An in vitro DNA Sensor-based Assay to Measure Receptor-specific Adhesion Forces of Eukaryotic Cells and Pathogens

Background

Motile cells and pathogens interact in many different ways with their environment (Parsons et al., 2010; Nan, 2017; Muthinja et al., 2018). For example, transmembrane receptors anchor single cells in their environment and allow them to interact with other cells (Hynes, 1992). Integrins, which are the main class of receptors connecting cells to the extracellular matrix, transmit forces in a bidirectional manner (Schoen et al., 2013). On the one hand, extracellular mechanical signals are transduced to the cytosol, where they trigger signaling cascades and thereby control various cellular functions like proliferation, differentiation and migration (Wang et al., 1993; Janmey and Miller, 2011; Totaro et al., 2017). On the other hand, forces generated by the cytoskeleton can be applied to the extracellular environment, causing tissue shape changes and modulate cellular motility in specialized cells (Iskratsch et al., 2014).

To test for integrin-specific adhesion of cells, here, we first describe a method “option I” to prepare substrates presenting cyclic Arginin-Glycin-Aspartic acid (cRGD) ligands while non-specific surface interactions being prevented by a polyethylene glycol (PEG)-based passivation layer (Figure 1A). Of note, this method can be easily adopted to other receptors by varying the ligand.

To characterize the different aspects of cellular force transmission like absolute tension values, direction or the spatiotemporal distribution of forces several methods exist, which are based on the deformation of hydrogels, micropillars or unfolding of molecules, which differ in their spatial resolution and force range (Polacheck and Chen, 2016). One of the latest approaches visualizes traction forces exerted on single ligands via fluorescent DNA-based tension sensors (Wang and Ha, 2013; Blakely et al., 2014; Zhang et al., 2014). These double-stranded (ds) DNA or hairpin (particularly self-annealing single stranded (ss) DNA structures) sensors are immobilized on an in vitro surface and carry a ligand (e.g., cRGD) that enables for receptor (integrin)-specific cell interactions. Upon force, the dsDNA regions can be unfolded, which is detected with a fluorescent sensor (Figure 1B). In combination with standard wide-field or total internal reflection microscopy the spatiotemporal distribution of forces can be determined at high resolution, which is only limited by the optical resolution (~0.2 µm). The mechanical stability of dsDNA depends on its length and the GC content (Zhang et al., 2015). The force F_1/2 that is required to unfold 50% of the DNA sensors, has been calibrated in previous single-molecule studies between ~4-60 pN for varying DNA sequences (Wang and Ha, 2013; Zhang et al., 2014).

The herein presented method “option II” builds on DNA hairpins, which allow to quantify single-molecular, force transmission in the range of 4-19 pN (Zhang et al., 2014; Liu et al., 2016). We modified the DNA strands to enable coupling of various ligands in a modular manner depending on the receptors of interest. In the closed configuration, the fluorescent tag is in close proximity to a quencher and thus dark. Above a specific threshold force the DNA-hairpins unzip and elongate, which lowers the quenching efficiency and gives rise to a digital ON/OFF signal (Figure 1B) (Woodside et al., 2006). They can refold if the force no longer persists (Bonnet et al., 1998; Woodside et al., 2006), which is accompanied by a decrease in fluorescence. This is especially important for the characterization of cells with high turnover of adhesion formations like motile pathogens. Here, DNA hairpin sensors were immobilized via gold nanoparticles, which serve as additional fluorescence quenchers. This reduces the background signals induced by thermodynamic fluctuations as well as the photobleaching in comparison to other sensor surfaces (Lang et al., 2004; Liu et al., 2016). If the range of forces a certain receptor can transmit is not previously known, it is advisable to start with the lowest force probe as it quantitively detects forces above its threshold. Next, the assay can be expanded by multiplexing sensors with different force thresholds and distinct fluorescent dyes to obtain semi-quantitative information on the distribution of cellular forces (Zhang et al., 2014).

Finally, we show how to combine substrate specific adhesion as established in “option I” with the molecular tension sensors “option II” allowing for traction measurements while assuring proper cell adhesion to the surface. This method “option III” can prevent frequent detaching of small and motile cells like Plasmodium sporozoites, even if they show low binding affinities for the ligands coupled to the force sensors.

By immobilizing the molecular tension sensors on such adaptable substrates, they can be used in a wide range of biophysical studies of cell adhesion to extracellular ligands. We recently employed molecular tension sensors to study the specific adhesion between pathogenic agents like viral particles and mammalian cells (Wiegand et al., 2020). Here we provide a protocol to study the interaction of different cell types, including Plasmodium sporozoites, with specific matrix ligands. The rapid movement of the Malaria parasites is a crucial part in the infection cycle (Frischknecht and Matuschewski, 2017) and has been investigated with traction force microscopy (Münter et al., 2009) and laser tweezers (Quadt et al., 2016). Note that the molecular sensors semi-quantitatively detect distribution of forces transduced via single receptors, while they do not report on the overall forces transmitted per cell (Goktas and Blank, 2017) nor the direction of force without integration of further fluorescence polarization techniques (Brockman et al., 2018). Since the methods are very sensitive to changes in the experimental procedure, we recommend to carefully control each step of the protocol.


Figure 1. Schematic of the experimental design and working principle of the in vitro DNA-sensor force assay. A. Step-by-step representation of the individual steps in this protocol to generate ligand-presenting surfaces (Opt. I), surfaces with DNA-based force sensors (Opt. II) and combination of cell adhesion ligands presented in the background of the surface and DNA-based force sensor bearing another type of ligand (Opt. III). Briefly, glass coverslips are cleaned, passivated with a PEG-layer and cellular ligands, gold nanoparticles and DNA strands are immobilized on top. For details, the indicated steps lead to the corresponding sections in the procedure chapter. B. Design of the DNA hairpin force sensors in relaxed conformation and under load. ‘Hairpin’ ssDNA (grey) forms the backbone of the sensors and is linked to the Gold nanoparticle at the bottom and a ligand at the top. Top ssDNA (orange) is linked to the fluorescent dye, bottom ssDNA (blue) is linked to a quencher. Additional bottom ssDNA (green) increases the quenching of background fluorescence. Annealing procedure of the DNA hairpin probe is described in Recipe 8. When a force is applied the partially self-annealing ‘hairpin’ strand unfolds by rupturing the hydrogen bonds between nucleotides, lowering the quenching efficiency between the two dyes, and thus local increase in the fluorescent signal. Figure is not drawn to scale.