Lysosomes play an essential role in intracellular degradation and cellular homeostasis, contributing to crucial functions within cellular metabolism. Ongoing research continues to expand our knowledge of the molecular biology and morphology of these organelles, revealing their diverse range of functions. The heterogeneity in lysosomal pH, catalytic activity, and localisation, within the lysosome regeneration cycle has been investigated in this work. This thesis provides evidence that during organelle remodelling in the lysosome regeneration cycle, lysosomal pH is regulated in fed cells by the reversible assembly/disassembly of the proton pumping V-ATPase and that the enzyme lysosomal phospholipase A2 (LPLA2/PLA2G15), a previously proposed target of cationic amphiphilic drugs (CADs), plays a role in bis(monoacylglycerol) phosphate (BMP) synthesis.

Chapter 1 of this thesis provides an introduction to lysosomes and the background to the experimental studies undertaken. It includes a description of endolysosome formation (ELF) as a result of the kissing and fusion of late endosomes with lysosomes as well as of lysosome reformation from endolysosomes (ELR), which together make up the regeneration cycle. It also includes a description of the sucrosome system used to study ELR.

Chapter 2 describes the materials and experimental methods used throughout my research.

In Chapter 3, I describe experiments utilising cathepsin activity probes, MRB and BMV109, to study the intracellular distribution of catalytically active endolysosomes and inactive terminal lysosomes. Using BMV109 in normal rat kidney (NRK) cells expressing EGFP-tagged lgp120 revealed how organelle size and function during ELF and ELR from sucrosomes is regulated. In cells expressing fluorescently tagged subunits of the V-ATPase and other proteins, I found that during the lysosome regeneration cycle, there was an increase in apparent recruitment (by colocalisation with the late endosomal/lysosomal marker lgp120/LAMP1) of the V1G1 subunit of V-ATPase to the lysosomal membrane during ELF (sucrosomes formation). However, colocalisation decreased after 4 hours of invertase-triggered reformation of terminal lysosomes from sucrosomes. In my investigation, I also found that the changes in Rab7a and RILP colocalisation with lgp120/LAMP1 during sucrosome formation (ELF) and ELR follow a similar time course to that observed for the V1-ATPase subunit V1G1. However, there was no change in the colocalisation of the Vo subcomplex subunit Voa3 or the small GTPase Arl8b with lgp120/LAMP1. This suggested the reversible assembly/disassembly of V-ATPase during ELF and ELR. I obtained quantitative evidence that peripheral terminal lysosomes were negative for V1G1 but positive for Voa3, whereas perinuclear endolysosomes were positive for both V1G1 and Voa3. Using pHLARE, a genetically-encoded pH probe, I demonstrated that increased colocalisation of V1G1 with lgp120 during ELF corresponds to decreased lysosomal pH, while the opposite was observed during ELR. In experiments with cells not treated with sucrose, in collaboration with Dr N. A. Bright, I was able to show that the recruitment of V1G1-EGFP to endolysosomes occurs shortly after kissing/fusion of late endosomes with lysosomes. Using fluorescence recovery after photobleaching, in collaboration with Dr N. A. Bright, I observed the dynamic equilibrium and rapid exchange between the cytosolic and membrane-bound pools of this subunit.

In Chapter 4, I used Western Blotting using pelleted membranes from NRK cells expressing Lgp120-mCherry/V1G1-EGFP to confirm that the changes in colocalisation of V1G1-EGFP and lgp120-mCherry during ELF (sucrosome formation) and ELR translated into changes in protein abundance at the lysosomal membrane. However, this process was time-consuming, labour-intensive, and yielded variable results. Therefore, lysosomes and sucrosomes were isolated from NRK cells, employing a magnetic isolation protocol. The distribution of V1 and Vo subunits during ELF (sucrosome formation) and ELR was assessed by analysing the magnetically enriched fractions Western blotting and aligned with the confocal microscopy data presented in the previous chapter. These findings support the reversible assembly and disassembly of V-ATPase during ELF and ELR from sucrosomes.

In Chapter 5, I describe experiments that showed that mammalian target of rapamycin complex 1 (mTORC1) activation and RILP were likely not necessary for V-ATPase disassembly during ELR in continuously fed cells. The colocalisation of lgp120-mCherry and V1G1-EGFP in NRK cells showed similar changes during ELF (sucrosome formation) and ELR, regardless of treatment with the mTORC1 inhibitor torin1. My conclusions were also supported when comparing the relative abundance of V1G1 and V1A1 subunits of the V1 subcomplex to the amount of the Vod1 subunit of the Vo subcomplex in magnetically isolated lysosomes before and after sucrosomes formation, as well as after a 4-hour invertase addition, both in presence or absence of torin1. Additionally, the siRNA knockdown of RILP did not affect the colocalisation pattern between lgp120-mCherry and V1G1-EGFP described in chapter 3. The role of Rab7a and its interactors’ associating with the V-ATPase may still play a significant role in regulating assembly during ELR.

Chapter 6 describes experiments focused on the relationship between lysosomal phospholipase A2 (LPLA2/PLA2G15) inhibition and drug induced phospholipidosis by cationic amphiphilic drugs (CADs). I was able to show, in human embryonic fibroblast (HEK) cells, that the increase in TFEB-GFP translocation after treatments with CADs corresponded to an increase in the fluorescent output of a commercially-available Phospholipidosis detection reagent, in a dose-dependent manner. I investigated whether the properties of LPLA2/PLA2G15 could be studied by expression in yeast cells. However, expressed human LPLA2/PLA2G15 was unable to rescue yeast growth in high oleate conditions and following starvation after depletion of its yeast ortholog, Lro1. Depletion of LPLA2/PLA2G15 in HEK cells, using two different siRNA oligonucleotides, led to an increase in the fluorescent output of the Phospholipidosis detection reagent and a decrease in the number of bis(monoacylglycerol) phosphate (BMP) puncta. This could be rescued after expressing LPLA2-mCherry in LPLA2-depleted HEK cells. This suggests that LPLA2/PLA2G15 is involved in BMP synthesis and its inhibition is linked to phospholipidosis. Lysosomal pH and catalytic activity remained unaffected following LPLA2/PLA2G15 depletion.

Chapter 7 is a final discussion including suggestions for future experiments.