Autophagy pp 315-329 | Cite as

Methods to Detect Loss of Lysosomal Membrane Integrity

  • Sonja AitsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)


Loss of lysosomal membrane integrity, often referred to as lysosomal membrane permeabilization (LMP), occurs in many instances of cell death either as an initiating or as an amplifying event. Currently, the best method for detecting LMP is the galectin puncta formation assay which can be used for a broad range of sample types, both fixed and live, is easy to perform, and highly sensitive. This method, which is similar to the widely used LC3 puncta formation assay for autophagy, is based on the translocation of galectins to damaged lysosomes resulting in a change from uniform to punctate staining pattern. Here, we provide protocols for the galectin puncta formation assay in fixed and live cells and for an alternative assay based on fluorescent dextran release from damaged lysosomes, which can be performed in parallel.

Key words

Lysosome Lysosomal membrane permeabilization Cell death Immunocytochemistry Galectin EGFP-galectin-3 Lysosome-associated membrane protein Dextran Microscopy 



The author is supported by the Danish Cancer Society, the Swedish Research Council, the Swedish Brain Foundation, the Längmanska Cultural Fund, the Einar Willumsen Foundation, the Segerfalk Foundation, the Sigurd and Elsa Golje's Memorial Foundation and the Thora and Viggo Groves Memorial Fund.


  1. 1.
    Luzio JP, Pryor PR, Bright NA (2007) Lysosomes: fusion and function. Nat Rev Mol Cell Biol 8(8):622–632. Scholar
  2. 2.
    Mrschtik M, Ryan KM (2015) Lysosomal proteins in cell death and autophagy. FEBS J 282(10):1858–1870. Scholar
  3. 3.
    Schroder BA, Wrocklage C, Hasilik A, Saftig P (2010) The proteome of lysosomes. Proteomics 10(22):4053–4076. Scholar
  4. 4.
    Aits S, Jäättelä M (2013) Lysosomal cell death at a glance. J Cell Sci 126(Pt 9):1905–1912. Scholar
  5. 5.
    Gomez-Sintes R, Ledesma MD, Boya P (2016) Lysosomal cell death mechanisms in aging. Ageing Res Rev 32:150–168. Scholar
  6. 6.
    Repnik U, Stoka V, Turk V, Turk B (2012) Lysosomes and lysosomal cathepsins in cell death. Biochim Biophys Acta 1824(1):22–33. Scholar
  7. 7.
    Appelqvist H, Waster P, Kagedal K, Ollinger K (2013) The lysosome: from waste bag to potential therapeutic target. J Mol Cell Biol 5(4):214–226. Scholar
  8. 8.
    Vanden Berghe T, Vanlangenakker N, Parthoens E, Deckers W, Devos M, Festjens N, Guerin CJ, Brunk UT, Declercq W, Vandenabeele P (2010) Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features. Cell Death Differ 17(6):922–930. Scholar
  9. 9.
    Brunk UT, Ericsson JL (1972) Cytochemical evidence for the leakage of acid phosphatase through ultrastructurally intact lysosomal membranes. Histochem J 4(6):479–491CrossRefGoogle Scholar
  10. 10.
    Aits S, Kricker J, Liu B, Ellegaard AM, Hamalisto S, Tvingsholm S, Corcelle-Termeau E, Hogh S, Farkas T, Holm Jonassen A, Gromova I, Mortensen M, Jaattela M (2015) Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay. Autophagy 11(8):1408–1424. Scholar
  11. 11.
    Aits S, Jaattela M, Nylandsted J (2015) Methods for the quantification of lysosomal membrane permeabilization: a hallmark of lysosomal cell death. Methods Cell Biol 126:261–285. Scholar
  12. 12.
    Repnik U, Cesen MH, Turk B (2016, 2016) Studying lysosomal membrane permeabilization by analyzing the release of preloaded bsa-gold particles into the cytosol. Cold Spring Harb Protoc (6). Scholar
  13. 13.
    Repnik U, Cesen MH, Turk B (2016, 2016) The use of lysosomotropic dyes to exclude lysosomal membrane permeabilization. Cold Spring Harb Protoc (5). Scholar
  14. 14.
    Schotte P, Declercq W, Van Huffel S, Vandenabeele P, Beyaert R (1999) Non-specific effects of methyl ketone peptide inhibitors of caspases. FEBS Lett 442(1):117–121CrossRefGoogle Scholar
  15. 15.
    Thurston TL, Wandel MP, von Muhlinen N, Foeglein A, Randow F (2012) Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion. Nature 482(7385):414–418. Scholar
  16. 16.
    Chauhan S, Kumar S, Jain A, Ponpuak M, Mudd MH, Kimura T, Choi SW, Peters R, Mandell M, Bruun JA, Johansen T, Deretic V (2016) TRIMs and galectins globally cooperate and TRIM16 and galectin-3 co-direct autophagy in endomembrane damage homeostasis. Dev Cell 39(1):13–27. Scholar
  17. 17.
    Hung YH, Chen LM, Yang JY, Yang WY (2013) Spatiotemporally controlled induction of autophagy-mediated lysosome turnover. Nat Commun 4:2111. Scholar
  18. 18.
    Kim BW, Hong SB, Kim JH, Kwon DH, Song HK (2013) Structural basis for recognition of autophagic receptor NDP52 by the sugar receptor galectin-8. Nat Commun 4:1613. Scholar
  19. 19.
    Li S, Wandel MP, Li F, Liu Z, He C, Wu J, Shi Y, Randow F (2013) Sterical hindrance promotes selectivity of the autophagy cargo receptor NDP52 for the danger receptor galectin-8 in antibacterial autophagy. Sci Signal 6(261):ra9. Scholar
  20. 20.
    Maejima I, Takahashi A, Omori H, Kimura T, Takabatake Y, Saitoh T, Yamamoto A, Hamasaki M, Noda T, Isaka Y, Yoshimori T (2013) Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury. EMBO J 32(17):2336–2347. Scholar
  21. 21.
    Pagliero RJ, D'Astolfo DS, Lelieveld D, Pratiwi RD, Aits S, Jaattela M, Martin NI, Klumperman J, Egan DA (2016) Discovery of small molecules that induce Lysosomal cell death in cancer cell lines using an image-based screening platform. Assay Drug Dev Technol 14(8):489–510. Scholar
  22. 22.
    Paz I, Sachse M, Dupont N, Mounier J, Cederfur C, Enninga J, Leffler H, Poirier F, Prevost MC, Lafont F, Sansonetti P (2010) Galectin-3, a marker for vacuole lysis by invasive pathogens. Cell Microbiol 12(4):530–544. Scholar
  23. 23.
    Chen X, Khambu B, Zhang H, Gao W, Li M, Yoshimori T, Yin XM (2014) Autophagy induced by calcium phosphate precipitates targets damaged endosomes. J Biol Chem 289(16):11162–11174. Scholar
  24. 24.
    Dupont N, Lacas-Gervais S, Bertout J, Paz I, Freche B, Van Nhieu GT, van der Goot FG, Sansonetti PJ, Lafont F (2009) Shigella phagocytic vacuolar membrane remnants participate in the cellular response to pathogen invasion and are regulated by autophagy. Cell Host Microbe 6(2):137–149. Scholar
  25. 25.
    Maier O, Marvin SA, Wodrich H, Campbell EM, Wiethoff CM (2012) Spatiotemporal dynamics of adenovirus membrane rupture and endosomal escape. J Virol 86(19):10821–10828. Scholar
  26. 26.
    Ray K, Bobard A, Danckaert A, Paz-Haftel I, Clair C, Ehsani S, Tang C, Sansonetti P, Tran GV, Enninga J (2010) Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuole rupture in real time. Cell Microbiol 12(4):545–556. Scholar
  27. 27.
    Giraldo AMV, Ollinger K, Loitto V (2017) Microscopic analysis of lysosomal membrane permeabilization. Methods Mol Biol 1594:73–92. Scholar
  28. 28.
    Hasegawa J, Maejima I, Iwamoto R, Yoshimori T (2015) Selective autophagy: lysophagy. Methods 75:128–132. Scholar
  29. 29.
    Uchimoto T, Nohara H, Kamehara R, Iwamura M, Watanabe N, Kobayashi Y (1999) Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester. Apoptosis 4(5):357–362CrossRefGoogle Scholar
  30. 30.
    Thiele DL, Lipsky PE (1985) Regulation of cellular function by products of lysosomal enzyme activity: elimination of human natural killer cells by a dipeptide methyl ester generated from L-leucine methyl ester by monocytes or polymorphonuclear leukocytes. Proc Natl Acad Sci U S A 82(8):2468–2472CrossRefGoogle Scholar
  31. 31.
    Thiele DL, Lipsky PE (1990) Mechanism of L-leucyl-L-leucine methyl ester-mediated killing of cytotoxic lymphocytes: dependence on a lysosomal thiol protease, dipeptidyl peptidase I, that is enriched in these cells. Proc Natl Acad Sci U S A 87(1):83–87CrossRefGoogle Scholar
  32. 32.
    Kagedal K, Zhao M, Svensson I, Brunk UT (2001) Sphingosine-induced apoptosis is dependent on lysosomal proteases. Biochem J 359(Pt 2):335–343CrossRefGoogle Scholar
  33. 33.
    Petersen NH, Olsen OD, Groth-Pedersen L, Ellegaard AM, Bilgin M, Redmer S, Ostenfeld MS, Ulanet D, Dovmark TH, Lonborg A, Vindelov SD, Hanahan D, Arenz C, Ejsing CS, Kirkegaard T, Rohde M, Nylandsted J, Jaattela M (2013) Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase. Cancer Cell 24(3):379–393. Scholar
  34. 34.
    Chu YP, Hung YH, Chang HY, Yang WY (2017) Assays to monitor lysophagy. Methods Enzymol 588:231–244. Scholar
  35. 35.
    Ellegaard AM, Dehlendorff C, Vind AC, Anand A, Cederkvist L, Petersen NH, Nylandsted J, Stenvang J, Mellemgaard A, Osterlind K, Friis S, Jaattela M (2016) Repurposing cationic amphiphilic antihistamines for cancer treatment. EBioMedicine 9:130–139. Scholar
  36. 36.
    Klutzny S, Lesche R, Keck M, Kaulfuss S, Schlicker A, Christian S, Sperl C, Neuhaus R, Mowat J, Steckel M, Riefke B, Prechtl S, Parczyk K, Steigemann P (2017) Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death. Cell Death Dis 8(3):e2709. Scholar
  37. 37.
    Pascua-Maestro R, Diez-Hermano S, Lillo C, Ganfornina MD, Sanchez D (2017) Protecting cells by protecting their vulnerable lysosomes: identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress. PLoS Genet 13(2):e1006603. Scholar
  38. 38.
    Sun L, Hu L, Cogdell D, Lu L, Gao C, Tian W, Zhang Z, Kang Y, Fleming JB, Zhang W (2017) MIR506 induces autophagy-related cell death in pancreatic cancer cells by targeting the STAT3 pathway. Autophagy 13(4):703–714. Scholar
  39. 39.
    Villanueva-Paz M, Cordero MD, Pavon AD, Vega BC, Cotan D, De la Mata M, Oropesa-Avila M, Alcocer-Gomez E, de Lavera I, Garrido-Maraver J, Carrascosa J, Zaderenko AP, Muntane J, de Miguel M, Sanchez-Alcazar JA (2016) Amitriptyline induces mitophagy that precedes apoptosis in human HepG2 cells. Genes Cancer 7(7-8):260–277.
  40. 40.
    Stegmayr J, Lepur A, Kahl-Knutson B, Aguilar-Moncayo M, Klyosov AA, Field RA, Oredsson S, Nilsson UJ, Leffler H (2016) Low or no inhibitory potency of the canonical galectin carbohydrate-binding site by pectins and galactomannans. J Biol Chem 291(25):13318–13334. Scholar
  41. 41.
    Bidere N, Lorenzo HK, Carmona S, Laforge M, Harper F, Dumont C, Senik A (2003) Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis. J Biol Chem 278(33):31401–31411. Scholar
  42. 42.
    Purschke M, Rubio N, Held KD, Redmond RW (2010) Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy. Photochem Photobiol Sci 9(12):1634–1639. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Cell Death and Lysosomes Group, Experimental Neuroinflammation Laboratory, Department of Experimental Medical ScienceLund UniversityLundSweden
  2. 2.Peter MacCallum Cancer CentreMelbourneAustralia

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