Trends in Biochemical Sciences, Journal Year: 2020, Volume and Issue: 45(12), P. 1080 - 1093

Published: Aug. 21, 2020

Autophagy is a fundamental process required for normal physiology and disruptions can cause disease; but measuring autophagy be challenging.By using specific flux assays mechanistic readouts, it possible to reliably interpret the status of in experimental systems.High-throughput small molecule genetic screens have accelerated discovery basic mechanisms as well potential drug targets.Identifying biomarkers use clinical development represents major opportunity future. lysosome-dependent intracellular degradation system various physiological processes dysregulated human disease. To understand its biological significance underlying mechanisms, autophagic activity (i.e., flux) critical. However, navigating which use, when, complicated at times results are often interpreted inappropriately. This review will summarize both advantages disadvantages currently available methods monitor autophagy. In addition, we discuss how these should used high-throughput identify autophagy-modulating drugs genes general features needed assess humans. that facilitates lysosomal components [1.Mizushima N. Komatsu M. Autophagy: renovation cells tissues.Cell. 2011; 147: 728-741Abstract Full Text PDF PubMed Scopus (2717) Google Scholar,2.Levine B. Kroemer G. Biological functions genes: disease perspective.Cell. 2019; 176: 11-42Abstract (283) Scholar]. There several types autophagy: macroautophagy (see Glossary) Scholar], microautophagy [3.Oku Sakai Y. Three distinct based on membrane dynamics molecular machineries.Bioessays. 2018; 40e1800008Crossref (55) chaperone-mediated (CMA) (and related called RNautophagy/DNautophagy) [4.Kaushik S. Cuervo A.M. The coming age autophagy.Nat. Rev. Mol. Cell Biol. 19: 365-381Crossref (184) Scholar,5.Fujiwara et al.Lysosomal nucleic acids-multiple pathways.J. Biochem. 2017; 161: 145-154PubMed Scholar] (Figure 1). important not only constitutive turnover components, also active elimination abnormal or potentially damaging materials access by-products degradation, such amino acids, during starvation Due role homeostasis, defects linked diseases [2.Levine Therefore, has been extensively studied across biomedical field demand accurate measure increasing. still easy simple, especially mammals [6.Mizushima Yoshimori T. How LC3 immunoblotting.Autophagy. 2007; 3: 542-545Crossref Scholar, 7.Mizushima al.Methods mammalian research.Cell. 2010; 140: 313-326Abstract (2901) 8.Klionsky D.J. al.Guidelines interpretation monitoring (3rd edition).Autophagy. 2016; 12: 1-222Crossref (2843) 9.Yoshii S.R. Mizushima Monitoring autophagy.Int. J. Sci. 18: 1865Crossref (225) summarizes (used interchangeably with hereafter) systems discover chemical probes modulate Finally, applied research development, timely need biomarkers. Autophagic typically measured biochemically amount cytoplasm-derived material lysosomes observed per unit time. order flux, necessary directly quantify autophagy-dependent cellular reporters capable representing cumulative degradation. Historically, radiometric long-lived protein were adapted determine rate proteolysis. Although no longer vogue, this remains one gold-standard approaches definitively endogenous proteolysis, including Electron microscopy observe characterize structures under different physio-pathological settings. misunderstood, number autophagosomes does necessarily indicate because autophagosome could increase either by induction reduced consumption lysosomes. latter occur due dysfunction. During early stages autophagy, cisterna, termed isolation phagophore, encloses portion cytoplasm eventually expands become an autophagosome, then fuses degrade sequestered A strategy shown Figure 2. If data suggest accumulation 2, Step 1), essential if there 2). After highly recommended probe autophagy-inducing signals confirm 3). Conversely, when decrease suggested, efficiency autophagosome–lysosome fusion function checked, addition signals. most commonly method autophagosomal ATG8 homologs GABARAP family proteins (collectively referred ATG8s) bind 1) [10.Mizushima ATG conjugation autophagy.Curr. Opin. 2020; 63: 1-10Crossref (22) Cytosolic (called ATG8-I) covalently conjugated phosphatidylethanolamine phagophore membranes through C terminal glycine residue becomes lipidated form ATG8-II. ATG8-II binds outer inner membranes, roughly correlates autophagosomes. lysosomes, gradually deconjugated recycled, whereas degraded estimated It noted may strictly macroautophagy; some adaptors sequestosome 1 (SQSTM1, known p62) NDP52 endosomal [11.Mejlvang al.Starvation induces rapid selective receptors microautophagy.J. 217: 3640-3655Crossref (39) single later) [12.Florey O. al.V-ATPase osmotic imbalances activate endolysosomal lipidation.Autophagy. 2015; 11: 88-99Crossref (65) Among ATG8s, LC3B was identified first widely (often simply LC3). Thus, generally 'LC3' unless information other homologs. When induced, example, starvation, increases 3A ). much actually determined comparing treated without inhibitors 3A). enhanced, difference between treatment groups high, inhibited, negligible For nutrient cell types, lysosome inhibition significant levels, indicating high flux. By contrast, change level, means low, even basally high. versatile performed immunoblot analysis against (e.g., caveats. First, case immunoblotting, semiquantitative dynamic range detection narrow. Second, prepare compare two samples inhibitors. More importantly, suppress mTOR activity, secondarily [13.Juhasz Interpretation bafilomycin, pH neutralizing protease inhibitor treatments experiments: novel considerations.Autophagy. 2012; 8: 1875-1876Crossref (0) Scholar,14.Li al.Suppression via feedback downregulation MTORC1 activity.J. Chem. 2013; 288: 35769-35780Crossref Third, care must taken selecting type concentration Commonly V-ATPase bafilomycin A1, lysosomotropic reagents chloroquine, enzyme pepstatin E64d. stress promote formation endolysosomes mechanism similar LC3-associated phagocytosis, from canonical 100 μM chloroquine LC3-II autophagy-independent manner Scholar,15.Jacquin E. al.Pharmacological modulators parallel noncanonical pathway driving unconventional 13: 854-867Crossref (41) significantly affecting measurement, low concentrations 25 μM) [15.Jacquin evaluated levels substrates 3B). Such recognized physically bound and, assay, substrate substrates, SQSTM1/p62 frequently used. emphasized expression regulated transcriptional level. SQSTM1 cases oxidative stress, represent overproduction [16.Sanchez-Martin P. p62/SQSTM1 - steering health disease.J. 131jcs222836Crossref ATG8-binding index mRNA induction. Alternatively, problem avoided pulse-labeling p62 tagged HaloTag [17.Benyounes A. al.A fluorescence-microscopic cytofluorometric p62/SQSTM1.Autophagy. 7: 883-891Crossref (27) As mentioned earlier, simple fluorescent protein-based GFP-LC3 sufficient overcome issue, tandem protein-tagged (tfLC3), RFP (or another red mCherry) GFP N terminus any protein) developed [18.Pankiv al.p62/SQSTM1 Atg8/LC3 facilitate ubiquitinated aggregates autophagy.J. 282: 24131-24145Crossref (2651) Scholar,19.Kimura al.Dissection maturation reporter protein, fluorescent-tagged LC3.Autophagy. 452-460Crossref 3C). emits green fluorescence appear yellow images merged. within acidic environment autolysosome, immediately quenched, leaving signal, unaffected pH. Following (indicating more autophagosomes) puncta autolysosomes). inhibition, dominate, indicates reduction and/or autolysosome. suppressed, reduced. 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