Mechanisms Of Organelle Identity and Adaptation In Immune Cells
thesisposted on 24.05.2021, 19:12 authored by Victoria Emily Barbosa Hipolito
Cells are exposed to diverse extracellular and intracellular cues, and coopt subcellular responses depending on their cellular state and functional demand; including upregulating signalling pathways or adapting organelle function and physiology. The immune system is a tightly regulated cohort of specialized cells with heterogeneous functions. Phagocytes, a type of immune cell, are challenged with disparate environmental stimuli and can adapt intracellularly to promote immunity. Due to their cellular plasticity, we aim to understand the molecular machinery that controls organelle identity and adaptation in immune cells, when challenged with immunostimulatory agents. First, we used a long tubular phagocytic cup, which provides the spatiotemporal resolution necessary to study the stages of phagocytosis. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the growing cup. Surprisingly, the early endosomal lipid, phosphatidylinositol-3-phosphate [PtdIns(3)P] persisted. We determined a novel pH-based mechanism that induces the dissociation of the Vps34 Class III phosphatidylinositol-3- kinase from tubular cups as they progressively acidify, when reaching 20 µm in length or upon phagosome closure. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for its turnover by PIKfyve. Given that PtdIns(3)P dependent signalling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes. Additionally, we examined how lysosomes, a kingpin organelle essential for pathogen killing, and antigen processing and presentation, adapt in response to phagocyte activation. During phagocyte activation, lysosomes are remodelled from dozens of globular structures to a tubular network, in a process that requires the PI3K-AKT-mTOR signalling pathway. We showed that lysosome tubulation is coupled with an increase in volume and holding capacity. Lysosome remodelling was dependent on de novo synthesis of lysosomal proteins, but independent of TFEB and TFE3 transcription factors, known to scale-up lysosome biogenesis. We demonstrate a novel mechanism of acute organelle expansion via mTORC1-S6K-4E-BP-dependent increase in lysosomal mRNA translation. This process was necessary for efficient and rapid antigen presentation to T-cells by dendritic cells (DCs). Moreover, lysosome remodelling was conserved in DCs activated with select adjuvants, additives used in vaccines to boost efficacy, providing evidence for its possible clinical applicability. Together, we have identified two novel mechanisms controlling organelle identity and adaptation in immune cells.