The spatiotemporal characterisation of signalling pathways in mammalian mitophagy

Mitochondria are double-membraned organelles fundamental to all mammalian life. Despite their importance, mammalian mitochondria are also a major biochemical hazard. Damaged mitochondria generate excessive quantities of reactive oxygen species (ROS), which can damage intracellular content and organelles. Without maintenance of mitochondrial health, ROSinduced mitochondrial damage can escalate into a catastrophic cascade of damage & dysfunction. This process has been implicated in the development of numerous diseases with poor clinical outcomes (Alzheimer's disease, amyotrophic lateral sclerosis, cancer metastasis, MELAS, etc.). Many of these diseases are incurable, but their therapeutic prevention may be possible through maintenance of mitochondrial health. Understanding the regulatory mechanisms of mitochondrial quality control is essential to this objective. My doctoral thesis aimed to characterise the signalling pathways that regulate the degradation of damaged mitochondria via mitophagy. Mitophagy is a mitochondrially targeted variant of macroautophagy, by which cytoplasmic content is sequestered within membranes and delivered to the lysosomes for degradation. Mitophagy is known to exist in mammalian cells, yet the underlying biochemistry has never been linked to an established mechanism of macroautophagy. To characterize the link between mitophagy and macroautophagy, I developed a suite of new genetically encoded mitochondrial probes and a procedure for live-cell correlative light electron microscopy (CLEM). These characterization techniques soon revealed fundamental discrepancies in the experimental basis of two leading mitophagy models, and led to the discovery of a new mechanism regulating the recovery of damaged mitochondria. This new mechanism represents an entirely new paradigm in the context of mitochondrial quality control. The work presented here describes the development of novel characterization techniques, and the unanticipated results yielded by those techniques.