Autophagy ('self-eating') is a cellular pathway that delivers cytoplasmic material, including organelles and protein aggregates, to the lysosome for degradation and recycling. The transport carrier for this pathway is a specialised double-membrane vesicle called an autophagosome which encapsulates target material and fuses with the lysosome. This process occurs in all eukaryotic organisms from yeast to man, and is important for cellular maintenance and adaptation to stress. Dysregulation of autophagy has been implicated in a broad range of diseases including cancer and neurodegeneration, therefore the modulation of autophagy has been proposed as a potential therapeutic strategy. The Ketteler lab has a long-standing interest in identifying genes that regulate autophagy as potential drug targets, as well as compounds that affect autophagy. In parallel to this, there are fundamental questions that are yet to be answered regarding the regulation of key genes already known to be involved in autophagy (the 'Atg' genes) in mammalian cells. A better understanding of the process in general will allow prediction of the effects of perturbation on autophagy, as well as more targeted approaches for therapy. The emergence of CRISPR-based genome editing technology has made the study of fundamental cell biology pathways in mammalian cells easier than ever before.
My PhD project focuses on understanding the function and regulation of the autophagy protease Atg4B using a human CRISPR knockout cell line. This protease is known to be involved in processing the LC3 family of proteins that are important in the formation of the autophagosome. However, there are two processing steps, and the effects of each step on autophagy have been difficult to distinguish and therefore are not fully understood. Furthermore, since LC3 is used as an autophagy marker protein, this presents the conundrum of being able to detect autophagy when the function of LC3 is perturbed. My project therefore makes use of the excellent electron microscopy (EM) facility available at the LMCB, since EM is a powerful tool to identify autophagosomes without the use of a marker protein. Importantly, this method can also be used to correlate fluorescent protein localisation by light microscopy with ultrastructure at the EM level (see image). I am also using high-content imaging approaches to obtain and analyse cellular phenotypic data, which the Ketteler lab as part of the High-content Biology platform , and working with the Bioinformatics Image Core (BIONIC), specialise in. Other techniques I use regularly include confocal microscopy on live and fixed cells, western blotting and biochemical assays. Overall, my project will answer questions about the basic biology of autophagy which could have wider implications for developing therapeutics.