Autophagy pp 281-293 | Cite as

Correlative Light and Electron Microscopy to Analyze LC3 Proteins in Caenorhabditis elegans Embryo

  • Céline Largeau
  • Renaud LegouisEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)


In this chapter, we present a protocol to perform correlative light and electron microscopy (CLEM) on Caenorhabditis elegans embryos. We use a specific fixation method which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins, then by transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows to determine the subcellular localization of the fluorescent protein. We have used this method to characterize the roles of autophagy in the phagocytosis of apoptotic cells in C. elegans embryos. We analyzed in apoptotic cell and phagocytic cell the localization of the two homologs of LC3/GABARAP proteins, namely, LGG-1 and LGG-2.

Key words

Green fluorescent protein LC3-associated phagocytosis High-pressure freezing Freeze substitution LGG-1 LGG-2 GMA resin 



The authors would like to thank the C. elegans community and the Caenorhabditis Genetics Center which is funded by the NIH National Center for Research Resources (NCRR) for C. elegans strains. We are grateful to Claire Boulogne for critical reading of the manuscript and helpful discussions. The present work has benefited from the expertise of Béatrice Satiat-Jeunemaitre, Cynthia Gillet, Jessica Marion and Claire Boulogne and the core facilities of Imagerie-Gif ( member of IBiSA (, supported by “France-BioImaging” (ANR-10-INBS-04-01), and the Labex “Saclay Plant Science” (ANR-11-IDEX-0003-02). This work was supported by the Agence Nationale de la Recherche (project EAT, ANR-12-BSV2-018) and the Association pour la Recherche contre le Cancer (SFI20111203826).


  1. 1.
    Ohsumi Y (2014) Historical landmarks of autophagy research. Cell Res 24(1):9–23CrossRefGoogle Scholar
  2. 2.
    Metzstein MM, Stanfield GM, Horvitz HR (1998) Genetics of programmed cell death in C. elegans: past, present and future. Trends Genet 14(10):410–416CrossRefGoogle Scholar
  3. 3.
    Henault J, Martinez J, Riggs JM, Tian J, Mehta P, Clarke L, Sasai M, Latz E, Brinkmann MM, Iwasaki A et al (2012) Noncanonical autophagy is required for type I interferon secretion in response to DNA-immune complexes. Immunity 37:986–997CrossRefGoogle Scholar
  4. 4.
    Martinez J, Almendinger J, Oberst A, Ness R, Dillon CP, Fitzgerald P, Hengartner MO, Green DR (2011) Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells. Proc Natl Acad Sci U S A 108:17396–17401CrossRefGoogle Scholar
  5. 5.
    Sanjuan MA, Dillon CP, Tait SWG, Moshiach S, Dorsey F, Connell S, Komatsu M, Tanaka K, Cleveland JL, Withoff S et al (2007) Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature 450:1253–1257CrossRefGoogle Scholar
  6. 6.
    Florey O, Kim SE, Sandoval CP, Haynes CM, Overholtzer M (2011) Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes. Nat Cell Biol 13:1335–1343CrossRefGoogle Scholar
  7. 7.
    Kim JY, Zhao H, Martinez J, Doggett TA, Kolesnikov AV, Tang PH, Ablonczy Z, Chan CC, Zhou Z, Green DR et al (2013) Noncanonical autophagy promotes the visual cycle. Cell 154:365–376CrossRefGoogle Scholar
  8. 8.
    Martinez J, Malireddi RKS, Lu Q, Cunha LD, Pelletier S, Gingras S, Orchard R, Guan JL, Tan H, Peng J et al (2015) Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins. Nat Cell Biol 17:893–906CrossRefGoogle Scholar
  9. 9.
    Manil-Ségalen M, Lefebvre C, Jenzer C, Trichet M, Boulogne C, Satiat-Jeunemaitre B, Legouis R (2014) The C. elegans LC3 acts downstream of GABARAP to degrade autophagosomes by interacting with the HOPS subunit VPS39. Dev Cell 28(1):43–55CrossRefGoogle Scholar
  10. 10.
    Jenzer C, Simionato E, Legouis R (2015) Tools and methods to analyze autophagy in C. elegans. Methods 75:162–171CrossRefGoogle Scholar
  11. 11.
    Kolotuev I, Schwab Y, Labouesse M (2009) A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms. Biol Cell 102(2):121–132CrossRefGoogle Scholar
  12. 12.
    Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71–94PubMedPubMedCentralGoogle Scholar
  13. 13.
    Lefebvre C, Largeau C, Michelet X, Fourrage C, Maniere X, Matic I, Legouis R, Culetto E (2016) The ESCRT-II proteins are involved in shaping the sarcoplasmic reticulum in C. elegans. J Cell Sci 129(7):1490–1499CrossRefGoogle Scholar
  14. 14.
    Watanabe S, Punge A, Hollopeter G, Willig KI, Hobson RJ, Davis MW, Hell SW, Jorgensen EM (2011) Protein localization in electron micrographs using fluorescence nanoscopy. Nat Methods 8(1):80–84CrossRefGoogle Scholar
  15. 15.
    Watanabe S, Richards J, Hollopeter G, Hobson RJ, Davis WM, Jorgensen EM (2012) Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy. J Vis Exp 70:e3995Google Scholar
  16. 16.
    Marion J, Le Bars R, Satiat-Jeunemaitre B, Boulogne C (2017) Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots. J Struct Biol 198(3):196–202CrossRefGoogle Scholar
  17. 17.
    Paul-Gilloteaux P, Heiligenstein X, Belle M, Domart MC, Larijani B, Collinson L, Raposo G, Salamero J (2017) eC-CLEM: flexible multidimensional registration software for correlative microscopies. Nat Methods 14(2):102–103CrossRefGoogle Scholar

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

Authors and Affiliations

  1. 1.Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-SaclayGif-sur-Yvette cedexFrance

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