Focal-adhesion components are enriched in ventral membranes isolated from transformed keratinocytes in culture

Abstract

The lack of procedures for isolating or reconstituting intact focal adhesions has hindered studies of how focal adhesions are organized and of how their assembly/disassembly is controlled. A method for isolating large quantities of the ventral portion of plasma membranes from transformed keratinocytes (A-431 cells) in culture is described. Plasma membranes are stabilized using Zn2+ and the ventral portion isolated attached to the culture substratum after the body of the cell has been sheared away. Compared with complete plasma membranes isolated from cells scraped from the dish, these ventral-membrane preparations are enriched 18.5-fold and 5.1-fold in the focal-adhesion components talin and vinculin respectively. While the epidermal-growth-factor receptor-kinase is less abundant in preparations of ventral membranes, a recently described tyrosine kinase which localizes to focal adhesions in mouse fibroblasts is enriched 19.9-fold. Extracellular matrix components, as well as their integrin receptors, are also enriched in these preparations of the ventral portion of plasma membranes compared with preparations of complete plasma membranes.