Spatial Gradients of E-Cadherin and Fibronectin in TGF-β1-Treated Epithelial Colonies Are Independent of Fibronectin Fibril Assembly

Epithelial to Mesenchymal Transition (EMT) is a dynamic, morphogenetic process characterized by a phenotypic shift in epithelial cells towards a motile and often invasive mesenchymal phenotype. We have previously demonstrated that EMT is associated with an increase in assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils. We have also demonstrated that Transforming Growth Factor-β1 (TGF-β1) localizes to FN fibrils, and disruption of FN assembly or disruption of TGF-β1 localization to FN fibrils attenuates EMT. Previous studies have shown that TGF-β1 induces spatial gradients of EMT in mammary epithelial cells cultured on FN islands, with cells at free edges of the island preferentially undergoing EMT. In the current work, we sought to investigate: (a) whether FN fibril assembly is also spatially patterned in response to TGF-β1, and (b) what effects FN fibril inhibition has on spatial gradients of E-Cadherin and FN fibrillogenesis. We demonstrate that mammary epithelial cells cultured on square micropatterns have fewer E-Cadherin-containing adherens junctions and assemble more FN fibrils at the periphery of the micropattern in response to increasing TGF-β1 concentration, indicating that TGF-β1 induces a spatial gradient of both E-Cadherin and FN fibrils. Inhibition of FN fibril assembly globally diminished E-Cadherin-containing adherens junctions and FN fibrillogenesis, but did not eliminate the spatial gradient of either. This suggests that global inhibition of FN reduces the degree of both FN fibrillogenesis and E-Cadherin-containing adherens junctions, but does not eliminate the spatial gradient of either, suggesting that spatial gradients of EMT and FN fibrillogenesis are influenced by additional factors.