Abstract
In conditions of cellular stress and nutrient shortage, macroautophagy (hereafter referred to as autophagy) assures the degradation of dysfunctional macromolecules and organelles as it liberates energy resources via the breakdown of dispensable cellular components. Morphologically, autophagy is characterized by the formation of double-membraned autophagosomes that facilitate the isolation of autophagic cargo for subsequent lysosomal degradation at low pH. Sequestosome-1 (SQSTM1, better known as ubiquitin-binding protein p62), is an autophagosomal cargo receptor that targets proteins for selective autophagic degradation. Indeed, the redistribution of tandem mCherry and enhanced green fluorescent protein (mCherry-EGFP)-conjugated p62 from the cytosol into nascent autophagosomes constitutes a phenotype applicable to microscopic analysis. Furthermore, the differential pH sensitivity of mCherry and EGFP allows the visualization of autophagic flux due to the selective decrease of the EGFP signal upon fusion of autophagosomes with lysosomes. Here, we describe a method employing automated confocal cellular imaging for the study of autophagic degradation that is amenable to systems biology approaches.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Levine B, Sinha S, Kroemer G (2008) Bcl-2 family members: dual regulators of apoptosis and autophagy. Autophagy 4:600–606. https://doi.org/10.4161/AUTO.6260
Levine B, Kroemer G (2019) Biological functions of autophagy genes: a disease perspective. Cell 176:11. https://doi.org/10.1016/J.CELL.2018.09.048
López-Otín C, Kroemer G (2021) Hallmarks of health. Cell 184:33–63. https://doi.org/10.1016/J.CELL.2020.11.034
Levy JMM, Towers CG, Thorburn A (2017) Targeting autophagy in cancer. Nat Rev Cancer 17:9 17:528–542. https://doi.org/10.1038/nrc.2017.53
Kim KH, Lee M-S (2014) Autophagy—a key player in cellular and body metabolism. Nat Rev Endocrinol 10:322–337. https://doi.org/10.1038/nrendo.2014.35
Deretic V, Kroemer G (2021) Autophagy in metabolism and quality control: opposing, complementary or interlinked functions? Autophagy. https://doi.org/10.1080/15548627.2021.1933742
Mizushima N (2020) The ATG conjugation systems in autophagy. Curr Opin Cell Biol 63:1–10. https://doi.org/10.1016/J.CEB.2019.12.001
Ponpuak M, Mandell MA, Kimura T et al (2015) Secretory autophagy. Curr Opin Cell Biol 35:106–116. https://doi.org/10.1016/J.CEB.2015.04.016
Katsuragi Y, Ichimura Y, Komatsu M (2015) p62/SQSTM1 functions as a signaling hub and an autophagy adaptor. FEBS J 282:4672–4678. https://doi.org/10.1111/FEBS.13540
Lamark T, Svenning S, Johansen T (2017) Regulation of selective autophagy: the p62/SQSTM1 paradigm. Essays Biochem 61:609–624. https://doi.org/10.1042/EBC20170035
Yang KC, Sathiyaseelan P, Ho C, Gorski SM (2018) Evolution of tools and methods for monitoring autophagic flux in mammalian cells. Biochem Soc Trans 46:97–110. https://doi.org/10.1042/BST20170102
Zhou C, Zhong W, Zhou J et al (2012) Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells. Autophagy 8:1215–1226. https://doi.org/10.4161/AUTO.20284
Bravo-San Pedro JM, Pietrocola F, Sica V et al (2017) High-throughput quantification of GFP-LC3+ dots by automated fluorescence microscopy. Methods Enzymol 587:71–86. https://doi.org/10.1016/BS.MIE.2016.10.022
Kepp O, Chen G, Carmona-Gutierrez D et al (2019) A discovery platform for the identification of caloric restriction mimetics with broad health-improving effects. Autophagy 16:188–189. https://doi.org/10.1080/15548627.2019.1688984
Wu Q, Tian A-L, Li B et al (2021) IGF1 receptor inhibition amplifies the effects of cancer drugs by autophagy and immune-dependent mechanisms. J Immunother Cancer 9:e002722. https://doi.org/10.1136/JITC-2021-002722
Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3:542–545. https://doi.org/10.4161/AUTO.4600
Yoshii SR, Mizushima N (2017) Monitoring and measuring autophagy. Int J Mol Sci 18:1865. https://doi.org/10.3390/IJMS18091865
Pankiv S, Clausen TH, Lamark T et al (2007) p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of Ubiquitinated protein aggregates by autophagy. J Biol Chem 282:24131–24145. https://doi.org/10.1074/JBC.M702824200
Klionsky DJ, Abdalla FC, Abeliovich H et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445–544. https://doi.org/10.4161/AUTO.19496
Acknowledgments
O.K. receives funding from the DIM Elicit (Ile de France) and the Institut National du Cancer (INCa). G.K. is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) – Projets blancs; AMMICa US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Association “Ruban Rose”; Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); a donation by Elior; Equipex Onco-Pheno-Screen; European Joint Programme on Rare Diseases (EJPRD); Gustave Roussy Odyssea, the European Union Horizon 2020 Projects Oncobiome and Crimson; Fondation Carrefour; Institut National du Cancer (INCa); Inserm (HTE); Institut Universitaire de France; LabEx Immuno-Oncology (ANR-18-IDEX-0001); the Leducq Foundation; the RHU Torino Lumière; Seerave Foundation; SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and SIRIC Cancer Research and Personalized Medicine (CARPEM). This study contributes to the IdEx Université de Paris ANR-18-IDEX-0001. The mCherry-EGFP-p62 plasmid was a kind gift of Terje Johansen.
Conflict of Interest
G.K. and O.K. are cofounders of Samsara Therapeutics. G.K. is a founder of everImmune and Therafast Bio.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Jin, H., Wu, Q., Kroemer, G., Kepp, O. (2022). A Fluorescence-Microscopic System for Monitoring the Turnover of the Autophagic Substrate p62/SQSTM1. In: Barcenilla, H., Diaz, D. (eds) Apoptosis and Cancer. Methods in Molecular Biology, vol 2543. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2553-8_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2553-8_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2552-1
Online ISBN: 978-1-0716-2553-8
eBook Packages: Springer Protocols