TECHNOLOGY FEATURE Autophagy: eat thyself, sustain thyself Vivien Marx A growing research community studies autophagy—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 cell 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 organelles or accumulated proteins 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 Nature America, Inc. All rights reserved. America, Inc. Nature poetic license to render the complex phases that the surging community will develop 5 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 © 201 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 yeast. 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 autophagosome 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- lysosome). Such studies necessitate multiple papers related to autophagy, many of them eases, cancer and neurodegeneration. 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 NATURE METHODS | VOL.12 NO.12 | DECEMBER 2015 | 1121 TECHNOLOGY FEATURE 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 diseases such as cancer. Fluorescent proteins such as GFP-LC3 Cytoplasm 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 autophagosomes in the answer,” says Klionsky. lined with a protein such as LC3 (microtu- cytosol 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 phosphatidylethanolamine, 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- Nature America, Inc. All rights reserved. America, Inc. Nature Autophagosomes are double-membraned imental approach 5 structures in which cellular components looks at conver- © 201 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. 1122 | VOL.12 NO.12 | DECEMBER 2015 | NATURE METHODS TECHNOLOGY FEATURE in tumor sections, Macleod sees scientists In contrast, when mitophagy 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 necrosis factor–α signaling. That such as mitophagy, in isolation.