Investigating how mitophagy is regulated by the metabolic context

Abstract

The selective degradation of mitochondria via macroautophagy, termed mitophagy, is a key quality control mechanism for mitochondrial network homeostasis, cell integrity and organism development. In mice, basal mitophagy is pervasive across mouse tissues, being particularly high in tissues of high metabolic demand. However, the molecular signalling regulating this basal mitophagy remains unclear as the known mitophagy regulators, PINK1 and Parkin, are not required, as will be confirmed in this thesis. The metabolic context of cells and tissues in vivo is likely to play a central role in the regulation of basal mitophagy. In line with this, basal and stress-induced mitophagy can be modulated in vitro by switching cellular metabolic dependencies between either a glycolytic or OXPHOS metabolic status. I implemented this metabolic modulation to address the aim in this thesis of identifying new factors regulating mitophagy by the metabolic context, which may provide new insights on the regulation of basal mitophagy in vivo. To achieve this aim, I established new tools to investigate mitophagy in vitro; including a new image analysis software to assess mitophagy with the mito-QC reporter and the ARPE-19 cell line as a new model system, from which I found initial hints for a new regulatory role of AMPK in mitophagy. I secondly developed a proteomic approach using APEX2 biotin proximity labelling to detect changes in mitophagy signalling from the mitochondria surface. I exploited the in vitro and in vivo validated mito-QC reporter to target APEX2 on the mitochondrial surface using a conditionally stable anti-GFP nanobody. Then, I implemented this mitochondrial APEX2 proteomics approach to screen for new proteins regulating hypoxia-induced mitophagy by a glycolytic or OXPHOS-promoting metabolic context in ARPE-19 cells. Here, I present the ongoing validation of selected candidates from the proteomics screen.

Date of Award	2020
Original language	English
Awarding Institution	University of Dundee
Supervisor	Ian Ganley (Supervisor)