Measuring Autophagic Cargo Flux with Keima-Based Probes

Methods Mol Biol. 2022;2445:99-115. doi: 10.1007/978-1-0716-2071-7_7.

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.

Keywords: Autophagic cargo flux; Autophagy; Bulk autophagy; Deferiprone; LDHB-mKeima; Mito-mKeima; Mitophagy; Selective autophagy; Torin1; mKeima.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy* / physiology
  • Microtubule-Associated Proteins* / metabolism
  • Mitochondria / metabolism
  • Mitophagy

Substances

  • Microtubule-Associated Proteins