Vinculin and talin: Turn-over rates, dynamics in living cells and binding site studies

Seung-won Lee, Purdue University

Abstract

The adhesion plaque is a multi-protein complex at a region of membrane where animal cells attach to the substrate. In the plaque, a series of proteins link intracellular actin filaments to extracellular matrix proteins. The growth, differentiation, mobility and shape of cells are thought to be restricted or regulated to some extent by such physical contacts with the environment. We have studied vinculin and talin, two proteins which are major cytoplasmic components in the adhesion plaque. We examined how the synthesis and degradation rates of both proteins are affected by cell density and viral transformation. In virally transformed cells, the synthesis rate of vinculin and talin were decreased by 40% and 30%, respectively. In both normal and transformed cells grown at high density, the synthesis rate of vinculin was decreased by 40%, but the synthesis rate of talin was unaffected. The degradation rates of both proteins (t$\sb{1/2} \sim 20$ hr) were not affected by cell density, but they were slightly increased in transformed cells (t$\sb{1/2} \sim 16$ hr). This indicates that protein synthesis, rather than degradation, mainly regulates the supply of vinculin and talin for construction of adhesion plaques under various conditions such as cell density and viral transformation. We determined the targeting rates of newly synthesized vinculin and talin to the adhesion plaque and the dissociation rates of both proteins from the adhesion plaque with pulse-chase experiments. In virally transformed cells, the cytoskeletal pools of both proteins turned over faster by having increased targeting and dissociation rates. This increased turn-over may relate to the increased motility of transformed cells. Vinculin and talin interact in vitro and presumably in vivo. By characterizing an anti-idiotypic vinculin antibody that recognizes talin, we were able to localize a vinculin binding site in the talin sequence by overlaying peptide maps of talin with the anti-idiotypic antibody. The existence of such an anti-idiotypic antibody strongly suggests that vinculin and talin do interact in vivo. Another vinculin binding site was identified with a $\sp{125}$I-vinculin blot overlay technique. This indicates that talin has two immunologically distinct vinculin binding sites which, in turn, suggests that vinculin may have two different talin binding sites. We speculate that the presence of two different binding domains mediating the talin-vinculin interaction may increase the stability of adhesion plaques.

Degree

Ph.D.

Advisors

Otto, Purdue University.

Subject Area

Biology

COinS