Thesis (Ph.D.) - University of St Andrews, January 2008.

Willin is a novel member of the Four-point-one Ezrin Radixin Moesin (FERM) protein superfamily, containing an N-terminal FERM domain most like the Ezrin-Radixin-Moesin (ERM) family but also the closely related protein Merlin. Willin was initially discovered as a yeast two-hybrid binding partner of neurofascin155, and this interaction has now been confirmed by both co-localisation studies and the use of two different biochemical methods. Like neurofascin155, Willin also localises to detergent resistant membranes, and like the ERM family, it is able to bind to phospholipids. The expression of Willin appears to be toxic as the production of cell-lines stably expressing Willin proved to be not possible and this appears to be because it induces apoptosis in cultured cells. This is a proliferation control function consistent with the suggestion that Willin is the human homologue of the Drosophila tumour suppressor ‘Expanded’. Three antibodies to Willin were also characterised and a novel splice variant, Willin2, subcloned into a GFP-tagged plasmid for comparison with the original form.

The Salvador/Warts/Hippo (Hippo) pathway defines a novel signalling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The Hippo pathway was initially utilised in D. melanogaster, where the Expanded protein acts in the Hippo signalling cascade to control organ size. Willin is the proposed human orthologue of Expanded and the aim of this thesis is to investigate whether willin can activate the mammalian Hippo signalling pathway. Ectopic willin expression causes an increase in phosphorylation of the core Hippo signalling pathway components MST1/2, LATS1 and YAP, an effect which can be antagonised by ezrin. In MCF10A cells, willin over-expression antagonises a YAP-induced epithelial-to-mesenchymal transition via the N- terminal FERM (Four-point-one Ezrin Radixin Moesin) domain of willin. Preliminary results show that willin is expressed within the sciatic nerve of rat and mice, and within the neuromast cells in the zebrafish; suggesting that willin and the Hippo pathway may play a vital role in the developmental regulation within the peripheral nervous system. To conclude, willin influences Hippo signalling activity by activating the core Hippo pathway kinase cassette in mammalian cells.