Autophagy is an essential process that cells utilize to degrade and recycle damaged material and fuel metabolism, especially under stress. The process is evolutionarily conserved and complex, relying on over 20 key proteins. Induction of autophagy is mediated by the formation of the ULK and BECLIN complexes, the latter of which includes BH3-containing proteins and AMBRA1, resulting in the formation of a double membrane phagophore structure. Damaged cytoplasmic material is engulfed in the elongating phagophore yielding an autophagosome: a step dependent on two ubiquitin-like conjugation systems including the conjugation of PE and LC3 to generate LC3-II and the conjugation of ATG5 and ATG12. Once the autophagosome is formed it can fuse with the lysosome and facilitate degradation of its constituents due to the low pH1.
Missregulation of autophagy plays a role in many diseases including neurological diseases and cancer1. While the process has been heavily studied, researchers are still in search of the most appropriate assay to quantify autophagy. One of the most commonly used assays relies on overexpression of a tandem LC3 molecule fused with mCherry and EGFP which takes advantage of the stable mCherry fluorescence in a low pH environment as compared to EGFP, a fluorescent molecule that is quenched in an acidic compartment2. The ratio of mCherry to GFP can be visualized with microscopy or quantified by flow cytommetry3. Although this assay can be quantitative and monitor autophagic flux, the most noteworthy caveat is the artificial overexpression of LC3.
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Immunocytochemistry/Immunofluorescence: p62/SQSTM1 Antibody (1478) [NBP2-43663] - Analysis of Samples: HeLa cells mock (left) and treated with 50uM Chloroquine for 24 hours (right) were fixed in 4% paraformaldehyde at RT for 15 min. (Green): SQSTM1 protein stained by SQSTM1 antibody [1478] diluted at 1:1000. (Red): phalloidin, a cytoskeleton marker, stained by phalloidin diluted at 1:200. (Blue): Hoechst 33342 staining.
Alternative assays monitor the expression of endogenous core autophagy proteins that are themselves degraded through the process, including LC3 and the autophagy receptor p62. With the correct controls, including autophagy induction and lysosomal inhibition, these assays can reliably monitor autophagic flux. It is critical, however, to have clean, specific antibodies. This is especially true in the case of LC3 as the cleavage product (LC3B-II) is the accurate read out for autophagy, as opposed to the uncleaved, LC3-I. Immunofluorescence is also used to monitor LC3-II puncta, and again is reliant on specific antibodies4. Recent reports have indicated specific roles for the different isoforms of LC3, including LC3A, LC3B, and LC3C5 underlying the importance of antibodies that are specific for each. Other valuable, yet imperfect assays that are commonly used to monitor autophagy include radiolabeling of long-lived proteins to measure their turn over, electron microscopy to identify double membrane autophagosome structures, and fluorescent labeling of core autophagic machinery, i.e. the snare protein STX17 that is necessary for autophagosome fusion with the lysosome2,6.
With all these assays, it is imperative to have appropriate controls. The most relevant negative control is inhibition of autophagy2. Genetic inhibition can be achieved with targeted RNAi oligos that can specifically and effectively target core autophagy proteins such as ATG5. Induction of autophagy can function as an appropriate positive control and is achieved by induced stress such as starvation. While all these assays have advantages and caveats, researchers believe that the combined use of several techniques can provide quantitative and accurate analysis of autophagy.
By Christina Towers, PhD
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