Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay

Abstract

Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation.

Keywords: C. elegans; breast involution; cell death; galectin; lysosome.

Figure 1.

Expression profiles of LGALS mRNAs and protein products. (A and B) Expression levels of LGALS1, LGALS3, LGALS8, and LGALS9 mRNA in indicated normal human tissues (A) and 44 cancer cell lines (B) were extracted from RNA-Seq data published in the Human Protein Atlas (www.proteinatlas.org). FPKM, fragments per kb of transcript per million mapped fragments. (C) Expression levels of LGALS1 and LGALS3 proteins in 51 human cancer cell lines were extracted from published mass spectrometry data.³³ CNS, central nervous system; NSCLC, non-small cell lung cancer.

Figure 2.

For figure legend, see page 1412.

Figure 3.

LGALS3 binds N-acetyllactosamine and colocalizes with LGALS1, LGALS8, and LGALS9 on damaged lysosomes. (A) Representative confocal images of MCF7 cells treated with 2 mM LLOMe for 6 h and costained with rabbit anti-LGALS1 (red) and mouse anti-LGALS3 antibodies (green), and Manders' coefficients for colocalization of LGALS1 with LGALS3 and vice versa. Error bars are SEMs for ≥ 26 cells. (B) Representative confocal images of U2OS-mCherry-LGALS3 cells (red) transiently transfected with plasmids coding for YFP-LGALS1, -8, -9 or EGFP-LGALS3 (green) and left untreated or treated with 2 mM LLOMe for 2 h before fixation. See also Movies S1–S4 for live-cell imaging. (C) Representative confocal images of U2OS cells transiently transfected with wild type EGFP-LGALS3, its R186S mutant (deficient in N-acetyllactosamine binding) or its G182A mutant (deficient in Galβ1-3GlcNAc binding), and treated with 2 mM LLOMe for 30 min before fixation. ***, P< 0.001. Scale bars: 10 µm.

Figure 4.

The galectin puncta assay is suitable for automated analysis of LMP. (A and B) U2OS-mCherry-LGALS3 cells were treated as indicated for 24 h (A) or for indicated times (B). After staining the nuclei with Hoechst 33342, cells were analyzed in a ScanR automated microscopy system to quantify the number of puncta inside a “cell mask” defined by a fixed radius around each nucleus. Representative images (A), quantification of percentage of cells with >3 mCherry-LGALS3 puncta (B, top), number of mCherry-LGALS3 puncta per cell (B, middle), and percentage of rounded cells (B, bottom) are shown. Error bars are SDs for means of 2 duplicate experiments with an average of > 400 cells/sample. (C and D) Representative confocal images of MCF7 cells treated with 200 nM thapsigargin or vehicle control (H₂O) for 24 h (C) or 300 nM vincristine or vehicle control (DMSO) for 48 h (D) and costained with mouse anti-LGALS3 (green) and rabbit anti-PDIA2 (red) or anti-GOLGA1 (red) antibodies. Nuclei are visualized with Hoechst 33342 (blue) in merged images. RGB color intensity for entire panel (D) was equally enhanced with Photoshop. Scale bars: 10 µm.

Figure 5.

The galectin puncta assay is more sensitive than existing methods to detect LMP. (A and B) Representative confocal images of U2OS (A) and MCF7 (B) cells that were treated with 2 mM LLOMe or 8 µM terfenadine for indicated times, fixed in ice-cold methanol and costained with mouse anti-CTSB antibodies (green) and rabbit anti-LGALS1 or rat anti-LGALS3 antibodies (red). Note that higher gain settings were used for terfenadine-treated cells. (C) U2OS cells transiently transfected with EGFP-LGALS3 (green) and preloaded for approximately 18 h with AF594-dextran (10 kDa; red) were treated with 2 mM LLOMe for indicated times and analyzed by live-cell confocal microscopy. Examples of cells with EGFP-LGALS3 puncta without clearly visible AF594-dextran in the cytosol are indicated by arrowheads. See also Movie S5. (D) U2OS-mCherry-LGALS3 cells (red) preloaded with fluorescein-dextran (10 kDa; green) for 6 h were treated with 1.5 mM LLOMe for 19 h and examined by live-cell confocal microscopy. Examples of cells with mCherry-LGALS3 puncta but without clearly visible fluorescein-dextran in the cytosol are indicated by arrowheads. (E) U2OS-mCherry-LGALS3 cells loaded with AF488-dextran (10 kDa) for approximately 18 h were treated with 1.5 mM LLOMe and analyzed in the ScanR automated microscopy system. Images were acquired every 60 ec. Scale bars: 10 µm.

Figure 6.

For figure legend, see page 1417.

Figure 7.

The galectin puncta assay detects LMP in paraffin-embedded tissue samples. (A) Sections of MCF7 breast cancer xenografts collected from SCID/FOX mice 48 h after p.o. treatment with 100 mg/kg terfenadine or siramesine or vehicle (200 µl of 0.5% methyl cellulose 15 in 0.9% NaCl solution) were costained with rat anti-LGALS3 (red) and mouse anti-LAMP1 antibodies (green) and Hoechst 33342 (blue) and analyzed by confocal microscopy. Representative maximum projection images of z-stacks are shown. Examples of puncta positive for both LGALS3 and LAMP1 are indicated by arrowheads. Two biological replicas showed similar results. See Figure S3A for images of galectin puncta formation in paraffin-embedded MCF7 cell pellets. (B) Sections of lactating (10 d) and involuting (24 h) mouse mammary gland tissues from C57B6/J wild type mice were stained with rabbit anti-LGALS1 or rat anti-LGALS3 antibodies. Representative images of chromogenic staining with HRP-linked secondary antibodies and diaminobenzidine (brown) and hematoxylin counterstain (blue) are shown. Black arrowheads indicate examples of cells with LGALS1 or LGALS3 puncta. Two biological replicas showed similar results. See Figure S3B for lower magnifications of the LGALS1 samples and control IgG staining. (C) Representative maximum projection images of confocal microscopy z-stacks of mammary gland sections [described in (B)] stained with rat anti-LGALS3 (red) and mouse anti-LAMP1 antibodies (green). Nuclei were labeled with Hoechst 33342 (blue; in merged images). Two biological replicas showed similar results. Scale bars: 10 µm.

Figure 8.

GFP-LGALS3 puncta mark heat-shock-induced leaky lysosomes in C. elegans. (A) L4 stage C. elegans larvae expressing the lysosomal marker NUC-1-mCherry were incubated at 20°C (control) or 37°C (heat shock) for 3 h, allowed to recover at 20°C for 3 h and imaged by confocal microscopy. Three randomly chosen intestinal areas of approximately 800 µm² in each worm were analyzed for the number of NUC-1-mCherry-positive lysosomes. Representative confocal images and average number of lysosomes per 100 µm² are shown. Error bars are SDs for means of 3 areas in ≥ 12 worms per condition. *, P< 0.05. (B) Adult C. elegans worms (L4 + 24 h) expressing NUC-1-mCherry and GFP-LGALS3 were treated and imaged as in (A) (recovery time was 4 h). The posterior parts of Hyp7 cells (n ≥ 24) were analyzed for the number of GFP-LGALS3 puncta. Representative images (left) and the percentage of cells with ≤5, 6 to 10, or ≥11 large (>1.5 µm²) GFP-LGALS3 puncta (right) are shown. White arrows indicate examples of GFP-LGALS3-positive leaky lysosomes with reduced NUC-1-mCherry content and the yellow arrow shows an example of a GFP-LGALS3-negative NUC-1-mCherry-positive intact lysosome. (C) The survival of worms treated as in (B) was analyzed by their responsiveness to picker touch at indicated time points after the heat shock. Error bars are SDs for means of 4 independent experiments with approximately 50 worms each. Scale bars: 10 µm.