All-Covalent Nuclease-Resistant and Hydrogel-Tethered DNA Hairpin Probes Map pN Cell Traction Forces

Abstract

Cells sense and respond to the physical properties of their environment through receptor-mediated signaling, a process known as mechanotransduction, which can modulate critical cellular functions such as proliferation, differentiation, and survival. At the molecular level, cell adhesion receptors, such as integrins, transmit piconewton (pN)-scale forces to the extracellular matrix, and the magnitude of the force plays a critical role in cell signaling. The most sensitive approach to measuring integrin forces involves DNA hairpin-based sensors, which are used to quantify and map forces in living cells. Despite the broad use of DNA hairpin sensors to study a variety of mechanotransduction processes, these sensors are typically anchored to rigid glass slides, which are orders of magnitude stiffer than the extracellular matrix and hence modulate native biological responses. Here, we have developed nuclease-resistant DNA hairpin probes that are all covalently tethered to PEG hydrogels to image cell traction forces on physiologically relevant substrate stiffness. Using HeLa cells as a model cell line, we show that the molecular forces transmitted by integrins are highly sensitive to the bulk modulus of the substrate, and cells cultured on the 6 and 13 kPa gels produced a greater number of hairpin unfolding events compared to the 2 kPa substrates. Tension signals are spatially colocalized with pY118-paxillin, confirming focal adhesion-mediated probe opening. Additionally, we found that integrin forces are greater than 5.8 pN but less than 19 pN on 13 kPa gels. This work provides a general strategy to integrate molecular tension probes into hydrogels, which can better mimic in vivo mechanotransduction.

Measuring integrin force loading rates using a two-step DNA tension sensor

“…As can be seen in SI Videos 1 and 2, and Figure 3d , the vast majority of shearing events ( F > 56 pN) occur near the periphery of the cell, a phenomenon repeatedly observed in past work. 17, 29, 40 In addition to the spatial information obtained from identifying shearing events, temporal information on the cells force loading rate can be derived from the fluorescence intensity traces. Ten representative traces exhibiting a shearing event are shown in Figure 3e , with a number 1-10 indexing their spatial coordinates according to Figure 3d above.…”

Traction Force Microscopy with DNA FluoroCubes

“…More recently developed techniques to measure cellular forces with high spatial resolution, albeit in a limited force range, rely on molecular force sensors that are directly linked to the adhesion molecule ligands and are grafted onto a rigid surface onto which cells are seeded [45][46][47][48][49] . Recently, such sensors have even been combined with soft hydrogel substrates 50 . DNA-based sensor molecules attached to a substrate can also allow local readout of various other cellular signals 51 .…”

Nuclease-Resistant L-DNA Tension Probes Enable Long-Term Force Mapping of Single Cells and Cell Consortia

“…[7] Cellular tension forces can thus be measured by direct appearance of fluorescence signals. The probes can either be designed for an irreversible (duplex) [12][13][14][15][16] or reversible (hairpin) [17][18][19][20] force detection, allowing to map cellular traction forces in real time. Similar probes can also be integrated into hydrogels to design mechano-reporting materials.…”