The role of mechanical tension in fibronectin matrix assembly

Many proteins experience mechanical forces in vivo and evidence is emerging that mechanical stretching can alter the functional states of proteins. Fibronectin (FN) is a large, extracellular matrix protein that is assembled by cells into elastic fibrils. Although most of FN's recognition sites are buried in the soluble state, these sites become expressed following cell-mediated assembly into matrices. The assembly of FN matrices is a complex, sequential process that is incompletely understood. We first investigated the role mechanical force in self-assembly separately from cell-mediated events by using a cell-free ex vivo experimental system. We found that FN assembles into extended fibrillar networks after adsorption to a dipalmitoyl phosphatidylcholine (DPPC) monolayer undergoing expansion. The resulting fibrils were characterized in situ by using fluorescence and light-scattering microscopy. There were striking similarities between fibrils produced under DPPC monolayers and those found on cellular surfaces, suggesting that self-assembly is triggered by mechanical force. To investigate structural alterations in FN during matrix assembly, we studied FN conformation in the mature matrices of cultured fibroblasts and during assembly using intramolecular fluorescence resonance energy transfer (FRET) between pairs of fluorophores attached to FN proteins. As the protein unfolds, nanometer-scale increases in distance between donor and acceptor fluorophores cause decreases in FRET measurable by spectroscopy or visible by fluorescence microscopy. The level of FRET was calibrated to FN extension and unfolding by progressively denaturing labeled FN in guanidium chloride solutions. Using FRET as an indicator of FN conformation, we found that FN diffusely bound is in a compact state, whereas FN in matrix fibrils is highly extended and partially unfolded. We then investigated the effect of cell-mediated mechanical tension on FN conformation in matrix fibrils by disrupting cell contractile forces with cytochalasin D. Our results show that FN extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension and that the basis for FN fibril elasticity may be the unraveling and refolding of FN type III domains.