Function follows form: how fibronectin matrix architecture controls cell behaviour

Abstract

Introduction Fibronectin (FN) matrix assembly is a tightly regulated stepwise process that is initiated by interactions between FN and cell‐surface integrin receptors. Assembly is affected by extracellular factors including availability of FN and the presence of other matrix molecules. In turn, FN matrix activates intracellular signalling pathways via integrin receptors, and these signals regulate cell adhesion, migration and survival. Two kinases immediately downstream of integrins are focal adhesion kinase (FAK) and pp60‐Src. Our results show that activation of these kinases regulates accumulation of FN matrix fibrils and that this process is affected by tenascin‐C, an ECM protein that modulates cell interactions with FN.Methods FN assembly was monitored by indirect immunofluorescence and by immunoblotting of deoxycholate (DOC)‐insoluble matrix material with anti‐FN antibodies. Wild‐type mouse fibroblasts and cells lacking FAKs or Src family kinases (SYF cells) were used. Kinase and phosphatase inhibitors were used to regulate enzyme activities.Results Mouse fibroblasts lacking FAK assemble significantly reduced amounts of FN fibrils and DOC‐insoluble matrix. FAK is phosphorylated by Src family kinases and fibroblasts lacking Src family kinases (SYF cells) or cells treated with PP1, an inhibitor of SYF kinases, also lack FN matrix. The effects of Src activity are most dramatic during the early stages of de novo assembly by fibroblasts implicating this kinase in the initiation process. While FN stimulates FAK, tenascin‐C, an ECM protein that is expressed at sites of cell movement, has the opposite effect on FAK activity. In fibroblasts on a 3D FN matrix, FAK is constitutively phosphorylated. In contrast, FAK is only transiently activated in the presence of tenascin‐C. Concomitant with the absence of FAK phosphorylation is an inability to assemble FN matrix fibrils.Conclusion Our results show that FAKs and Src kinases, which lie downstream of integrins, are essential for efficient initiation of FN matrix assembly. The effects of tenascin‐C on FN matrix and FAK phosphorylation suggest that this protein limits the extent of matrix deposition by regulating FAK activity. Thus, extracellular and intracellular events co‐operate to control FN matrix assembly.