TECHNOLOGY FEATURE

Autophagy: eat thyself, sustain thyself Vivien Marx

A growing research community studies —a process of cellular recycling and maintenance—but there are some hot-button methodological issues.

A student with his seeing-eye dog but it is also a balance: too much or too approached biologist Dan Klionsky of little autophagy can be harmful to the cell. the University of Michigan after one of his The field has grown to include scientists of talks. He thanked Klionsky for including the “all flavors,” as cancer biologist Kay Macleod musical score Macromusophagy in his pre- of the University of Chicago phrases it. She sentation, because it had allowed the young studies the role of autophagy in cancer and scientist to ‘hear’ the phases of autophagy, is developing ways to track the autophagic a process of cellular housekeeping in which process in vivo in mice. cells maintain themselves by degrading To study autophagy, researchers apply a faulty or accumulated number of methods such as electron and and recycling metabolites. Klionsky had fluorescence microscopy, western blotting invited composer Wendy Wan-Ki Lee to to track the processing of certain marker create that piece. proteins, and flow cytometry, but, says Macromusophagy is also part of Klionsky’s Macleod, “we all recognize that our ability collaboration with an illustrator and dancers. to monitor and measure autophagy is not Klionsky gave choreographer Peter Sparling as good as it should be.” She is optimistic poetic license to render the complex phases that the surging community will develop of autophagy in the language of modern and validate better tools to characterize the Wendy Wan-Ki Lee and Daniel J. Klionsky Daniel Lee and Wan-Ki Wendy

Nature America, Inc. All rights reserved. America, Inc. © 201 5 Nature dance. The piece was “tremendous fun” to process qualitatively and quantitatively. In develop and perform, says Klionsky. Macromusophagy is a musical score developed the meantime she is encountering a number When he is not integrating art and sci- at the Chinese University of Hong Kong and the of what she calls “hot-button issues.” One of ence, Klionsky studies autophagy in . University of Michigan. them is that researchers base their claims on npg He and 2,460 coauthors have finalized a findings from one method, instead of mul- paper now in press in Autophagy, of which react to environmental changes, including tiple methods. Klionsky is the editor, describing updated extreme situations such as starvation or Klionsky, too, identifies methodological guidelines for the use and interpretation of deprivation of amino acids. issues, pointing out that static measure- assays to monitor autophagy1. Researchers increasingly see that autoph- ments should not be used to draw conclu- agy has a role in many biological processes sions about autophagic flux (the entire Growing popularity such as development, aging and immunity. process of autophagy, from For the upcoming guideline paper, Klionsky Its misregulation appears to be a culprit in biogenesis through to degradation in the winnowed through a mountain of 16,000 conditions as diverse as inflammatory dis- ). Such studies necessitate multiple papers related to autophagy, many of them eases, cancer and . The measurements at different time points and on systems other than yeast, and many spectrum of findings is perhaps not surpris- the use of inhibitors to block the last stage of introducing new terms or methods. The last ing, says Klionsky. Autophagy is now appre- the process, for comparison. guidelines, published in 2012, had only half ciated as a fundamental cellular process that He draws an analogy to a city block on as many authors. Many factors have contrib- can affect physiology in many ways. which garbage has amassed. One may con- uted to this growth. What intrigues Goethe University clude that the area’s inhabitants have been Autophagy is a process of cellular house- researcher Ivan Dikic is the multitude and producing more garbage than is typical, keeping in which dysfunctional organelles, scope of ‘good’ autophagic processes, their but there might also be a strike. The out- aggregated macromolecules, misfolded pro- multiple roles in physiology and the fact come looks the same, but the causes are teins or pathogens are removed and degrad- that there can also be an “unexpected shift completely different. Similarly, cellular ed. Some of the metabolites are reused. toward pathological autophagy.” Autophagy autophagy might seem upregulated under Autophagy is also dynamic—it helps cells is a strategy for cellular self-fortification, certain conditions, but there might also be a

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Plasma back and upregulate LC3 at the transcrip- membrane tional level. In fact, a new focus in the field Lysosome is addressing how transcriptional regulators of autophagy and of lysosomal biogenesis Autophagosome contribute to the overall control of autoph- agy and responses to nutrient stress, she says. The nuclear levels of these transcrip- tion factors help scientists understand how Phagophore autophagy can be deregulated in such as cancer. Fluorescent proteins such as GFP-LC3 Autolysosome and mCherry-EGFP-LC3 help scientists Daniel J. Klionsky Daniel track LC3 with fluorescence microscopy. Klionsky thinks highly in particular of Autophagy is a process of cellular housekeeping, during which dysfunctional organelles, misfolded proteins or pathogens are removed and degraded. Some of the metabolites are then reused. the dual-tagged proteins for following LC3 in mammalian cells. Dual tags let a researcher follow autophagic flux because block in one of the last stages of the process. method, in his view, as it can be used to tell GFP fluorescence is quenched in the acidic “Looking at one time point will not tell the whether the structure in a micrograph is lysosome. Tagged in the answer,” says Klionsky. lined with a such as LC3 (microtu- appear yellow, but after they fuse Attentiveness to such issues can enable bule-associated protein 1 with light chain 3). with the lysosome, the green fluorescence comparison of data and analyses. Especially is quenched and an experimenter sees only in a growing field, says Macleod, having Reliance on LC3 red. Comparing the number of yellow to red comparable results from one lab to the next Autophagy researchers are reliant on dots gives some idea of flux, he says. is quite crucial. As Klionsky explains, the measurements of LC3, its isoforms and But these proteins need to be overex- guidelines aim to facilitate the review of processed variants thereof, says Macleod. pressed in the cell, which can itself induce manuscripts not just at his journal but, he Cytosolic LC3 is cleaved to LC3-I and conju- autophagy, says Macleod. “You’re actually hopes, at others as well. They can guide the gated to , which messing with the thing you are trying to community not as rigid rules but as recom- is recruited to the pre-autophagosomal measure, so that’s the problem there.” mendations. membrane as LC3-II. The different forms of In instances when LC3 cannot be mea- LC3 can be followed by western blotting; for sured in a reliable fashion, as can be the case Identifying structures instance, one exper- Autophagosomes are double-membraned imental approach structures in which cellular components looks at conver-

Nature America, Inc. All rights reserved. America, Inc. © 201 5 Nature are sequestered and readied for degradation. sion from LC3-I to But they are not always obvious to identify, LC3-II. The protein Klionsky says. can also be tagged Macleod regularly sees researchers over- with fluorophores, npg interpreting electron microscopy (EM) enabling a research- images and also forgetting that the images er to find autopha- are a view of one section in one crowded gosomes and to location of the cell. The cytoplasm has measure autophagic plenty of complex structures, she says. flux with fluores- Interpreting EM images to identify autoph- cence microscopy. agic structures with certainty takes much Scientists in this expertise2. field have long and EM is often called the gold standard rather turbulently for identifying structures involved in the discussed the inter- autophagic process and confirming find- pretation of western ings from fluorescence microscopy, says blots related to LC3, Klionsky. But “it is by its very nature static,” says Macleod3. An he says, whereas autophagy is a dynamic important issue too process. The autophagosome fuses with a often forgotten is lysosome, creating an autolysosome, where that LC3 levels can degradation occurs. EM can be used for change for many

autophagic flux measurements only if sci- reasons. When J. Klionsky Daniel and Yen Misuzu Baba, Wei-Lien entists image at multiple time points. Many autophagy is inhib- With electron microscopy, researchers can study autophagy. But it is not researchers also do not perform immuno- ited or modulated, always easy to identify autophagosomes, where cellular components are EM, says Klionsky. This is the preferable the cell can feed sequestered prior to degradation.

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in tumor sections, Macleod sees scientists In contrast, when in particu- measuring levels of the protein p62, which lar was inhibited in mouse models, tumors is turned over in autophagy. When autoph- metastasized more rapidly. Such findings, agy is inhibited, p62 levels rise, so accumu- says Macleod, show why scientists must be lated p62 is used as a proxy in autophagy circumspect in their interpretation of stud- measurements. But what is often underap- ies that involve inhibition of general autoph- preciated, she says, is that p62 is regulated agy. They also emphasize how important it by many pathways, including NF-κB and is to inhibit different types of autophagy, tumor factor–α signaling. That such as mitophagy, in isolation. If fluoro- means a change in p62 levels is not a sure- phores could be targeted to organelles and fire indication that a sample is showing also not interfere with the autophagy pro- autophagy defects or increases. “My feel- cess, they would provide a way to monitor ing on that is sometimes we have no choice, autophagy geographically within a cell,

but it’s clearly a measurement that’s open to Chicago of University Lab, Sharifi, Macleod Marina within tissue, in an organism and over time. caveats,” she says. Using levels of processed But until those tools are available and can A sensor called mito-mKeima helps researchers LC3, LC3-II and p62 can make researchers be validated, researchers need to be frank study mitophagy, a selective type of autophagy more confident about their data. that involves just mitochondria. Here, intact about their findings, says Macleod. Dikic also points to the challenges associ- mitochondria in MCF-7 breast cancer cells Scientists working with mammalian cells ated with overexpression when tagging pro- fluoresce blue, and mitochondria at the lysosome still lack what Klionsky and other yeast teins with fluorophores. He uses multiple undergoing mitophagy fluoresce red. The ratio of researchers have at their disposal: a truly methods to be sure about his data: biochem- red to blue gives a measure of mitophagy flux. quantitative assay. In yeast, there is nonse- ical assays that assess LC3 processing and lective and selective autophagy, as well as p62 levels, fluorescence microscopy using when they are trafficked to the autolysosome, the unique cytoplasm-to- targeting double-tagged GFP-mCherry fusion pro- the pH decreases and the sensor fluorescence (Cvt) pathway, which is selective as well as teins to assess LC3 levels, and EM. One may is bright red. “So you can actually see this biosynthetic, says Klionsky. One can mea- also use fluorescence-activated cell sorting quantitative shift in fluorescence that you can sure autophagy in yeast by monitoring the to look for the accumulation of processed, use as a very direct measure of mitochondrial activity of a modified phosphatase, Pho8, fluorophore-tagged LC3. “I like the double- turnover at the lysosome,” says Macleod. that is typically present in the cytosol in an tagged fusion proteins, as they indicate the Macleod believes mt-mKeima will inactive form but moves into the vacuole final delivery to the lysosome,” he says. improve researchers’ ability to interpret during autophagy. Autophagy activates that what autophagy—mitophagy specifically in enzyme, says Klionsky, which “gives us a Selective markers this case—is doing in tissues and systems, nice enzymatic readout.” Autophagy can be a quite selective pro- “but arguably we need something similar Pho8 is a vacuolar enzyme that normally cess. For example, only certain organelles for all other different forms of autophagy,” goes to the vacuole through the secretory

Nature America, Inc. All rights reserved. America, Inc. © 201 5 Nature such as and , the she says. Her wish list includes a marker for pathway. Klionsky and his team modify (ER) and part of ER and Pho8 to keep it in the cytosol—by removing the nuclear membrane may be autophago- turnover. She would its signal sequence—and can thus use it to cytosed. Macleod is particularly interested like to parse all the monitor nonselective autophagy. They used npg in the selective turnover of mitochondria different functions Pho8 for this purpose because, he says, there in autophagy (mitophagy) and is exploring of autophagy with are basically no great enzymatic assays for the signals that lead broken mitochondria markers, which is monitoring other vacuolar enzymes, such to be selected and then degraded. She is also not yet possible. as proteases. exploring the roles mitochondria play in Macleod is using Klionsky’s lab previously characterized cancer cells and metastasis. transgenic mice to aminopeptidase I, a vacuolar protease University of Chicago of University Macleod’s experimental challenge has been study autophagy that moves to the vacuole through the Cvt The autophagy field that she has needed to stain separately for in vivo. Here, too, pathway or autophagy6. Aminopeptidase has grown to include autophagosomes and for mitochondria and scientists of “all she cautions about I can be used to follow the normal secre- then look for overlap in the signals, “which, flavors,” says Kay overinterpreting tory pathway–independent itinerary to the when you’re doing that under the microscope, Macleod. results. For exam- vacuole, with gel electrophoresis used as is somewhat subjective,” she says. “You’re ple, in mouse mod- the readout. When Klionsky’s lab published looking for yellow in a sea of green and red.” els lacking the entire process of autophagy, this work, many scientists approached him Although software such as ImageJ can help defective mitochondria were interpreted for plasmids so they could try this approach with image analysis, she has begun exploring as contributors to impeded tumorigenesis. in their systems. But the method cannot another approach using mito-monomeric But, says Macleod, one cannot be sure that be readily transferred to other organisms, Keima (mt-mKeima), a fluorescent protein– defective mitochondria are causing the phe- given that it requires certain receptors and based sensor based on mKeima, both of which notype, because other organelles and func- a scaffold protein to be present. And, says were developed at RIKEN4,5. tions are damaged as well. The cause could Klionsky, there just may be no Cvt pathway When the mitochondria are in the cyto- have been reduced cycling at the in mammalian cells, although there are cer- sol, the sensor emits green fluorescence, but lysosome, she says. tainly other types of selective autophagy.

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Inhibiting, inducing ­scientists contact them even when they are In their experiments, scientists use small still in the experimental design stage as well molecules that act as both inhibitors and as when experiments are under way. This inducers of autophagy. Rapamycin can can help labs match the right reagents to induce autophagy, and, for example, 3-meth- their experiment. yladenine, and Jack Coleman, who directs biochem- inhibit it. As Macleod explains, many experi- istry at Enzo Life Sciences, explains that menters use these reagents for too long and his company sells a proprietary dye, an at too high a concentration, which leads to amphiphilic tracer that stains results that vary from one lab to the next. minimally while maximizing the fluo- The interpretation challenge is magni- rescence of autophagosomes, as part of a fied by the effect the inducer or inhibitor has kit called Cyto-ID. The kit can be used on protein levels, says Klionsky. LC3 levels to monitor autophagy in live cells. When might increase after a compound is added used with inhibitors, the dye’s specificity because autophagy is induced. But the com- helps researchers distinguish the inhibi- pound might also affect the later stages of tion of lysosomal fusion from an increase autophagy, which will also lead protein levels in autophagic flux, he says. Other dyes to rise, but for the too often show both lysosome and opposite reason; autophagosome, and, he says, his com- this risks experi- pany’s dye stays put, whereas other dyes menters drawing tend to disappear from the assay when the wrong conclu- certain inhibitors are used. sion from their The market has a broad selection of experiment. antibodies to LC3, but it also seems that What makes many of them fail. Enzo application sci- these misinterpre- entist Morgan Mathieu sees research-

University of Michigan of University tations especially ers sometimes struggling to distinguish treacherous is that between different forms of LC3. These It is better to not aspects of this isoforms can make it hard to interpret draw conclusions western blots, and researchers thus risk about autophagic research might flux from static one day lead to a drawing incorrect conclusions from the measurements, says drug candidate. obtained data. For experimental results to Dan Klionsky. The error could be informative, says Mathieu, the details be significant: a matter. Researchers need to prepare the

Nature America, Inc. All rights reserved. America, Inc. © 201 5 Nature drug candidate might be thought to induce sample in the right lysis buffer, they must autophagy when in fact it blocks autophagy. use the right controls, and they must have Labs are searching for ways to monitor the appropriate membranes for western autophagy in vivo, which could lead to the blotting, because of the lipophilic nature npg use of imaging methods to study drug can- of LC3-II. Any of these factors can con- didates. There is much interest in modulat- tribute to difficulties in analyzing LC3 by ing autophagy for clinical purposes, says western blotting. Klionsky, but the lack of approaches to deter- EMD Millipore scientist Chandra mine whether or to what extent a particular Mohan also notes that antibodies can react drug has affected autophagy makes it hard to to both LC3-I and LC3-II. Researchers modulate the process effectively. In neurode- need to conduct a sufficient number of generative conditions, for example, compro- experiments so they can study the western mised autophagy is considered a contributor blot bands, see the trends in their samples to neuronal cell . That makes it tempt- and learn which levels are rising. ing to explore how one might upregulate LC3-I is mainly in the cytosol, and its autophagy in a patient. But before that can predominance means the cell is not yet happen, basic biology research on autoph- fully committed to autophagy. LC3-II agy is needed. An approach cannot simply binds to the autophagosomal membrane involve maximal induction of autophagy, and is plentiful when the cell is firmly com- because, Klionsky says, “you can’t simply mitted to autophagy, says Mohan. EMD turn it on, period, because that can kill a cell.” Millipore sells a reagent kit that is tailored to Muse, the company’s flow cytometer. Commercial assays Scientists can label cells for the LC3 marker Companies offer many autophagy-related and then sort and count the cells to mea- products. Many companies propose that sure autophagy.

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Mohan sees a growing number of types and under different conditions to autophagy or track its changes over time researchers approaching his company for discover the minimal dosage needed, she and under different conditions. New tools reagents and kits related to autophagy, says. in this resurging field are bound to arrive. given the renaissance the field is experi- Another recommendation is to mea- Yet with both new and old approaches, sci- encing in both stem and can- sure cell viability and not just autophagy, entists will want to be sure to use multiple cer research. In stem cells, autophagy is as off-target effects can affect cell viability, approaches to home in on this complex and involved in maintaining quiescence, and says Bao. An experimenter might trigger dynamic process. it has a role in cellular remodeling dur- autophagy as well as or other sig- ing differentiation, he says. Cancer cells naling pathways inside the cell. 1. Klionsky, D. et al. Autophagy (in press). 2. Barth, S., Glick, D. & Macleod, K.F. J. Pathol. activate autophagy in response to cellular Mohan says he encounters researchers 221, 117–124 (2010). stress and increased metabolic demands who complain that they do not see inhibi- 3. Sharifi, M. et al. Methods Mol. Biol. 1292, for proliferation, he says. And the inhibi- tion when using a millimolar quantity of 129–150 (2015). 4. Kogure, T. et al. Nat. Biotechnol. 24, 577–581 tion of autophagy can restore the chemo- an inhibitor with a half-maximal inhibi- (2006). sensitivity of cancer cells. tory concentration (IC50) in the nanomolar 5. Katayama, H. et al. Chem. Biol. 18, 1042–1052 When it comes to inducers and inhibi- range. It is a common belief, says Mohan, (2011). 6. Lynch-Day, M.A. & Klionsky, D.J. FEBS Lett. 584, tors, Enzo scientist Jianjun Bao suggests that using a larger amount of an inducer 1359–1366 (2010). that customers use low dosages and low or an inhibitor will increase its effect. He incubation times, which will help them finds that he must repeatedly lay that mis- avoid the off-target effects that these conception to rest. Vivien Marx is technology editor for compounds can have. The company tests Autophagy researchers have no Nature and Nature Methods inducers and inhibitors in different cell ‘autophagometer’ with which to measure ([email protected]). Nature America, Inc. All rights reserved. America, Inc. © 201 5 Nature npg

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