Project No. 2370
Dr David Tumbarello – University of Southampton
Prof Georgios Giamas – University of Sussex
Cellular homeostasis is essential to ensure healthy ageing across the lifecourse of an organism.
Quality control mechanisms exist to minimise cellular damage resulting from mis-folded, aggregated proteins or dysfunctional organelles such as mitochondria, which contribute reactive oxygen species damaging cellular components. Autophagy plays a dominant role in ensuring organelle and protein quality control, and therefore defining mechanisms coordinating this selective degradation of cargo is required to understand what goes wrong during ageing. The primary aim is to use in situ proximity dependent biotin labelling coupled to mass spectrometry to define protein networks essential for whole mitochondrial clearance by autophagy, known as mitophagy. TurboID fusion proteins of key cargo adaptors in the mitophagy pathway will be produced using CRISPR-Cas9 knock-in approaches in established cell lines, which will subsequently be used to develop a local interactome map following mitochondrial induced damage. This will be coupled to streptavidin pull-downs and Western blotting, alongside cellular imaging, to validate candidate interaction partners. These cargo adaptors function as receptors containing ubiquitin-binding domains and LC3-interacting regions, which link ubiquitylated, damaged cargo to the growing LC3-positive autophagosomal membrane. In addition, there is coordination of individual autophagy receptors that not only identify cargo, but also recruit and activate essential autophagy regulators. Therefore, defining the local interactome of individual receptors will provide insight into the molecular mechanisms associated with mitophagy and how coordination of this machinery facilitates encapsulation by the autophagosome.
This project will focus on the autophagy adaptors TAX1BP1, optineurin, and NDP52, which are dominant receptors essential for mitophagy and widely associated with other selective autophagy pathways, such as aggrephagy and xenophagy. By comparing the local interactome of autophagy adaptors with both known redundant and non-redundant functions will allow us to develop a spatial and temporal map in response to severe mitochondrial stress and mitophagy induction.
Candidates with a strong interest in cell biology, CRISPR-Cas9 gene editing, microscopic imaging and proteomics are encouraged to apply