A DNA nanomachine chemically resolves lysosomes in live cells

Abstract

Lysosomes are multifunctional, subcellular organelles with roles in plasma membrane repair, autophagy, pathogen degradation and nutrient sensing. Dysfunctional lysosomes underlie Alzheimer's disease, Parkinson's disease and rare lysosomal storage diseases, but their contributions to these pathophysiologies are unclear. Live imaging has revealed lysosome subpopulations with different physical characteristics including dynamics, morphology or cellular localization. Here, we chemically resolve lysosome subpopulations using a DNA-based combination reporter that quantitatively images pH and chloride simultaneously in the same lysosome while retaining single-lysosome information in live cells. We call this technology two-ion measurement or 2-IM. 2-IM of lysosomes in primary skin fibroblasts derived from healthy individuals shows two main lysosome populations, one of which is absent in primary cells derived from patients with Niemann-Pick disease. When patient cells are treated with relevant therapeutics, the second population re-emerges. Chemically resolving lysosomes by 2-IM could enable decoding the mechanistic underpinnings of lysosomal diseases, monitoring disease progression or evaluating therapeutic efficacy.

Figure 1. Design and characterization of ChloropHore.

(a) Schematic of the working principle: A pH-induced change in FRET between Alexa546 (donor, orange sphere) and Alexa647 (acceptor, red star) reports pH ratiometrically. A Cl⁻ sensitive fluorophore (BAC, green triangle) and Alexa647 report Cl⁻ ratiometrically. (b) pH and Cl⁻ response profiles of ChloropHore: Normalized fluorescence intensity ratio (D/A) of donor (D) and acceptor (A) upon donor excitation in vitro as a function of pH and 50 mM Cl⁻ (red). Normalized fluorescence intensity ratio (R/G) of Alexa 647 (R) and BAC (G) as a function of Cl⁻ concentration at pH 7 (blue). Values were normalized 5 mM Cl⁻ for R/G or pH 4 for D/A. (c) Performance of the pH sensing module (red) at different [Cl⁻] and Cl⁻ sensing module at different pH (blue). Fold changes in D/A (red hatched bars) or R/G (blue hatched bars) for the pH and Cl⁻ sensing modules are shown. Stern Volmers constant (K_sv, solid blue bars) for Cl⁻ sensing at each pH. Calibration surface plot of the fluorescence intensity ratios of (d) D/A and (e) R/G of ChloropHore as a function of Cl⁻ and pH. Error bars indicate the mean ± s.e.m. of three independent measurements.

Figure 2. Trafficking pathway of ChloropHore in human dermal fibroblasts.

(a) Trafficking of ChloropHore along the scavenger receptor-mediated endocytic pathway. (b-c) ChloropHore uptake in primary skin fibroblasts (HDF cells) is competed out by excess maleylated BSA (mBSA, 10 μM). Cells are imaged in Alexa647 channel. AF: autofluorescence. (d-g) ChloropHore labels lysosomes in HDF cells. (d) Representative images of co-localization between lysosomes of HDF cells labeled with FITC Dextran (green) and LAMP-1 RFP (red) with the corresponding Pearson’s correlation coefficient (e). (f) Representative images of lysosomes of HDF cells labelled with TMR dextran (TMR; green) and ChloropHore (Alexa647, red). (g) Pearson’s correlation coefficient of colocalization between ChloropHore and lysosomes as a function of ChloropHore chase times. Experiments were performed in triplicate. Error bars indicate the mean of three independent experiments ± s.e.m. (n = 20 cells).

Figure 3. Intracellular calibration of ChloropHore and ChloropHore_Ly.

(a) Fluorescence images of primary HDF cells labeled with ChloropHore, clamped at the indicated pH and [Cl⁻], imaged in the donor (D), acceptor (A), reference (R), BAC (G) channel and the corresponding pseudocolour D/A (pH) and R/G (Cl⁻) maps. (b-c) In cell calibration surface corresponding to the pH and Cl⁻ response profiles of the sensing modules in ChloropHore at various [Cl⁻] and pH values respectively. (d) Representative scatter plot of D/A versus the R/G values of endosomes in primary HDF cells from a normal individual clamped at the indicated pH and [Cl⁻]. Each data point corresponds to a single endosome. (e) The scatter plot in (d) represented as a density plot in pseudocolour where red and blue correspond to populations with higher and lower frequencies of occurrence, i.e., a 2-IM profile. (f-h) 2-IM profiles of HDF cells clamped in indicated varying pH and fixed [Cl⁻]. (i-k) 2-IM profiles of HDF cells at fixed pH and increasing [Cl⁻]. a-k, experiments were performed in duplicate (n = 15 cells, n = 150 endosomes). (l) Single endosome clamping in HDF cells. ChloropHore labeled cells clamped at indicated pH (i) and Cl⁻ (ii) were clamped to a different indicated pH and same Cl⁻ (iv) (m) 2D-scatter plots and their projected histograms on a single axis of each clamping step of the same endosomes are shown. Gray arrow represents the direction of change in D/A (pH) values for each endosome. (n) Chlorophore_LY labeled HDF clamped at indicated pH (i) and Cl⁻ (ii) were clamped to the same pH (iii) but different [Cl⁻] (iv) (o) 2D”scatter plots and their projected histograms on a single axis for each clamping step. Gray arrow indicates direction of change in R/G (Cl⁻) values for each endosome. Scale bars, 10 μm. l-o, experiments were performed in duplicate (n = 30 endosomes).

Figure 4. 2-IM chemically resolves lysosome populations.

(a) Respective pseudocolour D/A and R/G map of HDF cells derived from normal individuals 1, NP-A patient 1 and NP-C patient 1 labeled with Chlorophore_LY. 2-IM profiles and the histograms of D/A, R/G ratios of (b,i) normal individual (N.I.) and in presence of (c) bafilomycin A1 or (d) NPPB. 2-IM profiles of lysosomes of primary HDF cells from the same normal individual showing three replicates (b,ii-vi), two different normal individuals 2–3, (e) NP-A, NP-B, NPC patients. (f) Scatter plots of lysosome sizes versus their R/G or D/A values in a normal individual and an NP-A patient. 2-IM profile and corresponding histograms of D/A, R/G ratios of (g) HDF cells treated with 65 μM amitriptyline (AH) or 20 μM U18666A (h) 2-IM profiles of NP-A, NP-B patient fibroblasts in the presence of 5 μg of acid sphingomyelinase (ASM) and NP-C patient fibroblasts in the presence of 50 μM of ß-CD. Experiments were performed in duplicate (n = 550 lysosome, = 55 cells). Scale bars 10 μm.