Connections Between Polarity Proteins and the Polarised Trafficking Machinery

Polarity is defined by molecular and morphological asymmetries in a cell that lead to distinct, functionally optimised cell geometries. Polarised cells are governed by many regulators, including polarity complexes. One of these, the Par complex, in part ensures the correct localisation of proteins and membrane components. Another set of proteins crucial to polarity regulation are GTPases. Both types of proteins function with the polarised trafficking machinery, including the cargo tethering exocyst complex. These machineries together deliver cargo to specific localisations, creating distinct asymmetries in cells. However, the mechanisms and regulation which connect polarity complexes, GTPases and the trafficking machinery are poorly understood. To address this niche, I have taken a protein biochemistry and cell biological approach. I first focused on direct interactions between the human Par complex and the exocyst. For the first time, I have purified full length Par3, which is an exciting reagent for in vitro biochemistry. I have interrogated its binding to the exocyst through both a single subunit and holocomplex approach, and harnessed both purified protein and in cell strategies. The Par3 protein enabled investigation on the conservation of a CDK1 phosphosite in humans. Following this I chose to investigate the RalA-exocyst interaction in the context of epithelial cells. Initially, using biochemical protein-protein interaction studies to both validate and further investigate RalA interacting with the exocyst. Furthermore, I utilised an NMuMG cell line to visualise RalA-exocyst colocalisation, and to probe the potential involvement of RalA in trafficking pathways including lysosomal and endosomal trafficking. I also used cell biology approaches to assess the membrane identity of RalA localisation in NMuMG cells. These studies provide the foundation for further investigation into the important intersection of polarised trafficking and the regulation of cell polarity.