Integrin receptors are transmembrane proteins responsible for linking the internal cytoskeleton of the cell with the outside extracellular matrix (ECM). Through this linkage, cells transmit their forces to the ECM to sense their surroundings. Past studies have been identified that cells sense and respond to the stiffness of their surroundings and alter their biochemical signaling, mechanotransduction. Using labile DNA duplex probes (TGTs) that irreversibly dissociate have shown that specific force thresholds at single-molecule levels are critical in controlling cell activation. Therefore, we were curious whether integrins can sense and distinguish unbreakable ECM ligands which behave thermodynamically identical yet kinetically different in molecular level.
To test this model, we have synthesized the ECM ligands covalently connected with single-stranded DNA (ssDNA) containing a hairpin or a pseudo-knot-like structure (Fig. 1a). The DNA hairpin probe (reversible unzipping) consists of two DNA strands covalently linked at their 5’ and 3’ ends, while the DNA strands that make up the DNA pseudo-knot-like probe (reversible shearing) are covalently linked at their 3’ and 3’ ends. Since the DNA probes consist of identical DNA duplex, they unfold the duplex identically in the thermodynamic process. In contrast, since the DNA duplexes dissociate in different manners, they showed different F_tol and force-extension behavior on oxDNA simulation (Fig. 1b). Importantly, further probe extension reaches the force range into nanonewtons because the probes behave similarly to ssDNA simulated as polyT₇₂ strand.
Using mouse-embryonic fibroblast (MEF) cells cultured on the probes, we have evaluated the cell-adhesion area, focal adhesion and cytoskeleton maturation, and YAP translocation as the mechanotransduction markers. As a result, we found that MEF cells cultured on the probes showed different responses in the markers, indicating that MEF cells distinguished the difference in the molecular force-extension curve of their ligands.

Figure 1 (a) Illustration of reversible unzipping (left) and shearing (right) probes, (b) oxDNA simulation of force-extension curves for reversible unzipping and shearing probes, and a polyT₇₂ chain as a ssDNA model.

Khalid Salaita

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