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.

FIGURE 1:

pHLARE localizes to lysosomes. (A) Schematic of pHLARE with rat LAMP1 tagged at the lumenal amino-terminus with sfGFP and at the cytoplasmic carboxyl-terminus with mCherry. (B) pHLARE stably expressed in human RPE cells, visualized with anti-mCherry antibodies, colocalizes with endogenous LAMP1, visualized with anti-human LAMP1 antibodies. (C) Fluorescence ratios of pHLARE in RPE cells in nigericin-containing buffers between pH 4.0 and 7.0. Data are means ± SEM of 15 cells from three separate cell preparations. Statistical analysis by Tukey–Kramer HSD indicates significant differences at all pH values except between pH 6.5 and 7.0. (D) Representative RFP immunoblot of three preparations of lysates from RPE cells, WT, and stably expressing pHLARE and untreated or treated with nigericin buffer at the indicated pH values for 5 min. The asterisks indicate nonspecific bands seen also in RPE WT cells not expressing pHLARE and GAPDH is used as a loading control. (E) Images of live RPE cells stably expressing pHLARE and stained with SiR-lysosome, a far-red pepstatin A that binds cathepsin D. The magenta outline in the insets at higher magnification indicate lysosomes detected in the SiR-lysosome channel showing overlap with pHLARE. (F) Analysis of the amount of pHLARE fluorescence signal associated with SiR-lysosome objects. As expected, the fluorescence distribution in the mCherry channel is insensitive to pH equilibration, while the SiR lysosome-associated sfGFP fluorescence increases. Statistical analysis by Tukey–Kramer HSD, n = 19 cells.

FIGURE 2:

Cell-based pHLARE pH calibration. (A) pHLARE-expressing RPE cell in NaHCO₃-containing buffer (described for pHi measurements) and in nigericin buffers of two different calibration pH values showing sfGFP (green) and mCherry (magenta) fluorescence. The three images are scaled identically showing the increase of sfGFP signal in lysosomes at near neutral pH equilibration and the dramatic overall decrease of sfGFP fluorescence at low pH. Insets show the indicated region at higher magnification. The yellow outline in the top row indicates lysosome objects detected in the mCherry channel. Note that lysosome size is near the resolution limit of optical microscopy and pHLARE lysosome membrane localization is only resolved in larger lysosomes. (B) Frequency histogram of lysosome object pH distribution in RPE cells obtained with the two different calibration methods as indicated. Bars are the average of n = 19 cells and error bars indicate 95% confidence interval. (C) Box plot of the median pHlys of the same set of cells. Statistical analysis by unpaired t test.

FIGURE 3:

Cell-specific pHlys. (A) Average steady-state pHlys in RPE cells stably expressing pHLARE (n = 74 cells) and in MCF10A (23 cells), MDCK (n = 17 cells), NIH-3T3 (15 cells), and CCL39 (n = 11 cells) transiently expressing pHLARE and maintained in growth medium. Box plots show median, first, and third quartile, with whiskers extending to observations within 1.5 times the interquartile range, and all individual data points include data for individual cells obtained from three to seven separate cell preparations. Statistical analysis by Tukey–Kramer HSD test. (B) Steady-state pHlys of lysosomes within 3 μm of the nuclear membrane (perinuclear) and 3 μm of the distal margin of membrane protrusions in RPE cells, indicated by hatched lines. Data are expressed as described in A and obtained from six separate cell preparations. (C–E) Lysosome motility, including mean speed (C), maximum speed (D), and displacement (E) of RPE control cells and RPE-pHLARE cells. Plots indicate mean and SEM with Student’s t test from three separate cell preparations.

FIGURE 4:

pHLARE measures dynamic changes in pHlys. (A, B) RPE cells stably expressing pHLARE were untreated (control, n = 27 cells) or treated with 100 nM of the V-ATPase inhibitor Bafilomycin A1 (n = 15 cells) or 100 μM chloroquine (n = 15 cells) for 2 h before acquiring images, which were used to calculate pHlys. (C) Lysosome size with indicated conditions determined by measuring more than 1,000 lysosomes in four cell preparations for controls and three cell preparations with bafilomycin and chloroquine treatment. (D, E) pHlys of RPE cells stably expressing pHLARE untreated (control, n = 39 cells) or treated with 250 nM Torin-1 (n = 50 cells) for 2 h before acquiring images. (F) Representative immunoblot of lysates from two separate RPE cell preparations untreated or treated with the indicated concentrations of Torin-1 and probed with antibodies to total and phosphorylated S6K. Box plots show median, first, and third quartile, with whiskers extending to observations within 1.5 times the interquartile range and all individual data points. Statistical analysis by Tukey–Kramer HSD test, with data obtained from three separate cell preparations in B, C, and F and four separate cell preparations in D.

FIGURE 5:

Differences in pHlys in breast cancer cells and with oncogene transformation. (A) The average pHlys of human untransformed MCF10A mammary epithelial cells (n = 36 cells), breast cancer MDA-MB-157 (n = 24 cells), MDA-MB-453 (n = 6 cells), and MCF7 (n = 21) cells, and MCF10A cells stably expressing H-RasV12 (n = 24 cells). (B, C) Representative images of MCF10A, MDA-MB-157, and MCF7 cells expressing pHLARE (B) and quantification of lysosomes within 3 μm of the plasma membrane indicated by mCherry fluorescence. (C, D) The average pHlys in canine MDCK epithelial cells WT (n = 23) and stably expressing K-RasV12 (n = 23 cells). Box plots show median, first, and third quartile, with whiskers extending to observations within 1.5 times the interquartile range and all individual data points. Statistical analysis by Tukey–Kramer HSD test, with data obtained from three to five separate preparations of all cells except for two preparations of MDA-MB-453.

FIGURE 6:

pHlys and pHi in cancer and untransformed cells. (A) Images of pHLARE and average pHlys of human untransformed HPDE cells (n = 19 cells) and pancreatic adenocarcinoma PANC-1 (n = 17 cells), BxPC3 (n = 13 cells), and MIA PaCa-2 (n = 20 cells) cells. (B) Images of pHLARE and average pHlys of human RPE untransformed cells (n = 12 cells), H1299 lung cancer cells (n = 19 cells), HCT116 colon cancer cells (n = 22 cells), and U-251 glioblastoma cells (n = 9 cells). (C) Steady-state pHi of the indicated cell types determined in a NaHCO₃-containing buffer. ***p < 0.001. (D) Average pHlys of RPE cells in the absence (Control; n = 20 cells) and presence of EIPA (10 μM, 18 h; n = 19 cells). Box plots show median, first, and third quartile, with whiskers extending to observations within 1.5 times the interquartile range and all individual data points. Statistical analysis by Tukey–Kramer HSD test, with data obtained from three to five separate cell preparations.