Stretch-dependent changes in molecular conformation in fibronectin nanofibers

Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein that plays an important role in a wide range of biological processes including embryonic development, wound healing, and fibrosis. Recent evidence has demonstrated that FN is mechanosensitive, where the application of force induces conformational changes within the FN molecule to expose otherwise cryptic binding domains. However, it has proven technically challenging to dynamically monitor how the nanostructure of FN fibers changes as a result of force-induced extension, due in part to the inherent complexity of FN networks within tissue and cell-generated extracellular matrix (ECM). This has limited our understanding of FN matrix mechanobiology and the complex bi-directional signaling between cells and the ECM, and de novo FN fiber fabrication strategies have only partially addressed this. Towards addressing this need, we have developed a modified surface-initiated assembly (SIA) technique to engineer FN nanofibers that we can uniaxially stretch to >7-fold extensions and subsequently immobilize them in the stretched state for high resolution atomic force microscopy (AFM) imaging. Using this approach, we analyzed how the nanostructure of FN molecules within the nanofibers changed with stretch. In fully contracted FN nanofibers, we observed large, densely packed, isotropically-oriented nodules. With intermediate extension, uniaxially-aligned fibrillar regions developed and nodules became progressively smaller. At high extension, the nanostructure consisted of highly aligned fibrils with small nodules in a beads-on-a-string arrangement. In summary, we have established a methodology to uniaxially stretch FN fibers and monitor changes in nanostructure using AFM. Our results provide new insight into how FN fiber extension can affect the morphology of the constituent FN molecules.