedulig, Thomas