Interactions between the plasma membrane and cytoskeleton of cultured fibroblasts

Observations of cultured cells made by double-fluorescence staining indicate that regions of the plasma membrane which are in close contact with actin-containing cytoplasmic fibers have characteristics different from other regions of the membrane. On fixed cells it is found that several integral membrane proteins are excluded from these regions of membrane-fiber apposition. If, however, these same integral proteins are clustered by their specific antibodies, the patches produced are rapidly lined up over the cytoplasmic fibers, resulting in a transmembrane linkage of clustered membrane proteins to the actin cytoskeleton. These observations have led us to predict the existence of a class of integral membrane proteins, X proteins, which are associated with actin fibers and are responsible for both the initial exclusion and then the transmembrane linkage of clusters of other integral membrane proteins. In an attempt to identify X proteins we have produced antisera against purified plasma membranes prepared from porcine intestinal brush borders. These antisera detect surface antigens on fixed human fibroblast cells which are initially lined up over actin fibers. These antigens are, thus, candidates for the hypothesized X proteins. Using the fluorescence microscope, we are attempting to isolate the potential X antigens with a staining absorption assay.