Autophagy pp 375-387 | Cite as

Autophagy Pathway Mapping to Elucidate the Function of Novel Autophagy Regulators Identified by High-Throughput Screening

  • Martina Wirth
  • Sharon A. ToozeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)


Autophagy is an evolutionarily conserved degradative pathway, and the core autophagy machinery acts in a highly regulated, hierarchical manner to engulf cytoplasmic material in a double-membrane-bound organelle and deliver it to the lysosome. High-throughput screening approaches lead to the identification of novel autophagy regulators, and we describe an autophagy pathway mapping strategy to determine the stage of the autophagy pathway at which these novel candidate proteins function.

Key words

Autophagy pathway mapping mTORC1 ULK complex WIPI2 ATG9 LC3 Autophagosome maturation Autophagic flux 


  1. 1.
    Gallagher LE, Williamson LE, Chan EYW (2016) Advances in autophagy regulatory mechanisms. Cell 5:24. Scholar
  2. 2.
    Lamb CA, Yoshimori T, Tooze SA (2013) The autophagosome: origins unknown, biogenesis complex. Nat Rev Mol Cell Biol 14:759–774. Scholar
  3. 3.
    McKnight NC, Jefferies HBJ, Alemu EA, Saunders RE, Howell M, Johansen T, Tooze SA (2012) Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC. EMBO J 31:1931–1946. Scholar
  4. 4.
    Lipinski MM, Zheng B, Lu T, Yan Z, Py BF, Ng A, Xavier RJ, Li C, Yankner BA, Scherzer CR et al (2010) Genome-wide analysis reveals mechanisms modulating autophagy in normal brain aging and in Alzheimer’s disease. Proc Natl Acad Sci U S A 107:14164–14169. Scholar
  5. 5.
    Kabeya Y (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728. Scholar
  6. 6.
    Yoshii SR, Mizushima N (2017) Monitoring and measuring autophagy. Int J Mol Sci 18:1–13. Scholar
  7. 7.
    Polson HEJ, de Lartigue J, Rigden DJ, Reedijk M, Urbé S, Clague MJ, Tooze SA (2010) Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation. Autophagy 6:506–522. Scholar
  8. 8.
    Dooley HC, Razi M, Polson HEJ, Girardin SE, Wilson MI, Tooze SA (2014) WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12–5-16L1. Mol Cell 55:238. Scholar
  9. 9.
    Young ARJ, Chan EYW, Hu XW, Köchl R, Crawshaw SG, High S, Hailey DW, Lippincott-Schwartz J, Tooze SA (2006) Starvation and ULK1-dependent cycling of mammalian Atg9 between the TGN and endosomes. J Cell Sci 119:3888–3900. Scholar
  10. 10.
    Orsi A, Razi M, Dooley HC, Robinson D, Weston AE, Collinson LM, Tooze SA (2012) Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Mol Biol Cell 23:1860–1873. Scholar
  11. 11.
    McAlpine F, Williamson LE, Tooze SA, Chan EYW (2013) Regulation of nutrient-sensitive autophagy by uncoordinated 51-like kinases 1 and 2. Autophagy 9:361–373. Scholar
  12. 12.
    Karanasios E, Walker SA, Okkenhaug H, Manifava M, Hummel E, Zimmermann H, Ahmed Q, Domart M-C, Collinson L, Ktistakis NT (2016) Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles. Nat Commun 7:12420. Scholar
  13. 13.
    Lamb CA, Nühlen S, Judith D, Frith D, Snijders AP, Behrends C, Tooze SA (2016) TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic. EMBO J 35:281–301. Scholar
  14. 14.
    Juhász G (2012) Interpretation of bafilomycin, pH neutralizing or protease inhibitor treatments in autophagic flux experiments: novel considerations. Autophagy 8:1875–1876. Scholar
  15. 15.
    Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM (2011) mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H+-ATPase. Science 334:678–683. Scholar
  16. 16.
    Florey O, Gammoh N, Kim SE, Jiang X, Overholtzer M (2015) V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation. Autophagy 11:88–99. Scholar
  17. 17.
    Kimura S, Noda T, Yoshimori T (2007) Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3:452–460CrossRefGoogle Scholar
  18. 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 64:835–849. Scholar
  19. 19.
    Lamb CA, Joachim J, Tooze SA (2017) Quantifying autophagic structures in mammalian cells using confocal microscopy. Methods Enzymol 587:21–42. Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Molecular Cell Biology of Autophagy LaboratoryThe Francis Crick InstituteLondonUK

Personalised recommendations