Research Thrust 1:
Single Cell-Cell Mechanical Interrogation and Strain Rate-Dependent Mechanical Behavior Characterization
Cell-cell adhesions are macromolecular structures that integrate individual cells into complex tissues by anchoring different cytoskeleton components. They have been the subjects of intensive scientific investigations due to their importance in cell and tissue mechanics. They are often subjected to mechanical strains of different rates and magnitudes in normal tissue function in our body. However, the strain rate-dependent mechanical behavior of individual cell-cell adhesions has not been fully characterized due to the lack of proper experimental techniques and therefore remains elusive. This knowledge is critical in understanding disease pathology and therapeutics. For instance, two of the biophysical hallmarks of cancer metastasis are the loss of cell-cell adhesion and anchorage-independent growth within the tensioned tumor micro-environment, with cell adhesion loss being the product of malignant transformations and cell interactions with the tensioned cellular microenvironment. Indeed, this clinical relevance supports the need for fundamental understanding of biophysical transformations when cells are subjected to load.
To address this knowledge gap, our group developed a single-cell adhesion micro tensile tester (SCAµTT) platform based on nanofabricated polymeric structures using two-photon polymerization (TPP). Two movable islands, supported with beams of known stiffness, are mechanically coupled through the formation of a mature junction between epithelial cells on each island. Integrating the polymeric microstructure with an atomic force microscopy (AFM) enables us to stretch the cell pair with precisely controlled strain rates, while the deformation of the supporting beams informs the resultant stress accumulated at the cell-cell junction (Figure 1a-d). To the best of our knowledge, this is the first platform that allows in situ investigation of stress-strain characteristics of a mature cell-cell junction through defined strains and strain rates. With SCAµTT, we reveal some interesting biophysical phenomena at the single cell-cell junction that were previously not possible to observe using existing techniques. We showed that cytoskeleton growth can effectively relax intercellular stress between an adherent cell pair in a strain-rate dependent manner. Along with cadherin clustering-induced bond strengthening, it prevents failure to occur at low strain rates. At high strain rates, insufficient relaxation leads to stress accumulation which results in cell-cell junction rupture. We show that a remarkably large strain can be sustained before junction rupture (> 200%), even at a strain rate as high as 50% s-1 (Figure 1e-h). Collectively, our characterization of the strain rate-dependent mechanical behavior of the cell-cell junction builds the foundation for a new mechanistic understanding of junction adaptation to external load, and potentially the spatiotemporal coordination of participating molecules at the cell-cell junction.
A single cell-cell adhesion interface mechanical characterization platform and its use for strain-rate dependent study. a. A single cell pair with junctional contacts is formed on Islands 1 and 2 (each cell residing on the scaffolds in each island). To apply mechanical strain to the cell-cell junction, an AFM-based manipulation system displaces Island 2 while Island 1 is fixed on the substrate. b. The applied displacement strains the mutual junction between the cells and bends the vertical beam under Island 1. A force-displacement relationship can be established by recording the bending, δ. c. The structure was fabricated on top of a glass substrate. A bowtie structure was also fabricated on top of each island for cell confinement. d. Cell deposition onto the fabricated structure shows the ability to place cells in the opposing bowtie confinement scaffolds. E-cadherin (E-cad) expression shows the cell-cell junction formation and actin filaments (Actin) show that cells spread over the structure. e-h. Representative image frames and the corresponding stress-strain curves are shown for stretch tests performed at strain rates of 0.5% s-1 (e-f) and 50% s-1 (g-h). For f, h, an average stress-strain curve (Ave. stress, dotted line) ± standard deviation (blue region), a representative stress-strain curve (Rep. stress, red curve), and an empirical fit for the average stress (Empirical fit, blue curve) are shown. Insets show the zoom-in images of the cell-cell junction at the indicated strain levels during the stretch process. Scale bars: c, 200 µm; d, 50 µm; e, 50 µm; g, 50 µm; Inset in f and h, 15 µm.
Amir Monemian Esfahani, Jordan Rosenbohm, Bahareh Safa, Nicolay V. Lavrik, Grayson Minnick, Quan Zhou, Fang Kong, Xiaowei Jin, Eujun Kim, Ying Liu, Yongfeng Lu, Jung Yul Lim, James K. Wahl, Ming Dao, Changjin Huang, Ruiguo Yang. Characterization of the Strain-Rate Dependent Mechanical Response of Single Cell-Cell Junctions. Proceedings of the National Academy of Sciences (PNAS) (2021), 118(7):1-12.