Multiple Cryptic Binding Sites are Necessary for Robust Fibronectin Assembly

Abstract

Assembly of elastic, insoluble fibronectin (FN) fibrils from soluble FN monomers is a crucial step in embryonic development and wound healing. However, the mechanism of FN fibrillogenesis is still poorly understood. FN fibril assembly requires cell-generated forces, applied to growing fibrils and exposing cryptic FN-FN binding sites buried in elastic Type III domains. The number and location of cryptic binding sites in Type III domains has been much debated, and there is increasing experimental evidence that multiple Type III domains may contain stretch-dependent FN-FN binding sites. The requirement of cell-dependent forces to generate fibrils limits the ability to recreate fibrils in cell-free experiments, restricting the investigation of the mechanisms underlying assembly. To address this, we use a recently developed biophysical model of FN fibrillogenesis (Weinberg et al, Biophys J, 2017), which allows us to simulate competing hypotheses for the location and properties of the cryptic FN-FN binding sites and quantify the effect of these molecular alterations on the morphology and biomechanical properties of the assembled fibril. Model predictions indicate that a single FN-FN binding site located in a domain N-terminal of the 10th Type III domain (which contains the integrin-binding motif RGD) facilitates negligible fibrillogenesis, while a single FN-FN binding site in a domain located C-terminal of the 10th Type III domain produces FN fibrils that are neither robust nor physiologically similar to actual FN fibrils. In contrast, the inclusion of multiple FN-FN binding sites predicts robust FN fibril assembly, which, importantly, minimally depends on the individual mechanical and chemical binding properties in FN with binding sites in all 15 Type III domains.