The identification and localization of fibronectin in cultured corneal endothelial cells: Cell surface polarity and physiological implications

Abstract

Bovine corneal endothelial cells can be maintained for over 200 generations in tissue culture provided that fibroblast growth factor is present in the medium. The cultured cells exhibited at confluence the morphology of a highly contact inhibited cell monolayer composed of flattened and closely apposed cells. Transmission electron microscopy of the confluent endothelial cell monolayer demonstrated that the cellular organelles and cytoskeletal elements had a morphological appearance and distribution indistinguishable from those seen in the corneal endothelium in vivo. The confluent cells were also capable of synthesizing a basement membrane. In both sparse and subconfluent cultures fibronectin detected by indirect immunofluorescence was distributed on the apical cell surface and in the regions of cell-cell contacts. In contrast, when the cultures reached confluence, fibronectin could no longer be detected on the apical cell surface, but rather accumulated in the extracellular meshwork underlying the cell monolayer. Metabolic labeling with [ 35S]-methionine demonstrated that at confluence fibronectin is deposited toward the extracellular matrix and secreted in large quantities into the tissue culture medium. Lactoperoxidase catalyzed iodination of confluent cultures showed that the fibronectin meshwork associated with the cell surface and the extracellular matrix is disulfide-bonded to form dimers and higher complexes. In contrast, actively growing cells that are not yet in contact showed little or no fibronectin on their cell surfaces and no production of an extracellular matrix. The apical surface of the confluent monolayer, which is not covered by fibronectin, is a non-thrombogenic surface, as indicated by the lack of platelet binding and release reaction. In contrast, the extracellular matrix, which is extensively coated with fibronectin, is thrombogenic. Our results therefore demonstrate that at confluence fibronectin becomes closely associated with the basel cell surface and can no longer be detected on top of the cells, thus maintaining the polarity of cell surfaces observed in vivo. It is suggested that the underlying meshwork of fibronectin may play a physiological role in maintaining the flattened and closely apposed morphology characteristic of differentiated corneal endothelial cells in vitro and in vivo and that the absence of fibronectin from the apical cell surface is related to the non-thrombogenic properties of these cells.