Investigating the role of lysosome positioning in inflammation

Abstract

Alongside their well-established degradative function, lysosomes act as signalling hubs for many different cellular processes. The position of lysosomes within cells, mediated by kinesin and dynein-dynactin motors, is an important regulator of these pathways. Previous research has shown that lysosome positioning plays a significant role in the regulation of lysosomal pH and autophagy, and is required for cancer growth, invasion and metastasis. However, the role of lysosome positioning in other cellular processes, such as lysosomal homeostasis, intracellular trafficking and cell death, as well as inflammatory pathways such as NLRP3 inflammasome activation and the cGAS-STING pathway, is poorly understood. Therefore, this thesis aimed to investigate the role of lysosome positioning in diverse cellular pathways, with a specific focus on inflammatory signalling. To do so, this thesis first characterised models of differential lysosome positioning by generating HeLa cells to overexpress Arl8b and TMEM55B, which induced lysosomal movement to the peripheral and perinuclear regions, respectively. Using the established cell models, this thesis found that, in accordance with previous studies, differential lysosome positioning regulated lysosomal pH, an essential mechanism to maintain lysosomal function. Furthermore, this thesis observed that differential lysosome positioning influenced lysosomal integrity and disrupted endolysosomal trafficking, suggesting that lysosome positioning plays an essential role in sustaining lysosomal homeostasis and modulating endosomal to lysosomal trafficking. With recent studies indicating that endosomes and lysosomes play a significant role in NLRP3 inflammasome assembly and activation, this thesis next used Arl8b-overexpressing mouse immortalised bone marrow-derived macrophages (iBMDM) and human monocyte-derived macrophages (THP-1) to assess the effects of peripheral lysosome positioning on inflammation. This research revealed that lysosome positioning plays a regulatory role in inflammatory pathways, including NLRP3 inflammasome priming and activation, TLR7 signalling and the cGAS-STING pathway. Furthermore, this thesis identified that lysosome positioning influenced both the subcellular location and size of the ASC speck, suggesting that lysosomes play an important role in NLRP3 inflammasome assembly. Together, the data presented in this thesis provide new insights into how lysosome positioning maintains cellular homeostasis and modulates inflammatory pathways, thus providing possible therapeutic avenues for inflammatory disease intervention.

Date of Award	29 May 2025
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Paula Seoane Denicola (Co Supervisor), Martin Lowe (Co Supervisor), David Brough (Main Supervisor) & Christopher Hoyle (Co Supervisor)