Autophagy Assays for Biological Discovery and Therapeutic Development
2020; Elsevier BV; Volume: 45; Issue: 12 Linguagem: Inglês
10.1016/j.tibs.2020.07.006
ISSN1362-4326
AutoresNoboru Mizushima, Leon O. Murphy,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoAutophagy is a fundamental process required for normal physiology and disruptions can cause disease; but measuring autophagy can be challenging.By using specific flux assays and mechanistic readouts, it is possible to reliably interpret the status of autophagy in experimental systems.High-throughput small molecule and genetic screens using autophagy assays have accelerated the discovery of basic mechanisms as well as potential drug targets.Identifying autophagy biomarkers for use in clinical development represents a major opportunity for the future. Autophagy is a lysosome-dependent intracellular degradation system required for various physiological processes and can be dysregulated in human disease. To understand its biological significance and underlying mechanisms, measuring autophagic activity (i.e., autophagic flux) is critical. However, navigating which assays to use, and when, is complicated and at times the results are often interpreted inappropriately. This review will summarize both advantages and disadvantages of currently available methods to monitor autophagy. In addition, we discuss how these assays should be used in high-throughput screens to identify autophagy-modulating drugs and genes and the general features needed for biomarkers to assess autophagy in humans. Autophagy is a lysosome-dependent intracellular degradation system required for various physiological processes and can be dysregulated in human disease. To understand its biological significance and underlying mechanisms, measuring autophagic activity (i.e., autophagic flux) is critical. However, navigating which assays to use, and when, is complicated and at times the results are often interpreted inappropriately. This review will summarize both advantages and disadvantages of currently available methods to monitor autophagy. In addition, we discuss how these assays should be used in high-throughput screens to identify autophagy-modulating drugs and genes and the general features needed for biomarkers to assess autophagy in humans. Autophagy is a process that facilitates lysosomal degradation of intracellular components [1.Mizushima N. Komatsu M. Autophagy: renovation of cells and tissues.Cell. 2011; 147: 728-741Abstract Full Text Full Text PDF PubMed Scopus (2717) Google Scholar,2.Levine B. Kroemer G. Biological functions of autophagy genes: a disease perspective.Cell. 2019; 176: 11-42Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar]. There are several types of autophagy: macroautophagy (see Glossary) [1.Mizushima N. Komatsu M. Autophagy: renovation of cells and tissues.Cell. 2011; 147: 728-741Abstract Full Text Full Text PDF PubMed Scopus (2717) Google Scholar,2.Levine B. Kroemer G. Biological functions of autophagy genes: a disease perspective.Cell. 2019; 176: 11-42Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar], microautophagy [3.Oku M. Sakai Y. Three distinct types of microautophagy based on membrane dynamics and molecular machineries.Bioessays. 2018; 40e1800008Crossref PubMed Scopus (55) Google Scholar], and chaperone-mediated autophagy (CMA) (and a related process called RNautophagy/DNautophagy) [4.Kaushik S. Cuervo A.M. The coming of age of chaperone-mediated autophagy.Nat. Rev. Mol. Cell Biol. 2018; 19: 365-381Crossref PubMed Scopus (184) Google Scholar,5.Fujiwara Y. et al.Lysosomal degradation of intracellular nucleic acids-multiple autophagic pathways.J. Biochem. 2017; 161: 145-154PubMed Google Scholar] (Figure 1). Autophagy is important not only for constitutive turnover of intracellular components, but also for the active elimination of abnormal or potentially damaging materials and to access the by-products of degradation, such as amino acids, during starvation [1.Mizushima N. Komatsu M. Autophagy: renovation of cells and tissues.Cell. 2011; 147: 728-741Abstract Full Text Full Text PDF PubMed Scopus (2717) Google Scholar,2.Levine B. Kroemer G. Biological functions of autophagy genes: a disease perspective.Cell. 2019; 176: 11-42Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar]. Due to its important role in homeostasis, defects in autophagy are linked to several human diseases [2.Levine B. Kroemer G. Biological functions of autophagy genes: a disease perspective.Cell. 2019; 176: 11-42Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar]. Therefore, autophagy has been extensively studied across the biomedical field and the demand for accurate methods to measure autophagic activity has been increasing. However, measuring autophagy is still not easy or simple, especially in mammals [6.Mizushima N. Yoshimori T. How to interpret LC3 immunoblotting.Autophagy. 2007; 3: 542-545Crossref PubMed Google Scholar, 7.Mizushima N. et al.Methods in mammalian autophagy research.Cell. 2010; 140: 313-326Abstract Full Text Full Text PDF PubMed Scopus (2901) Google Scholar, 8.Klionsky D.J. et al.Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).Autophagy. 2016; 12: 1-222Crossref PubMed Scopus (2843) Google Scholar, 9.Yoshii S.R. Mizushima N. Monitoring and measuring autophagy.Int. J. Mol. Sci. 2017; 18: 1865Crossref PubMed Scopus (225) Google Scholar]. This review summarizes various methods for measuring the activity of macroautophagy (used interchangeably with autophagy hereafter) in mammalian systems and the use of these assays to discover chemical probes and genes that modulate autophagy. Finally, for applied research and clinical development, it is also timely to discuss the need for autophagy biomarkers. Autophagic flux is typically measured biochemically and is the amount of degradation of cytoplasm-derived material in lysosomes observed per unit time. In order to measure flux, it is necessary to directly quantify the amount of autophagy-dependent degradation of cellular components, or to use reporters capable of representing the cumulative amount of degradation. Historically, radiometric long-lived protein degradation assays were adapted to determine the rate of autophagy-dependent proteolysis. Although no longer in vogue, this remains one of the gold-standard approaches to definitively measure endogenous proteolysis, including autophagy. Electron microscopy has also been used to observe and characterize autophagic structures under different physio-pathological settings. However, as is often misunderstood, the number of autophagosomes does not necessarily indicate flux because autophagosome number could increase either by induction of autophagy or by the reduced consumption of autophagosomes by lysosomes. The latter can occur due to lysosomal dysfunction. During the early stages of autophagy, a membrane cisterna, termed the isolation membrane or phagophore, encloses a portion of the cytoplasm and eventually expands to become an autophagosome, which then fuses with lysosomes to degrade sequestered materials (Figure 1). A general strategy for measuring autophagic activity is shown in Figure 2. If experimental data suggest an accumulation of autophagosomes (Figure 2, Step 1), it is then essential to measure autophagic flux to determine the rate of induction or if there is reduced consumption (Figure 2, Step 2). After measuring autophagic flux, it is highly recommended to probe specific autophagy-inducing signals to confirm the flux data (Figure 2, Step 3). Conversely, when a decrease in autophagic flux is suggested, the efficiency of autophagosome–lysosome fusion or lysosomal function should be checked, in addition to the autophagy-inducing signals. The most commonly used method to measure autophagic flux is to monitor the turnover rate of the autophagosomal protein ATG8 and its homologs such as LC3 and GABARAP family proteins (collectively referred to as ATG8s) that bind to the autophagosomal membrane (Figure 1) [10.Mizushima N. The ATG conjugation systems in autophagy.Curr. Opin. Cell Biol. 2020; 63: 1-10Crossref PubMed Scopus (22) Google Scholar]. Cytosolic ATG8 (called ATG8-I) is covalently conjugated to phosphatidylethanolamine in the phagophore and autophagosomal membranes through its C terminal glycine residue and becomes a lipidated form termed ATG8-II. ATG8-II binds to both the outer and inner autophagosomal membranes, and the amount of ATG8-II roughly correlates with the number of autophagosomes. After fusion with lysosomes, ATG8-II on the outer membrane is gradually deconjugated and recycled, whereas ATG8-II on the inner membrane is degraded (Figure 1). Therefore, the activity of autophagy can be estimated by monitoring the amount of ATG8 degradation. It should be noted that ATG8 may not be strictly specific to macroautophagy; ATG8 and some of the autophagy adaptors such as sequestosome 1 (SQSTM1, also known as p62) and NDP52 can be degraded by endosomal microautophagy [11.Mejlvang J. et al.Starvation induces rapid degradation of selective autophagy receptors by endosomal microautophagy.J. Cell Biol. 2018; 217: 3640-3655Crossref PubMed Scopus (39) Google Scholar] and ATG8 can also be conjugated to single membranes (see later) [12.Florey O. et al.V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation.Autophagy. 2015; 11: 88-99Crossref PubMed Scopus (65) Google Scholar] (Figure 2). Among ATG8s, LC3B was identified first and has been widely used (often referred to simply as LC3). Thus, we will generally use 'LC3' unless we have information on other homologs. When autophagy is induced, for example, by starvation, the number of autophagosomes and the amount of ATG8-II generally also increases (Figure 3A ). However, to monitor autophagic flux, we should measure how much ATG8-II is actually degraded in lysosomes. This can be determined by comparing the amount of ATG8-II in cells treated with and without lysosomal inhibitors (Figure 3A). When autophagic flux is enhanced, the difference between these treatment groups will be high, but when flux or consumption is inhibited, the difference will be negligible [6.Mizushima N. Yoshimori T. How to interpret LC3 immunoblotting.Autophagy. 2007; 3: 542-545Crossref PubMed Google Scholar]. For example, during nutrient starvation in most cell types, lysosome inhibition results in a significant increase in the ATG8-II levels, indicating high autophagic flux. By contrast, if lysosome inhibition does not change the ATG8-II level, it means that autophagic activity is low, even if the amount of ATG8-II is basally high. This method is highly versatile because it can be performed by immunoblot analysis against endogenous ATG8 proteins (e.g., LC3). However, there are some caveats. First, as is generally the case with immunoblotting, it is semiquantitative and the dynamic range of detection is narrow. Second, it is necessary to prepare and compare two samples with and without lysosome inhibitors. More importantly, lysosomal inhibition could suppress mTOR activity, which secondarily induces autophagy [13.Juhasz G. Interpretation of bafilomycin, pH neutralizing or protease inhibitor treatments in autophagic flux experiments: novel considerations.Autophagy. 2012; 8: 1875-1876Crossref PubMed Scopus (0) Google Scholar,14.Li M. et al.Suppression of lysosome function induces autophagy via a feedback downregulation of MTORC1 activity.J. Biol. Chem. 2013; 288: 35769-35780Crossref PubMed Scopus (0) Google Scholar]. Third, care must be taken in selecting the type and concentration of lysosomal inhibitors. Commonly used lysosomal inhibitors are V-ATPase inhibitors bafilomycin A1, lysosomotropic reagents such as chloroquine, and lysosomal enzyme inhibitors such as pepstatin and E64d. However, lysosomotropic reagents cause osmotic stress on lysosomes and promote ATG8-II formation on the single membrane of endolysosomes by a mechanism similar to LC3-associated phagocytosis, which is distinct from canonical autophagy [12.Florey O. et al.V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation.Autophagy. 2015; 11: 88-99Crossref PubMed Scopus (65) Google Scholar] (Figure 2). For example, 100 μM chloroquine increases LC3-II in an autophagy-independent manner [12.Florey O. et al.V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation.Autophagy. 2015; 11: 88-99Crossref PubMed Scopus (65) Google Scholar,15.Jacquin E. et al.Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation.Autophagy. 2017; 13: 854-867Crossref PubMed Scopus (41) Google Scholar], significantly affecting autophagic flux measurement, and should be used at low concentrations (e.g., 25 μM) [15.Jacquin E. et al.Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation.Autophagy. 2017; 13: 854-867Crossref PubMed Scopus (41) Google Scholar]. Autophagic flux can also be evaluated by monitoring the levels of selective autophagy substrates (Figure 3B). Such substrates are generally recognized and physically bound by autophagosomal ATG8 proteins and, similar to the ATG8 flux assay, substrate degradation can be measured using lysosomal inhibitors. Among known substrates, SQSTM1/p62 is frequently used. It should be emphasized that the expression of this protein is also highly regulated at the transcriptional level. For example, accumulation of SQSTM1 may indicate inhibition of autophagy, but in some cases such as oxidative stress, it may simply represent overproduction of SQSTM1 protein [16.Sanchez-Martin P. Komatsu M. p62/SQSTM1 - steering the cell through health and disease.J. Cell Sci. 2018; 131jcs222836Crossref PubMed Scopus (0) Google Scholar]. Therefore, when the amount of an ATG8-binding substrate is used as an index of autophagic activity, its mRNA levels should be measured to confirm that the change in protein is not due to transcriptional induction. Alternatively, this problem can be avoided by using pulse-labeling with p62 tagged with HaloTag [17.Benyounes A. et al.A fluorescence-microscopic and cytofluorometric system for monitoring the turnover of the autophagic substrate p62/SQSTM1.Autophagy. 2011; 7: 883-891Crossref PubMed Scopus (27) Google Scholar]. As mentioned earlier, simple detection of autophagic structures using fluorescent protein-based reporters such as GFP-LC3 is not sufficient to determine autophagic flux. To overcome this issue, tandem fluorescent protein-tagged LC3 (tfLC3), which has both RFP (or another related red fluorescent protein such as mCherry) and GFP at the N terminus of LC3 (or any other ATG8 family protein) was developed [18.Pankiv S. et al.p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy.J. Biol. Chem. 2007; 282: 24131-24145Crossref PubMed Scopus (2651) Google Scholar,19.Kimura S. et al.Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3.Autophagy. 2007; 3: 452-460Crossref PubMed Google Scholar] (Figure 3C). This reporter emits both red and green fluorescence such that autophagosomes will appear yellow when images are merged. However, within the acidic environment of the autolysosome, the GFP fluorescence is immediately quenched, leaving only the red fluorescent signal, which is unaffected by the low pH. Following induction of autophagy, there is an increase in yellow (indicating more autophagosomes) and red puncta (indicating more autolysosomes). However, when autophagy flux is reduced due to lysosomal inhibition, yellow ATG8 puncta dominate, which indicates a reduction in autophagosome–lysosome fusion and/or degradation within the autolysosome. When autophagy induction is suppressed, both yellow and red structures are reduced. Instead of LC3, selective substrates such as SQSTM1/p62 can also be used [18.Pankiv S. et al.p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy.J. Biol. Chem. 2007; 282: 24131-24145Crossref PubMed Scopus (2651) Google Scholar] and this complements the tfLC3 system because SQSTM1 is not (or only weakly) recruited to single membranes [12.Florey O. et al.V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation.Autophagy. 2015; 11: 88-99Crossref PubMed Scopus (65) Google Scholar,20.Romao S. et al.Autophagy proteins stabilize pathogen-containing phagosomes for prolonged MHC II antigen processing.J. Cell Biol. 2013; 203: 757-766Crossref PubMed Scopus (113) Google Scholar]. Flux determination using the tandem fluorescent reporters does not require lysosome inhibition and the tandem fluorescent (tf) module can be adopted to determine whether individual autophagic structures are degraded after fusion with lysosomes. Furthermore, this method can also be used to monitor the total cellular autophagic flux [21.Gump J.M. Thorburn A. Soting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry.Autophagy. 2014; 10: 1327-13234Crossref PubMed Scopus (0) Google Scholar, 22.Kaizuka T. et al.An autophagic flux probe that releases an internal control.Mol. Cell. 2016; 64: 835-849Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 23.Jia R. Bonifacino J.S. Negative regulation of autophagy by UBA6-BIRC6-mediated ubiquitination of LC3.Elife. 2019; 8e50034Crossref PubMed Scopus (6) Google Scholar]. However, the combination of GFP and RFP/mCherry in tandem reporters may not be optimal because, as fluorescence resonance energy transfer (FRET) occurs from GFP to RFP/mCherry, the RFP/mCherry (a FRET acceptor) signal becomes weaker after degradation of GFP (a FRET donor). This issue was recently solved by conjugating the lysosome-resistant FRET donor TOLLES with the lysosome-sensitive FRET acceptor YPet to produce a novel tandem construct named signal-retaining autophagy indicator (SRAI) (Box 1) [24.Katayama H. et al.Visualizing and modulating mitophagy for therapeutic dtudies of neurodegeneration.Cell. 2020; 181: 1176-1187Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar].Box 1Methods for Monitoring Flux of Selective AutophagySpecific proteins, organelles, and intracellular pathogens can be selectively recognized and degraded by autophagy [100.Gatica D. et al.Cargo recognition and degradation by selective autophagy.Nat. Cell Biol. 2018; 20: 233-242Crossref PubMed Scopus (215) Google Scholar], but their steady-state levels within the cell is a factor of synthesis and degradation. Some representative flux reporters for selective autophagy of mitochondria (mitophagy) and the endoplasmic reticulum (ER-phagy) are listed later, but these methods are applicable to all substrates.MitophagyThe delivery of mitochondria to lysosomes can be monitored by using mt-Keima (Keima with a mitochondria-targeting sequence) [27.Katayama H. et al.A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.Chem. Biol. 2011; 18: 1042-1052Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar,101.Kageyama Y. et al.Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain.EMBO J. 2014; 33: 2798-2813Crossref PubMed Scopus (197) Google Scholar, 102.Bingol B. et al.The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy.Nature. 2014; 510: 370-375Crossref PubMed Scopus (354) Google Scholar, 103.Mizumura K. et al.Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD.J. Clin. Invest. 2014; 124: 3987-4003Crossref PubMed Scopus (236) Google Scholar, 104.Ordureau A. et al.Global landscape and dynamics of Parkin and USP30-dependent ubiquitylomes in iNeurons during mitophagic signaling.Mol. Cell. 2020; 77: 1124-1142Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. However, Keima can only be used in live cells with lysosomal acidification intact. To overcome this problem, the mito-QC reporter was used [48.McWilliams T.G. et al.mito-QC illuminates mitophagy and mitochondrial architecture in vivo.J. Cell Biol. 2016; 214: 333-345Crossref PubMed Scopus (128) Google Scholar,105.Allen G.F. et al.Loss of iron triggers PINK1/Parkin-independent mitophagy.EMBO Rep. 2013; 14: 1127-1135Crossref PubMed Scopus (210) Google Scholar]. This is a mCherry-GFP tandem reporter that is fused to the mitochondria-targeting sequence from FIS1. Like the tfLC3 reporter, the GFP signal of mito-QC is quenched when mitochondria are delivered to lysosomes by autophagy. This reporter has the advantage that it can be used in fixed cell and tissue samples. Another mitophagy reporter that can be used in fixed samples is mito-SRAI [24.Katayama H. et al.Visualizing and modulating mitophagy for therapeutic dtudies of neurodegeneration.Cell. 2020; 181: 1176-1187Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. This is also a tandem fluorescent protein reporter but gives better fluorescent signals in lysosomes. To assess total mitochondrial mass, the amount of mitochondrial DNA or proteins in the inner membrane or matrix should be measured. It is not recommended to use outer membrane proteins as an indicator of the mitochondrial mass because these proteins can be selectively degraded by the proteasome in a ubiquitin- and p97-dependent manner [106.Tanaka A. et al.Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin.J. Cell Biol. 2010; 191: 1367-1380Crossref PubMed Scopus (799) Google Scholar, 107.Chan N.C. et al.Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy.Hum. Mol. Genet. 2011; 20: 1726-1737Crossref PubMed Scopus (607) Google Scholar, 108.Yoshii S.R. et al.Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane.J. Biol. Chem. 2011; 286: 19630-19640Crossref PubMed Scopus (379) Google Scholar].ER-phagyGiven that the total mass of the ER is large and its consumption by autophagy is relatively small, it is difficult to monitor ER-phagy by detecting changes in the levels of endogenous ER proteins. To this aim, it is important to use specific ER-phagy flux reporters such as the ER luminal reporter (ssGFP-RFP-KDEL [109.Chino H. et al.Intrinsically disordered protein TEX264 mediates ER-phagy.Mol. Cell. 2019; 74: 909-921Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]) and ER membrane reporters (mCherry-GFP-RAMP4 [110.Liang J.R. et al.Atlastins remodel the endoplasmic reticulum for selective autophagy.J. Cell Biol. 2018; 217: 3354-3367Crossref PubMed Scopus (24) Google Scholar,111.Liang J.R. et al.A genome-wide ER-phagy screen highlights key roles of mitochondrial metabolism and ER-resident UFMylation.Cell. 2020; 180: 1160-1177Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar] and mCherry-GFP-REEP5 [112.Chen Q. et al.ATL3 is a tubular ER-phagy receptor for GABARAP-mediated selective autophagy.Curr. Biol. 2019; 29: 846-855Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar]). ER-phagy adaptors can also be used as reporters by tagging them with tandem fluorescent proteins [113.Khaminets A. et al.Regulation of endoplasmic reticulum turnover by selective autophagy.Nature. 2015; 522: 354-358Crossref PubMed Scopus (349) Google Scholar] or Keima [29.An H. et al.TEX264 is an endoplasmic reticulum-resident ATG8-interacting protein critical for ER remodeling during nutrient stress.Mol. Cell. 2019; 74: 891-908Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar], but as these adaptors bind to ATG8, they can be degraded more efficiently than the ER itself. A HaloTag-Sec62 has been used to characterize the normalization of ER volume following ER stress [114.Loi M. et al.ESCRT-III-driven piecemeal micro-ER-phagy remodels the ER during recovery from ER stress.Nat. Commun. 2019; 10: 5058Crossref PubMed Scopus (10) Google Scholar]. Lysosomal delivery of ER-phagy reporters can be detected by either fluorescence microscopy or immunoblotting (i.e., measuring the proteolytic 'cleavage' of the reporter). ER-phagy can also be evaluated by immunoblotting to evaluate cleavage of mCherry-RAMP4 [110.Liang J.R. et al.Atlastins remodel the endoplasmic reticulum for selective autophagy.J. Cell Biol. 2018; 217: 3354-3367Crossref PubMed Scopus (24) Google Scholar] in mammalian cells and Sec63-mCherry (general ER marker), Hmg1-GFP (perinuclear ER), and Rtn1-GFP (cortical ER) in yeast cells [115.Mochida K. et al.Receptor-mediated selective autophagy degrades the endoplasmic reticulum and the nucleus.Nature. 2015; 522: 359-362Crossref PubMed Google Scholar]. It should be noted that the ER is not a homogeneous structure; autophagic degradation of specific regions can be differentiated by using region-specific reporters derived from the different ER phagy adaptors [116.Hubner C.A. Dikic I. ER-phagy and human diseases.Cell Death Differ. 2020; 27: 833-842Crossref PubMed Scopus (5) Google Scholar]. Specific proteins, organelles, and intracellular pathogens can be selectively recognized and degraded by autophagy [100.Gatica D. et al.Cargo recognition and degradation by selective autophagy.Nat. Cell Biol. 2018; 20: 233-242Crossref PubMed Scopus (215) Google Scholar], but their steady-state levels within the cell is a factor of synthesis and degradation. Some representative flux reporters for selective autophagy of mitochondria (mitophagy) and the endoplasmic reticulum (ER-phagy) are listed later, but these methods are applicable to all substrates. Mitophagy The delivery of mitochondria to lysosomes can be monitored by using mt-Keima (Keima with a mitochondria-targeting sequence) [27.Katayama H. et al.A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.Chem. Biol. 2011; 18: 1042-1052Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar,101.Kageyama Y. et al.Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain.EMBO J. 2014; 33: 2798-2813Crossref PubMed Scopus (197) Google Scholar, 102.Bingol B. et al.The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy.Nature. 2014; 510: 370-375Crossref PubMed Scopus (354) Google Scholar, 103.Mizumura K. et al.Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD.J. Clin. Invest. 2014; 124: 3987-4003Crossref PubMed Scopus (236) Google Scholar, 104.Ordureau A. et al.Global landscape and dynamics of Parkin and USP30-dependent ubiquitylomes in iNeurons during mitophagic signaling.Mol. Cell. 2020; 77: 1124-1142Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. However, Keima can only be used in live cells with lysosomal acidification intact. To overcome this problem, the mito-QC reporter was used [48.McWilliams T.G. et al.mito-QC illuminates mitophagy and mitochondrial architecture in vivo.J. Cell Biol. 2016; 214: 333-345Crossref PubMed Scopus (128) Google Scholar,105.Allen G.F. et al.Loss of iron triggers PINK1/Parkin-independent mitophagy.EMBO Rep. 2013; 14: 1127-1135Crossref PubMed Scopus (210) Google Scholar]. This is a mCherry-GFP tandem reporter that is fused to the mitochondria-targeting sequence from FIS1. Like the tfLC3 reporter, the GFP signal of mito-QC is quenched when mitochondria are delivered to lysosomes by autophagy. This reporter has the advantage that it can be used in fixed cell and tissue samples. Another mitophagy reporter that can be used in fixed samples is mito-SRAI [24.Katayama H. et al.Visualizing and modulating mitophagy for therapeutic dtudies of neurodegeneration.Cell. 2020; 181: 1176-1187Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. This is also a tandem fluorescent protein reporter but gives better fluorescent signals in lysosomes. To assess total mitochondrial mass, the amount of mitochondrial DNA or proteins in the inner membrane or matrix should be measured. It is not recommended to use outer membrane proteins as an indicator of the mitochondrial mass because these proteins can be selectively degraded by the proteasome in a ubiquitin- and p97-dependent manner [106.Tanaka A. et al.Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin.J. Cell Biol. 2010; 191: 1367-1380Crossref PubMed Scopus (799) Google Scholar, 107.Chan N.C. et al.Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy.Hum. Mol. Genet. 2011; 20: 1726-1737Crossref PubMed Scopus (607) Google Scholar, 108.Yoshii S.R. et al.Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane.J. Biol. Chem. 2011; 286: 19630-19640Crossref PubMed Scopus (379) Google Scholar]. ER-phagy Given that the total mass of the ER is large and its consumption by autophagy is relatively small, it is difficult to monitor ER-phagy by detecting changes in the levels of endogenous ER proteins. To this aim, it is important to use specific ER-phagy flux reporters such as the ER luminal reporter (ssGFP-RFP-KDEL [109.Chino H. et al.Intrinsically disordered protein TEX264 mediates ER-phagy.Mol. Cell. 2019; 74: 909-921Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]) and ER membrane reporters (mCherry-GFP-RAMP4 [110.Liang J.R. et al.Atlastins remodel the endoplasmic reticulum for selective autophagy.J. Cell Biol. 2018; 217: 3354-3367Crossref PubMed Scopus (24) Google Scholar,111.Liang J.R. et al.A genome-wide ER-phagy screen highlights key roles of mitochondrial metabolism and ER-resident UFMylation.Cell. 2020; 180: 1160-1177Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar] and mCherry-GFP-REEP5 [112.Chen Q. et al.ATL3 is a tubular ER-phagy receptor for GABARAP-mediated selective autophagy.Curr. Biol. 2019; 29: 846-855Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar]). ER-phagy adaptors can also be used as reporters by tagging them with tandem fluorescent proteins [113.Khaminets A. et al.Regulation of endoplasmic reticulum turnover by selective autophagy.Natu
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