A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag

  1. Willa Wen-You Yim
  2. Hayashi Yamamoto  Is a corresponding author
  3. Noboru Mizushima  Is a corresponding author
  1. University of Tokyo, Japan
  2. Nippon Medical School, Japan

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 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 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 Haloligand when delivered into lysosomes by autophagy. Hence, the amount of free Haloligand 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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1, S1, S2, 3, and 4.

Article and author information

Author details

  1. Willa Wen-You Yim

    1Department of Biochemistry and Molecular Biology, University of Tokyo, Tokyo, Japan
    Competing interests
    No competing interests declared.
  2. Hayashi Yamamoto

    Nippon Medical School, Tokyo, Japan
    For correspondence
    hayashi-yamamoto@nms.ac.jp
    Competing interests
    No competing interests declared.
  3. Noboru Mizushima

    Department of Biochemistry and Molecular Biology, University of Tokyo, Tokyo, Japan
    For correspondence
    nmizu@m.u-tokyo.ac.jp
    Competing interests
    Noboru Mizushima, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6258-6444

Funding

Japan Science and Technology Agency (Exploratory Research for Advanced Technology (ERATO),JPMJER1702)

  • Noboru Mizushima

Japan Society for the Promotion of Science (Transformative Research Areas (A),21H05256)

  • Hayashi Yamamoto

Japan Society for the Promotion of Science (Specially Promoted Research,22H04919)

  • Noboru Mizushima

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2022, Yim et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 13,068
    views
  • 2,757
    downloads
  • 89
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Willa Wen-You Yim
  2. Hayashi Yamamoto
  3. Noboru Mizushima
(2022)
A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag
eLife 11:e78923.
https://doi.org/10.7554/eLife.78923

Share this article

https://doi.org/10.7554/eLife.78923

Further reading

    1. Cell Biology
    2. Immunology and Inflammation
    Alejandro Rosell, Agata Adelajda Krygowska ... Esther Castellano Sanchez
    Research Article

    Macrophages are crucial in the body’s inflammatory response, with tightly regulated functions for optimal immune system performance. Our study reveals that the RAS–p110α signalling pathway, known for its involvement in various biological processes and tumourigenesis, regulates two vital aspects of the inflammatory response in macrophages: the initial monocyte movement and later-stage lysosomal function. Disrupting this pathway, either in a mouse model or through drug intervention, hampers the inflammatory response, leading to delayed resolution and the development of more severe acute inflammatory reactions in live models. This discovery uncovers a previously unknown role of the p110α isoform in immune regulation within macrophages, offering insight into the complex mechanisms governing their function during inflammation and opening new avenues for modulating inflammatory responses.

    1. Cell Biology
    Affiong Ika Oqua, Kin Chao ... Alejandra Tomas
    Research Article

    G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics, and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering, and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active vs inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these, Val229, to alanine on GLP-1R-cholesterol interactions, plasma membrane behaviours, clustering, trafficking and signalling in INS-1 832/3 rat pancreatic beta cells and primary mouse islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study (1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; (2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; (3) validates their functional relevance in beta cells; and (4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.