Höfs, Windie Lynntha; (2023) The role of E-cadherin in the translocation dynamics of Yes-associated Protein. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Yes-associated protein (YAP) transcriptional co-activator is a key mechanosensitive regulator of mammalian growth control. When cells are subjected to mechanical stimuli such as stretching or a change in substrate stiffness, YAP moves from the cytoplasm to the nucleus where it activates pro-growth, cytoskeletal, and stem cell identity genes. Intrinsic and extrinsic physical forces experienced by cells are sensed by mechanosensitive structures such as the cytoskeleton, focal adhesions, and adherens junctions. Past studies have shown that upon application of force, focal adhesions and cell–cell junctions both affect YAP localisation. However, the degree to which each mechanotransduction pathway is required for YAP nuclear localisation upon mechanical stimulation remains unexplored. To examine the relative contributions of focal adhesions and adherens junctions to YAP localisation, I developed mechanical assays monitoring live-YAP localisation in monolayer growth, dissociation (via epithelial-mesenchymal transition, EMT) and stretch. I used CRISPR/Cas9 to generate endogenously tagged YAP epithelial cell lines. By live-imaging these lines, I showed that high levels of the adherens junction component, E-cadherin, limits YAP nuclear localisation during monolayer growth. In contrast, cell scattering during epithelial to mesenchymal transition (EMT), leading to loss of polarized cell morphology and E-cadherin adhesions, triggered YAP nuclear translocation. This switch in localisation is dependent on both the ECM specific integrin engagement and Src signalling. Similarly, stretching monolayers solely by their adherens junction does not induce YAP nuclear localisation. Instead, strain-induced YAP nuclear localisation occurs when the cell-ECM contacts are present. In summary, YAP localisation to the nucleus upon mechanical deformation is mainly promoted by focal adhesion signalling, whereas E-cadherins limit nuclear YAP entry. This study therefore proposes that there is an antagonistic behaviour between cell-cell junctions and cell-ECM attachments which tightly regulates YAP to ensure growth homeostasis.

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