Measuring Autophagic Cargo Flux with Keima-Based Probes

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

  • Nikolai Engedal
  • Tonje Sønstevold
  • Carsten J. Beese
  • Sarvini Selladurai
  • Thea Melcher
  • Julia E. Simensen
  • Frankel, Lisa
  • Alfonso Urbanucci
  • Maria L. Torgersen

Autophagy and autophagy-associated genes are implicated in a growing list of cellular, physiological, and pathophysiological processes and conditions. Therefore, it is ever more important to be able to reliably monitor and quantify autophagic activity. Whereas autophagic markers, such as LC3 can provide general indications about autophagy, specific and accurate detection of autophagic activity requires assessment of autophagic cargo flux. Here, we provide protocols on how to monitor bulk and selective autophagy by the use of inducible expression of exogenous probes based on the fluorescent coral protein Keima. To exemplify and demonstrate the power of this system, we provide data obtained by analyses of cytosolic and mitochondrially targeted Keima probes in human retinal epithelial cells treated with the mTOR-inhibitor Torin1 or with the iron chelator deferiprone (DFP). Our data indicate that Torin1 induces autophagic flux of cytosol and mitochondria to a similar degree, that is, compatible with induction of bulk autophagy, whereas DFP induces a highly selective form of mitophagy that efficiently excludes cytosol.

Original languageEnglish
Title of host publicationAutophagy and Cancer : Methods and Protocols
Number of pages17
PublisherHumana Press
Publication date2022
Pages99-115
ISBN (Print)978-1-0716-2070-0
ISBN (Electronic)978-1-0716-2073-1
DOIs
Publication statusPublished - 2022
SeriesMethods in Molecular Biology
Volume2445
ISSN1064-3745

Bibliographical note

Publisher Copyright:
© 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

    Research areas

  • Autophagic cargo flux, Autophagy, Bulk autophagy, Deferiprone, LDHB-mKeima, Mito-mKeima, Mitophagy, mKeima, Selective autophagy, Torin1

ID: 342675542