Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts.

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Abstract
Information about the molecular structures present at cell-substratum and cell-cell contact sites formed by cultured [embryonic chick heart] fibroblasts was obtained. Double immunoelectron-microscopic, labeling experiments were carried out on ultrathin frozen sections cut through such contact sites to determine the absolute and relative dispositions of the 3 proteins fibronectin, vinculin and .alpha.-actinin with respect to these sites. Three types of cell-substratum and cell-cell contact sites familiar from plastic sections could also be discriminated in the frozen sections by morphological criteria alone, i.e., the gap distances between the 2 surfaces, and the presence of submembranous densities. These types were the following: focal adhesions (FA); close contacts (CC); and extracellular matrix contacts (ECM). This morphological typing of the contact sites allowed the recognition and assignment of and distinctive immunolabeling patterns for the 3 proteins to each type of site on the frozen sections. FA sites were immunolabeled intracellularly for vinculin and .alpha.-actinin, with vinculin labeling situated closer to the membrane than .alpha.-actinin. Fibronectin was not labeled in the narrow gap between the cell surface and the substratum or between 2 cells, at FA sites. Control experiments showed that this could not be ascribed to inaccessibility of the FA narrow gap to the immunolabeling reagents but indicated an absence or severe depletion of fibronectin from these sites. CC sites were labeled intracellularly for .alpha.-actinin but not vinculin and were labeled extracellularly for fibronectin. ECM sites were characterized by large separations (often > 100 nm) between the cell and substratum or between 2 cells, which were connected by long cables of extracellular matrix components, including fibronectin. In late (24-36 h) cultures, ECM contacts predominated over the other types. ECM sites appeared to be of 2 kinds, one labeled intracellularly for both .alpha.-actinin and vinculin, the other for .alpha.-actinin alone. From these and other results, a coherent but tentative scheme is proposed for the molecular ultrastructure of these contacts sites, and specific functional roles are suggested for fibronectin, vinculin and .alpha.-actinin in cell adhesion and in the linkage of intracellular microfilaments to membranes at the different types of contact sites.