Regulation of Cell Adhesion and Fibronectin Fibrillogenesis by Biomimetic Substrates

Abstract

Transmembrane integrin receptors modulate a wide variety of cellular functions, like survival, proliferation, adhesion and extracellular matrix (ECM) assembly. The major ligand of a5b1-integrin is fibronectin (FN), a key component of the ECM. This glycoprotein can be found as fibrillar network in different tissues (cellular FN, cFN) or circulating in plasma (plasma FN, pFN). These two physiologically ocurring types of FN also differ in their structure and composition due to alternative splicing. One objective of this work was to analyze how the different FNs affect cell spreading and the formation of adhesion sites. Therefore, glass substrates were coated with either pFN or cFN and cell responses were monitored by phase contrast or fluorescence microscopy. Cell spreading kinetics was similar on both substrates. However, it was observed for the first time that there were differences in the distribution and shape of adhesion sites; while different focal adhesion (FA) markers localized over the whole cell area on cFN, they were found preferentially at the cell periphery on pFN coatings. In addition, cFN favoured a more elongated shape of adhesion sites, a faster translocation of a5b1-integrin and FN fibril formation, showing that the molecular composition of this protein affects in turn FN assembly. Fibroblasts were seeded on gold nanopatterned surfaces biofunctionalized with cyclic RGD peptides targeting mainly avb3-integrin. On substrates presenting an inter-ligand distance of 58 nm, fibroblasts formed actin stress fibers and FN fibrils. In contrast, FN was only detected as dot-shaped accumulations on samples with a higher inter-ligand distance (73 nm or 110 nm), indicating that 58 nm is a critical inter-ligand distance for avb3-integrin that promotes both the formation of stress fibers and FN assembly. In this study I could show that distinct FNs and the spatial organization of integrin ligands differentially affect cell adhesion and FN deposition. In addition, these results reinforce the correlation between FA and fibrillar adhesion formation, indicating that FN assembly can be regulated by controlling cell adhesion through substrate functionalization.