Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

Abstract

The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG₇). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni²⁺-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG₇ underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII_7-10) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII_7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

Keywords: His-tag; fibronectin; integrin; patterning; photocleavage reaction.

Figure 1.

Schematic of a dynamic substrate that captures His-tagged proteins in response to light. (a) The surface of a gold substrate was functionalized with the SAMs of photocleavable PEG, NTA and EG₇ ligands. (Left) Before irradiation, the surface prevents adsorption of any proteins because of the presence of PEG brushes. (Right) Photoirradiation cuts off the PEG brushes, and the protein bearing a His-tag (protein A) is immobilized to the NTA group, while keeping the adsorption of non-His-tagged proteins (protein B) blocked by the EG₇ underbrushes. (b) A recombinant fibronectin fragment FNIII_7–10 bearing a His-tagged sequence is immobilized to the NTA ligand where it mediates cell adhesion by interacting with integrin α₅β₁ in the plasma membranes.

Figure 2.

Synthesis of disulfide ligands: (a) PEG-terminated disulfide bearing a photocleavable 2-nitrobenzyl ester, (b) NTA-terminated disulfide and (c) EG₇-terminated disulfide.

Figure 3.

His-tagged proteins used in this study. (a) Schematic of the constructs. (b) Sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified His-tagged proteins: (i) GST-His, (ii) GST-His-FNIII_7–10 and (iii) His-FNIII_7–10. The gels were stained with Coomassie Brilliant Blue. Molecular markers are also shown as reference. The His-tagged proteins are marked by asterisks.

Figure 4.

Contact angle on the gold substrate modified with disulfide ligand 1 versus irradiation time (in PBS, at λ=365 nm and 100 mW cm⁻²). Error bars show standard deviations among three different substrates.

Figure 5.

Effect of the surface composition on reducing undesired protein immobilization/adsorption in PBS. (a) Effect of the PEG chain lengths and content on the immobilization of GST-His to the non-irradiated regions. (b) Effect of the EG₇ content on the adsorption of BSA to the irradiated regions. The immobilized/adsorbed proteins were detected by the immunofluorescence methods, and the amount of each protein was evaluated from its fluorescence intensity. The %NTA was 0.5%.

Figure 6.

Photopatterning of His-FNIII_7–10 on the substrates with different EG₇ contents in PBS. (a–c) Immunofluorescence detection of FNIII_7–10 immobilized to the (a) 50%, (b) 70% and (c) 90% EG₇ substrate. Scale bars represent 50 μm. (d) Amount of immobilized FNIII_7–10 to the irradiated and non-irradiated regions of (a–c). The %NTA was 0.5%.

Figure 7.

Patterning of NIH3T3 cells on the substrates in a serum-containing medium. (a–c) Cell adhesion to the 70% EG₇ substrate (a) with or (b) without His-FNIII_7–10 (50 μg ml^-1) or (c) with pFN (50 μg ml^-1). The substrates were irradiated in a circular pattern. (d, e) Cell adhesion on the 50% EG₇ substrate (d) with or (e) without His-FNIII_7–10 (50 μg ml^-1). The substrates were irradiated in a striped pattern. Cells were allowed to attach for 4 h and phase-contrast images were obtained after removing the unattached cells. The %NTA was 5% for (a–c) and 0.5% for (d) and (e). Scale bars represent 50 μm.

Figure 8.

Identification of proteins involved in the cell adhesion to the photoirradiated regions on the 70% EG₇ substrate. (a) Effect of a soluble GRGDS peptide on the cell pattern formation. Cells were seeded in a serum-containing medium supplemented with 50 μg ml^-1 His-FNIII_7–10 and an excess GRGDS (100 μM) and allowed to attach for 6 h. (b, d) Immunofluorescence detection of the integrin (b) α₅ and (d) α_v subtypes. The integrin-FNIII_7–10 complex was cross-linked and then detected using the corresponding antibodies by the method described previously [18]. (c, e) Phase-contrast images of the cells shown in (b) and (d). The %NTA was 5%. Scale bars represent 50 μm.