In order to determine whether fibronexus morphogenesis is involved in the establishment of more normal cellular morphology and substrate adhesion patterns in Nil/HSV transformed fibroblasts induced by treatment with exogenous fibronectin (FN), this system was studied with electron microscopy (EM), immunocytochemistry, and interference reflection microscopy (IRM). EM analysis showed that cells grown in medium with 5% foetal bovine serum (FBS) had well-formed fibronexuses and enlarged actin-microfilament bundles at their dorsal surface by 1 h after FN addition. Expansion of the substrate-binding focal adhesions visualized with IRM, and increased cellular flattening, did not take place until at least 2 h later. These observations suggest that fibronexus induction and the initiation of actin-microfilament bundle enlargement occur as a direct result of FN attachment to the cell surface, with overt increases in substrate adhesion taking place subsequently. FN was not localized in focal contacts under these conditions. However, if fibronexus-reconstitution experiments were performed with Nil/HSV cultures maintained in medium with 0.3% FBS, then fibronectin fibres and fibronexuses were strikingly localized at focal contacts on the ventral cell surface. Fibronectin is evidently capable of exerting either a direct or an indirect influence on substrate adhesion, which is probably regulated by serum factors.

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