Monitoring Autophagy Flux and Activity: Principles and Applications

Macroautophagy is a major degradation mechanism of cell components via the lysosome. Macroautophagy greatly contributes to not only cell homeostasis but also the prevention of various diseases. Because macroautophagy proceeds through multi‐step reactions, researchers often face a persistent question...

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Bibliographic Details
Published in:BioEssays 2020-11, Vol.42 (11), p.e2000122-n/a
Main Authors: Ueno, Takashi, Komatsu, Masaaki
Format: Article
Language:English
Subjects:
Assaying
autolysosome
autophagosome
Autophagy
autophagy‐flux
Degradation
GABARAP protein
gamma‐aminobutyric acid receptor‐associated protein
Homeostasis
microtubule‐associated proteins 1A/1B light chain 3B
Monitoring
Monitoring methods
Phagocytosis
Principles
Proteins
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Summary:Macroautophagy is a major degradation mechanism of cell components via the lysosome. Macroautophagy greatly contributes to not only cell homeostasis but also the prevention of various diseases. Because macroautophagy proceeds through multi‐step reactions, researchers often face a persistent question of how macroautophagic activity can be measured correctly. To make a straightforward determination of macroautophagic activity, diverse monitoring assays have been developed. Direct measurement of lysosome‐dependent degradation of radioisotopically labeled cell proteins has long been applied. Meanwhile, indirect monitoring procedures have been developed. In these assays, autophagosome marker proteins, microtubule‐associated proteins 1A/1B light chain 3B‐II (LC3B‐II) and gamma‐aminobutyric acid receptor‐associated protein‐II (GABARAP‐II) have been analyzed and the validity of the assays strongly depends on appropriate assessment of the fluctuation of LC3‐II and/or GABARAP‐II levels in the presence or absence of lysosomal inhibitors. This article describes these monitoring methods, paying special attention to the principles and characteristics of each procedure. Upon induction of autophagy, autophagy flux can be measured by two categories of monitoring assays; one is a direct determination of amino acids released from autolysosomes via lysosomal inhibitor‐sensitive degradation (upper figure) and the other is an indirect determination of microtubule‐associated proteins 1A/1B light chain 3B‐II and/or gamma‐aminobutyric acid receptor‐associated protein‐II accumulating in the presence of lysosomal inhibitors (middle figure).
ISSN:0265-9247
1521-1878
DOI:10.1002/bies.202000122