Skip to main content

Correlative Light and Electron Microscopy (CLEM): Bringing Together the Best of Both Worlds to Study Neuronal Autophagy

  • 913 Accesses

Part of the Neuromethods book series (NM,volume 171)

Abstract

Autophagy is a key protein degradative pathway for primarily long-lived proteins and damaged organelles, contributing to cellular proteostasis. Neurons rely on a particularly effective autophagy machinery, due to their high metabolic activity, ATP demand, and terminally differentiated nature. Autophagy was originally identified with electron microscopy (EM), and it has remained one of the most important tools to study autophagy and its pathway intermediates. However, distinguishing between specific autophagic structures, such as autophagosomes, autolysosomes, and lysosomes, remains challenging, requires substantial expertise, and is often complicated by the large degree of membrane and cargo complexity, underpinned by the heterogeneous electron density within these structures. To accurately identify specific components of the autophagic machinery, correlative light and electron microscopy (CLEM) has emerged as a powerful tool. To discern between autophagic components, specific fluorescent tags can be assigned to macromolecules and structures of interest, and correlated to the ultrastructural detail and subcellular context provided by the electron micrograph. In doing so, a large degree of subjectivity is eliminated, leading to a more accurate characterization and depiction of the total cellular autophagic response and its biological role. This chapter will outline the advantage of CLEM for the study of neuronal autophagy and provide a methodological workflow for both two-dimensional and three-dimensional CLEM approaches.

Keywords

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Loos B, Toit du A, Hofmeyr J-HS (2014) Defining and measuring autophagosome flux—concept and reality. Autophagy 10:2087–2096. http://www.tandfonline.com/doi/full/10.4161/15548627.2014.973338

    Article  Google Scholar 

  2. Guo JY, Teng X, Laddha SV, Ma S, Van Nostrand SC, Yang Y, Khor S, Chan CS, Rabinowitz JD, White E (2016) Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells. Genes Dev 30:1704–1717

    Article  CAS  Google Scholar 

  3. Yin Z, Pascual C, Klionsky D (2016) Autophagy: machinery and regulation. Microb Cell 3:588–596. http://microbialcell.com/researcharticles/autophagy-machinery-and-regulation/

    Article  Google Scholar 

  4. Ziviani E, Tao RN, Whitworth AJ (2010) Drosophila parkin requires PINK1 for mitochondrial translocation and ubiquitinates mitofusin. Proc Natl Acad Sci U S A 107:5018–5023. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2841909&tool=pmcentrez&rendertype=abstract

    Article  CAS  Google Scholar 

  5. Lumkwana D, du Toit A, Kinnear C, Loos B (2017) Autophagic flux control in neurodegeneration: progress and precision targeting—where do we stand? Prog Neurobiol 153:64–85. https://doi.org/10.1016/j.pneurobio.2017.03.006

    Article  PubMed  Google Scholar 

  6. Yang Z, Klionsky DJ (2010) Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol 22:124–131

    Article  CAS  Google Scholar 

  7. Loos B, Klionsky DJ, Du Toit A, Hofmeyr JHS (2020) On the relevance of precision autophagy flux control in vivo–points of departure for clinical translation. Autophagy 16(4):750–762

    Article  CAS  Google Scholar 

  8. du Toit A, Hofmeyr J-HS, Gniadek TJ, Loos B (2018) Measuring autophagosome flux. Autophagy 14:1060–1071. https://doi.org/10.1080/15548627.2018.1469590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676. https://doi.org/10.1038/nmeth.2019

    Article  CAS  Google Scholar 

  10. Paul-Gilloteaux P, Heiligenstein X, Belle M, Domart M-C, Larijani B, Collinson L, Raposo G, Salamero J (2017) eC-CLEM: flexible multidimensional registration software for correlative microscopies. Nat Methods 14:102. https://doi.org/10.1038/nmeth.4170

    Article  CAS  PubMed  Google Scholar 

  11. De Chaumont F, Dallongeville S, Chenouard N, Hervé N, Pop S, Provoost T, Meas-Yedid V, Pankajakshan P, Lecomte T, Le Montagner Y et al (2012) Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods 9:690–696

    Article  Google Scholar 

  12. Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76

    Article  CAS  Google Scholar 

  13. Heintzmann, R., Cremer CG (1999) Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. In: Optical biopsies and microscopic techniques III 3568, pp 185–196

    Google Scholar 

  14. Russell MRG, Lerner TR, Burden JJ, Nkwe DO, Pelchen-Matthews A, Domart M-C, Durgan J, Weston A, Jones ML, Peddie CJ, Carzaniga R, Florey O, Marsh M, Gutierrez MG, Collinson LM (2017) 3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy. J Cell Sci 130(1):278–291

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Burel A, Lavault MT, Chevalier C, Gnaegi H, Prigent S, Mucciolo A, Dutertre S, Humbel BM, Guillaudeux T, Kolotuev I (2018) A targeted 3D EM and correlative microscopy method using SEM array tomography. Development 145(12):dev160879. https://doi.org/10.1242/dev.160879

    Article  CAS  PubMed  Google Scholar 

  16. Wacker I, Spomer W, Hofmann A, Thaler M, Hillmer S, Gengenbach U, Schröder RR (2016) Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues. BMC Cell Biol 17(1):38. https://doi.org/10.1186/s12860-016-0122-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Pietzsch T, Saalfeld S, Preibisch S et al (2015) BigDataViewer: visualization and processing for large image data sets. Nat Methods 12:481–483. https://doi.org/10.1038/nmeth.3392

    Article  CAS  PubMed  Google Scholar 

  18. Kaizuka T, Morishita H, Hama Y, Tsukamoto S, Matsui T, Toyota Y, Kodama A, Ishihara T, Mizushima T, Mizushima N (2016) An autophagic flux probe that releases an internal control. Mol Cell 64(4):835–849

    Article  CAS  Google Scholar 

  19. Micheva KD, Smith SJ (2007) Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55(1):25–36

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001999), the UK Medical Research Council (FC001999), and the Wellcome Trust (FC001999). Moreover, work was supported by the National Research Foundation South Africa (NRF), the South African Medical Research Council (SAMRC), and the Cancer Association South Africa (CANSA).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Lucy Collinson , Ben Loos or Lize Engelbrecht .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kriel, J. et al. (2022). Correlative Light and Electron Microscopy (CLEM): Bringing Together the Best of Both Worlds to Study Neuronal Autophagy. In: Loos, B., Wong, E. (eds) Imaging and Quantifying Neuronal Autophagy. Neuromethods, vol 171. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1589-8_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1589-8_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1588-1

  • Online ISBN: 978-1-0716-1589-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics