Organization of extracellular matrix by chick embryonic corneal epithelial cells in culture and the role of fibronectin in adhesion

Corneal epithelial cells from 15-day chick embryos produce an extensive extracellular matrix when cultured for up to 7 days on plastic, glass, and gelatin-coated substrata. Immunofluorescence studies revealed the presence of a fibrillar fibronectin matrix that became more extensive with time in culture. When cells were cultured in fibronectin-depleted medium little or no fibronectin was evident in the first 24 h, although the cells attached and spread normally when compared with controls. Considerably more fibronectin was associated with the cells after 48 h. Incubation of cells with [35S]methionine, followed by immunoprecipitation of cell extracts by specific anti-fibronectin antibody, confirmed that the cells synthesize fibronectin. Cells cultured in fibronectin-coated substrata were able to reorganize the fibronectin into fibrillar form as well as to endocytose fibronectin. It is suggested that cells ‘comb’ fibronectin from the substratum and organize it into fibrils. Interference reflection microscopy revealed no simple correlation between the distribution of fibronectin and focal contacts. However, some focal contacts seemed to lack fibronectin. In the presence of anti-fibronectin immunoglobulin G cells initially attached but failed to spread, and subsequently detached from glass and fibronectin-coated substrata. However, they attached and spread on collagen substrata. The results suggest that fibronectin is unnecessary for initial cell attachment but may be important in maintaining cell adhesion and spreading on certain substrata. However, it is not necessary for attachment and spreading on collagen. Cells on collagen may attach and spread directly on the substratum via specific collagen receptors or by using some other type of attachment factor(s). Electron microscopy revealed a densely staining fibrillar matrix between the basal cell layer and substratum as well as in the intercellular spaces between cells. Components of this matrix and the cell surface were strongly stained by ruthenium red, which indicates the presence of acidic groups such as glycosaminoglycans.