The physical basis of YAP signalling

Brief description of the context of the research including potential impact;
The control of tissue size during development underpins the evolution of animal size and shape and is of fundamental importance to our understanding of diseases involving cell growth such as cancer. Furthermore, understanding and manipulating tissue growth is essential for regenerative medicine. Although many signalling pathways influence final tissue size, how their activities change to signal growth arrest in a timely fashion remains unclear.
The conserved Hippo (Hpo) pathway represents the best candidate to mediate the effect of mechanical forces on size control. The pathway core consists of a kinase cascade, which inactivates the pro-growth transcriptional co-activator Yorkie (Yki - YAP/TAZ in mammals). Seminal work in cultured mammalian cells has shown that YAP/TAZ activity is correlated with microenvironment stiffness as well as extrinsic stresses applied across intercellular junctions. Activation of the Hippo pathway involves the cytoskeleton and signalling can take place either via the ECM or across intercellular junctions. However, it remains unclear whether the Hippo pathway responds to cellular density within the epithelium or intercellular tension.
Aims and objectives
A common downstream target of the Hippo pathway is the nuclear localization of the transcription factor YAP, which regulates the transcription of numerous genes involved in proliferation and survival. Nuclear exclusion of YAP occurs when the Hippo pathway is switched "on" and conversely nuclear localisation signifies Hippo signalling is switched "off". Seminal work in cultured mammalian cells has shown that YAP/TAZ activity is correlated with microenvironment stiffness as well as extrinsic stresses applied across intercellular junctions. Activation of the Hippo pathway involves the cytoskeleton and signalling can take place either via the ECM or across intercellular junctions. However, it remains unclear whether the Hippo pathway responds to cellular density within the epithelium or intercellular tension.
We will investigate the respective roles of physical forces and cellular density in regulating Hippo signalling.
We will generate monolayers expressing endogenous levels YAP-GFP using CRISPR/CAS9 approaches. This will allow us to observe in real time the movement of YAP in and out of the nucleus of cells within the monolayer, providing a readout for activation of the Hippo pathway.
To determine how Hippo signalling is activated, we will stretch cell monolayers at a constant tension using feedback control and monitor YAP localisation. As monolayers behave as viscoelastic materials, fast extensions result in high tensions and slow extensions in low tensions. If Hippo signalling is sensitive to cellular density, it will be activated for the same strain independent of tension. If the pathway is sensitive to tension, the activation strain will decrease for increasing tension. Next, we will investigate the response of the Hippo pathway to cyclic versus static mechanical stimulation. Finally, we will extend our experiments to cells grown on thin stretchable PDMS sheets where ECM signalling can take place in addition to signalling across intercellular junctions.
Novelty of the research methodology
We will combine novel mechanical testing tools from engineering developed in the LCN with molecular reporters generated in the Crick institute. The mechanical testing system relies on mammalian epithelial monolayers devoid of ECM and suspended between test rods. This experimental setup allows simultaneous application of stretch, measurement of monolayer tension, and high-magnification confocal imaging. A further advantage is that the absence of ECM signifies that all Hippo signalling occurs across intercellular junctions.