Vargas, Jose Norberto (Jobert) Sagullo; (2021) Spatiotemporal Control of ULK1 by NDP52 and TBK1 during selective autophagy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Selective autophagy enables cells to degrade specific intracellular cargo, such as damaged organelles, through autophagy. Selective autophagic targets, such as invading bacteria or damaged mitochondria are tagged by various “eat me” signals that allows for the autophagosome to be precisely targeted to cargos destined to be degraded. One of the most prevalent means for tagging intracellular cargo in eukaryotic cells is ubiquitination. For instance, during mitophagy, PINK1 kinase and Parkin E3 ligase is a quality control pathway that senses various mitochondrial insults leading to the feedforward phospho-ubiquitination of damaged mitochondria. The phospho-ubiquitin tagging of impaired mitochondria is followed by the recruitment of receptor proteins through their capacity to interact with ubiquitin chains. Although the hierarchical activation of the many ATG proteins involved in autophagy induction is well- characterized, there is a paucity in the understanding of what links cargo recognition with autophagosome biogenesis during selective autophagy. More specifically, it is unclear what provides the spatiotemporal control for the nucleation of the early autophagy machinery near the cargos destined to be degraded. Furthermore, it is not known how the ULK1 kinase complex, a key initiator of autophagosome biogenesis, is activated during selective autophagy. Recent evidence suggests that receptor proteins are able to recruit early autophagy components to cargo substrates, such as the ULK1 complex. Therefore, receptors could be the missing mechanistic link between cargo recognition and the spatiotemporal control of autophagosome formation. In this thesis, we utilized various CRISPR/Cas9 KO cell lines along with chemically- inducible dimerization (CID) assays to analyze the molecular basis of selective autophagy induction. We find that tethering of the receptor protein NDP52 to mitochondria is sufficient to trigger mitophagy via its ability to focally localize the ULK1 complex. NDP52 interacts with the ULK1 complex through FIP200, and this interaction is mediated by TBK1 kinase. Direct mitochondrial tethering of ULK1 complex also stimulates mitophagy and is able to bypass the requirement for functional receptor proteins and TBK1. Furthermore, we demonstrate that ULK1 autoactivates on cargo substrates, which can occur independent of AMPK and mTOR signalling. These findings provide a parsimonious model for the initiation of selective whereby ULK1 activation is coupled to its cargo localization, which in turn is mediated by receptor proteins and TBK1.

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