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Autophagy pp 295–303Cite as

Imaging Noncanonical Autophagy and LC3-Associated Phagocytosis in Cultured Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1880))

Abstract

Monitoring of ATG8 proteins by western blotting and immunofluorescence microscopy are the most common methods to monitor the autophagy pathway. However, it has recently been shown that ATG8 proteins can be lipidated to non-autophagosome, single-membrane compartments through a noncanonical autophagy pathway. This is commonly found to occur during macro-endocytic processes such as phagocytosis, where it has been termed LC3-associated phagocytosis, and upon lysosomotropic drug treatment. Therefore, care is required when interpreting data based on ATG8 in order to conclude whether a signal relates to the canonical or noncanonical pathway. Here we provide methods to monitor noncanonical autophagy through fluorescence microscopy.

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References

  1. Mizushima N et al (1998) A protein conjugation system essential for autophagy. Nature 395(6700):395–398

    Article  CAS  Google Scholar 

  2. Feng Y et al (2014) The machinery of macroautophagy. Cell Res 24:24–41

    Article  CAS  Google Scholar 

  3. Weidberg H et al (2010) LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis. EMBO J 29(11):1792–1802

    Article  CAS  Google Scholar 

  4. Lamark T, Svenning S, Johansen T (2017) Regulation of selective autophagy: the p62/SQSTM1 paradigm. Essays Biochem 61(6):609–624

    Article  Google Scholar 

  5. Nguyen TN et al (2016) Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation. J Cell Biol 215(6):857–874

    Article  CAS  Google Scholar 

  6. Virgin HW, Levine B (2009) Autophagy genes in immunity. Nat Immunol 10(5):461–470

    Article  CAS  Google Scholar 

  7. Sprenkeler EG, Gresnigt MS, van de Veerdonk FL (2016) LC3-associated phagocytosis: a crucial mechanism for antifungal host defence against Aspergillus fumigatus. Cell Microbiol 18(9):1208–1216

    Article  CAS  Google Scholar 

  8. Florey O, Overholtzer M (2012) Autophagy proteins in macroendocytic engulfment. Trends Cell Biol 22(7):374–380

    Article  CAS  Google Scholar 

  9. Sanjuan MA et al (2007) Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature 450(7173):1253–1257

    Article  CAS  Google Scholar 

  10. Martinez J et al (2015) Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins. Nat Cell Biol 17(7):893–906

    Article  CAS  Google Scholar 

  11. Martinez J et al (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(42):17396–17401

    Article  CAS  Google Scholar 

  12. Florey O et al (2011) Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes. Nat Cell Biol 13(11):1335–1343

    Article  CAS  Google Scholar 

  13. Martinez J et al (2016) Noncanonical autophagy inhibits the autoinflammatory, lupus-like response to dying cells. Nature 533(7601):115–119

    Article  CAS  Google Scholar 

  14. Ma J et al (2012) Dectin-1-triggered recruitment of light chain 3 protein to phagosomes facilitates major histocompatibility complex class II presentation of fungal-derived antigens. J Biol Chem 287(41):34149–34156

    Article  CAS  Google Scholar 

  15. Lee HK et al (2010) In vivo requirement for Atg5 in antigen presentation by dendritic cells. Immunity 32(2):227–239

    Article  CAS  Google Scholar 

  16. Henault J et al (2012) Noncanonical autophagy is required for type I interferon secretion in response to DNA-immune complexes. Immunity 37(6):986–997

    Article  CAS  Google Scholar 

  17. Fletcher K et al (2018) The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes. EMBO J 37(4):e97840

    Article  Google Scholar 

  18. Jacquin E et al (2017) Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation. Autophagy 13(5):854–867

    Article  CAS  Google Scholar 

  19. Florey O et al (2015) V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation. Autophagy 11(1):88–99

    Article  Google Scholar 

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Correspondence to Oliver Florey .

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Jacquin, E., Fletcher, K., Florey, O. (2019). Imaging Noncanonical Autophagy and LC3-Associated Phagocytosis in Cultured Cells. In: Ktistakis, N., Florey, O. (eds) Autophagy. Methods in Molecular Biology, vol 1880. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8873-0_19

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  • DOI: https://doi.org/10.1007/978-1-4939-8873-0_19

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8872-3

  • Online ISBN: 978-1-4939-8873-0

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