Vinculin interaction with permeabilized cells: disruption and reconstitution of a binding site.
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.
D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D002463:Cell Membrane Permeability; D002645:Chickens; D005455:Fluorescent Antibody Technique; D005895:Gizzard, Avian / Q000648:ultrastructure; D051379:Mice; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D016596:Vinculin
D009124:Muscle Proteins; D016596:Vinculin