Dataset Information

PHLARE: a new biosensor reveals decreased lysosome pH in cancer cells.

ABSTRACT: 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 (pH Lysosomal Activity REporter), 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.

SUBMITTER: Webb BA

PROVIDER: S-EPMC8120692 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Webb Bradley A BA Aloisio Francesca M FM Charafeddine Rabab A RA Cook Jessica J Wittmann Torsten T Barber Diane L DL

Molecular biology of the cell 20201125 2

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 l ...[more]

PMID: 33237838

Similar Datasets

Project description:Disorders of lysosomal physiology have increasingly been found to underlie the pathology of a rapidly growing cast of neurodevelopmental disorders and sporadic diseases of aging. One cardinal aspect of lysosomal (dys)function is lysosomal acidification in which defects trigger lysosomal stress signaling and defects in proteolytic capacity. We have developed a genetically encoded ratiometric probe to measure lysosomal pH coupled with a purification tag to efficiently purify lysosomes for both proteomic and in vitro evaluation of their function. Using our probe, we showed that lysosomal pH is remarkably stable over a period of days in a variety of cell types. Additionally, this probe can be used to determine that lysosomal stress signaling via TFEB is uncoupled from gross changes in lysosomal pH. Finally, we demonstrated that while overexpression of ARL8B GTPase causes striking alkalinization of peripheral lysosomes in HEK293 T cells, peripheral lysosomes per se are no less acidic than juxtanuclear lysosomes in our cell lines.Abbreviations: ARL8B: ADP ribosylation factor like GTPase 8B; ATP: adenosine triphosphate; ATP5F1B/ATPB: ATP synthase F1 subunit beta; ATP6V1A: ATPase H+ transporting V1 subunit A; Baf: bafilomycin A1; BLOC-1: biogenesis of lysosome-related organelles complex 1; BSA: bovine serum albumin; Cos7: African green monkey kidney fibroblast-like cell line; CQ: chloroquine; CTSB: cathepsin B; CYCS: cytochrome c, somatic; DAPI: 4',6-diamidino -2- phenylindole; DIC: differential interference contrast; DIV: days in vitro; DMEM: Dulbecco's modified Eagle's medium;‎ E8: embryonic day 8; EEA1: early endosome antigen 1; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; ER: endoplasmic reticulum; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GTP: guanosine triphosphate; HEK293T: human embryonic kidney 293 cells, that expresses a mutant version of the SV40 large T antigen; HeLa: Henrietta Lacks-derived cell; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP: horseradish peroxidase; IGF2R/ciM6PR: insulin like growth factor 2 receptor; LAMP1/2: lysosomal associated membrane protein 1/2; LMAN2/VIP36: lectin, mannose binding 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PCR: polymerase chain reaction; PDL: poly-d-lysine; PGK1p: promotor from human phosphoglycerate kinase 1; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PPT1/CLN1: palmitoyl-protein thioesterase 1; RPS6KB1/p70: ribosomal protein S6 kinase B1; STAT3: signal transducer and activator of transcription 3; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TGN: trans-Golgi network; TGOLN2/TGN46: trans-Golgi network protein 2; TIRF: total internal reflection fluorescence; TMEM106B: transmembrane protein 106B; TOR: target of rapamycin; TRPM2: transient receptor potential cation channel subfamily M member 2; V-ATPase: vacuolar-type proton-translocating ATPase; VPS35: VPS35 retromer complex component.

Dataset Information

PHLARE: a new biosensor reveals decreased lysosome pH in cancer cells.

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets