Targeting of cytoskeletal linker proteins to focal adhesion complexes is reduced in fibroblasts adhering to laminin-1 when compared to fibronectin

Abstract

Cellular interactions with the extracellular matrix determine to a large extent cell behavior, including cell migration. These interactions take place at specialized cellular structures, the focal adhesions, which have a substrate-specific morphology. To determine the molecular and functional relevance of this observation, the composition of isolated focal adhesions developed by fibroblasts adhering to fibronectin or laminin-1 was analyzed by indirect immunofluorescence and immunoblotting with or without stabilization of the structures by cross-linking. In the absence of cross-linking, integrins, talin, vinculin and, to a lower extent, paxillin remained associated with the focal adhesions formed on both substrates, indicating a tight association of these proteins with the extracellular matrix support. By contrast, α-actinin, FAK, and actin were apparently loosely maintained within focal adhesions and were found associated to these structures only after stabilization by crosslinking. Interestingly, although both substrates induced clustering and aggregation of all these proteins, their relative concentration, with the exception of α-actinin, was lower within the focal adhesions formed on laminin-1 than in those formed on fibronectin. Moreover, as assessed in migration assays, the locomotory speed of fibroblasts was higher on laminin-1 than on fibronectin. Altogether these results indicate that integrins involved in cellular interactions with fibronectin or laminin-1 trigger the formation of focal adhesion structures which differ by molecular organization, concentration in several adhesion plaque components, and function.