High-Frequency Mechanostimulation of Cell Adhesion

Abstract

Cell adhesion is regulated by molecularly defined protein interactions and by mechanical forces, which can activate a dynamic restructuring of adhesion sites. Previous attempts to explore the response of cell adhesion to forces have been limited to applying mechanical stimuli that involve the cytoskeleton. In contrast, we here apply a new, oscillatory type of stimulus through push-pull azobenzenes. Push-pull azobenzenes perform a high-frequency, molecular oscillation upon irradiation with visible light that has frequently been applied in polymer surface relief grating. We here use these oscillations to address single adhesion receptors. The effect of molecular oscillatory forces on cell adhesion has been analyzed using single-cell force spectroscopy and gene expression studies. Our experiments demonstrate a reinforcement of cell adhesion as well as upregulated expression levels of adhesion-associated genes as a result of the nanoscale "tickling" of integrins. This novel type of mechanical stimulus provides a previously unprecedented molecular control of cellular mechanosensing.

Keywords: azobenzene; cell adhesion; integrins; mechanosensing; single-cell force spectroscopy.

Figure 1

Chemistry and photoresponse of the RGD push–pull azobenzene layer. a) Functionalization of glass with PEG2000 and RGD push–pull azobenzene. b) Illustration of the RGD‐coupled azobenzene monolayer. If the layer is irradiated with light, the push–pull azobenzene molecules oscillate.

Figure 2

Cell adhesion as a function of RGD push–pull azobenzene oscillation. a) A cell is immobilized on a tipless cantilever and an atomic force microscope is used to measure the deflection of the cantilever during approach and retraction of the cell. b) Representative force–distance curves in the dark and under continuous irradiation with light (530 nm). F _OFF and F _ON are the forces needed to detach cells from the surface when the light is switched off and on, respectively. F _S is the force associated with the last rupture event. c) Normalized cell detachment force in subsequent irradiation cycles for both 1 s and 3 s contact times. The increase in detachment force due to azobenzene oscillation is statistically significant (Student's t‐test, p<0.001). Data from a control experiment (1 s contact time on bare glass) are also shown. Each square represents the mean value of ≥40 force curves; error bars denote standard deviation. Each color symbolizes an independent experiment. Cell detachment forces increase considerably if the molecules oscillate.

Figure 3

Relative frequencies of the force (F _s) associated with the last rupture event in force–distance curves. a,b) Force distributions for 1 s and 3 s cell‐surface contact times. c,d) Boxplots of the force distributions (interquartile range; line in each box: median; dot: mean) for 1 s and 3 s contact times. The force shifts due to RGD push–pull azobenzene oscillations are statistically significant (Student's t‐test, p<0.001, number of analyzed rupture events: 868 (ON) and 1318 (OFF) for 1 s, 392 (ON) and 559 (OFF) for 3 s).

Figure 4

Gene expression as a function of RGD push–pull azobenzene oscillation. a) Experimental steps showing four different types of experiments with different light stimulation protocols (color codes). b) Average fold changes in gene expression in response to different light stimulation protocols on RGD push–pull azobenzene surfaces. Three samples were analyzed with at least two technical replicates for each experiment setting. Error bars denote standard deviation.