Analysis of E-Cadherin as a Tension Sensor at Epithelial Cell-Cell Junctions

Abstract

Cells are mechanosensitive systems in which the detection of external mechanical forces by proteins that span the plasma membrane leads to changes in cell organization, differentiation and proliferation. Cell-cell contacts are thought to be sites of mechano-transduction, and a major cell adhesion protein E-cadherin is a candidate protein to transduce forces between cells under tension. To directly test whether force transduction occurs through E-cadherin, we inserted a force sensor module into the E-cadherin cytoplasmic domain. The force sensor (TSMod) is a 32-aa-long flexible peptide capped by mTFP and Venus that together form a FRET pair. FRET efficiency scales inversely with linker extension, and thus reports the tension exerted on the sensor within the host protein. Using FRET intensity ratio imaging in live MDCK cells, we show that the E-cadherin/TSmod construct is recruited to cell-cell contacts like its endogenous counterpart. We observe decreased FRET both at cell-cell contacts and at the plasma membrane distant from intercellular junctions, indicating that the E-cadherin cytoplasmic domain is under tension regardless of its recruitment to intercellular contacts. Significantly, we observe increased FRET upon actin cytoskeleton disruption or depletion of alpha-catenin, a putative linker between actin and E-cadherin, regardless of subcellular location. These results indicate that the E-cadherin complex is under tension through the catenin complex and actin cytoskeleton, and that this is a constitutive state that is independent of cell-cell adhesion.