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Redox Report : Communications in Free Radical Research logoLink to Redox Report : Communications in Free Radical Research
. 2026 Feb 4;31(1):2621497. doi: 10.1080/13510002.2026.2621497

Time-resolved simultaneous imaging of mitochondrial reactive oxygen species and lysosomal permeabilization to determine organelle-centred cell death

P J Jain Tiffee a,b, Aswathy Sivasailam a,c, Kiran S Kumar a, Shine Varghese Jancy a, Aparna Geetha Jayaprasad a, Aman Munirpasha Halikar a,b, Aijaz Ahmed Rather a,b, Nithin Satheesan Sinivirgin a, K G Anurup a, T R Santhoshkumar a,CONTACT
PMCID: PMC12879504  PMID: 41639612

ABSTRACT

Background

Mitochondria and lysosomes are pivotal in dictating cell survival or death outcomes. While mitochondrial damage and ROS production are key events in mitochondrial cell death, lysosome membrane permeabilization and cathepsin B release mark lysosomal cell death. We aimed to generate a live-cell approach to concurrently monitor mitochondrial redox alterations and lysosomal permeabilization. This would provide mechanistic insight into their dynamic interplay during cell death and enable the discovery of organelle-specific death inducers.

Methods

A dual cell sensor, stably expressing tdTomato-CathepsinB and mitochondria-targeted redox GFP (mt-roGFP), was successfully engineered, and simultaneous imaging of both events by real-time confocal imaging was carried out with selected drugs.

Results

This platform faithfully reported the chronological sequence of organelle-specific events with the progression of cell death, with good temporal and spatial resolution at the single-cell level. Moreover, we have identified and categorised potential lead compounds that predominantly induce lysosomal cell death or mitochondrial cell death, as well as a subset that elicit both events concomitantly.

Conclusion

The study provided evidence that both organelles contribute to cell death in a context-dependent manner, and the temporal analysis of both events is critical in understanding unique organelle-centred cell death.

KEYWORDS: Lysosomal membrane permeabilization, mitochondrial oxidation, live cell imaging, cathepsin B, mito-roGFP, live cell sensor, high-throughput drug screening, bax activation

Introduction

Mitochondria, often called the ‘powerhouse of the cell,’ are essential for producing ATP, which maintains a cell's structure and function. However, programmed cell death involving mitochondrial permeabilization-dependent caspase activation helps in tissue homeostasis [1]. Similarly, lysosomes, the suicidal bag of a cell, regulate the intracellular degradation necessary for nutrient recycling, autophagy, and homeostasis. Lysosomes also provide pathways of distinct cell death through controlled lysosomal permeabilization by releasing an array of proteases, like cathepsins, into the cytoplasm [2]. Interestingly, multiple means of cell death, such as apoptosis, necrosis, inflammatory cell death, necroptosis, etc., always proceed through the coordinated action of both mitochondria and lysosomes [3]. Even though the primary pathway of cell death is through apoptosis, many anti-cancer and cytotoxic drugs are known to induce lysosomal permeabilization [4].

Since many forms of cell death proceed with a well-ordered sequence of events from initiation to culmination with complex and differing temporal kinetics involving multiple organelles, it is difficult to identify cell death pathways influenced only by mitochondria or lysosomes. Many of the previously identified mitochondrial cell death inducers are known to target lysosomes [4,5]. Similarly, a class of compounds known as lysosomotropic agents utilizes mitochondrial permeabilization to complete the cell death process [5]. The major challenge in identifying organelle-dependent cell death is the complex and variable temporal control of these two events once initiated at a single cell level, which is difficult to capture with conventional chemical probes. A specialized live cell approach for simultaneous visualization of organelle damage or its integrity under cell death in real time is essential to address such challenging aspects at the single-cell level. The primary contribution of mitochondria in apoptosis involves the release of cytochrome c and Smac (Second mitochondria-derived activator of caspase) from the intermembrane space to the cytosol, which further activates caspases. The loss of mitochondrial transmembrane potential and its permeabilization are key events that trigger the mitochondrial pathway of cell death. Recently, we have demonstrated that mitochondrial integrity and permeabilization events during diverse forms of cell death accompany mitochondrial oxidation, which is detectable by mitochondrial-targeted roGFP (mt-roGFP) in live cells [6]. Similarly, the lysosomes undergo selective permeabilization, releasing an array of proteases that include cathepsins such as cathepsin B and D. In the current study, we have stably expressed cathepsin B as a tdTomato fluorescent fusion protein in HeLa cells, which served as a reliable lysosomal membrane permeabilization (LMP) indicator in live cells with good temporal resolution under normal and stress conditions. Further, expressing mt-roGFP into HeLa tdTomato-CathepsinB cells helped us to create a versatile dual cell sensor for live cell detection of both organelles’ contributions to cell death, with good temporal and spatial resolution, resulting in the generation of the first live-cell sensor for identifying organelle-centred cell death inducers.

Materials and methods

Cell lines and expression vectors

Cervical cancer cell lines, HeLa, SiHa expressing EGFP-Gal3 and ECFP-Bax, and SiHa expressing EGFP-Gal3 were obtained from the Central Cell Line (CCL) Repository of BRIC-Rajiv Gandhi Centre for Biotechnology. The cells were maintained in DMEM medium (Cat no. 12800017, Gibco, ThermoFisher Scientific, USA) containing 10% Fetal Bovine Serum (Cat No. 26140079, Gibco, ThermoFisher Scientific, USA) and 1X antibiotic-antimycotic (Cat No. 15240062, Gibco, ThermoFisher Scientific, USA). The cells were used within 10 passages after revival from the original stock. The mammalian expression vector tdTomato-CathepsinB-6 was a gift from Prof. Michael Davidson (Addgene plasmid # 58075; http://n2t.net/addgene:58075; RRID: Addgene_58075), and Mitochondrial-targeted roGFP2 (mt-roGFP) was procured from Prof. S. James Remington (Institute of Molecular Biology, University of Oregon, Eugene, OR) [7].

Generation of a stable cell line expressing tdTomato-CathepsinB as a live cell sensor for lysosomal permeabilization

For transfection with tdTomato-CathepsinB-6 (Addgene #58075), 0.2 × 106 HeLa cells were seeded in a 12-well plate (Cat No. 3513, Corning, USA). At 60% confluency, cells were transfected using Lipofectamine™ LTX plus reagent (Cat No. 15338100, Invitrogen, ThermoFisher Scientific, USA) as per the manufacturer’s protocol, and cells were maintained in 800 µg/ml of G418 (Cat No.10131035, Gibco, ThermoFisher Scientific, USA) containing medium for four weeks. The heterogeneous populations of HeLa tdTomato-CathepsinB cells were further sorted based on the intensity of tdTomato (excitation/emission 561/610 ± 10 nm) using FACSAriaIII (Becton Dickinson) for enriching cells with relatively homogenous expression. The cells were further maintained in selection media until stable expression was confirmed. Only clones expressing a properly targeted probe were expanded for further experiments.

For confocal imaging, cells were stained with 1 µg/ml of the nucleic acid dye Hoechst 33342 (Cat No. H1399, Molecular Probes, ThermoFisher Scientific, USA), incubated for 10 min, and washed twice with PBS. Cells were imaged with a 60X oil objective in the Nikon AIR confocal imager. Excitation/emission settings were 560/600 ± 20 nm for tdTomato-CathepsinB and for Hoechst 405 nm/420 ± 20 nm.

Confirmation of tdTomato-CathepsinB targeting using lysotracker deep red and mitotracker deep red staining

To verify the localization of tdTomato-CathepsinB to lysosomes, both lysosomes and mitochondria were stained independently using organelle-specific probes. HeLa tdTomato-CathepsinB cells were seeded in an 8-well chambered cover glass (Cat No.177402, Nunc, USA) and stained with 50 nM lysotracker deep red, (Cat No. L12492, Invitrogen, ThermoFisher Scientific, USA) and 50nM Mitotracker Deep Red, (Cat No. M22426, Invitrogen, ThermoFisher Scientific, USA) for 30 min at 37 °C at 5% CO2. Cells were imaged with a 60X oil objective in the Nikon AIR confocal imager. Excitation/emission settings were 640/690 ± 30 nm for both LysoTracker Deep Red and MitoTracker Deep Red, and 560/600 ± 20 nm for tdTomato-CathepsinB. Pearson’s Correlation analysis was performed using Nikon’s NIS Element software for colocalization analysis.

Functional analysis of cathepsin B release in HeLa tdTomato-CathepsinB cells

For the functional evaluation of the HeLa tdTomato-CathepsinB, cells seeded in 96-well optical bottom plates (Cat No. 165305, Nunc, USA) at 70% density were treated with known lysosomotropic agent, bafilomycin (10nM, Cat No. B1793, Sigma-Aldrich, USA), and an apoptotic inducer, etoposide (50 µM, Cat No. E1383, Sigma-Aldrich, USA). End-point images were captured after 24 h using a laser scanning confocal inverted microscope (Nikon A1R). Imaging settings for tdTomato-CathepsinB were followed as described above.

Further to understand the cathepsin B release in real-time at a single cell level, in response to lysosomotropic agent bafilomycin, live cell imaging was performed using a Nikon A1R laser scanning confocal microscope, aided with an on-stage live-cell incubation chamber from Tokai Hit (Japan). The temperature was maintained at 37 0C, and humidified CO2 (5%) was supplied continuously to maintain ideal conditions. Confocal imaging was carried out for 48 h with an interval of 6 h.

Development of a dual sensor for simultaneous visualization of mitochondrial ROS and lysosomal permeabilization

For the simultaneous imaging of mitochondrial ROS and lysosomal permeabilization, 0.2 × 106 HeLa tdTomato-CathepsinB cells were seeded in a 12-well plate (Cat No. 3513, Corning, USA) and transfected with mt-roGFP as described earlier [8]. roGFP, the genetically encoded GFP-based reduction–oxidation sensitive probe, detects glutathione redox potential by exhibiting dual excitation characteristics. This technique has made it possible to monitor thiol-based redox changes in a variety of physiological and pathological states, as well as within different cellular organelles[9,10]. The transfected cells were maintained in 800 µg/ml of G418 selection pressure for four weeks, followed by flow cytometry sorting to enrich cells homogenously expressing both tdTomato-CathepsinB and mt-roGFP probe using FACSAriaIII. For sorting mt-roGFP, the cells were excited with 405 and 488 nm lasers, and the single emission at 520 ± 20 nm was collected in 405/488 nm ratio mode. The sorting condition for tdTomato-CathepsinB is mentioned above.

Functional evaluation of HeLa tdTomato-CathepsinB mt-roGFP

For the functional evaluation of the HeLa tdTomato-CathepsinB mt-roGFP, cells were seeded in 96-well optical bottom plates (Cat No. 165305, Nunc, USA) at 70% density, and were treated with a known apoptosis inducer, camptothecin (25 µM, Cat No. C9911, Sigma-Aldrich, USA). End-point images were captured after 48 h using a laser scanning confocal inverted microscope (Nikon A1R). For imaging mt-roGFP, the cells were sequentially excited at 405 and 488 nm, and the emission was collected at 520 ± 20 nm. tdTomato was imaged with settings detailed earlier. To track time-resolved alterations in mitochondrial oxidation and lysosomal permeabilization in real-time, confocal live cell imaging was carried out for 60 h with an interval of 6 h.

Real-time confocal imaging for drug screening with HeLa tdTomato-CathepsinB mt-roGFP

For real-time imaging, cells grown in 96-well optical bottom plates (Cat No. 165305, Nunc, USA) for 24 h were treated with compounds at appropriate working concentrations (Table S1). Thirty drugs that could induce apoptosis, mitochondrial oxidation, and lysosome destabilization were selected for the drug screening study based on the available literature.

Images were captured using a Leica SP8 WLL confocal laser scanning microscope, aided with an on-stage live-cell incubation chamber, maintaining ideal conditions as previously mentioned. The imaging was initiated four hours after drug treatment and continued for 44 h with an interval of 1 h using a 20X objective with a numerical aperture of 0.75. Photo bleaching was avoided by reducing the excitation laser intensity to minimum levels. The images were captured and analyzed in 405/488 nm ratio mode for mt-roGFP (excitation at 405 and 488 nm, emission collected at the range of 500-550 nm) and tdTomato-CathepsinB, excited at 561 nm and emission collected at the range of 580-640 nm. Analysis was performed using LAS X software.

Dose and time-dependent alterations in mitochondrial oxidation analysed using MitoSOX™ red staining and cell death by annexinV-BFP

HeLa tdTomato-CathepsinB mt-roGFP and the parental HeLa cells were stained with MitoSOX™ Red after 24 and 48 h of drug treatment, and were analyzed by FACS. In brief, HeLa tdTomato-CathepsinB mt-roGFP and HeLa cells were seeded in a 12-well plate (Cat No. 3513, Corning, USA) and treated with bafilomycin (10nM and 20nM), camptothecin (12.5 and 25 µM), ursolic acid (10 and 20 µM) for 24 and 48 h, trypsinised and stained with 50nM MitoSOXTM Red (Cat No. M36008, Invitrogen, ThermoFisher Scientific, USA) for 10 min at 37 °C at 5% CO2. Analysis was performed using FACSAriaIII (Becton Dickinson) with appropriate filter sets for the Hoechst red channel (Ex/Em 355/675 ± 25 nm). Histograms were plotted using Kaluza software.

To analyze drug-induced apoptosis, HeLa tdTomato-CathepsinB mt roGFP cells and parental HeLa cells, grown in 96-well plates, were treated with varying concentrations of drugs as mentioned above. For apoptosis detection, the cells were incubated with Annexin V-Blue Fluorescent Protein (Cat No. PLMAX02-50, Primordia Life Sciences Pvt Ltd, India) at a final concentration of 2.5 µg/mL. Imaging was performed from 6 h after drug treatment for 48 h with an interval of 1 h. Confocal imaging was performed using a 20X objective in the Nikon AIR confocal imager. For Annexin V-BFP, excitation/emission settings were 407/450 ± 25 nm; tdTomato-CathepsinB and mt-roGFP were imaged using the settings described earlier.

Western blot analysis of lamp1 protein in HeLa tdtomato-CathepsinB mt-roGFP cells and parental cells

Western blot analysis was performed using HeLa tdTomato-CathepsinB mt-roGFP and HeLa cells to compare LAMP1 protein levels following the standard protocol. After electrophoresis, the separated protein was transferred to a PVDF membrane and probed with specific primary and HRP-conjugated secondary antibodies. Rabbit monoclonal antibody against LAMP1 (#9091) and polyclonal antibody β-Actin (#IMG-5142A) were procured from Cell Signaling, USA, and Novus Biologicals, USA, respectively. Chemiluminescent signals were developed using Pierce ECL Western Blotting Substrate and imaged with Amersham ImageQuant 8000.

Validation of the lead compounds using EGFP-Gal3 and ECFP-Bax overexpressed cells

To further validate lysosomal dominance over mitochondrial cell death, SiHa EGFP-Gal3 ECFP-Bax cells were seeded in 96-well optical bottom plates (Cat No. 165305, Nunc, USA), and after 24 h, compounds to be tested were added (Table S1). Live-cell imaging was performed using a Nikon A1R laser scanning confocal microscope. For imaging EGFP-Gal3, cells were excited at 488 nm, emission was collected at 525 ± 25 nm, and for ECFP-Bax, cells were excited at 408 nm, collecting 450 ± 30 nm emission. Confocal imaging was initiated 2 h after drug treatment and continued for 30 h with an interval of 1 h, under the incubation conditions previously described.

Statistical analysis

Statistical analysis performed using Two-way analysis of variance (ANOVA) using GraphPad Prism software. Values represent mean ± SD; p-values are described in the figure legends.

Results

Development and validation of a live cell sensor for lysosomal permeabilization

The lysosomal permeabilization event culminates in the cytoplasmic release of an array of proteases belonging to the cathepsin family. To develop a live cell indicator for lysosomal permeabilization, HeLa cells were transfected with tdTomato-CathepsinB. Multiple clones of cells were expanded independently, eliminating mistargeted clones. A confocal image of stable HeLa tdTomato-CathepsinB cells is shown in Figure 1A. The selected clones were independently stained using Mitotracker Deep Red and Lysotracker Deep Red to confirm the localization of the probe (Figure 1B and D). The localization of tdTomato-CathepsinB to the lysosome is evident with lysotracker; however, the mito-tracker failed to show any localization of tdTomato-CathepsinB, suggesting the proper targeting of the probe within the lysosome in the selected clones. The Pearson correlation analysis further substantiated the results with a strong correlation coefficient of 0.8 for the lysotracker and 0.27 for the mitotracker, with tdTomato-CathepsinB (Figure 1C and E). Only clones that express tdTomato-CathepsinB with a visible lysosomal appearance were used for further experiments. The cells were imaged after 24 h of treatment with bafilomycin and etoposide for the functional evaluation. The untreated cells demonstrated a granular expression pattern of tdTomato-CathepsinB, while the cells exposed to bafilomycin showed a diffuse expression pattern with a significant increase in tdTomato-CathepsinB signal intensity, confirming the cytoplasmic release of cathepsin B in most of the cells. However, etoposide treatment induced cathepsin B release only in a few cells; even dead cells maintained intact tdTomato-CathepsinB granularity (Figure 1F). Since we observed increased and diffuse expression of tdTomato-CathepsinB in bafilomycin-treated cells at 24 h, real-time confocal imaging was carried out at an interval of 6 h for 48 h to see the kinetics at the single-cell level (Figure 1G, Video S1). As seen from the image and video, from 12 h onwards, a gradual increase in tdTomato-CathepsinB intensity and the diffuse expression of the protein is observed in most of the cells, even before any evidence of cell death. The untreated cells maintained intact lysosomal cathepsin B throughout the imaging period. As shown, the stable cells maintained their proliferative state without any significant cell cycle inhibition, suggesting the nontoxic nature of the probe (Video S2). This is further evident from the average doubling time of HeLa tdTomato-CathepsinB calculated from the real-time imaging as 29 h, which is similar to the reported parental HeLa cell doubling time [11]. The results confirmed that tdTomato-CathepsinB cells retained their intact targeting and functional capability to report lysosomal permeabilization in real time.

Figure 1.

Figure 1.

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Development and validation of a cell sensor for lysosomal permeabilization. (A) Confocal images showing HeLa cells homogenously expressing tdTomato-CathepsinB, respective channels for tdTomato-CathepsinB, Hoechst, and overlay are also shown. (B) Confocal images of HeLa tdTomato-CathepsinB stained with lysotracker deep red (Ex./Em. 640/690 ± 30 nm) showing lysosomal localisation of tdTomato-CathepsinB. (C) The Scatter plot of the colocalization map also confirms the lysosomal localization of cathepsin B. (D) Confocal images of HeLa tdTomato-CathepsinB stained with mitotracker deep red (Ex./Em. 640/690 ± 30 nm) showed the absence of mitochondrial localization of tdTomato-CathepsinB. (E) The Scatter plot of the colocalization map also confirms the absence of mitochondrial localization of tdTomato-CathepsinB. (F) Confocal images of HeLa tdTomato-CathepsinB cells treated with lysosomotropic agent, bafilomycin, and apoptotic inducer, etoposide, for 24 h showed cathepsinB release in bafilomycin treatments. (G) Time-lapse confocal imaging of HeLa tdTomato-CathepsinB cells treated with bafilomycin for 48 h. Cells showing cathepsin B release are marked with yellow arrows. Respective channels for tdTomato-CathepsinB, brightfield, and overlay are shown.

A dual sensor for lysosomal permeabilization and mitochondrial damage supported long-time tracking of both events with cell death

The tdTomato-CathepsinB stable cells were further transfected with the mt-roGFP to generate a dual sensor for lysosomal and mitochondrial damage. Figure 2A shows the respective channels of tdTomato-CathepsinB, 405, and 488 nm of mt-roGFP, along with the mt-roGFP ratio (405/488 nm), and overlay, which confirms the stable expression of both sensors. For the functional validation of the sensor cells, we analyzed the changes in mitochondrial ROS and cathepsin B release by end-point imaging at 48 h of camptothecin treatment. As shown in Figure 2B, compared to control cells, camptothecin induced mitochondrial oxidation with a marked increase in the 405/488 ratio. In the majority of dying cells, the release of tdTomato-CathepsinB with diffuse red fluorescence is well pronounced, and that also correlated with a high 405/488 ratio by 48 h. Interestingly, camptothecin induced an increase in tdTomato-CathepsinB intensity in many of the treated cells. To track time-resolved alterations in mitochondrial oxidation and lysosomal permeabilization in live cells, we have done real-time imaging for 60 h with an interval of 6 h (Figure 2C). The full video showing the progression of organelle alterations is shown in Video S3. There was a time-dependent oxidation of mitochondria and release of cathepsin B from 24 h, which progressed until 60 h. More interestingly, real-time imaging revealed rare surviving cells with upregulated cathepsin B without a change in mt-roGFP ratio, with pseudo-senescent morphology.

Figure 2.

Figure 2.

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Development and functional validation of a dual sensor for lysosomal permeabilization and mitochondrial damage. (A) Confocal images of HeLa tdTomato-CathepsinB cells homogenously expressing mt-roGFP; respective tdTomato, mt-roGFP (488 and 405 nm), ratio-metric image (405/488), and overlay are shown. (B) End point imaging of HeLa tdTomato-CathepsinB mt-roGFP cells exposed to the drug camptothecin for 48 h. (C) Time-lapse confocal images of HeLa tdTomato-CathepsinB mt-roGFP cells with camptothecin treatment for 60 h. Cells showing higher mitochondrial oxidation are marked with red arrows. Respective channels for ratio images (405/488), tdTomato, and brightfield are shown.

Further, the imaging demonstrated biphasic mitochondrial oxidation, as recently described [6]. Overall, the cell system developed proved a highly efficient model system for the characterization of organelle-centred cell death and also for long-time real-time imaging applications.

We also assessed the impact of overexpressed cathepsin B on lysosomal mass in HeLa tdTomato-CathepsinB mt-roGFP cells. Lysosomal density was compared in HeLa tdTomato-CathepsinB mt-roGFP and parental HeLa cells using lysotracker deep red staining. The staining revealed similar and comparable red fluorescence intensity in the cathepsin B-overexpressing cells and parental cells (Supplementary Figure S2 A-B). This was further substantiated by Western blot analysis of lysosomal-associated membrane protein 1 (LAMP1) levels in the two cells, with β-actin used as a loading control (Supplementary Figure S2 C).

Multi-well plate imaging revealed the organelle-centred response of cytotoxic compounds and potential application in the functional grouping of drugs

To verify the potential utility of the dual sensor to screen compound libraries to identify organelle-centred responses of drugs with a temporal relationship, 96-well plate real-time imaging was carried out by confocal imaging. The imaging was started after four hours of drug treatment and continued for 44 h with an interval of 1 h. The cellular events of cathepsin B release, cathepsin intensity increase, and mitochondrial ROS generation were systematically analyzed, with their timing of occurrence, at the single-cell level for each compound. The cell death observed from DIC further enabled the determination of the extent of cytotoxicity. The integrated schematics of the working principle of the developed sensor, the experimental setup, and categorization of drugs into four groups based on single-cell response are shown in Figure 3A. The first group consists of drugs that induced cathepsin B release before the evident mt-roGFP ratio change, the second group consists of drugs that induced both events at almost the same time point, within 1 h of the imaging interval, and the third group consists of compounds that have only induced mitochondrial oxidation with minimal change in lysosome integrity. The fourth group of compounds represents a pronounced increase in cathepsin B fluorescence intensity without mitochondrial oxidation. A representative time sequence of control and a promising candidate belonging to each group is shown in Figure 3B. From Group 1, the representative 48-hour image of Ursolic acid is shown. The full sequence of events for the two drugs belonging to this group, Ursolic acid and Thapsigargin, depicting cathepsin B release before mitochondrial ROS generation, is shown in Video S4. From the second group, representative 48-hour images of camptothecin are shown. The full video of the two drugs belonging to this group, camptothecin and MG-132, is shown in Video S5. As shown in the 48-hour image and from the full sequence video, both compounds triggered cathepsin B release and mitochondrial ROS generation almost simultaneously. Without detectable cathepsin B release or intensity alteration, group 3 compounds induced significant mitochondrial ROS change, with noticeable cell death. From Group 3, representative images of diflunisal, 48-hour post-drug treatment images are shown. The whole video sequence of two compounds belonging to this group, diflunisal and CoCl2, is shown in Video S6. Group 4 compounds significantly increased cathepsin intensity without any detectable mitochondrial ROS generation. 48-hour post-drug treatment images of Nigericin, which represent Group 4 compounds, are shown. As shown in Video S7, both Nigericin and cyclophosphamide have triggered visible cathepsin B intensity without its release and ratio change up to 48 h of treatment. Interestingly, these compounds were relatively less toxic at the concentration used. An integrated quantitative representation of all the drugs in groups 1, 2, and 3 is shown in Figure 3C. Overall, the study demonstrated that the cellular model is capable of imaging the impact of lysosomal damage and mitochondrial damage at the single-cell level with sufficient temporal resolution in a multiwell format, enabling large-scale screening to systematically characterize drugs as lysosomal dominant or mitochondrial dominant.

Figure 3.

HeLa tdTomato-CathepsinB mt-roGFP for the simultaneous visualization of lysosome destabilization and mitochondrial oxidation: (A) Schematic representation of the experimental setup, including the working principle of the developed dual sensor and categorization of drugs based on the functional attributes of lysosome destabilization and mitochondrial oxidation. (B) Real-time confocal imaging of drug screening using HeLa tdTomato-CathepsinB mt-roGFP cells for 44 h, imaging was initiated after 4 h of drug treatment. Representative images of a single drug from each group with respective tdTomato, mt-roGFP ratio, and brightfield images are shown. Ursolic acid, a drug from Group-1, shows prominent cathepsinB release over mitochondrial oxidation. Camptothecin, a drug from Group 2, shows simultaneous mitochondrial oxidation and cathepsinB release. Diflunisal, a drug from Group 3, shows prominent mitochondrial oxidation over cathepsinB release. Nigericin, from Group 4, showed increased intensity of tdTomato-CathepsinB without significant release and mitochondrial oxidation. (C) Bar diagram representing quantitative analysis of the percentage of cells with cathepsin B release and mt-roGFP ratio change of all compounds from groups 1, 2, and 3, respectively. Values ± SD were taken. Two-way analysis of variance (ANOVA) was used to determine statistically significant differences (GraphPad Prism). ****P < 0.0001, ***P < 0.001, **P < 0.01.

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Dose and time-dependent mitochondrial oxidation response was also analysed using MitoSOX Red to compare the response between parental HeLa and HeLa tdTomato-CathepsinB mt-roGFP cells. Studies showed HeLa tdTomato-CathepsinB mt-roGFP cells exhibited a similar response pattern to that of their parental HeLa cells, both at lower and higher doses of the drugs, and at 24 and 48 h. However, a moderate increase in oxidation was observed in HeLa tdTomato-CathepsinB mt-roGFP cells treated with camptothecin at 48 h. Notably, the dose-dependent response to bafilomycin, as shown by both cell types, in consensus, showed less oxidation at higher doses of the drug. Ursolic acid failed to induce evident mitochondrial oxidation in both cells at the indicated concentration of the drugs (Supplementary Figure S1).

Accordingly, the dose – and time-dependent cell death response was also compared in HeLa tdTomato-CathepsinB mt-roGFP cells versus parental HeLa cells by Annexin V-BFP staining. Both cell lines exhibited similar and comparable dose – and time-dependent apoptotic responses, demonstrating that the overexpression of cathepsin B does not influence overall drug-induced cell death. A representative image of Annexin V-BFP staining at 48 h, treated with bafilomycin, camptothecin, and ursolic acid, is shown in Supplementary Figure S2.

Confirmation of lysosomal permeabilization upstream of bax activation using SiHa EGFP-Gal3 ECFP-Bax cells

The potential application of the sensor cells, HeLa tdTomato-CathepsinB mt-roGFP, is to identify lysosomal permeabilization-inducing compounds and compounds that specifically induce lysosomal-mediated cell death with minimal involvement of mitochondria. The real-time image using selected compounds generally supported the co-existence of both events in dying cells with differing time scales. However, the first group of compounds seems to be unique in that they target lysosomes more than mitochondria in killing the cells. So, we decided to test group-1 compounds, with additional models to confirm their lysosomal dominance. At present, the best marker used for the lysosomal membrane integrity breach is galectin 3 (Gal-3) translocation to the damaged lysosomes. Similarly, a critical mitochondrial permeabilization event is marked by Bax activation and its translocation to mitochondria. Logically, any compound that triggers lysosomal-mediated cell death is supposed to elicit lysosomal permeabilization before Bax activation and its translocation to mitochondria. Since the time scale of Bax activation and Gal-3 translocation is to be determined with good temporal resolution, a real-time probe for these two events is needed. To achieve this, we have used a new sensor cell line, SiHa EGFP-Gal3 ECFP-Bax, for detecting Bax activation and Gal-3 translocation to lysosomes in live cells. To functionally validate, group-1 compounds for their lysosomal dominance and group-3 compounds for their mitochondrial dominance, cells were exposed to these compounds, and real-time imaging was performed for spatio-temporal alterations of Bax and Gal-3. As shown in Figure 4A, out of group-1 drugs, despite the proapoptotic nature of Bax, ursolic acid induced Gal-3 translocations within 5 h of drug treatment before detectable Bax activation (Video S8). Interestingly, many cells demonstrated cell death without significant Bax activation, suggesting Bax-independent cell death. However, diflunisal, from group-3 compounds, has induced Bax aggregation in 17 h without any detectable Gal-3 puncta formation (Video S9). Overall, the results confirmed that our single-cell sensor, HeLa tdTomato-CathepsinB mt-roGFP, and the real-time imaging approach are sufficient to accurately identify lysosome-mediated cell death inducers.

Figure 4.

Lysosomal permeabilization upstream of Bax activation in Bax-overexpressing conditions using SiHa EGFP-Gal3 ECFP-Bax cells (A) Time-lapse images of SiHa EGFP-Gal3 ECFP-Bax cells treated with ursolic acid and diflunisal. Imaging was initiated 2 h post-drug treatment; the respective EGFP-Gal3 and ECFP-Bax channels are shown. The red arrow shows cell death with prominent Gal3 puncta formation but without Bax activation in ursolic acid treatment. The yellow arrow shows cells undergoing Bax-dependent death without Gal3 puncta formation. Cropped images in insets show Gal-3 puncta formation or Bax activation. Images were analysed for the sequelae of Gal3 puncta formation and Bax activation. (B) Time-lapse images of the effect of ursolic acid on Gal3 puncta formation in SiHa EGFP-Gal3 ECFP-Bax and SiHa EGFP-Gal3 cells. Imaging was initiated 2 h post-drug treatment. Inset images of two regions of both cells show the initiation of Gal3 puncta formation (Red arrows) at three hours.

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Since Bax is a dominant pro-apoptotic protein, we further compared the effect of ursolic acid on Gal3 puncta formation in Bax overexpressed and non-overexpressed conditions using SiHa EGFP-Gal3 and SiHa EGFP-Gal3 ECFP-Bax cells. Even though both cells initiated Gal3 puncta formation at 3 h, Gal3 alone cells showed prominent Gal3 puncta formation (Figure 4B and Video S10).

Discussion

The primary mode of mammalian cell death is apoptosis via extrinsic or intrinsic routes that require mitochondria as the key player. Mitochondria provide the signal for caspase activation through their controlled permeabilization, leading to cytochrome c release. Lysosomes are also emerging as a key mediator of lysosomal cell death and contribute to apoptosis, necrosis, paraptosis, and necroptosis in distinct ways, serving as an additional layer of cell demise [12,13]. The primary function of the lysosome is intracellular degradation, a cellular process required for cell survival and homeostasis. Phagocytic degradation of damaged organelles, biomacromolecules, toxins, and pathogens is managed by the lysosome for nutrient cycling. Increasing studies reveal complex cross-talk between these two organelles in diverse forms of cell death, pathology, as well as survival [13,14]. Lysosomal leakage of proteases, such as cathepsins, cleaves proapoptotic protein Bid and anti-apoptotic Bcl-2 to promote mitochondrial membrane permeabilization, facilitating caspase activation [14,15]. Caspase-independent cell death with hallmarks of apoptosis induced by quinolone antibiotics, by lysosomal damage, was also reported [15]. Even though many studies have demonstrated coordinated activity of both organelles in cell death, lysosomal permeabilization-mediated cell death as an independent cell demise mechanism, corroborating suicidal activity, is also emerging [16]. However, lysosomal permeabilization-mediated cell death independent of mitochondria is difficult to characterize at the cellular level because of the complex spatiotemporal control of these events in dying cells on a fast time scale once initiated. A system for the detection of initiation and progression of both events with good temporal resolution and long-term imaging is important to address such a complex sequence of cellular events at the single-cell level. In the current work, we describe an approach to visualize two decisive events of mitochondrial and lysosomal organelle damage along with cell death in unperturbed growing cells, and also when cells are exposed to diverse forms of stress, including anticancer drugs. The stable cells expressing mt-roGFP are highly sensitive to detect the depletion of glutathione in mitochondria, which is a hallmark of mitochondrial permeabilization [17]. Compared to many fluorescent protein-based permeabilization indicators, such as cytochrome c or Smac, that work based on fluorescent translocation as a readout, the ratio-metric imaging for mitochondrial ROS using roGFP targeted to mitochondria is more compatible for the detection of mitochondrial damage. We also compared the performance of mt-roGFP with a similar chemical probe, MitoSOX. The mt-roGFP probe, being genetically encoded and stably expressed, supports ratiometric repeated, longitudinal measurements of redox changes in the same cells without the need for dye reloading [8,18,19]. MitoSOX is light-sensitive, prone to auto-oxidation, and can perturb mitochondria at higher doses. Its use for long-term imaging is heavily constrained and requires very careful optimization [20–22]. Further cell death-dependent organelle damage could compromise dye loading and the detection of dyes, affecting the interpretation of the true oxidation status of organelles. Acidic extracellular or cytosolic pH, induced by drugs, is known to collapse or reduce mitochondrial membrane potential [20,21] and alter TPP+ partitioning, resulting in less MitoSOX accumulation in the matrix, independent of superoxide levels [22,23]. This could be a reason for the observed low MitoSOX fluorescence in the higher concentration of bafilomycin treatment.

We have expressed tdTomato-CathepsinB fluorescent fusion protein considering its compatibility with roGFP. More importantly, the signal intensity of tdTomato is not affected by low lysosomal pH and provides sufficient signal for imaging. In our system, where tdTomato-cathepsinB is overexpressed and normally retained within the acidic milieu of lysosomes, where protease activity is optimal, the tdTomato signal remains detectable as a punctate appearance owing to its basic, low acid-sensitivity with a relatively low pKa value [24,25].Upon lysosomal membrane permeabilization and its redistribution to the neutral cytosol, it is clearly observed as a diffuse fluorescence pattern. Rather than a pH-dependent change in fluorescence, visualizing the temporal change and translocation-based readout reflects the release of cathepsin B, a mechanistic hallmark of lysosome membrane permeabilization. A pure pH sensor can report luminal neutralization even ratiometrically, too, but cannot distinguish full rupture from partial permeabilization without additional markers. There is mounting evidence that cathepsins have biological roles in environments with non-acidic pH, so a translocation-based readout remains a better option than the pH-dependent sensor [26–28].

In short, tdTomato was selected because it is bright, photostable, and has low acid sensitivity in the pH range encountered upon lysosomal damage, making it a suitable stable reporter of cathepsin B localization. The pH-sensitive probes, such as pHluorin or recently developed lysosomal pH biosensors (e.g. ratiometric Cherry pHluorin and FIRE pHLy), are indeed powerful means for quantifying pH alterations [29,30] and, in the future, may be combined with our cathepsin tdTomato reporter to further disentangle membrane permeabilization from pH alterations.

Functional fluorescent dyes such as LysoTracker and TMRM (Tetramethyl rhodamine Methyl Ester) and single-cell imaging approaches were described in earlier publications [31]. Lysotracker red is not compatible with long-time imaging and is not a direct indicator of its permeabilization. Similarly, many chemical-based ROS probes also suffer from their light-induced ROS generation, with toxicity and bleaching. All these shortcomings are addressed in genetically encoded sensors as they allow stable expression with full confidence to report the desired activity. Real-time imaging using the reporter revealed unique response patterns of these two organelles towards multiple stress conditions and enabled the identification of compounds that can induce early lysosomal permeabilization before mitochondrial damage. Such unique compounds have many applications in addressing chemoresistance, often mediated by loss of apoptosis signalling, preventing mitochondrial permeabilization. Many solid tumours are inherently resistant to intrinsic cell death signalling through alterations in apoptotic regulatory proteins such as Bcl2 and BclxL [32,33]. Similarly, there are signals that even compromise cell death downstream of mitochondrial permeabilization, such as XIAP, IAPS (X-linked inhibitor of apoptosis protein, Inhibitors of Apoptosis Proteins), and apoptosis-resistant tumour stem cells [34]. In the future, the compounds identified in Group 1 (lysosomal dominant drugs) could be tested against drug resistance in such cell systems. Ursolic acid (UA) may be counted as a potential candidate from this category. The compound UA is an FDA-approved small molecule widely studied and recommended for the treatment of arthritis [35]. UA exhibits strong anti-inflammatory and antioxidant properties, and for that reason, it has been spotlighted for extensive research aiming to develop preventive and therapeutic strategies for many other metabolic diseases, such as obesity, hypertension, diabetes, cardiovascular disease, liver and kidney dysfunction, and cancer [36,37]. UA can induce cell death in breast carcinoma cells, compromising lysosomal membrane stability and disrupting lysosomal function [38]. More interestingly, the result of our current study agrees and is consistent with this study, which demonstrated that ursolic acid triggered lysosomal membrane permeabilization before Bax activation. This further confirms the sensitivity of the sensor system developed. Lysosomal density and activity are upregulated in many cancers as survival signalling, which promotes autophagy compared to normal cells [39–41]. So, the identification of specific lysosomal destabilizing agents with proapoptotic activity with minimal involvement of mitochondria could be a therapeutic strategy for drug-resistant cancers. Also, combining similar compounds with apoptosis-inducing activity could induce rapid cancer cell elimination even in drug-resistant conditions. A combination of venetoclax, a Bcl2 protein inhibitor, along with lysosome-disrupting agents, is reported to augment cell death [42]. Interestingly, our study also identified a group of compounds that induce upregulation of cathepsin B intensity without its release and negligible cell death. Since increased lysosomal biogenesis invariably contributes to an increase in lysosomal density, an increase in cathepsin B could reflect lysosomal biogenesis. Further studies are required on whether the probe can be used as an indicator of lysosomal biogenesis. The two prominent compounds that belong to this category are Endoxifen and tamoxifen, both of which are known to induce autophagy and lysosomal biogenesis transcription factor EB (TFEB) [43,44].

Multiple studies have demonstrated that the generation of mitochondrial ROS is a very early event preceding the loss of mitochondrial potential and its permeabilization [45]. An increase in ROS caused by LMP-dependent cathepsin-D release on mitochondria has also been demonstrated [46]. Recent work demonstrates that Newcastle disease virus infection triggers LMP, leading to the translocation of cathepsin B and D and subsequent mitochondria-ROS-dependent apoptosis [47]. These studies suggest the contribution of mitochondrial ROS in lysosomal cell death, even though the time scale of these events is not analyzed. So, the cell models described here have great application in large-scale screening as well as in understanding the interrelationship between these organelles in the modulation of cell death by drugs and pathologies. We have used an interval of 1 h with high-throughput imaging mode in real time, which is sufficient to characterise compounds based on the temporal sequence of events. It is also possible to image with shorter intervals if required, as the expression of cathepsin B with tdTomato demonstrated sufficient signal with minimal photo bleaching.

The cell model is also useful to dissect the implications of mitochondrial ROS with other cell fates, such as autophagy and mitophagy. Increased and sustained levels of mitochondrial ROS can lead to the activation of mitophagy pathways via PINK1/Parkin and redox-sensitive autophagy receptors, such as NDP52 and OPTN [48]. Lysosomal membrane permeabilization or loss of membrane integrity can inhibit autophagy [49–52]. Since autophagy completion requires functional lysosomes, the autophagy lysosome degradation pathway is compromised by defective or permeabilised lysosomes. Misregulation of lysosome positioning [53,54] or defects in signaling molecules associating with lysosomes (e.g. MTORC1 and TFEB) [55] are known to interfere with autophagy. A recent study further revealed that bleomycin-induced ROS mediates defects in autophagic degradation, with implications in cellular senescence, both in vitro and in vivo conditions involving lysosomal membrane permeabilization [56].

Altogether, the study describes robust, real-time genetically encoded sensor cells that distinguish the two major organelle-centred cell death events with a good temporal resolution for the identification of unique compounds with mitochondria or lysosomal-specific activity. The cell system has potential utility for the systematic screening of large compound libraries in the future to expand the number of compounds belonging to each category as defined here.

Conclusion

Lysosome-dependent cell death is emerging as a new therapeutic pathway of cell death with implications in diseases and as a new target for cancer chemotherapy. Many lysosomal-disrupting agents have been identified; however, because of the simultaneous engagement of organelles such as mitochondria in decisive events of death, it is difficult to characterise lysosomal-specific cell death. We have developed stable cells expressing mt-roGFP and tdTomato-CathepsinB as reporter cell systems for sensing both organelle damage in real time and demonstrating their potential utility to dissect organelle-centred cell death signalling. The cell line is high-throughput adaptable, and limited screening led to the systematic grouping of compounds based on their impact on mitochondria, lysosomes, or both.

Supplementary Material

SUPPLEMENTARY_FILE_clean.docx
YRER_A_2621497_SM3191.docx (4.4MB, docx)

Acknowledgments

The authors thank Prof. S. James Remington (Institute of Molecular Biology, University of Oregon, Eugene, OR, USA) for the expression vector for mitochondrial redox-sensitive GFP (mito-roGFP) and Prof. Michael Davidson for the expression vector for tdTomato-CathepsinB-6.

Funding Statement

This work was supported by a Lifetime Imaging Facility Research Grant from the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India, to TRS (BT/INF/22/SP33090/2019). JTPJ is supported by UGC-CSIR. AS and AH are supported by the University Grants Commission.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets used or analyzed during this study are available from the corresponding author on reasonable request.

Supplemental Material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/13510002.2026.2621497.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

SUPPLEMENTARY_FILE_clean.docx
YRER_A_2621497_SM3191.docx (4.4MB, docx)

Data Availability Statement

The datasets used or analyzed during this study are available from the corresponding author on reasonable request.

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