Lysosomes in Cancer-At the Crossroad of Good and Evil.

Although it has been known for decades that lysosomes are central for degradation and recycling in the cell, their pivotal role as nutrient sensing signaling hubs has recently become of central interest. Since lysosomes are highly dynamic and in constant change regarding content and intracellular position, fusion/fission events allow communication between organelles in the cell, as well as cell-to-cell communication via exocytosis of lysosomal content and release of extracellular vesicles. Lysosomes also mediate different forms of regulated cell death by permeabilization of the lysosomal membrane and release of their content to the cytosol. In cancer cells, lysosomal biogenesis and autophagy are increased to support the increased metabolism and allow growth even under nutrient- and oxygen-poor conditions. Tumor cells also induce exocytosis of lysosomal content to the extracellular space to promote invasion and metastasis. However, due to the enhanced lysosomal function, cancer cells are often more susceptible to lysosomal membrane permeabilization, providing an alternative strategy to induce cell death. This review summarizes the current knowledge of cancer-associated alterations in lysosomal structure and function and illustrates how lysosomal exocytosis and release of extracellular vesicles affect disease progression. We focus on functional differences depending on lysosomal localization and the regulation of intracellular transport, and lastly provide insight how new therapeutic strategies can exploit the power of the lysosome and improve cancer treatment.

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange.

Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.

Lysosomal Function and Intracellular Position Determine the Malignant Phenotype in Malignant Melanoma.

Lysosomes are central in cell homeostasis and participate in macromolecular degradation, plasma membrane repair, exosome release, cell adhesion/migration, and apoptosis. In cancer, alterations in lysosomal function and spatial distribution may facilitate disease progression. In this study, we show enhanced lysosomal activity in malignant melanoma cells compared with that in normal human melanocytes. Most lysosomes show perinuclear location in melanocytes, while they are more dispersed in melanoma, with retained proteolytic activity and low pH also in the peripheral population. Rab7a expression is lower in melanoma cells than in melanocytes, and by increasing Rab7a, lysosomes are relocated to the perinuclear region in melanoma. Exposure to the lysosome-destabilizing drug L-leucyl-L-leucine methyl ester causes higher damage in the perinuclear subset of lysosomes in melanomas, whereas differences in subpopulation susceptibility cannot be found in melanocytes. Interestingly, melanoma cells recruit the endosomal sorting complex required for transport-III core protein CHMP4B, involved in lysosomal membrane repair, rather than initiate lysophagy. However, when the perinuclear lysosomal position is promoted by Rab7a overexpression or kinesore treatment, lysophagy is increased. In addition, Rab7a overexpression is accompanied by reduced migration capacity. Taken together, the study emphasizes that alterations in lysosomal properties facilitate the malignant phenotype and declares the targeting of lysosomal function as a future therapeutic approach.

Amyloid-ß induced membrane damage instigates tunneling nanotube-like conduits by p21-activated kinase dependent actin remodulation.

Alzheimer's disease (AD) pathology progresses gradually via anatomically connected brain regions. Direct transfer of amyloid-ß1-42 oligomers (oAß) between connected neurons has been shown, however, the mechanism is not fully revealed. We observed formation of oAß induced tunneling nanotubes (TNTs)-like nanoscaled f-actin containing membrane conduits, in differentially differentiated SH-SY5Y neuronal models. Time-lapse images showed that oAß propagate from one cell to another via TNT-like structures. Preceding the formation of TNT-like conduits, we detected oAß-induced plasma membrane (PM) damage and calcium-dependent repair through lysosomal-exocytosis, followed by massive endocytosis to re-establish the PM. Massive endocytosis was monitored by an influx of the membrane-staining dye TMA-DPH and PM damage was quantified by propidium iodide influx in the absence of Ca2+. The massive endocytosis eventually caused accumulation of internalized oAß in Lamp1 positive multivesicular bodies/lysosomes via the actin cytoskeleton remodulating p21-activated kinase1 (PAK1) dependent endocytic pathway. Three-dimensional quantitative confocal imaging, structured illumination superresolution microscopy, and flowcytometry quantifications revealed that oAß induces activation of phospho-PAK1, which modulates the formation of long stretched f-actin extensions between cells. Moreover, the formation of TNT-like conduits was inhibited by preventing PAK1-dependent internalization of oAß using the small-molecule inhibitor IPA-3, a highly selective cell-permeable auto-regulatory inhibitor of PAK1. The present study reveals that the TNT-like conduits are probably instigated as a consequence of oAß induced PM damage and repair process, followed by PAK1 dependent endocytosis and actin remodeling, probably to maintain cell surface expansion and/or membrane tension in equilibrium.

Apoptosis in idiopathic inflammatory myopathies with partial invasion; a role for CD8+ cytotoxic T cells?

Polymyositis and inclusion body myositis are idiopathic inflammatory myopathies, with a pathology characterized by partial invasion of non-necrotic muscle fibres by CD8+ cytotoxic T-cells, leading to fibre degeneration. Although the main effector pathway of CD8+ T-cells is to induce apoptosis of target cells, it has remained unclear if apoptosis occurs in these diseases, and if so, if it is mediated by CD8+ T-cells. In consecutive biopsy sections from 10 patients with partial invasion, muscle fibres and inflammatory cells were assessed by immunohistochemistry and apoptotic nuclei by the TUNEL assay. Analysis of muscle fibre morphology, staining pattern and quantification were performed on digital images, and they were compared with biopsies from 10 dermatomyositis patients and 10 controls without muscle disease. Apoptotic myonuclei were found in muscle with partial invasion, but not in the invaded fibres. Fibres with TUNEL positive nuclei were surrounded by CD8+ T-cells, granzyme B+ cells and macrophages, but lacked FAS receptor expression. In contrast, apoptotic myonuclei were rare in dermatomyositis and absent in controls. The findings confirm that apoptosis occurs in idiopathic inflammatory myopathies and support that it is mediated by CD8+ cytotoxic T- cells, acting in parallel to the process of partial invasion.

Restoration of lysosomal function after damage is accompanied by recycling of lysosomal membrane proteins.

Lysosomes are central organelles for cellular degradation and energy homeostasis. In addition, lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal content to the cytosol can initiate programmed cell death. The extent of LMP and available repair mechanisms determine the cell fate after lysosomal damage. In this study, we aimed to investigate the premises for lysosomal membrane repair after LMP and found that lysosomal membrane damage initiated by L-leucyl-L-leucine methyl ester (LLOMe) caused caspase-dependent apoptosis in almost 50% of the cells, while the rest recovered. Immediately after LLOMe addition, lysosomal proteases were detected in the cytosol and the ESCRT-components ALIX and CHMP4B were recruited to the lysosomal membrane. Next, lysophagic clearance of damaged lysosomes was evident and a concentration-dependent translocation of several lysosomal membrane proteins, including LAMP2, to the cytosol was found. LAMP2 was present in small vesicles with the N-terminal protein chain facing the lumen of the vesicle. We conclude that lysophagic clearance of damaged lysosomes results in generation of lysosomal membrane protein complexes, which constitute small membrane enclosed units, possibly for recycling of lysosomal membrane proteins. These lysosomal membrane complexes enable an efficient regeneration of lysosomes to regain cell functionality.

Interactions of the Lysosomotropic Detergent O-Methyl-Serine Dodecylamide Hydrochloride (MSDH) with Lipid Bilayer Membranes-Implications for Cell Toxicity.

O-methyl-serine dodecylamine hydrochloride (MSDH) is a detergent that accumulates selectively in lysosomes, a so-called lysosomotropic detergent, with unexpected chemical properties. At physiological pH, it spontaneously forms vesicles, which disassemble into small aggregates (probably micelles) below pH 6.4. In this study, we characterize the interaction between MSDH and liposomes at different pH and correlate the findings to toxicity in human fibroblasts. We find that the effect of MSDH on lipid membranes is highly pH-dependent. At neutral pH, the partitioning of MSDH into the liposome membrane is immediate and causes the leakage of small fluorophores, unless the ratio between MSDH and lipids is kept low. At pH 5, the partitioning of MSDH into the membrane is kinetically impeded since MSDH is charged and a high ratio between MSDH and the lipids is required to permeabilize the membrane. When transferred to cell culture conditions, the ratio between MSDH and plasma membrane lipids must therefore be low, at physiological pH, to maintain plasma membrane integrity. Transmission electron microscopy suggests that MSDH vesicles are taken up by endocytosis. As the pH of the endosomal compartment progressively drops, MSDH vesicles disassemble, leading to a high concentration of increasingly charged MSDH in small aggregates inside the lysosomes. At sufficiently high MSDH concentrations, the lysosome is permeabilized, the proteolytic content released to the cytosol and apoptotic cell death is induced.

Extracellular vesicles released by melanocytes after UVA irradiation promote intercellular signaling via miR21.

Skin pigmentation is controlled by complex crosstalk between melanocytes and keratinocytes and is primarily induced by exposure to ultraviolet (UV) irradiation. Several aspects of UVA-induced signaling remain to be explored. In skin cells, UVA induces plasma membrane damage, which is repaired by lysosomal exocytosis followed by instant shedding of extracellular vesicles (EVs) from the plasma membrane. The released EVs are taken up by neighboring cells. To elucidate the intercellular crosstalk induced by UVA irradiation, EVs were purified from UVA-exposed melanocytes and added to keratinocytes. Transcriptome analysis of the keratinocytes revealed the activation of TGF-ß and IL-6/STAT3 signaling pathways and subsequent upregulation of microRNA (miR)21. EVs induced phosphorylation of ERK and JNK, reduced protein levels of PDCD4 and PTEN, and augment antiapoptotic signaling. Consequently, keratinocyte proliferation and migration were stimulated and UV-induced apoptosis was significantly reduced. Interestingly, melanoma cells and melanoma spheroids also generate increased amounts of EVs with capacity to stimulate proliferation and migration upon UVA. In conclusion, we present a novel intercellular crosstalk mediated by UVA-induced lysosome-derived EVs leading to the activation of proliferation and antiapoptotic signaling via miR21.

Lipid membranes accelerate amyloid formation in the mouse model of AA amyloidosis.

INTRODUCTION: AA amyloidosis develops as a result of prolonged inflammation and is characterized by deposits of N-terminal proteolytic fragments of the acute phase reactant serum amyloid A (SAA). Macrophages are usually found adjacent to amyloid, suggesting their involvement in the formation and/or degradation of the amyloid fibrils. Furthermore, accumulating evidence suggests that lipid membranes accelerate the fibrillation of different amyloid proteins. METHODS: Using an experimental mouse model of AA amyloidosis, we compared the amyloidogenic effect of liposomes and/or amyloid-enhancing factor (AEF). Inflammation was induced by subcutaneous injection of silver nitrate followed by intravenous injection of liposomes and/or AEF to accelerate amyloid formation. RESULTS: We showed that liposomes accelerate amyloid formation in inflamed mice, but the amyloidogenic effect of liposomes was weaker compared with AEF. Regardless of the induction method, amyloid deposits were mainly found in the marginal zones of the spleen and coincided with the depletion of marginal zone macrophages, while red pulp macrophages and metallophilic marginal zone macrophages proved insensitive to amyloid deposition. CONCLUSIONS: We conclude that increased intracellular lipid content facilitates AA amyloid fibril formation and show that the mouse model of AA amyloidosis is a suitable system for further mechanistic studies.

Lipid vesicles affect the aggregation of 4-hydroxy-2-nonenal-modified α-synuclein oligomers.

Parkinson's disease (PD) and other synucleinopathies are characterized by accumulation of misfolded aggregates of α-synuclein (α-syn). The normal function of α-syn is still under investigation, but it has been generally linked to synaptic plasticity, neurotransmitter release and the maintenance of the synaptic pool. α-Syn localizes at synaptic terminals where it can bind to synaptic vesicles as well as to other cellular membranes. It has become clear that these interactions have an impact on both α-syn functional role and its propensity to aggregate. In this study, we investigated the aggregation process of α-syn covalently modified with 4-hydroxy-2-nonenal (HNE). HNE is a product of lipid peroxidation and has been implicated in the pathogenesis of different neurodegenerative diseases by modifying the kinetics of soluble toxic oligomers. Although HNE-modified α-syn has been reported to assemble into stable oligomers, we found that slightly acidic conditions promoted further protein aggregation. Lipid vesicles delayed the aggregation process in a concentration-dependent manner, an effect that was observed only when they were added at the beginning of the aggregation process. Co-aggregation of lipid vesicles with HNE-modified α-syn also induced cytotoxic effects on differentiated SHSY-5Y cells. Under conditions in which the aggregation process was delayed cell viability was reduced. By exploring the behavior and potential cytotoxic effects of HNE-α-syn under acidic conditions in relation to protein-lipid interactions our study gives a framework to examine a possible pathway leading from a physiological setting to the pathological outcome of PD.

UV radiation promotes melanoma dissemination mediated by the sequential reaction axis of cathepsins-TGF-ß1-FAP-α.

BACKGROUND: Ultraviolet radiation (UVR) is the major risk factor for development of malignant melanoma. Fibroblast activation protein (FAP)-α is a serine protease expressed on the surface of activated fibroblasts, promoting tumour invasion through extracellular matrix (ECM) degradation. The signalling mechanism behind the upregulation of FAP-α is not yet completely revealed. METHODS: Expression of FAP-α was analysed after UVR exposure in in vitro co-culture systems, gene expression arrays and artificial skin constructs. Cell migration and invasion was studied in relation to cathepsin activity and secretion of transforming growth factor (TGF)-ß1. RESULTS: Fibroblast activation protein-α expression was induced by UVR in melanocytes of human skin. The FAP-α expression was regulated by UVR-induced release of TGF-ß1 and cathepsin inhibitors prevented such secretion. In melanoma cell culture models and in a xenograft tumour model of zebrafish embryos, FAP-α mediated ECM degradation and facilitated tumour cell dissemination. CONCLUSIONS: Our results provide evidence for a sequential reaction axis from UVR via cathepsins, TGF-ß1 and FAP-α expression, promoting cancer cell dissemination and melanoma metastatic spread.

Microscopic Analysis of Lysosomal Membrane Permeabilization.

Lysosomes and lysosomal proteases have been found to participate during several forms of cell death pathways including apoptosis. A critical step in the mediation of apoptotic signaling is the release of cathepsins to the cytosol, a process known as lysosomal membrane permeabilization (LMP). In this chapter, we describe immunofluorescence detection of LMP in cell cultures stained for cathepsin B and LAMP-2 using three confocal techniques namely laser scanning, spinning disk, and aperture correlation spinning disk confocal to obtain images. Image analysis is performed using Huygens software for deconvolution. LMP results in a decrease in the fraction of cathepsin B colocalizing with LAMP-2, which is quantified through Manders' colocalization coefficient. Analysis of the images obtained by the three techniques show the same trend but the magnitude of the decrease differs due to the axial resolution. The observations emphasize the use of highest possible resolution when determining colocalization.

Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells.

The acidic environment of the lysosomal lumen provides an optimal milieu for the acid hydrolases and is also essential for fusion/fission of endo-lysosomal compartments and sorting of cargo. Evidence suggests that maintaining lysosomal acidity is essential to avoid disease. In this chapter, we describe a protocol for analyzing the lysosomal pH in cultured cells using the fluorescent probe fluorescein isothiocyanate (FITC)-dextran together with a dual-emission ratiometric technique suitable for flow cytometry. Fluorescence-labeled dextran is endocytosed and accumulated in the lysosomal compartment. FITC shows a pH-dependent variation in fluorescence when analyzed at maximum emission wavelength and no variation when analyzing at the isosbestic point, thereby the ratio can be used to determine the lysosomal pH. A standard curve is obtained by equilibrating intralysosomal pH with extracellular pH using the ionophore nigericin. The protocol also includes information regarding procedures to induce lysosomal alkalinization and lysosomal membrane permeabilization.

Impact of high cholesterol in a Parkinson's disease model: Prevention of lysosomal leakage versus stimulation of α-synuclein aggregation.

Parkinson's disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated α-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), we found that MPP+-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of α-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP+-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect α-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinson's disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of α-synuclein accumulation.

Evaluation of tubulin ß-3 as a novel senescence-associated gene in melanocytic malignant transformation.

Malignant melanoma might develop from melanocytic nevi in which the growth-arrested state has been broken. We analyzed the gene expression of young and senescent human melanocytes in culture and compared the gene expression data with a dataset from nevi and melanomas. A concordant altered gene expression was identified in 84 genes when comparing the growth-arrested samples with proliferating samples. TUBB3, which encodes the microtubule protein tubulin ß-3, showed a decreased expression in senescent melanocytes and nevi and was selected for further studies. Depletion of tubulin ß-3 caused accumulation of cells in the G2/M phase and decreased proliferation and migration. Immunohistochemical assessment of tubulin ß-3 in benign lesions revealed strong staining in the superficial part of the intradermal components, which faded with depth. In contrast, primary melanomas exhibited staining without gradient in a disordered pattern and strong staining of the invasive front. Our results describe an approach to find clinically useful diagnostic biomarkers to more precisely identify cutaneous malignant melanoma and present tubulin ß-3 as a candidate marker.

Spontaneous Vesiculation and pH-Induced Disassembly of a Lysosomotropic Detergent: Impacts on Lysosomotropism and Lysosomal Delivery.

Lysosomotropic detergents (LDs) selectively rupture lysosomal membranes through mechanisms that have yet to be characterized. A consensus view, currently, holds that LDs, which are weakly basic, diffuse across cellular membranes as monomers in an uncharged state, and via protonation in the acidic lysosomal compartment, they become trapped, accumulate, and subsequently solubilize the membrane and induce lysosomal membrane permeabilization. Here we demonstrate that the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) spontaneously assembles into vesicles at, and above, cytosolic pH, and that the vesicles disassemble as the pH reaches 6.4 or lower. The aggregation commences at concentrations below the range of those used in cell studies. Assembly and disassembly of the vesicles was studied via dynamic light scattering, zeta potential measurements, cryo-TEM, and fluorescence correlation spectroscopy and was found to be reversible via control of the pH. Aggregation of MSDH into closed vesicles under cytosolic conditions is at variance with the commonly held view of LD behavior, and we propose that endocytotic pathways should be considered as possible routes of LD entry into lysosomes. We further demonstrate that MSDH vesicles can be loaded with fluorophores via a solution transition from low to high pH, for subsequent release when the pH is lowered again. The ability to encapsulate molecular cargo into MSDH vesicles together with its ability to disaggregate at low pH and to permeabilize the lysosomal membrane presents an intriguing possibility to use MSDH as a delivery system.

Extracellular vesicles are transferred from melanocytes to keratinocytes after UVA irradiation.

Ultraviolet (UV) irradiation induces skin pigmentation, which relies on the intercellular crosstalk of melanin between melanocytes to keratinocytes. However, studying the separate effects of UVA and UVB irradiation reveals differences in cellular response. Herein, we show an immediate shedding of extracellular vesicles (EVs) from the plasma membrane when exposing human melanocytes to UVA, but not UVB. The EV-shedding is preceded by UVA-induced plasma membrane damage, which is rapidly repaired by Ca(2+)-dependent lysosomal exocytosis. Using co-cultures of melanocytes and keratinocytes, we show that EVs are preferably endocytosed by keratinocytes. Importantly, EV-formation is prevented by the inhibition of exocytosis and increased lysosomal pH but is not affected by actin and microtubule inhibitors. Melanosome transfer from melanocytes to keratinocytes is equally stimulated by UVA and UVB and depends on a functional cytoskeleton. In conclusion, we show a novel cell response after UVA irradiation, resulting in transfer of lysosome-derived EVs from melanocytes to keratinocytes.

Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances.

Exocytosis of lysosomal contents from platelets has been speculated to participate in clearance of thrombi and vessel wall remodelling. The mechanisms that regulate lysosomal exocytosis in platelets are, however, still unclear. The aim of this study was to identify the pathways underlying platelet lysosomal secretion and elucidate how this process is controlled by platelet inhibitors. We found that high concentrations of thrombin induced partial lysosomal exocytosis as assessed by analysis of the activity of released N-acetyl-ß-glucosaminidase (NAG) and by identifying the fraction of platelets exposing the lysosomal-associated membrane protein (LAMP)-1 on the cell surface by flow cytometry. Stimulation of thrombin receptors PAR1 or PAR4 with specific peptides was equally effective in inducing LAMP-1 surface expression. Notably, lysosomal exocytosis in response to thrombin was significantly reduced if the secondary activation by ADP was inhibited by the P2Y12 antagonist cangrelor, while inhibition of thromboxane A2 formation by treatment with acetylsalicylic acid was of minor importance in this regard. Moreover, the NO-releasing drug S-nitroso-N-acetyl penicillamine (SNAP) or the cyclic AMP-elevating eicosanoid prostaglandin I2 (PGI2) significantly suppressed lysosomal exocytosis. We conclude that platelet inhibitors that mimic functional endothelium such as PGI2 or NO efficiently counteract lysosomal exocytosis. Furthermore, we suggest that secondary release of ADP and concomitant signaling via PAR1/4- and P2Y12 receptors is important for efficient platelet lysosomal exocytosis by thrombin.

Melanoma growth and progression after ultraviolet a irradiation: impact of lysosomal exocytosis and cathepsin proteases.

Ultraviolet (UV) irradiation is a risk factor for development of malignant melanoma. UVA-induced lysosomal exocytosis and subsequent cell growth enhancement was studied in malignant melanoma cell lines and human skin melanocytes. UVA irradiation caused plasma membrane damage that was rapidly repaired by calcium-dependent lysosomal exocytosis. Lysosomal content was released into the culture medium directly after irradiation and such conditioned media stimulated the growth of non-irradiated cell cultures. By comparing melanocytes and melanoma cells, it was found that only the melanoma cells spontaneously secreted cathepsins into the surrounding medium. Melanoma cells from a primary tumour showed pronounced invasion ability, which was prevented by addition of inhibitors of cathepsins B, D and L. Proliferation was reduced by cathepsin L inhibition in all melanoma cell lines, but did not affect melano-cyte growth. In conclusion, UVA-induced release of cathepsins outside cells may be an important factor that promotes melanoma growth and progression.

Sunbathing: What've lysosomes got to do with it?

Solar radiation is an important risk factor for skin cancer, the incidence of which is increasing, especially in the fair-skinned populations of the world. While the ultraviolet (UV)B component has direct DNA damaging ability, UVA-induced effects are currently mainly attributed to the production of reactive oxygen species. In our recent study, we compared the effects of UVA and UVB radiation on human keratinocytes and found that UVA-induced plasma membrane damage was rapidly repaired by lysosomal exocytosis, which was detected based on the expression of lysosomal membrane associated protein-1 (LAMP-1) on the plasma membrane of non-permeabilized cells. Later, the keratinocytes died through caspase-8 mediated apoptosis. In contrast, the plasma membranes of keratinocytes exposed to UVB showed no LAMP-1 expression, and, although the cells died by apoptosis, no initial caspase-8 activity was detected. We have also demonstrated the occurrence of UVA-induced lysosomal exocytosis in reconstructed skin and shown the relocation of lysosomes from the center of cells to the vicinity of the plasma membrane. Thus, we suggest that lysosomal exocytosis also occurs in keratinocytes covered by the stratum corneum following exposure to UVA. Our findings provide new insight into the mechanism of UVA-induced skin damage.