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The role of CASM in the endolysosomal damage response https://doi.org/10.17863/CAM.127207 Cross, Jake Florey, Oliver Lysosomes are the major degradative organelle within the cell, where they play integral roles in degradative processes such as macroautophagy and phagocytosis, in addition to engaging in other key cellular processes such as antigen presentation. Endolysosomal (hereafter referred to as lysosomal) damage can occur in response to various cell intrinsic and extrinsic factors, such as infection, neurotoxic aggregates, and lysosomotropic drugs. Given the importance of lysosomal function to the overall function of the cell, and the deleterious effects of the wider cellular environment being exposed to the highly acidic, degradative environment of lysosomes, it is essential that lysosomal damage can be efficiently and effectively resolved. This project explores and establishes the role of Conjugation of ATG8 to Single Membranes (CASM) in the lysosomal damage response. Findings conclusively showed that following lysosomal damage, CASM was rapidly and potently activated, and subsequently mediated the lipidation of ATG8 (e.g. LC3A) family proteins directly onto damaged lysosome membranes. Specifically, this damage-induced lipidation occurs via the central V-ATPase – ATG16L1 axis, and onto phosphatidylserine (PS), a highly specific hallmark of CASM. This was in stark contrast to the previous consensus, where all ATG8 lipidation observed following damage was assumed to be present on autophagosomes facilitating lysophagy. Excitingly, we comprehensively challenged this, showing that not only did ATG8 lipidation directly onto damaged lysosomes via CASM occur, but represented the majority of the early (i.e., < 30 minutes) ATG8 response, not lysophagy. Interestingly, we go onto show that LC3A interacts with the bridge-like lipid transfer proteins ATG2A/B following damage in a CASM-dependent manner. This presents a model whereby CASM-dependent ATG8 lipidation following lysosomal damage provides a recruitment platform for effector proteins that could promote resolution of damage via a variety of mechanisms downstream of lipidation. We also show data suggesting CASM more generally is required for efficient lysosomal repair, and, in an important methodological advancement, have developed a novel method by which to measure ER – lysosome lipid transfer directly in mammalian cells. Overall, this work has presented CASM as a novel lysosomal repair pathway. University of Cambridge 2025-09-29 Thesis Doctoral Doctor of Philosophy (PhD) eng N/A https://www.repository.cam.ac.uk/handle/1810/398326 https://www.repository.cam.ac.uk/bitstreams/e5eeeb36-1d78-456f-b0c0-54db2712b406/download 015a1ef0f3fcc28cf23e321d1692c342 https://www.repository.cam.ac.uk/bitstreams/1c75bd1c-26b8-4346-8956-1678a47f4b9f/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved CASM Lysosome damage ATG8 Lipid transfer ATG2