Next generation imaging to investigate the mechanisms of mtDNA escape

Background: Mitochondria are energy-producing organelles. They form a network inside cells, and their health is critical to cell function. Mitochondria also carry their own tiny genome; a 16kb circular double helix that encodes 13 proteins. Despite being localised to the innermost compartment of the mitochondria, it is becoming increasingly apparent that mtDNA can escape to the cytoplasm, and the extracellular environment, where it is a potent agonist of the innate immune system. More than 60 studies have documented increased circulating mtDNA levels in myriad disease settings including SLE, sepsis, liver failure, HIV, myocardial infarct, stroke, cancer and rheumatoid arthritis. We recently published the first example of mtDNA escape to the cytoplasm of cells in real-time, via a process we termed mitochondrial herniation. Within these images, we discovered mitochondrial-derived vesicles which contained mtDNA, however their formation, frequency and function remains a mystery. We hypothesise that this novel entity might be one mechanism by which mtDNA is shuttled around the cell and out into the extracellular environment. Correlative Light and Electron Microscopy (CLEM) combines the strengths of both light and electron microscopy, allowing the analysis of biological events at the highest resolution possible, whilst maintaining cellular context. When combined with cryo-focused ion beam (FIB) milling, which can cut windows into thicker parts of the cell, this will allow all-access fluorescence-directed high-resolution imaging of cellular events. Aims: This project aims to develop a combined Cryo-CLEM and Cryo-FIB-milling approach to characterise the frequency, formation and fate of DNA-containing mitochondrial-derived vesicles. Such an approach will be an Australian-first, and the biological question it will be applied to, will generate significant interest in the cell biology, mitochondria and cell death fields. Techniques: This project will combine techniques at the very forefront of both Electron and Light Microscopy, whilst also utilizing standard biochemical and cell biology techniques. This will include all techniques associated with sample preparation, Cryo-CLEM, Cryo-FIB-milling, cell culture, CRISPR-Cas9 gene editing and multiple methods for image/data analysis and visualisation. Supervisors: This project will be co-supervised by Dr Georg Ramm, Dr Kate McArthur and Professor Benjamin Kile. Dr Georg Ramm is the head of the Monash Ramaciotti Centre for Cryo-Electron Microscopy. He is an internationally recognized specialist in developing new imaging techniques and the application of cutting-edge optical/electron microscopy to biological questions. Dr Kate McArthur is an early career researcher with expertise in the fields of imaging, cell death, immune signalling and mitochondrial biology. Dr McArthur’s major publications have combined cell biology and advanced light microscopy to demonstrate that mtDNA triggers the cGAS/STING pathway during apoptosis through the newly defined process of mitochondrial herniation. Prof Benjamin Kile is the Executive Dean of the Faculty of Health and Medical Sciences at The University of Adelaide. Prof Kile is an internationally recognised expert on cell death, with over 100 primary research papers, amounting to more than 6,800 citations. These works include the two publications providing the key background for this project, (White et al, Cell 2014) & (McArthur et al, Science 2018).