Using conjugated fibronectins in cell microscopy and molecular biology

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Using conjugated fibronectins in cell microscopy and molecular biology

Using conjugated fibronectins in cell microscopy and molecular biology

How does the ECM affect cells in health and disease?

The three-dimensional meshwork of proteins and glycans, known as the extracellular matrix (ECM), functions as structural support for cells, but also produces chemical and mechanical signaling cues that drive a multitude of cellular processes^{(reviewed in 1, 2)}. Not surprisingly, the ECM influences cell migration, adhesion, and mechanotransduction signaling, but it also regulates cell invasion, growth, survival, differentiation, metabolism, and many other processes.

The ECM is thought to play an essential role in nearly all aspects of metazoan physiology, and may have an equally important role in many diseases^{(reviewed in 2-5)}. It has been heavily investigated for its role in cancer invasion and metastasis, and has also been implicated in other diseases including fibrosis, muscular dystrophy, connective tissue disorders, dysfunctional tissue repair, vascular, and musculoskeletal diseases.

ECM properties such as its architecture, viscoelasticity, composition, synthesis, remodeling, stiffness, and degradation can all be regulated to control cellular phenotypes that have profound effects both physiologically and pathologically^{(reviewed in 1, 2, 5)}. However, the precise mechanisms that modulate these ECM properties as well as the cell’s response to these changes are still under intense investigation.

Key ECM proteins include collagen, fibronectin, laminin, elastin, and fibrillin. Fibronectin has been shown to play important roles in many physiological processes, and has been implicated in many diseases such as cancer^{(reviewed in 6, 7)}. Effectively studying fibronectin is key to defining its role in the ECM in health and disease. Below, we discuss several applications where our purified labeled fibronectin was used to define the ECM’s role in a multitude of experimental models.

Protocol image — Figure Legend: Schematic depicting the core steps needed to make Fibronectin-micropatterned surfaces.

How are investigators using purified labeled fibronectin to investigate the role of the ECM?

Labeled fibronectins are important ECM research tools that are used to facilitate the investigation of many pressing ECM questions. Scientists use labeled fibronectin, a key component of the ECM, to coat substrates, study mechanisms that regulate ECM properties, and investigate downstream cellular processes like force generation and adhesion.

Below we highlight four applications where our labeled fibronectin (i.e., biotinylated or fluorescent) was utilized to investigate critical questions about the ECM.

Studying ECM patterning:

Understanding how ECM micropatterned substrates affect cell force, function, and signaling is critical to defining its role in disease progression. Investigators use micropatterning approaches involving silicon wafer molds etched with surface features using standard photolithography techniques and polydimethylsiloxane substrate stamps to create specific ECM patterns for experimental testing⁸ (see Figure). Other ECM micropatterning approaches utilizing fluorescent fibronectin have also been reported, including photopatterned substrates⁹ and quartz chrome masks coated with PMOXA¹⁰.

These substrate stamps are then layered with fluorescently labeled fibronectin and stamped onto activated coverslips or culture dishes. ECM micropatterning approaches have recently been used with our FNR01 and FNR02 fluorescent fibronectin proteins in a wide array of experimental models, including developing in vitro pericyte contraction cytometry models¹¹, wound healing experiments¹², nuclear elongation in fibroblast models¹³, 2D traction force measurements^{14, 15}, and to study spontaneous coherent motion¹⁶.

Studying ECM degradation:

Modulation of ECM levels through mechanisms like synthesis and degradation occurs both physiologically and pathologically. ECM degradation has been implicated in several diseases, specifically in cancer. ECM degradation assays are useful for assessing how specific tumor-promoting proteins affect the ECM. For example, investigators used an ECM degradation assay to elucidate how the protein complex of LCN2, LOXL2, and MMP9 promoted metastasis in an esophageal cancer model¹⁷. The fluorescent-fibronectin degradation assay was performed via confocal microscopy on FNR02-coated coverslips with or without cell seeding.

Gibson et al. used the fluorescent-fibronectin degradation assay with our FNR01 purified fibronectin in combination with 1089-seeded-cells to examine how the protease ADAMTS3 affected ECM remodeling and cancer invasion¹⁸. Another example utilized this ECM degradation assay to understand how cancer drugs like EGFR inhibitors target specific proteins to alter ECM degradation¹⁹.

Studying fibronectin uptake and recycling:

Another mechanism to control fibronectin levels via a transcription-independent mechanism is through cellular uptake of fibronectin that can then be recycled for matrix incorporation²⁰. Previous studies utilized biotinylated fibronectin (FNR03) to perform their fibronectin recycling studies^{20, 21}. Post-uptake, these cells were processed, and analysis was performed via immunoblot with a streptavidin-conjugated IR Dye. More recently, biotinylated fibronectin was used in an uptake experiment, but rather than immunoblotting, the group used 488-labeled streptavidin and analyzed the uptake via IF staining²².

Studying the ECM in adhesion and migration assays:

Understanding how ECM properties like stiffness and viscoelasticity affect cell adhesion and migration is of great interest. To assess this, investigators used far-red HiLyte-647-labeled fibronectin (FNR04) to coat the substrate surface and visualized integrin and actin in attached cells to understand its effect on focal adhesion formation²³. Single-cell fluorescence microscopy images showed that cells with more viscous substrates remodeled fibronectin into enriched agglomerations.

In another study, rhodamine-labeled fibronectin (FNR01) was used to confirm effective deposition of the fibronectin for the concentration ranges used to study fibronectin and laminin’s differential effects on macrophage adhesion and migration²⁴. In a third example, investigators used HiLyte-488-labeled fibronectin (FNR02) to confirm coating of surfaces that was used to assess how fibroblast “tracks” (secreted soluble factors, rearranged ECM, and membranous tubular network) promoted cancer cell adhesion and migration²⁵.

References

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2. Zhao, T., et al., Extracellular Matrix Signaling Cues: Biological Functions, Diseases, and Therapeutic Targets. MedComm (2020), 2025. 6(8): p. e70281.

3. Iozzo, R.V. and M.A. Gubbiotti, Extracellular matrix: The driving force of mammalian diseases. Matrix Biol, 2018. 71-72: p. 1-9.

4. Karamanos, N.K., Extracellular matrix: key structural and functional meshwork in health and disease. FEBS J, 2019. 286(15): p. 2826-2829.

5. Dzobo, K. and C. Dandara, The Extracellular Matrix: Its Composition, Function, Remodeling, and Role in Tumorigenesis. Biomimetics (Basel), 2023. 8(2).

6. Lin, T.C., et al., Fibronectin in Cancer: Friend or Foe. Cells, 2019. 9(1).

7. Guerrero-Barbera, G., N. Burday, and M. Costell, Shaping Oncogenic Microenvironments: Contribution of Fibronectin. Front Cell Dev Biol, 2024. 12: p. 1363004.

8. von Philipsborn, A.C., et al., Microcontact printing of axon guidance molecules for generation of graded patterns. Nat Protoc, 2006. 1(3): p. 1322-8.

9. Pezzotti, M., et al., A vertically integrated system for tracking and assessing cell-cycle-aware phenotypes under confinement. APL Bioeng, 2026. 10(1): p. 016114.

10. Caillier, A., et al., T cells use focal adhesions to pull themselves through confined environments. J Cell Biol, 2024. 223(10).

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12. Thiagarajan, R., et al., Pulsations and flows in tissues as two collective dynamics with simple cellular rules. iScience, 2022. 25(10): p. 105053.

13. Dickinson, R.B., et al., Viscous shaping of the compliant cell nucleus. APL Bioeng, 2022. 6(1): p. 010901.

14. Lichtenberg, J.Y., et al., Leader cells mechanically respond to aligned collagen architecture to direct collective migration. PLoS One, 2024. 19(1): p. e0296153.

15. Tran, S.K., et al., P-cadherin-dependent adhesions are required for single lumen formation and HGF-mediated cell protrusions during epithelial morphogenesis. iScience, 2025. 28(2): p. 111844.

16. Lo Vecchio, S., et al., Spontaneous rotations in epithelia as an interplay between cell polarity and boundaries. Nature Physics, 2024. 20(2): p. 322-331.

17. Xia, Q., et al., A protein complex of LCN2, LOXL2 and MMP9 facilitates tumour metastasis in oesophageal cancer. Mol Oncol, 2023. 17(11): p. 2451-2471.

18. Gibson, S.V., et al., ADAMTS3 restricts cancer invasion in models of early breast cancer progression through enhanced fibronectin degradation. Matrix Biol, 2023. 121: p. 74-89.

19. Mao, X., et al., EGFR inhibition promotes functional engraftment and graft survival via donor-derived CHI3L1 in fetal RPE suspension transplantation. Cell Rep Med, 2026. 7(8): p. 102963.

20. Varadaraj, A., et al., TGF-beta triggers rapid fibrillogenesis via a novel TbetaRII-dependent fibronectin-trafficking mechanism. Mol Biol Cell, 2017. 28(9): p. 1195-1207.

21. Varadaraj, A., C. Magdaleno, and K. Mythreye, Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells. Bio Protoc, 2018. 8(16).

22. El-Mansi, S., et al., Endothelial PIEZO1 regulates Weibel-Palade body exocytosis. Blood, 2026.

23. Voigt, J.L., J. Timmer, and E.A. Cavalcanti-Adam, Substrate Stress Relaxation Regulates Cell-Mediated Assembly of Extracellular Matrix. Advanced Functional Materials, 2026. 36(40): p. e09352.

24. Stinson, M.W., et al., Macrophage migration is differentially regulated by fibronectin and laminin through altered adhesion and myosin II localization. Mol Biol Cell, 2024. 35(2): p. ar22.

25. Baschieri, F., et al., Fibroblasts generate topographical cues that steer cancer cell migration. Sci Adv, 2023. 9(33): p. eade2120.