Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity.
Introduction
Many researchers, especially those new to the field, need to determine which criteria are essential for demonstrating autophagy, either for the purposes of their own research, or in the capacity of a manuscript or grant review.1 Acceptable standards are an important issue, particularly considering that each of us may have his/her own opinion regarding the answer. Unfortunately, the answer is in part a “moving target” as the field evolves.2 This can be extremely frustrating for researchers who may think they have met those criteria, only to find out that the reviewers of their papers have different ideas. Conversely, as a reviewer, it is tiresome to raise the same objections repeatedly, wondering why researchers have not fulfilled some of the basic requirements for establishing the occurrence of an autophagic process. Several fundamental points must be kept in mind as we establish guidelines for the selection of appropriate methods to monitor autophagy.2 Importantly, there are no absolute criteria for determining autophagic status that are applicable in every biological or experimental context. This is because some assays are inappropriate, problematic or may not work at all in particular cells, tissues or organisms.3-6 For example, autophagic responses to drugs may be different in transformed versus nontransformed cells, and in confluent versus nonconfluent cells, or in cells grown with or without glucose.4
In addition, these guidelines are likely to evolve as new methodologies are developed and current assays are superseded. Nonetheless, it is useful to establish guidelines for acceptable assays that can reliably monitor autophagy in many experimental systems. It is important to note that in this set of guidelines the term “autophagy” generally refers to macroautophagy; other autophagy-related processes are specifically designated when appropriate. For the purposes of this review, the autophagic compartments (Fig. 1) are referred to as the sequestering (pre-autophagosomal) phagophore (PG; previously called the isolation or sequestration membrane5,6),7 the autophagosome (AP),8 the amphisome (AM; generated by the fusion of autophagosomes with endosomes),9 the lysosome, the autolysosome (AL; generated by fusion of autophagosomes or amphisomes with a lysosome), and the autophagic body (AB; generated by fusion and release of the internal autophagosomal compartment into the vacuole in fungi and plants). Except for cases of highly stimulated autophagic sequestration (Fig. 2), autophagic bodies are not seen in animal cells, because lysosomes/autolysosomes are typically smaller than autophagosomes.6,8,10 One critical point is that autophagy is a highly dynamic, multi-step process. Like other cellular pathways, it can be modulated at several steps, both positively and negatively. An accumulation of autophagosomes (measured by transmission electron microscopy [TEM] image analysis,11 as green fluorescent protein [GFP]-MAP1LC3 [GFP-LC3] puncta, or as changes in the amount of lipidated LC3 [LC3-II] on a western blot), could, for example, reflect a reduction in autophagosome turnover,12-14 or the inability of turnover to keep pace with increased autophagosome formation (Fig. 1B).15
For example, inefficient fusion with endosomes and/or lysosomes, or perturbation of the transport machinery,16 would inhibit autophagosome maturation to amphisomes or autolysosomes (Fig. 1C), whereas decreased flux could also be due to inefficient degradation of the cargo once fusion has occurred.17 Moreover, GFP-LC3 puncta and LC3 lipidation can reflect the induction of a different/modified pathway such as LC3-associated phagocytosis (LAP),18 and the noncanonical destruction pathway of the paternal mitochondria after fertilization.
Transmission electron microscopy
Autophagy was first detected by TEM in the 1950s (reviewed in ref. 6). It was originally observed as focal degradation of cytoplasmic areas performed by lysosomes, which remains the hallmark of this process. Later analyses revealed that it starts with the sequestration of portions of the cytoplasm by a special double-membrane structure (now termed the phagophore), which matures into the autophagosome, still bordered by a double membrane. Subsequent fusion events expose the cargo to the lysosome (or the vacuole in fungi or plants) for enzymatic breakdown. The importance of TEM in autophagy research lies in several qualities. It is the only tool that reveals the morphology of autophagic structures at a resolution in the nm range; shows these structures in their natural environment and position among all other cellular components; allows their exact identification; and, in addition, it can support quantitative studies if the rules of proper sampling are followed.11
Autophagy can be both selective and nonselective, and TEM can be used to monitor both. In the case of selective autophagy, the cargo is the specific substrate being targeted for sequestration—bulk cytoplasm is essentially excluded. In contrast, during nonselective autophagy, the various cytoplasmic constituents are sequestered randomly, resulting in autophagosomes in the size range of normal mitochondria. Sequestration of larger structures (such as big lipid droplets, extremely elongated or branching mitochondria or the entire Golgi complex) is rare, indicating an apparent upper size limit for individual autophagosomes. However, it has been observed that under special circumstances the potential exists for the formation of huge autophagosomes, which can even engulf a complete nucleus.25 Cellular components that form large confluent areas excluding bulk cytoplasm, such as organized, functional myofibrillar structures, do not seem to be sequestered by macroautophagy. The situation is less clear with regard to glycogen.45-4
Western blotting and ubiquitin-like protein conjugation systems
The Atg8/LC3 protein is a ubiquitin-like protein that can be conjugated to PE (and possibly to phosphatidylserine137). In yeast and several other organisms, the conjugated form is referred to as Atg8–PE. The mammalian homologs of Atg8 constitute a family of proteins subdivided in 2 major subfamilies: MAP1LC3/LC3 and GABARAP. The former consists of LC3A, B, B2 and C, whereas the latter family includes GABARAP, GABARAPL1 and GABARAPL2/GATE-16.138
After cleavage of the precursor protein mostly by the cysteine protease ATG4B,139,140 the nonlipidated and lipidated forms are usually referred to respectively as LC3-I and LC3-II, or GABARAP and GABARAP–PE, etc. The PE-conjugated form of Atg8/LC3, although larger in mass, shows faster electrophoretic mobility in SDS-PAGE gels, probably as a consequence of increased hydrophobicity. The positions of both Atg8/LC3-I (approximately 16–18 kDa) and Atg8–PE/LC3-II (approximately 14–16 kDa) should be indicated on western blots whenever both are detectable. The differences among the LC3 proteins with regard to function and tissue-specific expression are not known. Therefore, it is important to indicate the isoform being analyzed just as it is for the GABARAP subfamily.The mammalian Atg8 homologs share from 29% to 94% sequence identity with the yeast protein and have all, apart from GABARAPL3, been demonstrated to be involved in autophagosome biogenesis.141 The LC3 proteins are involved in phagophore formation, with participation of GABARAP subfamily members in later stages of autophagosome formation, in particular phagophore elongation and closure.142 Some evidence, however, suggests that at least in certain cell types the LC3 subfamily may be dispensable for bulk autophagic sequestration of cytosolic proteins, whereas the GABARAP subfamily is absolutely required.143 Due to unique features in their molecular surface charge distribution,144 emerging evidence indicates that LC3 and GABARAP proteins may be involved in recognizing distinct sets of cargoes for selective autophagy.145-147
Nevertheless, in most published studies, LC3 has been the primary Atg8 homolog examined in mammalian cells and the one that is typically characterized as an autophagosome marker per se. Note that although this protein is referred to as “Atg8” in many other systems, we primarily refer to it here as LC3 to distinguish it from the yeast protein and from the GABARAP subfamily. LC3, like the other Atg8 homologs, is initially synthesized in an unprocessed form, proLC3, which is converted into a proteolytically processed form lacking amino acids from the C terminus, LC3-I, and is finally modified into the PE-conjugated form, LC3-II (Fig. 6). Atg8–PE/LC3-II is the only protein marker that is reliably associated with completed autophagosomes, but is also localized to phagophores. In yeast, Atg8 amounts increase at least 10-fold when autophagy is induced.148 When dealing with animal tissues, western blotting of LC3 should be performed on frozen biopsy samples homogenized in the presence of general protease inhibitors (C. Isidoro, personal communication; see also Human).197 Caveats regarding detection of LC3 by western blotting have been covered in a review.26 For example, PVDF membranes may result in a stronger LC3- II retention than nitrocellulose membranes, possibly due to a higher affinity for hydrophobic proteins (Fig. 6B; J. Kovsan and A. Rudich, personal communication), and Triton X-100 may not efficiently solubilize LC3-II in some systems.198 Heating in the presence of 1% SDS, or analysis of membrane fractions,44 may assist in the detection of the lipidated form of this protein. This observation is particularly relevant for cells with a high nucleocytoplasmic ratio, such as lymphocytes.
Under these constraints, direct lysis in Laemmli loading buffer, containing SDS, just before heating, greatly improves LC3 detection on PVDF membranes, especially when working with a small number of cells (F. Gros, unpublished observations).199 Analysis of a membrane fraction is particularly useful for brain where levels of soluble LC3-I greatly exceed the level of LC3-II. One of the most important issues is the quantification of changes in LC3-II, because this assay is one of the most widely used in the field and is often prone to misinterpretation. Levels of LC3-II should be compared to actin (e.g., ACTB), but not to LC3-I (see the caveat in the next paragraph), and, ideally, to more than one “housekeeping” protein (HKP). Actin and other HKPs are usually abundant and can easily be overloaded on the gel200 such that they are not detected within a linear range. Moreover, actin levels may decrease when autophagy is induced in many organisms from yeast to mammals. For any proteins used as “loading controls” (including actin, tubulin and GAPDH) multiple exposures of the western blot are generally necessary to ensure that the signals are detected in the linear range. An alternative approach is to stain for total cellular proteins with Coomassie Brilliant Blue and Ponceau Red,201 but these methods are generally less sensitive and may not reveal small differences in protein loading. Stain-Free gels, which also stain for total cellular proteins, have been shown to be an excellent alternative to HKPs.202
Turnover of LC3-II/Atg8–PE
Autophagic flux is often inferred on the basis of LC3-II turnover, measured by western blot (Fig. 6C) 174 in the presence and absence of lysosomal, or vacuolar degradation.
However, it should be cautioned that such LC3 assays are merely indicative of autophagic “carrier flux”, not of actual autophagic cargo/substrate flux. It has, in fact, been observed that in rat hepatocytes, an autophagic-lysosomal flux of LC3-II can take place in the absence of an accompanying flux of cytosolic bulk cargo.223 The relevant parameter in LC3 assays is the difference in the amount of LC3-II in the presence and absence of saturating levels of inhibitors, which can be used to examine the transit of LC3-II through the autophagic pathway; if flux is occurring, the amount of LC3-II will be higher in the presence of the inhibitor.174 Lysosomal degradation can be prevented through the use of protease inhibitors (e.g., pepstatin A, leupeptin and E-64d), compounds that neutralize the lysosomal pH such as bafilomycin A1, chloroquine or NH4Cl,16,149,158,164,224,225 or by treatment with agents that block the fusion of autophagosomes with lysosomes (note that bafilomycin A1 will ultimately cause a fusion block as well as neutralize the pH,156 but the inhibition of fusion may be due to a block in ATP2A/SERCA activity226).155-157,227 Alternatively, knocking down or knocking out LAMP2 (lysosomal-associated membrane protein 2) represents a genetic approach to block the fusion of autophagosomes and lysosomes (for example, inhibiting LAMP2 in myeloid leukemic cells results in a marked increase of GFP-LC3 dots and endogenous LC3-II protein compared to control cells upon autophagy induction during myeloid differentiation [M.P. Tschan, unpublished data]).
228 This approach, however, is only valid when the knockdown of LAMP2 is directed against the mRNA region specific for the LAMP2B spliced variant, as targeting the region common to the 3 variants would also inhibit chaperone-mediated autophagy, which may result in the compensatory upregulation of macroautophagy.Due tothe advances in time-lapse fluorescence microscopy and the development of photoswitchable fluorescent proteins, autophagic flux can also be monitored by assessing the half-life of the LC3 protein240 post-photoactivation or by quantitatively measuring the autophagosomal pool size and its transition time.241 These approaches deliver invaluable information on the kinetics of the system and the time required to clear a complete autophagosomal pool. Nonetheless, care must be taken for this type of analysis as changes in translational/transcriptional regulation of LC3 might also affect the readout. Finally, autophagic flux can be monitored based on the turnover of LC3-II, by utilizing a luminescence-based assay. For example, a reporter assay based on the degradation of Renilla reniformis luciferase (Rluc)-LC3 fusion proteins is well suited for screening compounds affecting autophagic flux.242 In this assay, Rluc is fused N-terminally to either wild-type LC3 (LC3WT) or a lipidation-deficient mutant of LC3 (G120A). Since Rluc-LC3WT, in contrast to Rluc-LC3G120A, specifically associates with the autophagosomal membranes, Rluc-LC3WT is more sensitive to autophagic degradation. A change in autophagy-dependent LC3 turnover can thus be estimated by monitoring the change in the ratio of luciferase activities between the 2 cell populations expressing either Rluc-LC3WT
GFP-Atg8/LC3 fluorescence microscopy
LC3B, or the protein tagged at its N terminus with a fluorescent protein such as GFP (GFP-LC3), has been used to monitor autophagy through indirect immunofluorescence or direct fluorescence microscopy (Fig. 10), measured as an increase in punctate LC3 or GFP-LC3.269,270 The detection of GFP-LC3/ Atg8 is also useful for in vivo studies using transgenic organisms such as Caenorhabditis elegans, 271 Dictyostelium discoideum, 272 filamentous ascomycetes,273-277 Ciona intestinalis, 278 Drosophila melanogaster, 279-281 Arabidopsis thaliana, 282 Zea mays, 283 Trypanosoma brucei, 221,284,285 Leishmania major286-288 and mice.153 It is also possible to use anti-LC3/Atg8 antibodies for immunocytochemistry or immunohistochemistry (IHC),197,289-294 procedures that have the advantages of detecting the endogenous protein, obviating the need for transfection and/or the generation of a transgenic organism, as well as avoiding potential artifacts resulting from overexpression.For example, high levels of overexpressed GFP-LC3 can result in its nuclear localization, although the protein can still relocate to the cytosol upon starvation. The use of imaging cytometry allows rapid and quantitative measures of the number of LC3 puncta and their relative number in individual or mixed cell types, using computerized assessment, enumeration, and data display (e.g., see refs. 44, 295). In this respect, the alternative use of an automated counting system may be helpful for obtaining an objective number of puncta per cell.
For this purpose, the WatershedCounting3D plug-in for ImageJ may be useful.296,297 Changes in the number of GFP-Atg8 puncta can also be monitored using flow cytometry (see Autophagic flux determination using flow and multispectral imaging cytometry).221
Autophagic flux determination using flow and multispectral imaging cytometry
Whereas fluorescence microscopy, in combination with novel autophagy probes, has permitted single-cell analysis of autophagic flux, automation for allowing medium- to high-throughput analysis has been challenging. A number of methods have been developed that allow the determination of autophagic flux using flow cytometry,225,311,327,358-361 and commercial kits are now available for monitoring autophagy by flow cytometry. These approaches make it possible to capture data or, in specialized instruments, high-content, multiparametric images of cells in flow (at rates of up to 1,000 cells/sec for imaging, and higher in nonimaging flow cytometers), and are particularly useful for cells that grow in suspension. Optimization of image analysis permits the study of cells with heterogeneous LC3 puncta, thus making it possible to quantify autophagic flux accurately in situations that might perturb normal processes (e.g., microbial infection).360,362 Since EGFP-LC3 is a substrate for autophagic degradation, total fluorescence intensity of EGFP-LC3 can be used to indicate levels of autophagy in living mammalian cells.358 When autophagy is induced, the decrease in total cellular fluorescence can be precisely quantified in large numbers of cells to obtain robust data.
In another approach, soluble EGFPLC3-I can be depleted from the cell by a brief saponin extraction so that the total fluorescence of EGFP-LC3 then represents that of EGFP-LC3-II alone (Fig. 13A).326,327 Since EGFP-LC3 transfection typically results in high relative levels of EGFP-LC3-I, this treatment significantly reduces the background fluorescence due to nonautophagosome-associated reporter protein. By comparing treatments in the presence or absence of lysosomal degradation inhibitors, subtle changes in the flux rate of the GFPLC3 reporter construct can be detected. If it is not desirable to treat cells with lysosomal inhibitors to determine rates of autophagic flux, a tandem mRFP/mCherry-EGFP-LC3 (or similar) construct can also be used for autophagic flux measurements in flow cytometry experiments (see Tandem mRFP/mCherry-GFP fluorescence microscopy).359
Immunohistochemistry
Immunodetection of ATG proteins (particularly LC3 and BECN1) has been reported as a prognostic factor in various human carcinomas, including lymphoma,197,366 breast carcinoma,367 endometrial adenocarcinoma,368,369 head and neck squamous cell carcinoma,370-372 hepatocellular carcinoma,373,374 gliomas,375 non-small cell lung carcinomas,376 pancreatic377 and colon adenocarcinomas,378-380 as well as in cutaneous and uveal melanomas.381,382 Unfortunately, the reported changes often reflect overall diffuse staining intensity rather than appropriately compartmentalized puncta. Therefore, the observation of increased levels of diffuse LC3 staining (which may reflect a decrease in autophagy) should not be used to draw conclusions that autophagy is increased in cancer or other tissue samples.
Importantly, this kind of assay should be performed as recommended by the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK).383 As we identify new drugs for modulating autophagy in clinical applications, this type of information may prove useful in the identification of subgroups of patients for targeted therapy.
TOR/MTOR, AMPK and Atg1/ULK1
Atg1/ULK1 are central components in autophagy that likely act at more than one stage of the process. There are multiple ULK isoforms in mammalian cells including ULK1, ULK2, ULK3, ULK4 and STK36.452 ULK3 is a positive regulator of the Hedgehog signaling pathway,453 and its overexpression induces both autophagy and senescence.454 Along these lines, ectopic ULK3 displays a punctate pattern upon starvation-induced autophagy induction.454 ULK3, ULK4 and STK36, however, lack the domains present on ULK1 and ULK2 that bind ATG13 and RB1CC1/FIP200.455 Thus, ULK3 may play a role that is restricted to senescence and that is independent of the core autophagy machinery. ULK2 has a higher degree of identity with ULK1 than any of the other homologs, and they may have similar functions that are tissue specific. However, ULK1 may be the predominant isoform involved in autophagy, as knockdown of ULK2 does not affect movement of ATG9.456 Similarly, pharmacological inhibition of ULK1 and ULK2, with the compound MRT68921, blocks macroautophagy and expression of a drug-resistant ULK1 mutant is sufficient to rescue this block.457
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