Structure-function analyses of the BORC

For many years one thought that lysosomes are only intracellular garbage depots. However, our understanding of lysosomal functions has significantly changed in the last years. Lysosomal malfunctions are implicated in many human pathologies, including neurodegeneration, cancer, infection, immunodeficiency and obesity. Lysosomes are now considered as important intracellular signaling platforms thereby orchestrating many essential cellular functions. Over the years, our team has significantly contributed towards the identification and functional characterization of a pentameric lysosomal protein complex named LAMTOR. This complex acts as a critical signaling hub coordinating MAPK and mTORC signaling from the lysosomal membrane to control biogenesis and positioning of lysosomes. Lysosomal motility and positioning requires the integration of extracellular and intracellular signals that converge on a competition between plus- and minus-end directed motor proteins. We have recently shown that LAMTOR negatively regulates BORC dependent Arl8b recruitment to lysosomes, thereby regulating kinesin dependent organelle movement. However, the structural mechanisms how these essential protein machineries couple to each other and how they then coordinate endosomal positioning and biogenesis are presently unknown and will, therefore, be the main focus of this research proposal. Interestingly cancer progression and metastasis are tightly associated with lysosomal alterations, including changes in organelle numbers, size, luminal pH and intracellular distribution. Moreover, it has been recently proposed that drugs targeting the regulation of lysosomal motility might have anti- metastatic potential. The proposed work will deliver the first structural analysis of the human and the C. elegans BORC complex bound to either LAMTOR or Arl8b. This will be complemented with cellular assays and genome editing to determine how the LAMTOR and BORC functions are executed and coordinated. LAMTOR exists in at least three states on the limiting membrane of lysosomes; (1) LAMTOR alone, (2) LAMTOR/BORC, (3) LAMTOR/Rags. Importantly, the BORC component Lyspersin binds LAMTOR via the same structural region used to recruit the RagGTPases. We will investigate the following key questions in lysosomal biology: (1) solve the structures of the human and the C. elegans BORC complexes, (2) perform a comparative analysis between the LAMTOR-BORC and the LAMTOR-RagGTPases complexes, (3) determine the orientation of both LAMTOR and BORC towards membranes, (4) investigate the regulatory mechanisms determining the association of LAMTOR to either BORC or the RagGTPases and of BORC to ARl8b. Therefore, this project will add structural and mechanistic insight into the role(s) of both BORC and LAMTOR complexes in the coordination of signaling, endosomal biogenesis and organelle positioning.

Lysosomes are specialized membrane-enclosed structures within cells that play critical roles in various cellular functions, including protein degradation and control of cellular metabolism. Diseases ranging from genetic disorders that affect storage within the cell to more complex conditions such as brain disorders, metabolic problems, cancer and aging are associated with problems with lysosomes. Due to their importance, cells have developed unique mechanisms to control lysosomal numbers, size, movement, degradative capacity, interaction with other intracellular structures, and even selective degradation of dysfunctional lysosomes. The outer membrane of lysosomes supports important signaling molecules that regulate many of their functions. In particular, the LAMTOR and BORC complexes play a critical role. BORC is involved in three key activities: positioning lysosomes within the cell, influencing their shape and size, and facilitating the fusion of lysosomes with other structures known as autophagosomes. Meanwhile, the LAMTOR complex acts as a central hub, attracting and activating various proteins that further influence lysosomal operations. The interaction between LAMTOR and BORC is particularly important because it helps localize lysosomes to specific areas within the cell. In our study, we focused on how LAMTOR and BORC coordinate and control lysosomal functions. We began by studying the BORC complex in a simple model organism, C. elegans, using techniques such as cross-linking mass spectrometry and cryo-electron microscopy. These studies confirmed our predicted 3D models of the BORC complex, showing a high degree of similarity across species, and highlighted key regions of specific BORC components that keep the complex together. We then tested how changes in these regions affect the BORC's assembly and interactions with other proteins, including LAMTOR. This is part of our broader effort to understand how these proteins work together under different cellular conditions. Through our research, we have also discovered that BORC can interact with other protein complexes under nutrient-deficient conditions, suggesting a versatile approach by which cells adapt to different needs. This interaction suggests that specific components of BORC may have unique roles depending on the cellular environment. We also investigated how LAMTOR function is affected by a process called phosphorylation, a chemical modification. We found that this modification affects how LAMTOR interacts with other proteins involved in lysosomal signaling, highlighting its dynamic role in cellular response mechanisms. Our findings shed light on the complex architecture and regulatory mechanisms of lysosomes, emphasizing their critical role in cell health and disease. This research not only advances our understanding of cell biology, but also points to potential therapeutic targets for diseases associated with lysosomal dysfunction