No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of Dil

In regions of focal adhesion, cells adhere to a substrate through the interaction of extracellular matrix proteins and transmembrane integrins which are coupled to the cell skeleton. It is generally assumed that the plasma membrane is brought to close proximity to the substrate there. We used the novel method of fluorescence interference contrast (FLIC) microscopy to measure the distance of the plasma membrane of GD25 fibroblasts on silica coated with fibronectin. We correlated the distance map with the distribution of vinculin tagged with green fluorescent protein. We found that the major part of the membrane was separated by 50 nm from the substrate. With respect to this plateau, we found spots of upward deformation and of close adhesion as well as a general ruffling of the membrane. There was no correlation between the areas of close adhesion and the distribution of vinculin. We conclude that focal adhesion does not imply a close attachment of membrane and substrate.