Lysosomes are intracellular organelles that were considered for a long time to be simply an acidic and hydrolytically active end point of trafficking routes for degradation, in the last 20 years, light has been shed on their functional heterogeneity and striking role in signalling and nutrient homeostasis. While the dynamic nature and variety of lysosomal functions are now better appreciated, the mechanisms governing lysosomal fusion, reformation, signalling, and homeostasis remain to be fully elucidated, and are investigated here. In this study, endolysosomes which formed by fusion of late endosomes with lysosomes and are thought to be the predominant site of hydrolytic activity, were further characterised. Using live cell imaging and fluorescent labelling, the proportion of endolysosomes in the total pool of lysosomes was estimated using probes to their acidity and cathepsin activity, and their larger size compared to storage lysosomes was observed. The endolysosomal membrane was also shown to be marked by Rab7, Rab9, PI(3,5)P2 supporting the role of endolysosomes a highly active and dynamic principal site of hydrolase activity. The contributions of VAMP7 and VAMP8 to endolysosome fusion, measured by delivery of endocytosed cargo from late endosomes to endolysosomes, were analysed by CRISPR-Cas9 mediated knockout. Cells lacking VAMP7 and VAMP8 had no effect on delivery to endolysosomes, however at EM level, they displayed extensive tethering between late endocytic organelles, and accumulated small tethered vesicles. YKT6 knockdown impeded delivery to endolysosomes in VAMP7+VAMP8 knockout cells, which was rescued by VAMP7 expression, suggesting YKT6 substituted for VAMP7 in lysosome fusion. Following the hypothesis that reversible dissociation of V1 and Vo sectors of the V- ATPase may control the increase in pH of reforming storage lysosomes, cells expressing tagged V1G1 and Voa3 were generated. These markers of both sectors are present on endolysosomal membranes, and on the emerging endolysosomal tubules, suggesting the V1 and Vo sectors remain associated at this earliest stage of lysosome reformation, but these markers are still in development. IV Two assays were developed to give a readout of, and assess lysosomal stress. Firstly, an assay measuring TFEB-GFP translocation to the nucleus gave a robust and quantifiable readout of lysosomal perturbation. Secondly, a qPCR assay was developed to measure lysosomal gene upregulation as a downstream reporter of TFEB-activating lysosomal perturbations, however this assay, despite being more lysosome-specific, lacked the consistency and dynamic range of the TFEB translocation quantification. In summary, lysosomes are a heterogeneous collection of organelles, which have been better characterised primarily according to their acidity and hydrolytic capacity. Additionally, more SNAREs appear to be involved in lysosome fusion in cells than suggested by cell free assays, and I have developed tools to trace the V-ATPase during reformation of lysosomes after fusion to form endolysosomes. Lastly, I have developed a robust, reporter for a range of lysosomal stress-inducing conditions, providing a broad indication of their effects on lysosomal signalling and homeostasis.