Professor The Ohio State University, United States
Introduction:: Transmission of cell-generated (i.e., endogenous) tension at cell-cell contacts is crucial for tissue shape changes during morphogenesis and adult tissue repair in tissues like epithelia. E-cadherin-based adhesions at cell-cell contacts are the primary means by which endogenous tension is transmitted between cells. The E-cadherin-β-catenin-α-catenin complex mechanically couples to the actin cytoskeleton (and thereby the cell’s contractile machinery) both directly and indirectly. The α-catenin-vinculin complex in particular is essential for the mechanotransduction response to high forces. However, the key mechanical links required for substantial endogenous force transmission at these adhesions in cell-cell contacts remains unclear. Identification of crucial mechanical linkers is essential for understanding cell shapes changes during physiological processes and events like cancer metastasis. The objective of our study is to directly test the hypothesis that α-catenin is more essential for high endogenous force transmission than downstream linkers like vinculin.
Materials and Methods:: Madin-Darby Canine Kidney (MDCK) II cells were cultured in DMEM with 10% FBS. MDCK cells were plated overnight onto collagen I-coated soft silicone of Young’s modulus 8.7±2.0 kPa atop 22 mm square No.1.5 coverslips in 35 mm culture dishes and then used for experiments. To generate knockout (KO) cells, CRISPR/Cas9 was used with previously validated gRNA sequences in the Sigma All-in-One U6-gRNA/CMV-Cas9-tGFP Vector. Clones were screened for vinculin or α-catenin loss using Western blotting. For the double (α-catenin and vinculin) KO, vinculin KO cells were used to generate an additional KO of α-catenin. α-catenin with its vinculin binding site (amino acids 316-405 in α-catenin) replaced by a homologous similar sequence from vinculin (amino acids 514-606 in vinculin), called α-catenin DVBS (delta vinculin binding site) was used to generate the α-catenin DVBS in α-catenin KO cell line. A Leica DMi8 epifluorescence microscope was used to image live and fixed cells. Traction Force Microscopy (TFM) was used to obtain traction maps and the Traction Force Imbalance Method (TFIM) was used to compute the inter-cellular force at a cell-cell contact within a cell pair from the vector sum of traction forces under each cell within the cell pair. Biaxial stretch of epithelial Islands on silicone sheets was used to assess cell-cell contact adhesion strength. For statistical analysis, t-tests or ANOVA was used.
Results, Conclusions, and Discussions:: We found that the cell-cell tension was significantly less for vinculin KO cell pairs (fig.1) than that for WT cell pairs. Since it is α-catenin that is considered to be the primary recruiter of vinculin to E-cadherin adhesions, we surmised that α-catenin would be at least as important as vinculin in force transmission through cell-cell contacts. To our surprise, we found that the inter-cellular tension for α-catenin KO cell-cell contacts was not (statistically) significantly lesser than that for WT contacts (fig.1).
Next, we wanted to test the specific role of the α-catenin-vinculin interaction. We found that the inter-cellular force for MDCK α-catenin-DVBS cell-cell contacts (where the α-catenin-vinculin interaction is prevented) was also not (statistically) significantly lesser than that for WT contacts (fig. 1). Furthermore, the cell-cell tension at MDCK α-catenin-vinculin double KO contacts was also very low– similar to that for vinculin KO contacts and significantly less than that for WT contacts (fig. 1). Thus, it is vinculin rather than α-catenin that is essential for transmitting high endogenous forces at cell-cell contacts.
Given the essential role that we found for vinculin in the exertion of high endogenous inter-cellular forces, we wanted to know if vinculin also performs a similar essential role in protecting cell-cell contact integrity under mechanical challenges. We found that (fig. 2), upon external stretch, MDCK vinculin KO cell-cell contacts ruptured at over twice the rate as that of MDCK cell-cell contacts. Thus, vinculin is not only essential for high endogenous force transmission, but also for maintaining cell-cell contact integrity under high external forces.
To conclude, our results not only suggest that the α-catenin-vinculin complex is not necessary for transmitting high endogenous tension through cell-cell contacts, but also that α-catenin’s interaction with other proteins like afadin, EPLIN or ZO-1 is not an essential mechanical link for transmitting high inter-cellular tension. Vinculin’s ability to support high junctional tension as well as high contact strength, as shown here, is consistent with its essential role not only at cell-cell contacts in epithelia, but also other tissues undergoing dynamic events, both during normal and disease states.
