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Autophagy pp 307-313 | Cite as

Measurement of Bulk Autophagy by a Cargo Sequestration Assay

  • Nikolai EngedalEmail author
  • Morten Luhr
  • Paula Szalai
  • Per O. SeglenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)

Abstract

The nonselective bulk sequestration of cytoplasm and its subsequent delivery to lysosomes for degradation was originally defined as autophagy or macroautophagy. However, both terms are now increasingly being applied in a generic sense to encompass the many recently described mechanisms for selective sequestration and degradation of individual cellular elements. We will therefore use the term bulk autophagy to denote the non-exclusive and largely nonselective process.

Bulk autophagy can be measured directly by a cargo sequestration assay, using a cargo marker representative of total cytoplasm. The assay described here measures the sequestration and accumulation of the ubiquitous cytosolic protein lactate dehydrogenase (LDH) in the sedimentable autophagic vacuoles of cells incubated with an inhibitor of intravacuolar degradation such as bafilomycin or leupeptin. Electrodisruption of the plasma membrane followed by centrifugal sedimentation of the “cell corpses” (which retain their organelles in an intact state, bound to the cytoskeleton) is a convenient, efficient, and reproducible way to separate the small fraction of sequestered, sedimentable LDH from the major pool of cytosolic LDH.

Key words

Autophagy Bulk autophagy Bafilomycin Cargo assay Cell corpse Cell culture Electrodisruption Lactate dehydrogenase Lysosome Phagophore Sequestration 

Notes

Acknowledgments

This work has been generously supported by grants from the Norwegian Cancer Society, the Research Council of Norway, the University of Oslo, the Nansen Foundation, the Blix Family Fund, and Astri and Birger Torsted's Legacy.

Disclosure: No conflict of interest was disclosed.

References

  1. 1.
    Gordon PB, Seglen PO (1982) Autophagic sequestration of [14C]sucrose, introduced into isolated rat hepatocytes by electropermeabilization. Exp Cell Res 142:1–14CrossRefGoogle Scholar
  2. 2.
    Seglen PO, Gordon PB (1984) Amino acid control of autophagic sequestration and protein degradation in isolated rat hepatocytes. J Cell Biol 99:435–444CrossRefGoogle Scholar
  3. 3.
    Seglen PO, Gordon PB, Tolleshaug H, Høyvik H (1986) Use of [3H]raffinose as a specific probe of autophagic sequestration. Exp Cell Res 162:273–277CrossRefGoogle Scholar
  4. 4.
    Høyvik H, Gordon PB, Seglen PO (1986) Use of a hydrolysable probe, [14C]lactose, to distinguish between pre-lysosomal and lysosomal steps in the autophagic pathway. Exp Cell Res 166:1–14CrossRefGoogle Scholar
  5. 5.
    Kopitz J, Kisen GØ, Gordon PB, Bohley P, Seglen PO (1990) Non-selective autophagy of cytosolic enzymes in isolated rat hepatocytes. J Cell Biol 111:941–953CrossRefGoogle Scholar
  6. 6.
    Seglen PO, Øverbye A, Sætre F (2009) Sequestration assays for mammalian autophagy. Methods Enzymol 452:63–83CrossRefGoogle Scholar
  7. 7.
    Engedal N, Torgersen ML, Guldvik IJ, Barfeld SJ, Bakula D, Sætre F, Hagen LK, Patterson JB, Proikas-Cezanne T, Seglen PO, Simonsen A, Mills IG (2013) Modulation of intracellular calcium homeostasis blocks autophagosome formation. Autophagy 9:1475–1490CrossRefGoogle Scholar
  8. 8.
    Seglen PO, Luhr M, Mills IG, Sætre F, Szalai P, Engedal N (2015) Macroautophagic cargo sequestration assays. Methods 75:25–36CrossRefGoogle Scholar
  9. 9.
    Luhr M, Szalai P, Sætre F, Gerner L, Seglen PO, Engedal N (2017) A simple cargo sequestration assay for quantitative measurement of nonselective autophagy in cultured cells. Methods Enzymol 587:351–364CrossRefGoogle Scholar
  10. 10.
    Strømhaug PE, Berg TO, Fengsrud M, Seglen PO (1998) Purification and characterization of autophagosomes from rat hepatocytes. Biochem J 335:217–224CrossRefGoogle Scholar
  11. 11.
    Berg TO, Fengsrud M, Strømhaug PE, Berg T, Seglen PO (1998) Isolation and characterization of rat liver amphisomes: Evidence for fusion of autophagosomes with both early and late endosomes. J Biol Chem 273:21883–21892CrossRefGoogle Scholar
  12. 12.
    Øverbye A, Sætre F, Hagen LK, Johansen HT, Seglen PO (2011) Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10) generated in amphisomes by the asparaginyl proteinase, legumain. Autophagy 7:1011–1027CrossRefGoogle Scholar
  13. 13.
    Klionsky DJ, Elazar Z, Seglen PO, Rubinsztein DC (2008) Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Autophagy 4:849–850CrossRefGoogle Scholar
  14. 14.
    Szalai P, Hagen LK, Sætre F, Luhr M, Sponheim M, Øverbye A, Mills IG, Seglen PO, Engedal N (2015) Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs. Exp Cell Res 333:21–38CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Centre for Molecular Medicine Norway (NCMM), Nordic EMBL PartnershipUniversity of OsloOsloNorway

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