Measuring Autophagic Cargo Flux with Keima-Based Probes

Publikation: Bidrag til bog/antologi/rapportBidrag til bog/antologiForskningfagfællebedømt

  • 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.

OriginalsprogEngelsk
TitelAutophagy and Cancer : Methods and Protocols
Antal sider17
ForlagHumana Press
Publikationsdato2022
Sider99-115
ISBN (Trykt)978-1-0716-2070-0
ISBN (Elektronisk)978-1-0716-2073-1
DOI
StatusUdgivet - 2022
NavnMethods in Molecular Biology
Vol/bind2445
ISSN1064-3745

Bibliografisk note

Funding Information:
The following core facilities at Oslo University Hospital, Institute for Cancer Research, are acknowledged for providing access to equipment and expertise: The Core Facility for Confocal Microscopy and the Core Facility for Flow Cytometry. This work was supported by The Research Council of Norway (NANO2021; project number 274574 to MLT and TS), The Norwegian Cancer Society (Project number 198016-2018 to AU and NE), and The South-Eastern Norway Regional Health Authority (AUTOprost; Project number 2021088 to NE and JES).

Funding Information:
The following core facilities at Oslo University Hospital, Institute for Cancer Research, are acknowledged for providing access to equipment and expertise: The Core Facility for Confocal Microscopy and the Core Facility for Flow Cytometry. This work was supported by The Research Council of Norway (NANO2021; project number 274574 to MLT and TS), The Norwegian Cancer

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

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