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Distinguishing Necroptosis from Apoptosis

  • Inbar ShlomovitzEmail author
  • Sefi Zargarian
  • Ziv Erlich
  • Liat Edry-Botzer
  • Motti GerlicEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1857)

Abstract

Apoptosis was the first programmed cell death to be defined—highly regulated and immunologically silent, as apoptotic bodies are being removed without triggering inflammation. Few decades later, necroptosis was discovered—uniquely regulated but inflammatory. As these two programmed cell death pathways may be initiated via similar pathways (death receptors and intracellular receptors) while being differently regulated and resulting in distinctive physiological consequences, the need for distinguishing apoptosis from necroptosis is required. Here we describe a series of distinguishing assays that use apoptotic- and necroptotic-distinct response to pharmacological interventions with specific death inhibitors, morphology and death-specific proteins involvement. The procedure includes cell death kinetics assessment and morphology monitoring of stimulated and pharmacologically treated-cells using flow cytometry and live imaging, with the detection of death-specific proteins using Immunoblot. The procedure described here is simple and thus can be adjusted to various experimental systems, enabling apoptosis to be distinguished from necroptosis in one’s system of interest, without the need for more complex reagents such as genetic knockout models.

Key words

Cell death Apoptosis Necroptosis Caspase 3 MLKL 

References

  1. 1.
    Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26(4):239–257CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ravichandran KS, Lorenz U (2007) Engulfment of apoptotic cells: signals for a good meal. Nat Rev Immunol 7(12):964–974. https://doi.org/10.1038/nri2214CrossRefPubMedGoogle Scholar
  3. 3.
    Fink SL, Cookson BT (2005) Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun 73(4):1907–1916. https://doi.org/10.1128/IAI.73.4.1907-1916.2005CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Galluzzi L, Kroemer G (2008) Necroptosis: a specialized pathway of programmed necrosis. Cell 135(7):1161–1163. https://doi.org/10.1016/j.cell.2008.12.004CrossRefPubMedGoogle Scholar
  5. 5.
    Shlomovitz ISZ, Gerlic M (2017) Mechanisms of RIPK3-induced inflammation. Immunol Cell Biol 95(2):166–172CrossRefPubMedGoogle Scholar
  6. 6.
    Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B, Tschopp J (2000) Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1(6):489–495CrossRefPubMedGoogle Scholar
  7. 7.
    Rickard JA, O'Donnell JA, Evans JM, Lalaoui N, Poh AR, Rogers T, Vince JE, Lawlor KE, Ninnis RL, Anderton H, Hall C, Spall SK, Phesse TJ, Abud HE, Cengia LH, Corbin J, Mifsud S, Di Rago L, Metcalf D, Ernst M, Dewson G, Roberts AW, Alexander WS, Murphy JM, Ekert PG, Masters SL, Vaux DL, Croker BA, Gerlic M, Silke J (2014) RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis. Cell 157(5):1175–1188. https://doi.org/10.1016/j.cell.2014.04.019CrossRefPubMedGoogle Scholar
  8. 8.
    Pasparakis M, Vandenabeele P (2015) Necroptosis and its role in inflammation. Nature 517(7534):311–320. https://doi.org/10.1038/nature14191CrossRefPubMedGoogle Scholar
  9. 9.
    Silke J, Rickard JA, Gerlic M (2015) The diverse role of RIP kinases in necroptosis and inflammation. Nat Immunol 16(7):689–697. https://doi.org/10.1038/ni.3206CrossRefPubMedGoogle Scholar
  10. 10.
    Taylor RC, Cullen SP, Martin SJ (2008) Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol 9(3):231–241. https://doi.org/10.1038/nrm2312CrossRefPubMedGoogle Scholar
  11. 11.
    Oberst A, Dillon CP, Weinlich R, McCormick LL, Fitzgerald P, Pop C, Hakem R, Salvesen GS, Green DR (2011) Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature 471(7338):363–367. https://doi.org/10.1038/nature09852CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kaiser WJ, Upton JW, Long AB, Livingston-Rosanoff D, Daley-Bauer LP, Hakem R, Caspary T, Mocarski ES (2011) RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature 471(7338):368–372. https://doi.org/10.1038/nature09857CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, Chan FK (2009) Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137(6):1112–1123. https://doi.org/10.1016/j.cell.2009.05.037CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    He S, Wang L, Miao L, Wang T, Du F, Zhao L, Wang X (2009) Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 137(6):1100–1111. https://doi.org/10.1016/j.cell.2009.05.021CrossRefPubMedGoogle Scholar
  15. 15.
    Murphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S, Lewis R, Lalaoui N, Metcalf D, Webb AI, Young SN, Varghese LN, Tannahill GM, Hatchell EC, Majewski IJ, Okamoto T, Dobson RC, Hilton DJ, Babon JJ, Nicola NA, Strasser A, Silke J, Alexander WS (2013) The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39(3):443–453. https://doi.org/10.1016/j.immuni.2013.06.018CrossRefPubMedGoogle Scholar
  16. 16.
    Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X, Wang X (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148(1–2):213–227. https://doi.org/10.1016/j.cell.2011.11.031CrossRefPubMedGoogle Scholar
  17. 17.
    Hildebrand JM, Tanzer MC, Lucet IS, Young SN, Spall SK, Sharma P, Pierotti C, Garnier JM, Dobson RC, Webb AI, Tripaydonis A, Babon JJ, Mulcair MD, Scanlon MJ, Alexander WS, Wilks AF, Czabotar PE, Lessene G, Murphy JM, Silke J (2014) Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proc Natl Acad Sci U S A 111(42):15072–15077. https://doi.org/10.1073/pnas.1408987111CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Dondelinger Y, Declercq W, Montessuit S, Roelandt R, Goncalves A, Bruggeman I, Hulpiau P, Weber K, Sehon CA, Marquis RW, Bertin J, Gough PJ, Savvides S, Martinou JC, Bertrand MJ, Vandenabeele P (2014) MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep 7(4):971–981. https://doi.org/10.1016/j.celrep.2014.04.026CrossRefPubMedGoogle Scholar
  19. 19.
    Cai Z, Jitkaew S, Zhao J, Chiang HC, Choksi S, Liu J, Ward Y, Wu LG, Liu ZG (2014) Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol 16(1):55–65. https://doi.org/10.1038/ncb2883CrossRefPubMedGoogle Scholar
  20. 20.
    Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J (2005) Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 1(2):112–119. https://doi.org/10.1038/nchembio711CrossRefPubMedGoogle Scholar
  21. 21.
    Welz PS, Wullaert A, Vlantis K, Kondylis V, Fernandez-Majada V, Ermolaeva M, Kirsch P, Sterner-Kock A, van Loo G, Pasparakis M (2011) FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation. Nature 477(7364):330–334. https://doi.org/10.1038/nature10273CrossRefPubMedGoogle Scholar
  22. 22.
    Kaiser WJ, Sridharan H, Huang C, Mandal P, Upton JW, Gough PJ, Sehon CA, Marquis RW, Bertin J, Mocarski ES (2013) Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J Biol Chem 288(43):31268–31279. https://doi.org/10.1074/jbc.M113.462341CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Zargarian S, Shlomovitz I, Erlich Z, Hourizadeh A, Ofir-Birin Y, Croker BA, Regev-Rudzki N, Edry-Botzer L, Gerlic M (2017) Phosphatidylserine externalization, "necroptotic bodies" release, and phagocytosis during necroptosis. PLoS Biol 15(6):e2002711. https://doi.org/10.1371/journal.pbio.2002711CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Su Z, Yang Z, Xie L, DeWitt JP, Chen Y (2016) Cancer therapy in the necroptosis era. Cell Death Differ 23(5):748–756. https://doi.org/10.1038/cdd.2016.8CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Mandal P, Berger SB, Pillay S, Moriwaki K, Huang C, Guo H, Lich JD, Finger J, Kasparcova V, Votta B, Ouellette M, King BW, Wisnoski D, Lakdawala AS, DeMartino MP, Casillas LN, Haile PA, Sehon CA, Marquis RW, Upton J, Daley-Bauer LP, Roback L, Ramia N, Dovey CM, Carette JE, Chan FK, Bertin J, Gough PJ, Mocarski ES, Kaiser WJ (2014) RIP3 induces apoptosis independent of pronecrotic kinase activity. Mol Cell 56(4):481–495. https://doi.org/10.1016/j.molcel.2014.10.021CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lawlor KE, Khan N, Mildenhall A, Gerlic M, Croker BA, D'Cruz AA, Hall C, Kaur Spall S, Anderton H, Masters SL, Rashidi M, Wicks IP, Alexander WS, Mitsuuchi Y, Benetatos CA, Condon SM, Wong WW, Silke J, Vaux DL, Vince JE (2015) RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nat Commun 6:6282. https://doi.org/10.1038/ncomms7282CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Magidson V, Khodjakov A (2013) Circumventing photodamage in live-cell microscopy. Methods Cell Biol 114:545–560. https://doi.org/10.1016/B978-0-12-407761-4.00023-3CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Clinical Microbiology and Immunology, Sackler Faculty of MedicineTel Aviv UniversityTel AvivIsrael

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