Lysosomal membrane dynamics during stress - ABSTRACT Lysosomes are round-shaped acidic organelles full of hydrolases. They play multiple roles within the cell, including degradation, recycling, or signaling. Notably, lysosomal damage is a hallmark in various human diseases, including lysosomal storage disorders, neurodegeneration, and immune-related diseases. A prevalent outcome of lysosomal dysfunction is the rupture of the lysosomal membrane. During this process, protons and hydrolases escape into the cytosol, potentially triggering cell death if left unaddressed. Recently, new cellular responses to lysosomal membrane damage have been identified. Lysosomes activate various pathways to counteract membrane damage and prevent cell death, including lysosomal membrane repair and the clearance of damaged lysosomes through autophagy. Nevertheless, additional pathways likely exist and await identification. Through the integration of unbiased proteomics and advanced microscopy, we have uncovered a novel cellular process triggered by lysosomal membrane damage, which we named LYsosomal Tubulation and sorting driven by LRRK2 (LYTL). LYTL is orchestrated by Leucine-rich repeat kinase 2 (LRRK2), a large kinase typically located in the cytosol that becomes active upon recruitment to membranes. Mutations in LRRK2 are associated with neurological disorders such as Parkinson’s disease and Progressive supranuclear palsy, as well as immune-related disorders like Crohn's disease, Leprosy, and Tuberculosis. Once recruited to lysosomes, LRRK2 phosphorylates and recruits several RAB GTPases. Phosphorylated- RABs (pRABs) subsequently recruit two effectors, C-JNK-Interacting Protein 4 (JIP4) and RILP-like protein 1 (RILPL1). Both proteins bind to motor proteins upon membrane recruitment, regulating the elongation and retraction of LYTL tubules. Despite LYTL vesicles contacting healthy lysosomes, likely delivering undegraded cargo, the precise cellular role of LYTL in cellular homeostasis remains elusive. This proposal aims to unravel lysosomal membrane dynamics under stress conditions, providing a better mechanistic understanding of lysosomal quality control and potential therapeutic applications. In the first aim, we will elucidate the role of LYTL in cellular homeostasis by precisely tracking the destination of LYTL vesicles and identifying their cargo through unbiased proteomics and lipidomics. The second aim focuses on studying a novel response to lysosomal membrane damage driven by a subset of Annexin A proteins (ANXA4/5/6/7/11 or ANXA4-11). Lastly, we will address a crucial and unknown question in the field: How do disease-relevant cell types respond to lysosomal membrane damage? To answer this question, we will study the response to lysosomal membrane damage in human macrophages differentiated from induced pluripotent stem cells (iPSC).
