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Regulation of lysosome biogenesis by phosphoinositides and phagocytosis

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posted on 22.05.2021, 16:30 by Christopher Choy
Lysosomes are acidic organelles responsible for molecular degradation, energy balance, and pathogen clearance. Consequently, lysosome dysfunction is linked to numerous diseases, including lysosome storage diseases. Notably, enhancing lysosome biogenesis ameliorates cell function and helps clear metabolites. The transcription factor EB (TFEB) is a master regulator of lysosome biogenesis, and thus a potential therapeutic target. Among known regulators of TFEB, the mammalian target of rapamycin complex 1 (mTORC1) is best understood. In nutrient-rich cells, mTORC1 is activated and represses TFEB by phosphorylation. Upon starvation, mTORC1 is inactivated and TFEB enters the nucleus, upregulating lysosomal gene expression to enhance cellular degradation for energy recovery. Numerous other TFEB-dependent pathways have been identified. We aim to understand how TFEB is regulated in two additional contexts: in lysosome enlargement during phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] depletion and in phagocytosis. First, PtdIns(3,5)P2 is required for maintaining lysosome size by an incompletely understood mechanism. We hypothesized that TFEB-mediated lysosome biogenesis contributes de novo lysosomal material. Acute depletion of PtdIns(3,5)P2-synthesizing kinase PIKfyve induced TFEB nuclear accumulation. Despite increases in transcription, little to no protein translation was observed. Furthermore, tfeb-/-cells and cells blocked with cycloheximide were similar to wild-type cells, with regard to the number and size of lysosomes during PIKfyve inhibition cells, suggesting biosynthesis is not necessary for lysosome enlargement. However, TFEB still becomes active by an known mechanism. We show that TFEB nuclear localization during PIKfyve inhibition was not due to mTORC1 inactivation but may result from GSK3 inhibition. Secondly, phagocytosis allows immune cells to sequester potential pathogens by engulfing them into phagosomes. These phagosomes are then degraded by the lysosome. We postulated that phagocytosis would enhance TFEB-mediated lysosome biogenesis to promote pathogen killing. Fcγ receptor-mediated phagocytosis activated TFEB and increased biosynthesis of select lysosomal genes, augmenting existing lysosomes and enhancing proteolysis. To understand how TFEB was activated by the Fcγ receptor, we inhibited key signaling and trafficking mediators. Particle internalization, phagosome formation, and phagosome maturation appear to be necessary for TFEB activation. Overall, our work uncovers two additional mechanisms that may govern TFEBactivation.





Doctor of Philosophy


Molecular Science

Granting Institution

Ryerson University

LAC Thesis Type