A novel fibronectin-binding peptide reveals dynamic intermediate structures during matrix assembly.

The extracellular matrix (ECM) is an expansive network of polymers that regulates cell adhesion, migration, and tissue morphogenesis, and that becomes dysregulated in fibrotic disease states. Of the many ECM proteins, fibronectin (FN) is foundational and FN fibrils are instrumental in promoting deposition of many other ECM proteins. FN fibrils form by cell receptor-mediated polymerization of secreted FN dimers. To provide a deeper understanding of the FN assembly process, we sought to identify FN-binding peptides that could be used to target FN during matrix assembly. Here we report the isolation of a FN-binding peptide, S2, by M13 phage display. When displayed within a GST-S2-EGFP fusion protein, S2 directs incorporation of the fluorescent fusion protein into FN fibrils during fibroblast matrix assembly, enabling direct visualization of fibril formation and accumulation. The S2 fusion protein remains stably associated with FN in decellularized matrices. Live cell imaging with GST-S2-EGFP revealed events in matrix formation and maturation and highlighted distinct features of FN assembly such as nascent fibril nucleation and elongation from coalescing FN aggregates. This S2 peptide fusion protein represents a new tool for real-time analysis of the ECM, for generation of fluorescent scaffolds, and for directing functional proteins to FN matrix.