Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

Biomechanics

Biomechanics - Poster Session E

Poster P9 - CHARACTERIZATION OF THE STRAIN-RATE DEPENDENT ACTIVE MECHANICAL RESPONSE OF CELL-CELL JUNCTIONS

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Cell-cell junctions are essential components of multicellular structures and often experience strains of various magnitudes and rates during their normal physiological function. However, their mechanical behavior is currently underexplored due to the lack of techniques to quantitatively characterize junctional stress-strain relationships. We developed a polymeric microstructure to strain the mutual junction of a single cell pair while simultaneously recording the junction stress. This structure enabled us to investigate the adaptation mechanisms at play in epithelial cell pairs under various regimens of mechanical tests. By subjecting epithelial cell pairs to ramp strains, we captured a previously unseen strain-rate dependent junction response where stress relaxation due to cytoskeleton growth could mitigate stress buildup and prevent junction failure at low strain rates. In contrast, high strain rates led to synchronized junction failures at remarkably large strain levels. This study and previous studies on epithelial monolayers have demonstrated the profound role of stress relaxation in mitigating the risk of cell damage. However, experiencing large, rapidly applied, and sustained strain may lead to dangerously large deformations that compromise homeostasis, requiring cells to actively tension. By applying controlled sustained strains, we showed that epithelial cell pairs exhibit active tensioning in addition to stress relaxation, depending on the rate and magnitude of strain. Our findings suggest a new mode of stress response in which epithelial cells actively generate stress to reduce strain and potentially prevent fracture. Collectively, our work provides a foundation for future research in the mechanobiological investigation of adaptive behaviors in epithelial cells.

Materials and

Methods:  We designed and fabricated a novel single cell adhesion micro tensile tester (SCAµTT) using two-photon polymerization and performed displacement-controlled tensile tests of individual pairs of adherent epithelial cells with a mature cell-cell adhesion (Figure 1.A-B). These micro-devices consist of a double-island spring-like structure. One island is used for applying micro-ranged displacement while the other island functions as a force sensor that can measure the force applied to the cell junction with a resolution in the nano-Newton range. The device is integrated with atomic force microscopy (AFM) system to precisely apply the strain and strain rate. To limit the migration of cells on the structure, we engineered a bowtie cell containment that not only prevents the cell from moving from one island to another but also increases the chance of forming a mature junction between the gap. A single A431 cell is picked using the Eppendorf single cell isolation system and placed on one of the islands and an additional cell is placed on the other island. When a mature junction forms between the cell pair, for studies involving ramp strains, the cell pair is subjected to increasing strain until failure to assess mechanical strength. For studies involving sustained strains, a predetermined strain is applied at a controlled rate and maintained for two minutes. The stress within the structure is determined by monitoring the deformation of the beams. Utilizing this novel platform, we can evaluate the strength of adhesion junctions with different mutations or in the presence of modifying reagents.

Results, Conclusions, and Discussions: To interrogate the response of epithelial cell pairs under ramp strains, we stretched the junction under three main strain rates. At the lowest strain rate (0.5%s^(-1)), we observed no junction rupture even at strains higher than 200% (Figure 2.A). At the intermediate strain rate (5%s^(-1)), cell-cell junctions began to show some signs of rupture through a gradual necking process (Figure 2.B). 47% of cell pairs stretched at this rate experienced complete junction rupture. However, a 50%s^(-1) strain rate led to a sudden rupture in all cell pairs due to stress accumulation (Figure 2.C). To elucidate the rate-dependent response, we developed an active mechanosensing model and demonstrated that cytoskeleton growth plays an essential role in dissipating stress at low strain rates and protects the integrity of the junction. The model revealed that this stress dissipation mechanism is less effective at high strain rates because the cells have less time to adapt to external stimuli.
Therefore, to explore the adaptation mechanism at higher strain rates, we performed another set of experiments by applying ramp strains at different rates and maintaining the strain constant for 2 minutes (Figure 3.A). Interestingly, we observed a different mode of strain-rate dependent response (Figure 3.B). First, stress increased with the ramp deformation and peaked when the target deformation was reached. Then, regardless of the strain rate, stress briefly relaxed in the first ~10s. After this short dip in stress, the temporal evolution of stress in the following 2 minutes depended greatly on the applied strain rate. For strain rates ≤5%s^(-1), the stress remained approximately constant at the stress achieved after the relaxation phase, indicative of a solid-like behavior. However, for strain rates ≥10%s^(-1), in some cell pairs, the stress rose linearly (i.e., tensioning) reaching values larger than the stress at t = 0 s. Also, the rate of stress enhancement increased with strain rate. In conjunction with the stress increase, we also observed a decrease in strain at t = 2 min (Figure 3.C). These results suggest that cells are actively reacting to the loading conditions and exhibit varied responses dependent on those conditions.

Acknowledgements (Optional):  We acknowledge fundings from the NSF (award #1826135, #2143997) and the NIH National Institutes of General Medical Sciences (R35GM150623). Design and fabrication of the TPP structures were conducted at the Center for Nanophase Materials Sciences (CNMS) at ORNL, which is a DOE Office of Science User Facility.