Fibronectin (FN) is a foundational extracellular matrix (ECM) molecule that plays essential roles in cell adhesion, migration, and differentiation. It serves as a scaffold for cells and other ECM proteins, influencing tissue organization and cellular signaling. FN is essential for embryonic development and plays key roles in tissue morphogenesis. Dysregulation of FN assembly has been implicated in a wide range of pathological conditions including fibrosis, cancer, skeletal abnormalities and cardiovascular disease.
FN exists as a large glycoprotein composed of multiple functional domains that facilitate interactions with integrins, growth factors, and other ECM molecules. It is secreted as a soluble dimer and undergoes fibrillogenesis, a process driven by cell-FN interactions that converts soluble FN into insoluble fibrils. This dynamic assembly is crucial for ECM deposition and organization. Despite its importance, visualizing FN matrix formation in live-cell systems remains a technical challenge, limiting our ability to study its real-time dynamics and cellular interactions.
Traditional methods for studying FN matrix include immunofluorescence staining and addition or expression of fluorescently-tagged proteins. While these approaches provide valuable insights, they each have drawbacks for studying ECM dynamics. For example, immunofluorescence staining often requires fixation, which halts the assembly process, whereas genetically-tagged ECM proteins limit study of FN to the organisms/cells in which the tag exists. Addition of fluorescently-tagged FN to cell cultures marks sites where assembly is occurring but may give incomplete labeling that may bias analysis. As such, a probe that could be deployed in diverse systems and allow for live imaging of FN would facilitate analysis of FN matrix assembly and help further our understanding of FN in both healthy and pathological contexts.
Adherent cultured cells such as fibroblasts or endothelial cells deposit FN-rich matrices that mimic aspects of tissue microenvironments. These and other in vitro models have been instrumental in understanding FN’s role in physiological and pathological conditions. FN visualization through super-resolution microscopy and molecular probes that differentially stain stressed versus relaxed FN fibrils have advanced our understanding of specific features of the ECM. A tool that stably incorporates into FN fibrils would be very useful for studying FN matrix assembly, remodeling, and cell-matrix interactions in static and real-time situations.
To address this goal, we leveraged phage display technology to develop a novel GFP-tagged FN-binding protein that integrates into FN fibrils. We identified a unique peptide that allows for live imaging of FN matrix formation and offers the unique advantage of matrix retention after decellularization, enabling the study of fluorescent FN matrices in both culture systems and cell-derived scaffolds. Here, we describe the development and application of this tool for visualizing FN matrix dynamics in both fixed and live-cell environments.
We screened an M13 phage display library for peptides that bind to a recombinant N-terminal 70 kDa fragment of human FN. The 70 kDa fragment is composed of two domains, the assembly domain with the FN-binding site that is required for matrix assembly and the gelatin/collagen-binding domain. The phage display library displaying 12 amino acid-long peptides was panned against plates coated with the 70 kDa fragment following the workflow in Supplementary Figure S1. After 3 rounds of panning, 47 phages were randomly selected for sequencing, which yielded 26 unique peptide sequences. 11 of the 26 sequences were found in multiple phage isolates. Binding of individual phage clones to 70 kDa was confirmed with an enzyme linked immunosorbent assay (ELISA), using pooled unbound phage from the first pan as a negative control. All but 2 of the 26 unique sequences generated a positive ELISA signal, as defined by a mean signal higher than 3x the average negative control signal.
FN-binding peptides identified by phage display. (A) M13 peptide phages were screened against surfaces coated with the 70 kDa fragment of human FN. Phages arbitrarily numbered S1 through S47 were used for DNA sequencing. Each unique peptide sequence and the phage clone numbers are listed (left and middle columns). Note that some sequences were present in more than one phage isolate. Phage binding to 70 kDa was confirmed in an ELISA, with “+” indicating a signal at least 3 times the negative control. (B) NIH 3T3 cells grown on glass coverslips were fixed, blocked with BSA, and then incubated with 1 × 10 11 pfu/mL phage for 1 h before immunostaining for FN (left, green) and M13 phage protein (middle, red) with the merged images shown on the right. Examples show representative photomicrographs of three phages and depict two phages (S2, S12) that demonstrated positive binding and S33 which did not bind. Scale bar = 200 μm.
To determine whether the peptides can bind to FN fibrils, we used NIH 3T3 fibroblasts which assemble a dense fibrillar FN matrix. Cultures were fixed and incubated with individual phage clones, followed by co-staining with antibodies against FN and an M13 phage protein. Phage binding was scored as positive if phage signal both resembled and overlayed the fibrillar structure of the FN matrix. Three of the phage clones (S2, S12, S39) exhibited a fibrillar pattern with extensive colocalization with the FN matrix. The other M13 phage staining resembled the negative control (wild-type M13 phage, WT), with very little phage signal shown in two examples (S33 and S37). We conclude that while most of the peptides can target phages to FN-coated plastic, only 3 peptides showed the capacity to bind phage to FN fibrils in this assay.
The observation that most of the peptides did not bind to FN fibrils after assembly and fixation suggests that the binding sites might not be accessible or may be affected by fixation. Phage clones were incubated with growing fibroblast cultures during matrix assembly for 48 h followed by fixation and staining. Since our phage display screening was against human 70 kDa, we used human WI-38 fibroblasts in this experiment to assess incorporation of phage into FN matrix. As with the matrix binding experiment with mouse cells, phages with peptide S2, S12, or S39 showed significant incorporation into the human FN matrix. The WT negative control phage showed very little association with FN fibrils. In contrast to the binding results, a number of phage pept
