Mechanism of Lysophosphatidylcholine-Induced Lysosome Destabilization

Lysosomal destabilization is critical for the organelle and living cells. Phospholipase A₂ (PLA₂) was shown to be able to destabilize lysosomes under some conditions. By what mechanism the enzyme affects lysosomal stability is not fully studied. In this study, we investigated the effects of lysophosphatidylcholine (lysoPC), a PLA₂-produced lipid metabolite, on lysosomal ion permeability, osmotic sensitivity and stability. By measuring lysosomal β-hexosaminidase free activity, membrane potential, proton leakage and their enzyme latency loss in hypotonic sucrose medium, we established that lysoPC could increase the lysosomal permeability to both potassium ions and protons and enhance lysosomal osmotic sensitivity. These changes in lysosomal membrane properties promoted entry of potassium ions into lysosomes via K⁺/H⁺ exchange. The resultant osmotic imbalance across the membranes led to losses of lysosomal integrity. The enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. These results suggest that lysoPC may play a key role in PLA₂-induced lysosomal destabilization.     

Chaperone‐mediated autophagy is defective in mucolipidosis type IV

Mucolipidosis type IV (MLIV) is a lysosomal storage disorder caused by mutations in the MCOLN1 gene, a member of the transient receptor potential (TRP) cation channel gene family. The encoded protein, transient receptor potential mucolipin‐1 (TRPML1), has been localized to lysosomes and late endosomes but the pathogenic mechanism by which loss of TRPML1 leads to abnormal cellular storage and neuronal cell death is still poorly understood. Yeast two‐hybrid and co‐immunoprecipitation (coIP) experiments identified interactions between TRPML1 and Hsc70 as well as TRPML1 and Hsp40. Hsc70 and Hsp40 are members of a molecular chaperone complex required for protein transport into the lysosome during chaperone‐mediated autophagy (CMA). To determine the functional relevance of this interaction, we compared fibroblasts from MLIV patients to those from sex‐ and age‐matched controls and show a defect in CMA in response to serum withdrawal. This defect in CMA was subsequently confirmed in purified lysosomes isolated from control and MLIV fibroblasts. We further show that the amount of lysosomal‐associated membrane protein type 2A (LAMP‐2A) is reduced in lysosomal membranes of MLIV fibroblasts. As a result of decreased CMA, MLIV fibroblasts have increased levels of oxidized proteins compared to control fibroblasts. We hypothesize that TRPML1 may act as a docking site for intralysosomal Hsc70 (ly‐Hsc70) allowing it to more efficiently pull in substrates for CMA. It is also possible that TRPML1 channel activity may be required for CMA. Understanding the role of TRPML1 in CMA will undoubtedly help to characterize the pathogenesis of MLIV. J. Cell. Physiol. 219: 344–353, 2009. © 2008 Wiley‐Liss, Inc.     

Control of mitosis,inflammation, and cell motility by limited leakage of lysosomes

Lysosomal membrane permeabilization and subsequent leakage of lysosomal hydrolases into the cytosol are considered as the major hallmarks of evolutionarily conserved lysosome-dependent cell death. Contradicting this postulate, new sensitive methods that can detect a minimal lysosomal membrane damage have demonstrated that lysosomal leakage does not necessarily equal cell death. Notably, cells are not only able to survive minor lysosomal membrane permeabilization, but some of their normal functions actually depend on leaked lysosomal hydrolases. Here we discuss emerging data suggesting that spatially and temporally controlled lysosomal leakage delivers lysosomal hydrolases to specific subcellular sites of action and controls at least three essential cellular processes, namely mitotic chromosome segregation, inflammatory signaling, and cellular motility.     

Exposure of cells to nonlethal concentrations of hydrogen peroxide induces degeneration-repair mechanisms involving lysosomal destabilization

The cytotoxicity of hydrogen peroxide is, at least partly, mediated by the induction of intralysosomal iron-catalyzed oxidative reactions with damage to lysosomal membranes and leakage of destructive contents. We hypothesize that minor such leakage may be nonlethal, and the ensuing cellular degeneration repairable. Consequently, we investigated, using a model system of cultured J-774 cells, the effects of hydrogen peroxide in moderate concentrations on cellular viability, lysosomal membrane integrity, morphology, and ATP and reduced glutathione concentrations. These parameters were initially estimated directly after a 30 min exposure to a bolus dose of hydrogen peroxide in phosphate buffered saline at 37°C, and then again following subsequent recovery periods of different lengths under ordinary culture conditions. All cells survived an exposure to 250 μM hydrogen peroxide for 30 min, whereas 350 and 500 μM exposure was lethal to a small fraction of cells. The oxidative stress caused early, time- and dose-dependent, partial relocalization of the lysosomotropic weak base acridine orange from the lysosomal compartment to the cytosol. This phenomenon is known to parallel leakage of damaging lysosomal contents such as hydrolytic enzymes. There were also signs of cellular damage in the form of surface blebbing and increased autophagocytosis, more marked with the higher doses of hydrogen peroxide. Also found was a rapid depletion of ATP and GSH. These alterations were all reversible, as long as cells were exposed to nonlethal amounts of hydrogen peroxide. Based on these and previous findings, we suggest that lysosomes are less stable organelles than has hitherto been assumed. Restricted lysosomal leakage might be a common event, for example, during sublethal oxidative stress, causing reversible, degenerative alterations, which are repaired by autophagocytosis.     

Ultrastructural and functional abnormalities of mitochondria in cultivated fibroblasts from α -mannosidosis patients

α-Mannosidosis is a lysosomal storage disorder caused by α-mannosidase deficiency. Clinical course of the disease ranges from severe infantile to milder juvenile type and includes mental retardation, skeletal deformities, coarse facies, hepatomegaly and hearing loss. The aim of the study was to analyse mitochondrial ultrastructure and function in cultivated fibroblasts from three patients with α-mannosidosis. All patients were homozygous for the c.2248C>T mutation in the MAN2B1 gene encoding lysosomal α-mannosidase. The mutation results in incorrect protein folding and severe decrease of α-mannosidase activity. The misfolded protein is retained by the control system of endoplasmic reticulum (ER). In analysed fibroblasts, we observed dilated ER, higher amount of aberrant mitochondria and reduced mitochondrial mass compared to controls. Respiratory chain complex IV, cytochrome c oxidase (COX), activity and the ratio between COX and citrate synthase (control enzyme) were significantly increased in comparison to controls (P < 0.05). Furthermore, the activity at least from one of other respiratory chain complexes was increased in each studied cell line. Mitochondrial membrane potential as well as reactive oxygen species production were comparable with controls. Based on our results, we hypothesize more profound effect of swelled and damaged mitochondria and ER dilatation on tissues with higher energy demand than fibroblasts have.     

Poliovirus-induced Cellular Injury

Protein leakage was used as a quantitative measure of poliovirus-induced cellular injury under suspended cell culture conditions. The requirements for protein leakage were studied in detail and it was established that events early in the infectious cycle which depend upon viral protein synthesis were responsible for cell damage. Extralysosomal β-glucuronidase appeared in infected cells before the onset of protein leakage and release of newly synthesized virus. Hydrocortisone treatment of infected cells resulted in only a slight delay in the release of β-glucuronidase from lysosomes and protein and virus from cells. These results suggest that events associated with poliovirus synthesis trigger the release of lysosomal hydrolases which in turn injure the plasma membrane, allowing cytoplasmic proteins and virus to leak out of the cell.     

Improvement in Lipid and Protein Trafficking in Niemann‐Pick C1 Cells by Correction of a Secondary Enzyme Defect

Different primary lysosomal trafficking defects lead to common alterations in lipid trafficking, suggesting cooperative interactions among lysosomal lipids. However, cellular analysis of the functional consequences of this phenomenon is lacking. As a test case, we studied cells with defective Niemann‐Pick C1 (NPC1) protein, a cholesterol trafficking protein whose defect gives rise to lysosomal accumulation of cholesterol and other lipids, leading to NPC disease. NPC1 cells also develop a secondary defect in acid sphingomyelinase (SMase) activity despite a normal acid SMase gene (SMPD1). When acid SMase activity was restored to normal levels in NPC1‐deficient CHO cells through SMPD1 transfection, there was a dramatic reduction in lysosomal cholesterol. Two other defects, excess lysosomal bis‐(monoacylglycerol) phosphate (BMP) and defective transferrin receptor (TfR) recycling, were also markedly improved. To test its relevance in human cells, the acid SMase activity defect in fibroblasts from NPC1 patients was corrected by SMPD1 transfection or acid SMase enzyme replacement. Both treatments resulted in a dramatic reduction in lysosomal cholesterol. These data show that correcting one aspect of a complex lysosomal lipid storage disease can reduce the cellular consequences even if the primary genetic defect is not corrected.     

Lethal hydrogen peroxide toxicity involves lysosomal iron-catalyzed reactions with membrane damage

Secondary lysosomes contain low-molecular weight iron-complexes as a consequence of normal autophagocytotic degradation of various metallo-proteins. Thus, entry of hydrogen peroxide into these organelles may induce ironcatalyzed oxidative reactions with ensuing damage to lysosomal membranes and leakage of destructive contents. The amount of lysosomal reactive iron and the cellular capacity to degrade hydrogen peroxide would then be important determining factors in cellular resistance to oxidative stress. The effects of hydrogen peroxide on cell viability and, in particular, on lysosomal membrane integrity, evaluated by acridine orange, lucifer yellow, neutral red, and cathepsin D relocalization, were investigated in a model system of cultured J-774 cells. The protective effect of the iron-chelator desferal was studied after exposure to the drug under ordinary culture conditions and after inhibition of cellular endocytosis. Hydrogen peroxide-exposure (500 μM in PBS, 37°C, 5–90 min) was manifested as a time-dependent decrease in cell viability. This was preceded by a rapid reduction of the proton gradient across the lysosomal membranes, as judged by relocalization of acridine orange. Another early sign of damage was plasma membrane blebbing, found on many cells within minutes after the initiation of hydrogen peroxide-exposure. The cells also showed a partial redistribution of the lysosomal markers lucifer yellow, neutral red, and cathepsin D, indicating lysosomal destabilization. The pre-exposure of cells to desferal in culture prevented all these phenomena, unless endocytotic uptake of the drug was prevented.     

Contact-mediated changes in the fluidity of membrane lipids in normal and malignant transformed mammalian fibroblasts

The degree of microviscosity, gh, (fluidity/rigidity behavior) of membrane lipids of normal and transformed mammalian fibroblasts obtained from mice, hamsters and rats was quantitatively monitored by fluorescence polarization, P, analysis of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH) when embedded in lipid regions of cellular membranes of intact viable cells. Analysis of membrane microviscosity of six different cell populations and of individual cells in each cell population have indicated that the membrane microviscosity of all cell types, both normal and transformed fibroblasts, changes as a function of the cell density in the growing cultures. The membrane microviscosity was found to be low (high lipid fluidity) in sparse conditions but high (high lipid rigidity) in dense conditions. The induced changes in membrane microviscosity are practically reversible for all cell types and a complete reversion can be obtained within a few hours after changing the cell density conditions from sparse to dense and vice versa.Comparative studies with normal and transformed fibroblasts have shown that transformed fibroblasts have a more rigid lipid layer in their cellular membranes than normal or untransformed fibroblasts. The difference in membrane microviscosity between transformed and normal fibroblasts is higher in confluent conditions as compared with subconfluent cultures. These differences in the degree of fluidity of membrane lipids that are controlled by possible differences in the cellto-cell contact in normal and transformed fibroblasts may play a major role in determining the growth behavior of normal and malignant cells that are growing as a solid tissue and may have a direct effect on the control mechanisms that determine the presence or absence of the “density dependent inhibition” of growth.     

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time -a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift -was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonie medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 μM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.     

Lysosomal changes in the response of molluscan hepatopancreatic cells to extracellular signals

Summary Marine mussels (Mytilus edulis, a bivalve mollusc) are increasingly used as environmental sentinels in pollution biomonitoring. Pathological reactions of the lysosomal system in hepatopancreatic cells have proven to be sensitive bioindicators of pollutant effect. However, if such reactions are to be used as biomarkers, then they must be clearly distinguishable from any hormonally-induced changes linked to normal seasonal activity such as the reproductive cycle. The aim of the present study was to test the effects of several cell-to-cell signalling compounds on the lysosomes of the hepatopancreatic digestive cells.In vitro incubation of tissue slices showed that epinephrine, acetylcholine and prostaglandin F₂ reduced lysosomal membrane stability and latency of -N-acetylhexosaminidase and -glucuronidase. These results indicate the presence of cell surface receptors for all three hormones. The observed changes in lysosomal fragility were less pronounced than those induced by pollutants and when considered together with other published data, indicate that hormonal regulation of digestive cell lysosomes is unlikely to pose a problem for the use of lysosomal reactions as biomarkers for chemical insult.     

Evidence of lysosomal membrane permeabilization in mucopolysaccharidosis type I: Rupture of calcium and proton homeostasis

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of α‐iduronidase (IDUA), which leads to intralysosomal accumulation of glysosaminoglycans. Patients with MPS I present a wide range of clinical manifestations, but the mechanisms by which these alterations occur are still not fully understood. Genotype–phenotype correlations have not been well established for MPS I; hence, it is likely that secondary and tertiary alterations in cellular metabolism and signaling may contribute to the physiopathology of the disease. The aim of this study was to analyze Ca²⁺ and H⁺ homeostasis, lysosomal leakage of cysteine proteases, and apoptosis in a murine model of MPS I. After exposition to specific drugs, cells from Idua?/? mice were shown to release more Ca²⁺ from the lysosomes and endoplasmic reticulum than Idua+/+ control mice, suggesting a higher intraorganelle store of this ion. A lower content of H⁺ in the lysosomes and in the cytosol was found in cells from Idua?/? mice, suggesting an alteration of pH homeostasis. In addition, Idua?/? cells presented a higher activity of cysteine proteases in the cytosol and an increased rate of apoptotic cells when compared to the control group, indicating that lysosomal membrane permeabilization might occur in this model. Altogether, our results suggest that secondary alterations—as changes in Ca²⁺ and H⁺ homeostasis and lysosomal membrane permeabilization—may contribute for cellular damage and death in the physiopathology of MPS I. J. Cell. Physiol. 223: 335–342, 2010. © 2010 Wiley‐Liss, Inc.     

Cardiotoxicity of Jamesoni's mamba (Dendroaspis jamesoni) venom and its fractionated components in primary cultures of rat myocardial cells

Summary Primary cultures of spontaneously beating myocardial cells isolated from neonatal rat hearts were used to screen the cardiotoxic effects of Jamesoni's mamba (Dendroaspis jamesoni) venom and components isolated from the venom by gel filtration and ion exchange chromatography. Cardiotoxicity was evaluated on the basis of leakage of lactate dehydrogenase (LDH), changes in morphology, cell membrane lysis, cellular viability, and alterations in spontaneous beating activity. The whole venom caused dose- and time-dependent leakage of LDH, disruption of the cell monolayer, decreases in viability, and inhibition of beating activity. Gel filtration of the venom yielded eight fractions (DjI to DjVIII). DjI (30 μg/ml), DjII (20 μg/ml), and DjV (20 μg/ml) caused significant (P<0.001) leakage of LDH, extensive morphologic damage, and decreases in viability. At lower concentrations DjI to DjVIII caused progressive inhibition of spontaneous beating activity. The main fraction (DjV), which was the most toxic, was further separated into 14 polypeptides (Dj1 to Dj14) by ion-exchange chromatography using Bio-Rex 70. Based on the ability to induce LDH leakage, produce morphologic damage, lyse cell membranes, and arrest beating activity, four categories of polypeptides were identified: cardiotoxins, Dj1 and Dj2; cardiotoxinlike polypeptides, Dj3 to Dj8; less active membrane lytic polypeptides, Dj9 to Dj13; and membrane lytic polypeptide, Dj14. This study was supported in part by the Fulbright Scholar Program (1986–1987) and the Burroughs Wellcome Fund. D. A. is a Burroughs Wellcome Scholar in Toxicology.     

Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content

Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.     

Lipophilic cationic drugs increase the permeability of lysosomal membranes in a cell culture system

Lysosomes accumulate many drugs several fold higher compared to their extracellular concentration. This mechanism is believed to be responsible for many pharmacological effects. So far, uptake and release kinetics are largely unknown and interactions between concomitantly administered drugs often provoke mutual interference. In this study, we addressed these questions in a cell culture model. The molecular mechanism for lysosomal uptake kinetics was analyzed by live cell fluorescence microscopy in SY5Y cells using four drugs (amantadine, amitriptyline, cinnarizine, flavoxate) with different physicochemical properties. Drugs with higher lipophilicity accumulated more extensively within lysosomes, whereas a higher pK_a value was associated with a more rapid uptake. The drug‐induced displacement of LysoTracker was neither caused by elevation of intra‐lysosomal pH, nor by increased lysosomal volume. We extended our previously developed numerical single cell model by introducing a dynamic feedback mechanism. The empirical data were in good agreement with the results obtained from the numerical model. The experimental data and results from the numerical model lead to the conclusion that intra‐lysosomal accumulation of lipophilic xenobiotics enhances lysosomal membrane permeability. Manipulation of lysosomal membrane permeability might be useful to overcome, for example, multi‐drug resistance by altering subcellular drug distribution. J. Cell. Physiol. 224:152–164, 2010 © 2010 Wiley‐Liss, Inc.     

Cell growth and substrate effects on characteristics of a lysosomal enzyme (cathepsin C) in Duchenne muscular dystrophy fibroblasts

Skin fibroblasts from normal males and males suffering from Duchenne muscular dystrophy were studied in culture over a 10-week period. The lysosomal enzyme cathepsin C (dipeptidyl aminopeptidase I; EC 3.4.14.1), defined by the chloride-dependent hydrolysis of dipeptide-beta-naphthylamide (dipeptide-beta-NA) substrates at pH 5.1, was significantly lower in Duchenne cell sonicates and cell lysosomal preparations. The apparent difference in activity tended to increase with in vitro cell culture age, with the Duchenne cells being found also to grow faster and yield a greater number of cells at confluence. An analysis of all 10 cell lines as a group indicated that cathepsin C activity was related to growth rate. In addition, while analyses of cell homogenization and fractionation showed that the yield of cathepsin C was not different in Duchenne lysosomal preparations, the enzyme showed significantly lower latent activity in the Duchenne lysosomes with Gly-Phe-NA used as substrate. However, despite significant differences in specific activity compared with normal lysosomal preparations, no latency difference was observed if three other substrates were used (Gly-Arg-, Pro-Arg-, and Pro-Phe-NAs). The expression of this enzyme can thus be differentially influenced by cell growth and its latency characteristics can be influenced by the substrate used in assays.     

Temporal relationship of free radical-induced lipid peroxidation and loss of latent enzyme activity in highly enriched hepatic lysosomes

Loss of latency due to membrane lipid peroxidation induced in vitro was studied in highly purified rat liver lysosomes. Enriched fractions of lysosomes were isolated by free flow electrophoresis. Lipid peroxidation of lysosomes, assayed as malondialdehyde formation, was catalyzed by a radical generating system consisting of dihydroxyfumaric acid and Fe3+-ADP. The peroxidation reaction occurred readily at 37 degrees C and reached a plateau at 10 min; however, the loss of lysosomal latency, determined as increased percentage free beta-N-acetylglucosaminidase activity, occurred more gradually and reached a maximum after 30 min. Scavengers of superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl radicals did not inhibit the peroxidation reaction nor prevent the loss of lysosomal latency. However, preincubation of the lysosomes with alpha-tocopherol effectively blocked the induction of peroxidation and substantially reduced the loss of lysosomal latency. These results indicate that the lysosomal membrane is susceptible to free radical-induced lipid peroxidation; further, this process may be the immediate cause of the subsequent disintegration of the lysosome. The nature of the protective effect of alpha-tocopherol is unclear but may be due to its interaction with the unsaturated membrane lipids and the subsequent interruption of the chain-reaction initiated by free radicals.     

A novel antimicrobial peptide M1-8 targets the lysosomal pathway to inhibit autolysosome formation and promote apoptosis in liver cancer cells

Lysosomes, a central regulator of autophagy, play a critical role in tumour growth. Lysosomal protease cathepsin D can initiate apoptosis when released from lysosomes into the cytosol. In this study, we observed that Musca domestica cecropin (Mdc) 1–8 (M1-8), a small anti-tumour peptide derived from Mdc, inhibits hepatoma cell growth by blocking autophagy–lysosome fusion. This effect is likely achieved by targeting lysosomes to activate lysosomal protease D. Additionally, we examined whether lysosomal content and cathepsin D release were involved in M1-8-induced apoptosis. After exposure to M1-8, human hepatoma HepG2 cells rapidly co-localized with lysosomes, disrupted lysosomal integrity, caused leakage of lysosomal protease cathepsin D, caspase activation and mitochondrial membrane potential changes; and promoted cell apoptosis. Interestingly, in M1-8-treated HepG2 cells, autophagic protein content increased and the lysosome–autophagosome fusion was inhibited, suggesting that M1-8 can cause apoptosis through autophagy and lysosomes. This result indicates that a small accumulation of autophagy and autolysosome inhibition in cells can cause cell death. Taken together, these data suggest a novel insight into the regulatory mechanisms of M1-8 in autophagy and lysosomes, which may facilitate the development of M1-8 as a potential cancer therapeutic agent.     

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry. Effect of silver lactate and hypotonic conditions

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time - a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift - was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonic medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 microM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.