A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag
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Curated by eLife
Evaluation Summary:
This paper will be of interest to researchers in the autophagy field. It provides a useful tool to accurately measure autophagy flux, providing a useful alternative to the existing assay. The key claims of the manuscript are well supported by the data, and the approaches used are thoughtful and rigorous.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Monitoring autophagic flux is necessary for most autophagy studies. The autophagic flux assays currently available for mammalian cells are generally complicated and do not yield highly quantitative results. Yeast autophagic flux is routinely monitored with the green fluorescence protein (GFP)-based processing assay, whereby the amount of GFP proteolytically released from GFP-containing reporters (e.g. GFP-Atg8), detected by immunoblotting, reflects autophagic flux. However, this simple and effective assay is typically inapplicable to mammalian cells because GFP is efficiently degraded in lysosomes while the more proteolytically resistant red fluorescent protein (RFP) accumulates in lysosomes under basal conditions. Here, we report a HaloTag (Halo)-based reporter processing assay to monitor mammalian autophagic flux. We found that Halo is sensitive to lysosomal proteolysis but becomes resistant upon ligand binding. When delivered into lysosomes by autophagy, pulse-labeled Halo-based reporters (e.g. Halo-LC3 and Halo-GFP) are proteolytically processed to generate Halo ligand when delivered into lysosomes by autophagy. Hence, the amount of free Halo ligand detected by immunoblotting or in-gel fluorescence imaging reflects autophagic flux. We demonstrate the applications of this assay by monitoring the autophagy pathways, macroautophagy, selective autophagy, and even bulk nonselective autophagy. With the Halo-based processing assay, mammalian autophagic flux and lysosome-mediated degradation can be monitored easily and precisely.
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Evaluation Summary:
This paper will be of interest to researchers in the autophagy field. It provides a useful tool to accurately measure autophagy flux, providing a useful alternative to the existing assay. The key claims of the manuscript are well supported by the data, and the approaches used are thoughtful and rigorous.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript, authors found Halo tag become resistant to lysosome degradation upon ligand binding, using this unique property, they developed a highly sensitive assay to monitor the autophagy flux. Measuring autophagy flux is one of the most important assays for studying autophagy, there are a few widely used assays to monitor the autophagy flux, such as p62 degradation, and LC3 processing, however, each of them has its own limitation, which is well known in the field. In this regard, this assay provides a simple, straight forward and sensitive assay for measuring autophagy flux, which I personally think is very likely it will be widely used by the autophagy community. This is a well-controlled, rigorous study and the manuscript is clearly written.
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Reviewer #2 (Public Review):
Yim et al have utilised the HaloTag system to generate tools and assays to measure autophagy flux. The assays are highly accessible and straight forward to conduct. The study does not have any major weaknesses, with all key conclusions strongly supported by clear data. A major strength of the study is the robustness of the assay and its ease of use across SDS-PAGE and imaging techniques that I expect will help with its uptake by the research community. The assay utilises the HaLo tag and its inherent stability within lysosomes once pulsed with a HaLo ligand. This enables analysis of autophagy flux over a set period of time. The approach is highly complementary to the recently published study by Rudinskiy et al (2022) MBoC, but also includes additional tools to measure different types of selective autophagy …
Reviewer #2 (Public Review):
Yim et al have utilised the HaloTag system to generate tools and assays to measure autophagy flux. The assays are highly accessible and straight forward to conduct. The study does not have any major weaknesses, with all key conclusions strongly supported by clear data. A major strength of the study is the robustness of the assay and its ease of use across SDS-PAGE and imaging techniques that I expect will help with its uptake by the research community. The assay utilises the HaLo tag and its inherent stability within lysosomes once pulsed with a HaLo ligand. This enables analysis of autophagy flux over a set period of time. The approach is highly complementary to the recently published study by Rudinskiy et al (2022) MBoC, but also includes additional tools to measure different types of selective autophagy and bulk autophagy. The inclusion of limitations of their approach within the discussion will be very useful for researchers planning to use the assay in their work. Overall, this is an excellent study that has generated very valuable tools for the study of autophagy.
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Reviewer #3 (Public Review):
Monitoring autophagy induction and flux in mammalian cells is challenging and depends largely on the mammalian ATG8 proteins (LC3 and GABARAP), typically tagged at the N-terminus with a small tag (HA, flag, myc) or a range of fluorescent tags. When autophagy is induced these ATG8 proteins get captured into autophagosomes and delivered to lysosomes for degradation. Monitoring flux by western blots relies on a molecular weight shift caused by lipidation, and quantification of loss of signal from degradation (analysis of initiation), or accumulation by the addition of inhibition of lysosomal inhibitors (analyses of flux). Fluorescent tags provide similar results but the measurements rely on counting degradation sensitive or resistant fluorescent signals. Image-based analysis is more challenging than western …
Reviewer #3 (Public Review):
Monitoring autophagy induction and flux in mammalian cells is challenging and depends largely on the mammalian ATG8 proteins (LC3 and GABARAP), typically tagged at the N-terminus with a small tag (HA, flag, myc) or a range of fluorescent tags. When autophagy is induced these ATG8 proteins get captured into autophagosomes and delivered to lysosomes for degradation. Monitoring flux by western blots relies on a molecular weight shift caused by lipidation, and quantification of loss of signal from degradation (analysis of initiation), or accumulation by the addition of inhibition of lysosomal inhibitors (analyses of flux). Fluorescent tags provide similar results but the measurements rely on counting degradation sensitive or resistant fluorescent signals. Image-based analysis is more challenging than western blot but both require significant optimization. In this manuscript these existing assays are modified by the use of a probe (Halo tag) again appended to the N-terminus of ATG8s which becomes resistant to lysosomal degradation after binding a ligand (TMR). The ligand can be pulsed-in to allow detection of acute induction of autophagy eliminating the background from basal accumulation. Generation of the Halo-TMR is then monitored by western blot or using an in gel-fluorescent assay. The authors present data which shows the adaptability of the system for imaging analysis, and for both quantitative analysis using western blot and imaging of selective autophagy or bulk, non-selective autophagy. The authors have developed a robust, useful alternative to existing assay and present the results in a careful, well described brief manuscript. These modifications are important for the field and for those who require quantitative results. The drawbacks are similar to existing assays and will usually require the generation of stable cell lines because over-expressed ATG8s can aggregate and confound the measurements.
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