Skip to main content
SpringerLink
Log in

Quantification of Autophagosome Size and Formation Rate by Electron and Fluorescence Microscopy in Baker’s Yeast

  • Protocol
  • First Online:
Imaging and Quantifying Neuronal Autophagy

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

  • 891 Accesses

Abstract

The use of both transmission electron microscopy and fluorescence microscopy have provided tremendous advances to our understanding of autophagosome formation in baker’s yeast, Saccharomyces cerevisiae. In the last decade, parallel techniques have been developed for both types of microscopy that allow the quantification of the rate of autophagosome formation. Importantly, these techniques, unlike other measures of total autophagic flux, allow a researcher to distinguish between effects on autophagosome size and autophagosome number. This has led to the discovery that certain autophagy proteins (e.g., Atg8) contribute primarily to the control of autophagosome size, whereas others (e.g., Atg9) are principally involved in controlling autophagosome number, suggesting different roles for these proteins in the autophagosome formation process.

In this chapter, we present two methods for quantifying autophagosome formation in yeast. One, based on electron microscopy analysis of autophagic bodies in the vacuole, can give estimates of both autophagosome size and number. The other, based on live-cell imaging of growing autophagosomes labeled with GFP-Atg8, can provide information on the rate of autophagosome formation. Together they provide a robust toolbox for analyzing the roles of different proteins in the process of autophagosome formation.

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

Access this chapter

Log in via an institution
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

Institutional subscriptions

References

  1. Eskelinen E-L, Reggiori F, Baba M et al (2011) Seeing is believing: the impact of electron microscopy on autophagy research. Autophagy 7:935–956. https://doi.org/10.4161/auto.7.9.15760

    Article  CAS  PubMed  Google Scholar 

  2. Takeshige K, Baba M, Tsuboi S et al (1992) Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J Cell Biol 119(2):301–311

    Article  CAS  Google Scholar 

  3. Baba M, Takeshige K, Baba N, Ohsumi Y (1994) Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization. J Cell Biol 124:903–913

    Article  CAS  Google Scholar 

  4. Baba M, Osumi M, Scott SV et al (1997) Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome. J Cell Biol 139(7):1687–1695

    Article  CAS  Google Scholar 

  5. He C, Song H, Yorimitsu T et al (2006) Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast. J Cell Biol 175:925–935. https://doi.org/10.1083/jcb.200606084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Baba M, Tomonaga S, Suzuki M et al (2019) A nuclear membrane-derived structure associated with Atg8 is involved in the sequestration of selective cargo, the Cvt complex, during autophagosome formation in yeast. Autophagy 15:423–437. https://doi.org/10.1080/15548627.2018.1525475

    Article  CAS  PubMed  Google Scholar 

  7. Gómez-Sánchez R, Rose J, Guimarães R et al (2018) Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores. J Cell Biol 217:2743–2763. https://doi.org/10.1083/jcb.201710116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Noda T, Klionsky DJ (2008) Chapter 3. The quantitative Pho8Δ60 assay of nonspecific autophagy. Methods Enzymol 451:33–42. https://doi.org/10.1016/S0076-6879(08)03203-5

    Article  CAS  PubMed  Google Scholar 

  9. Guimaraes RS, Delorme-Axford E, Klionsky DJ, Reggiori F (2015) Assays for the biochemical and ultrastructural measurement of selective and nonselective types of autophagy in the yeast Saccharomyces cerevisiae. Methods 75:141–150. https://doi.org/10.1016/j.ymeth.2014.11.023

    Article  CAS  PubMed  Google Scholar 

  10. Torggler R, Papinski D, Kraft C (2017) Assays to monitor autophagy in Saccharomyces cerevisiae. Cell 6(3):23. https://doi.org/10.3390/cells6030023

    Article  CAS  Google Scholar 

  11. Xie Z, Nair U, Klionsky DJ (2008) Atg8 controls phagophore expansion during autophagosome formation. Mol Biol Cell 19:3290–3298. https://doi.org/10.1091/mbc.E07-12-1292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nakatogawa H, Ichimura Y, Ohsumi Y (2007) Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell 130:165–178. https://doi.org/10.1016/J.CELL.2007.05.021

    Article  CAS  PubMed  Google Scholar 

  13. Jin M, He D, Backues SK et al (2014) Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation. Curr Biol 24:1314–1322. https://doi.org/10.1016/j.cub.2014.04.048

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Legakis JE, Yen W-L, Klionsky DJ (2007) A cycling protein complex required for selective autophagy. Autophagy 3:422–432. https://doi.org/10.4161/auto.4129

    Article  CAS  PubMed  Google Scholar 

  15. Tucker KA, Reggiori F, Dunn WA, Klionsky DJ (2003) Atg23 is essential for the cytoplasm to vacuole targeting pathway and efficient autophagy but not pexophagy. J Biol Chem 278:48445–48452. https://doi.org/10.1074/jbc.M309238200

    Article  CAS  PubMed  Google Scholar 

  16. Yao Z, Delorme-Axford E, Backues SK, Klionsky DJ (2015) Atg41/Icy2 regulates autophagosome formation. Autophagy 11(12):2288–2299. https://doi.org/10.1080/15548627.2015.1107692

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Feng Y, Backues SK, Baba M et al (2016) Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation. Autophagy 12(4):648–658. https://doi.org/10.1080/15548627.2016.1157237

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Cawthon H, Chakraborty R, Roberts JR, Backues SK (2018) Control of autophagosome size and number by Atg7. Biochem Biophys Res Commun 503:651–656. https://doi.org/10.1016/J.BBRC.2018.06.056

    Article  CAS  PubMed  Google Scholar 

  19. Xie Z, Nair U, Geng J et al (2009) Indirect estimation of the area density of Atg8 on the phagophore. Autophagy 5:217–220. https://doi.org/10.4161/auto.5.2.7201

    Article  CAS  PubMed  Google Scholar 

  20. Backues SK, Chen D, Ruan J et al (2014) Estimating the size and number of autophagic bodies by electron microscopy. Autophagy 10:155–164

    Article  CAS  Google Scholar 

  21. Suzuki K, Kirisako T, Kamada Y et al (2001) The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. EMBO J 20:5971–5981. https://doi.org/10.1093/emboj/20.21.5971

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kim J, Huang W-P, Stromhaug PE, Klionsky DJ (2002) Convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicle formation. J Biol Chem 277:763–773. https://doi.org/10.1074/jbc.M109134200

    Article  CAS  PubMed  Google Scholar 

  23. Segarra VA, Boettner DR, Lemmon SK (2015) Atg27 tyrosine sorting motif is important for its trafficking and Atg9 localization. Traffic 16:365–378. https://doi.org/10.1111/tra.12253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yamamoto H, Kakuta S, Watanabe TM et al (2012) Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J Cell Biol 198:219–233. https://doi.org/10.1083/jcb.201202061

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Backues SK, Orban DP, Bernard A et al (2015) Atg23 and Atg27 act at the early stages of Atg9 trafficking in S. cerevisiae. Traffic 16:172–190. https://doi.org/10.1111/tra.12240

    Article  CAS  PubMed  Google Scholar 

  26. Suzuki K, Kubota Y, Sekito T, Ohsumi Y (2007) Hierarchy of Atg proteins in pre-autophagosomal structure organization. Genes Cells 12:209–218. https://doi.org/10.1111/j.1365-2443.2007.01050.x

    Article  CAS  PubMed  Google Scholar 

  27. Reggiori F, Tucker KA, Stromhaug PE, Klionsky DJ (2004) The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the pre-autophagosomal structure. Dev Cell 6:79–90. https://doi.org/10.1016/S1534-5807(03)00402-7

    Article  CAS  PubMed  Google Scholar 

  28. Cheong H, Yorimitsu T, Reggiori F et al (2005) Atg17 regulates the magnitude of the autophagic response. Mol Biol Cell 16:3438–3453. https://doi.org/10.1091/mbc.E04-10-0894

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Feuerverger A, Menzinger M, Atwood HL, Cooper RL (2000) Statistical methods for assessing the dimensions of synaptic vesicles in nerve terminals. J Neurosci Methods 103:181–190

    Article  CAS  Google Scholar 

  30. Bernard A, Jin M, González-Rodríguez P et al (2015) Rph1/KDM4 mediates nutrient-limitation signaling that leads to the transcriptional induction of autophagy. Curr Biol 25:546–555. https://doi.org/10.1016/j.cub.2014.12.049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Klionsky DJ (2011) For the last time, it is GFP-Atg8, not Atg8-GFP (and the same goes for LC3). Autophagy 7:1093–1094. https://doi.org/10.4161/auto.7.10.15492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, Eastern Michigan University, Ypsilanti, MI, USA

    Steven K. Backues

  2. Department of Molecular, Cellular and Developmental Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    Daniel J. Klionsky

Authors
  1. Steven K. Backues
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel J. Klionsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Daniel J. Klionsky .

Editor information

Editors and Affiliations

  1. Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South Africa

    Ben Loos

  2. Healthy Longevity Translational Research Programme, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Healthy Longevity National University Health System, Singapore, Singapore

    Esther Wong

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

Backues, S.K., Klionsky, D.J. (2022). Quantification of Autophagosome Size and Formation Rate by Electron and Fluorescence Microscopy in Baker’s Yeast. 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_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1589-8_1

  • 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

Search

Navigation