Quantitative in vivo analysis of ligand-receptor interactions in the Wnt signalling network

Abstract

The Wnt family of proteins are secreted glycoproteins with significant palmitoleate post-translation modification that renders each Wnt protein hydrophobic. Wnt proteins are a class of morphogens whose role is to be secreted and dispersed within the tissue to elicit a wide range of cellular responses based on the concentration of the ligand. However, as Wnt proteins are hydrophobic, standard diffusion through the aqueous extracellular environment is severely reduced. Indeed, alternative methods are required to aid dispersal and prevent aggregation. One such method involves active extension and protrusion of cell surface membrane by actin rich structures called cytonemes. These cytonemes have been characterised in the literature and several proteins are known to play an important role in their function and regulation. Indeed, Wnt protein over-expression and reception directly modulates cytoneme number, length and polarity of the cell to bias cytoneme direction in growth. However, to date, there has been no unbiased identification of the proteome surrounding cytoneme mediated Wnt handover. To address this, I developed a novel GFP-binding nanobody-directed biotin ligase system to extract and identify proteins specifically on cell surface membrane and cytoneme protrusions involved in the Wnt handover. I generated this assay from the ground up; exploring different cell cultures, biotin ligase constructs, methods of application and optimisations. This methods development was subject to a series of mass spectrometry analyses, which culminated in the identification of Caveolin1 as a protein hit to Wnt5a. Further protein hits were also suggested and investigated for possible co-localisation to Wnt5a, demonstrating the success of the assay. While further improvements are necessary, this thesis demonstrates the success of the novel nanobody-biotin ligase assay and its applicability to a wide range of proteins of interest.