LC3, an Autophagosome Marker, Can be Incorporated into Protein Aggregates Independent of Autophagy: Caution in the Interpretation of LC3 Localization

By Akiko Kuma, M. Matsui, N. Mizushima. Autophagy, 2007

595 citations recorded by the source corpus.

Autophagy is an intracellular bulk degradation system, through which a portion of the cytoplasm is delivered to lysosomes to be degraded. Microtuble-associated protein light chain 3 (LC3), a mammalian homolog of yeast Atg8, has been used as a specific marker to monitor autophagy. Upon induction of autophagy, LC3 is conjugated to phosphatidylethanolamine and targeted to autophagic membranes. Therefore, changes in LC3 localization have been used to measure autophagy. However, this method has some limitations. In this report, we show that LC3 protein tends to aggregate in an autophagy-independent manner when it is transiently overexpressed by transfection. In addition, LC3 is easily incorporated into intracellular protein aggregates, such as inclusion bodies induced by polyQ expression or formed in autophagy-deficient hepatocytes, neurons, or senescent fibroblasts. These findings demonstrate that punctate dots containing LC3 do not always represent autophagic structures. Therefore, LC3 localization should be carefully interpreted, particularly if LC3 is overexpressed by transient transfection or if aggregates are formed within cells.

Subfields: Epidemiology, Physiology

View Paper: https://www.semanticscholar.org/paper/5d5fb30a41e0817fd56223097de7b548940031cf

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Citations in the corpus

Measuring in vivo mitophagy (2015, background): “Given the transient nature of the interaction between autophagosome and mitochondria and the fact that LC3-based fluorescent probes may have a high rate of false positive signals (Kuma et al., 2007), these methods can suffer from a lack of sensitivity and specificity.”
Molecular definitions of autophagy and related processes (2017, unknown): “Importantly, LC3 can accumulate at protein aggregates in a p62-dependent but autophagosome-independent manner (Kuma et al , 2007; Shvets & Elazar, 2008).”
Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. (2008, background): “In HeLa cells stably expressing GFP-LC3, GFP-LC3 was mostly present in the nucleus under normal conditions, although its physiological significance is unknown (Kuma et al., 2007).”
Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. (2009, background): “GFP-LC3 was detected diffusely and enriched in the nucleus under normal conditions, although its physiological significance remains unknown (Kuma et al., 2007).”
FoxO3 controls autophagy in skeletal muscle in vivo. (2007, background): “aggregation rather than autophagy (Kuma et al., 2007).”
Guidelines for the use and interpretation of assays for monitoring autophagy (2012, background): “An important caveat in the use of GFP-LC3 is that this chimera can associate with aggregates, especially when expressed at high levels in the presence of aggregate-prone proteins, which can lead to a misinterpretation of the results.(224) Of note, GFP-LC3 can associate with ubiquitinated protein agg”
Toll‐like receptors control autophagy (2008, methodology): “…is a reflection of autophagy activation &2008 European Molecular Biology Organization The EMBO Journal VOL 27 | NO 7 | 2008 1111 and not nonspecific protein aggregation (Kuma et al, 2007), we employed a control based on GFP fusion with a mutant LC3 form, LC3DC22,G120A (Kabeya et al, 2000).”
HDAC6 controls autophagosome maturation essential for ubiquitin‐selective quality‐control autophagy (2010, background): “Importantly, this staining is not caused by non-specific incorporation of LC3 into protein aggregates (Kuma et al, 2007), as no LC3positive structures were observed in ATG5-deficient MEFs subject to the same treatment (Figure 1D and Supplementary Figure S1A).”