Harnessing the Fibronectin-Binding Peptide S2 as a Probe of Fibronectin Matrix Assembly

Abstract

The extracellular matrix (ECM) is a dynamic network of proteins and proteoglycans that provide structural and functional support to the cells they encompass. One of the most abundant and foundational proteins in the ECM is fibronectin (FN). FN plays many important roles in wound healing, cell-ECM interactions, and matrix assembly and maintenance. FN matrix assembly is a critical cell-driven process that forms a scaffold upon which other ECM proteins assemble, ensuring proper development of tissues and organs in multicellular organisms. To gain insight into specific details of matrix assembly and to observe this process in diverse systems, we have developed a FN-binding peptide, S2, to visualize and label FN matrix as part of an S2 fusion protein. Solid-phase binding assays revealed the binding specificity of the S2 fusion proteins for the N-terminal 70kD fragment of FN, but not for other ECM proteins or FN domains, and a high binding affinity of S2 for FN was calculated. Labeling of FN matrix by S2 fusion proteins in cell culture is comparable to post-hoc antibody labeling, but an inverse relationship exists between cell density and the extent of co-labeled fibrils, suggesting that the S2 peptide harnesses a unique FN-labeling mechanism. Greater incorporation into FN fibrils was observed with longer incubations with the S2 fusion protein. Proximity labeling to deduce protein-FN interactions was also demonstrated by S2-BioID2, with the majority of labeled ECM proteins identified to be FN. Overall, these findings display the unique FN-labeling capabilities of the S2 fusion proteins, thus advancing our knowledge of FN matrix assembly and the overall organization of the ECM with implications that span species, cell types, and diseases.