Optical techniques for the investigation of a mechanical role for FRMD6/Willin in the Hippo signalling pathway
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The mammalian hippo signalling pathway controls cell proliferation and apoptosis via transcriptional co-activators YAP and TAZ, and as such is a key regulator of organ and tissue growth. Multiple cellular components converge in this pathway, including the actin cytoskeleton, which is required for YAP/TAZ activity. The precise mechanism by which the mechanical actomyosin network regulates Hippo signalling, however, is unknown. Optical methods provide a non-invasive way to image and study the biomechanics of cells. In the past two decades, super-resolution fluorescence microscopy techniques that break the diffraction limit of light have come to the fore, enabling visualisation of intracellular detail at the nanoscale level. Optical trapping, on the other hand, allows precise control of micron-sized objects such as cells. Here, super resolution structured illumination microscopy (SIM) and elastic resonator interference stress microscopy (ERISM) were used to investigate a potential role for the FERM-domain protein FRMD6, or Willin, in the mechanical control of the Hippo pathway in a neuronal cell model. A double optical trap was also integrated with the Nikon-SIM with the aim of cell stretching. Willin expression was shown to modify the morphology, neuronal differentiation, actin cytoskeleton and forces of SH-SY5Y cells. Optical trapping from above the SIM objective, however, was demonstrated to be ineffective for manipulation of adherent cells. The results presented here indicate a function for Willin in the assembly of actin stress fibres that may be the result of an interaction with the Hippo pathway regulator AMOT. Further investigation, for example by direct cell stretching, is required to elucidate the exact role of Willin in the mechanical control of YAP/TAZ.
Thesis, PhD Doctor of Philosophy
Embargo Date: 2021-02-15
Embargo Reason: Thesis restricted in accordance with University regulations. Print and electronic copy restricted until 15th February 2021
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