Scanning electron microscopy of focal contacts on the substratum attachment surface of fibroblasts adherent to fibronectin

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We have examined the cell-to-substratum attachment surface of hamster fibroblasts with scanning EM, and describe the surface ultrastructure of focal contacts and microspikes during cellular attachment and spreading on fibronectin. Nil 8 fibroblasts were seeded onto fibronectin-coated glass coverslips in serum-free medium, fixed, and the fibroblast-fibronectin monolayer was separated from the glass and inverted for scanning electron microscopic (EM) analysis. Focal contact development was detected by interference reflection microscopy and correlated with the immunofluorescence microscopic distribution of fibronectin receptor antigens. The cell undersurface appeared smooth and featureless at 0.5 h when focal contacts were undetectable and fibronectin receptors were distributed diffusely. By 1-2 h, undersurface membrane impressions of focal contacts were detected with scanning EM; their size, shape and distribution matched that of focal contacts seen with interference reflection microscopy (IRM). These contacts had smooth external surfaces and were often arranged in chevron-shaped complexes. However, at 4-6 h, the surface texture of focal contacts became fibrous and the contact periphery was delineated with the orifices of membrane-associated vesicles. Development of this filamentous substructure is correlated with the maximum concentration of fibronectin receptors and fibronectin at focal contacts, suggesting that these molecules are involved in the maturation and stabilization of focal contacts.