Elucidating the Mechanosensitive Properties of Fibronectin Using Surface-Initiated Assembly

The extracellular matrix (ECM) plays an integral role in many biological processes such as embryonic development, wound healing, homeostasis, and even disease progression. While the physical and chemical properties of the ECM are known to influence cell behavior and modulate gene expression, emerging evidence suggests that ECM proteins also have mechanosensitive properties. Specifically, the ECM protein fibronectin (FN) undergoes strain-dependent conformational changes that modulate it’s self-assembly as well as binding to a number of ligands including transmembrane integrin receptors, growth factors, and other ECM proteins. The goal of this thesis is to investigate how strain affects the structural and biological properties of FN nanofibers assembled through a surface-initiated assembly technique (SIA). First, atomic force microscopy (AFM) was used to morphometrically track the micro- and nanoscale structures of FN nanofibers during large deformations associated with the SIA process. While there were significant changes in length, width, and thickness during this process, the nanofiber volume remained constant suggesting that they behaved as incompressible materials over these strain ranges. In addition, AFM at high resolution revealed that there were distinct morphological differences within the nanostructure of FN nanofibers in pre- and post-release states. Next, the FN nanofibers were uniaxially strained to track how the structural morphology of FN nanofibers from fully contracted to highly strained states. The structure of fully contracted FN nanofibers was predominantly comprised of large, isotropically-oriented nodules that became progressively smaller with increasing strain. At maximum strain, the nanostructure was highly aligned and contained small nodules in a ‘beads-on-a-string’ arrangement. Finally, a Patterning-on-Topography (PoT) method was developed to investigate how FN strain affects C2C12 adhesion and α5 integrin activation. Using this approach, it was found that while a decrease in FN density likely promoted less cell spreading and α5 integrin expression, the observed behaviors were further enhanced by FN in a highly strained state. Collectively, the work presented in this thesis demonstrates how the SIA and PoT can be leveraged to study the mechanosensitive properties of iv FN to ultimately progress our knowledge for how cells are capable of dynamically manipulating and responding to the ECM particularly during tissue morphogenesis and disease progression.