Characterization of the adhesion plaque proteins vinculin and talin: Their localization and interaction with each other and with actin in vitro

Mary Ellen Groesch, Purdue University

Abstract

Adhesion plaques are complexes of proteins found at certain regions of cell-substrate contact where actin filaments are linked to the plasma membrane. We have studied vinculin and talin, adhesion plaque proteins that have been proposed to be involved in linking the cytoskeleton to the membrane. Polyclonal antibodies were raised against vinculin and talin and used in immunofluorescence studies to confirm that talin and vinculin are localized in adhesion plaques. Immunoblotting showed that both proteins are present in detergent -soluble and -insoluble fractions of fibroblasts. Because of their location at regions of microfilament-plasma membrane association, talin or vinculin might have a role in linking actin to the membrane. We used falling ball viscometry to try to detect interactions between polymerized actin and vinculin and/or talin in vitro and found no evidence of any interaction, suggesting that neither protein directly links actin to the membrane. Since it was known that vinculin and talin interact, studies were carried out to localize the talin-binding site on the vinculin molecule. Vinculin was digested with Staphylococcus aureus V8 protease into two major fragments of 85 kDa and 30 kDa. We were able to purify both of the V8 protease-generated vinculin fragments. Nitrocellulose overlays with $\sp{125}$I-talin and $\sp{125}$I-85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin-binding domain was localized to the 85 kDa vinculin fragment. Quantification of $\sp{125}$I-talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. These results suggest that the 30 kDa fragment may affect vinculin-talin interaction.

Degree

Ph.D.

Advisors

Otto, Purdue University.

Subject Area

Biology

COinS