pHLARE: a new biosensor reveals decreased lysosome pH in cancer cells

• Published in: Molecular biology of the cell Vol. 32; no. 2; pp. 131 - 142
• Main Authors: Webb, Bradley A, Aloisio, Francesca M, Charafeddine, Rabab A, Cook, Jessica, Wittmann, Torsten, Barber, Diane L
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 15-01-2021
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.E20-06-0383

Many lysosome functions are determined by a lumenal pH of ∼5.0, including the activity of resident acid-activated hydrolases. Lysosome pH (pHlys) is often increased in neurodegenerative disorders and predicted to be decreased in cancers, making it a potential target for therapeutics to limit the progression of these diseases. Accurately measuring pHlys, however, is limited by currently used dyes that accumulate in multiple intracellular compartments and cannot be propagated in clonal cells for longitudinal studies or used for in vivo determinations. To resolve this limitation, we developed a genetically encoded ratiometric pHlys biosensor, pHLARE (
ysosomal
ctivity
porter), which localizes predominantly in lysosomes, has a dynamic range of pH 4.0 to 6.5, and can be stably expressed in cells. Using pHLARE we show decreased pHlys with inhibiting activity of the mammalian target of rapamycin complex 1 (mTORC1). Also, cancer cells from different tissue origins have a lower pHlys than untransformed cells, and stably expressing oncogenic RasV12 in untransformed cells is sufficient to decrease pHlys. pHLARE is a new tool to accurately measure pHlys for improved understanding of lysosome dynamics, which is increasingly considered a therapeutic target.