Abstract
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.
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Acknowledgments
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).
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Engedal, N. et al. (2022). Measuring Autophagic Cargo Flux with Keima-Based Probes. In: Norberg, H., Norberg, E. (eds) Autophagy and Cancer. Methods in Molecular Biology, vol 2445. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2071-7_7
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DOI: https://doi.org/10.1007/978-1-0716-2071-7_7
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Online ISBN: 978-1-0716-2071-7
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