Skip to main content

Tools to Measure Autophagy Using High Content Imaging and Analysis

  • Protocol
  • First Online:
High Content Screening

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1683))

Abstract

Macroautophagy, hereafter referred to as autophagy, is a predominately pro-survival catabolic process responsible for the degradation of long-lived or aggregated proteins, invading microorganisms and damaged or redundant intracellular organelles. Removal of these entities is achieved through encompassment of the target by the autophagosome and subsequent delivery to the lysosome. The use of fluorescence microscopy is a common method to investigate autophagy through monitoring the spatial and temporal recruitment both of autophagosomal markers and cargo to the autophagosome. In this section, we will discuss the use of high content imaging (HCI) and analysis in the study of autophagy with reference to commonly used markers of autophagosomal formation.

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

Access this chapter

Institutional subscriptions

References

  1. Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728–741

    Article  CAS  Google Scholar 

  2. Klionsky DJ, Abdelmohsen K, Abe A et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12:1–122

    Article  Google Scholar 

  3. Feng Y, Mitchison TJ, Bender A et al (2009) Multi-parametric phenotypic profiling: using cellular effects to characterize small-scale compounds. Nat Rev Drug Discov 8:567–578

    Article  CAS  Google Scholar 

  4. Zanella F, Lorens JB, Link W (2010) High content screening: seeing is believing. Trends Biotechnol 28(5):237–245

    Article  CAS  Google Scholar 

  5. Nyfeler B, Bergman P, Wilson CJ et al (2012) Quantitative visualization of autophagy induction by mTOR inhibitors. Methods Mol Biol 821:239–250

    Article  CAS  Google Scholar 

  6. Joachim J, Jiang M, McKnight NC et al (2015) High-throughput screening methods to identify regulators of mammalian autophagy. Methods 75:96–104

    Article  CAS  Google Scholar 

  7. Mcknight NC, Jefferies HBJ, Alemu EA et al (2012) Genome wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC. EMBO J 31:1931–1946

    Article  CAS  Google Scholar 

  8. Ovredahl A, Sumpter R Jr, Xiao G et al (2011) Image-based genome-wide siRNA identifies selective autophagy factors. Nature 480:113–117

    Article  Google Scholar 

  9. Buchser WJ, Laskow TC, Pavlik PJ et al (2012) Cell-mediated autophagy promotes cancer cell survival. Cancer Res 12:2970–2979

    Article  Google Scholar 

  10. Zhang L, Yu J, Pan H et al (2007) Small molecule regulators of autophagy identified by an image-based high-throughput screen. Proc Natl Acad Sci U S A 104:19023–19028

    Article  CAS  Google Scholar 

  11. Underwood BR, Imarisio S, Fleming S et al (2010) Antioxidants can inhibit basal autophagy and enhance neurodegeneration in models of polyglutamine disease. Hum Mol Genet 19:3413–3429

    Article  CAS  Google Scholar 

  12. Hasson SA, Kane LA, Yamano K et al (2013) High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy. Nature 504:3675–3688

    Article  Google Scholar 

  13. Klionsky DJ, Abdalla FC, Abeliovich H et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445–544

    Article  CAS  Google Scholar 

  14. Miyawaki A (2011) Proteins on the move: insights gained from fluorescent protein technologies. Nat Rev Mol Cell Biol 23:656–668

    Article  Google Scholar 

  15. Schnell U, Dijk F, Sjollema KA et al (2012) Immunolabeling artifacts and the need for live-cell imaging. Nat Methods 9:152–158

    Article  CAS  Google Scholar 

  16. Mizushima N, Yoshimori T, Levine B (2011) Methods in mammalian autophagy research. Cell 140:313–326

    Article  Google Scholar 

  17. Wright AV, Nuñez JK, Doudna JA (2016) Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164:29–44

    Article  CAS  Google Scholar 

  18. Hancock MK, Hermanson SB, Dolman NJ (2012) A quantitative TR-FRET plate reader immunoassay for measuring autophagy. Autophagy 8:1227–1244

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Bhaskar S. Mandavilli .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Dolman, N.J., Samson, B.A., Chambers, K.M., Janes, M.S., Mandavilli, B.S. (2018). Tools to Measure Autophagy Using High Content Imaging and Analysis. In: Johnston, P., Trask, O. (eds) High Content Screening. Methods in Molecular Biology, vol 1683. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7357-6_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7357-6_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7355-2

  • Online ISBN: 978-1-4939-7357-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics