Centrioles are critical organelles within eukaryotic cells that organise centrosomes, which serve as the primary microtubule-organizing centres (MTOCs). These structures are essential for ensuring the fidelity of cell division and cellular signalling and the formation of cilia, specialised structures involved in mechanosensation, motility, and cellular asymmetry. The multifunctional role of centrosomes arises from their dynamic architecture, characterised by a proximal region, a central core, and a distal end. Proteome of Centriole 1 A (POC1A) is a vertebrate protein vital for maintaining centriole integrity, particularly within the core. POC1A mutations have been linked to a rare ciliopathy, SOFT syndrome, underscoring its functional significance. Its homolog, POC1B, is similarly essential and has been shown to stabilise the inner scaffold that spans the core and proximal regions of the centriole, including the A-C linker. The redundancy between POC1A and POC1B, as suggested by POC1 depletion studies, indicates they may co-occupy similar spatial regions within the centriole. Yet, the precise molecular mechanisms by which POC1A performs its structural and regulatory roles remain unclear.
POC1 proteins consist of an N-terminal WD40 domain followed by a coiled-coil motif. The WD40 domain is a highly conserved structural motif across diverse species and serves as a critical platform for protein-protein interactions (PPIs) of centriolar and cilia processes. Despite this knowledge, the exact PPI landscape mediated by POC1A within the centriole architecture still needs to be fully delineated.
In this thesis, I use Affimers—a class of non-antibody scaffold proteins—targeted against the WD40 domain of POC1A (POC1AWD40) to systematically investigate how POC1A mediates its interactions with other centriolar proteins. I propose that POC1A forms a complex with key centriolar constituents such as FAM161A, CEP44, POC1B, and POC5 through its conserved WD40 domain. I further demonstrate that the WD40 domain may share a common interaction interface on the top and side surfaces of the domain. I found that POC5 and CEP44 share the same interaction interface of POC1AWD40, suggesting that these interactions are mutually exclusive and may regulate the centriole assembly process. This finding is particularly intriguing as POC5 and CEP44 are localised to the core and proximal regions of the centriole, respectively. These results provide novel insights into how POC1A may select binding partners based on its position within the centriole, contributing to its multifunctional role in centriole stability and biogenesis.
