Vinculin interaction with permeabilized cells: disruption and reconstitution of a binding site.

Abstract

Abstract. Fluorescently labeled vinculin binds to focal contact areas in permeabilized cells independent of actin (Avnur, Z., J. V. Small, and B. Geiger, 1983, J. Cell Biol., 96:1622-1630, but the nature of the binding site is unknown. In this study we have examined the interaction of vinculin with these sites in permeabilized L6 myoblasts to define conditions that perturb the binding and subsequently to reconstitute it. Mild treatment with low concentrations of protease prevents vinculin incorporation without gross changes in the cytoskeleton or extensive protein breakdown. Exposure to buffers of moderate ionic strength also reduces subsequent vinculin binding without large morphological effects. These extraction conditions were used to obtain a fraction from gizzard which was able to restore the vinculin localization. Talin, actin, and vinculin itself were able to alter the binding of labeled vinculin to permeabilized cells and each also interacted with vinculin in gel overlays; however, they were unable to reconstitute the binding site in treated permeabilized cells. The results show a requirement for an as yet unidentified protein to capacitate vinculin binding to focal contact sites and suggest that this protein is peripheral and interacts directly with the binding site.

Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.

“…To specifically focus on associations with the CSK framework of the FAC, cells bound to beads were extracted with detergent (0.5% Triton X-100) in a CSK-stabilizing buffer to remove membranes and soluble cytosolic components before fixation. Past studies have shown that cellular proteins do not nonspecifically associate with the cytoskeleton when extracted with Triton under these conditions (Burr et al, 1980;Fey et al, 1984;Ball, 1986).…”

“…The extraction buffers we used for FAC isolation were adapted from a CSK isolation procedure that was previously used to demonstrate that active pp6Ov-src associates with the CSK (Burr et al, 1980). The presence of calcium and magnesium in this buffer is required to preserve the structural integrity of the CSK as well as the FAC (Ball et al, 1986), however, it leaves open the potential for degradation of FACassociated molecules by cation-dependent proteases. For example, pp6Oc-src is a substrate for the calpain II (Oda et al, 1993), a calcium-dependent protease that has been reported to localize to the FAC (Beckerle et al, 1987).…”

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

“…Another major finding of this study is the reconstitution and modulation by [Ca 2ϩ ] of the distribution of a partially reconstituted receptor complex in actin-depleted VPMs. Reconstitution of the binding of focal adhesion proteins to permeabilized or partially disrupted cells has been reported (Avnur et al, 1983;Ball et al, 1986). In our system we can test the binding of purified components to VPMs in which ␤1 receptors affinity for the ligand can be modulated.…”

Suppression of tumorigenicity in transformed cells after transfection with vinculin cDNA.

“…The Triton X-100-soluble fraction was removed and the insoluble fraction, enriched in membrane-cytoskeletal complexes (Ben-Ze'ev et al, 1979) and adhesion plaques (Ball et al, 1986), was scraped into 1 ml of the same buffer. Equal volumes of the two fractions were analyzed by PAGE, followed by immunoblotting with the broad-range antivinculin antibody.…”