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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is ∼4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis–induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

Mitochondrial dysfunction is linked to apoptosis, aging, cancer, and a number of neurodegenerative and muscular disorders. The interplay between mitophagy and mitochondrial dynamics has been linked to the removal of dysfunctional mitochondria ensuring mitochondrial quality control. An open question is what role mitochondrial fission plays in the removal of mitochondria after mild and transient oxidative stress; conditions reported to result in moderately elevated reactive oxygen species (ROS) levels comparable to physical activity. Here we show that applying such conditions led to fragmentation of mitochondria and induction of mitophagy in mouse and human cells. These conditions increased ROS levels only slightly and neither triggered cell death nor led to a detectable induction of non-selective autophagy. Starvation led to hyperfusion of mitochondria, to high ROS levels, and to the induction of both non-selective autophagy and to a lesser extent to mitophagy. We conclude that moderate levels of ROS specifically trigger mitophagy but are insufficient to trigger non-selective autophagy. Expression of a dominant-negative variant of the fission factor DRP1 blocked mitophagy induction by mild oxidative stress as well as by starvation. Taken together, we demonstrate that in mammalian cells under mild oxidative stress a DRP1-dependent type of mitophagy is triggered while a concomitant induction of non-selective autophagy was not observed. We propose that these mild oxidative conditions resembling well physiological situations are thus very helpful for studying the molecular pathways governing the selective removal of dysfunctional mitochondria.

Autophagy, a highly regulated programme found in almost all eukaryotes, is mainly viewed as a catabolic process that degrades nonessential cellular components into molecular building blocks, subsequently available for biosynthesis at a lesser expense than de novo synthesis. Autophagy is largely known to be regulated by nutritional conditions. Here we show that, in yeast cells grown under nonstarving conditions, autophagy can be induced by mitochondrial dysfunction. Electron micrographs and biochemical studies show that an autophagic activity can result from impairing the mitochondrial electrochemical transmembrane potential. Furthermore, mitochondrial damage-induced autophagy results in the preferential degradation of impaired mitochondria (mitophagy), before leading to cell death. Mitophagy appears to rely on classical macroautophagy machinery while being independent of cellular ATP collapse. These results suggest that in this case, autophagy can be envisioned either as a process of mitochondrial quality control, or as an ultimate cellular response triggered when cells are overwhelmed with damaged mitochondria.

Mitophagy has been recently described as a mechanism of elimination of damaged organelles. Although the regulation of the amount of mitochondria is a core issue concerning cellular energy homeostasis, the relationship between mitochondrial degradation and energetic activity has not yet been considered. Here, we report that the stimulation of mitochondrial oxidative phosphorylation enhances mitochondrial renewal by increasing its degradation rate. Upon high oxidative phosphorylation activity, we found that the small GTPase Rheb is recruited to the mitochondrial outer membrane. This mitochondrial localization of Rheb promotes mitophagy through a physical interaction with the mitochondrial autophagic receptor Nix and the autophagosomal protein LC3-II. Thus, Rheb-dependent mitophagy contributes to the maintenance of optimal mitochondrial energy production. Our data suggest that mitochondrial degradation contributes to a bulk renewal of the organelle in order to prevent mitochondrial aging and to maintain the efficiency of oxidative phosphorylation.

The translocation of Bax ␣, a pro-apoptotic member of the BCL-2 family from the cytosol to mitochondria, is a central event of the apoptotic program. We report here that the N-terminal (NT) end of Bax ␣, which contains its first ␣ helix (⌯␣1), is a functional mitochondrial-addressing signal both in mammals and in yeast. Similar results were obtained with a newly described variant of Bax called Bax , which lacks the first 20 amino acids of Bax ␣ and is constitutively associated with mitochondria. Deletion of ⌯␣1 impairs the binding of Bax to mitochondria, whereas a fusion of the N terminus of Bax ␣, which contains ⌯␣1 with a cytosolic protein, results in the binding of the chimeric proteins to mitochondria both in a cell-free assay and in vitro. More importantly, the mitochondria-bound chimeric proteins inhibit the interaction of Bax with mitochondria as well as Baxapoptogenic properties. The mutations of the ⌯␣1, which inhibit Bax ␣ and Bax translocation to mitochondria, also block the subsequent activation of the execution phase of apoptosis. Conversely, a deletion of the C terminus does not appear to influence Bax ␣ and Bax mitochondrial addressing. Taken together, our results suggest that Bax is targeted to mitochondria by its NT and thus through a pathway that is unique for a member of the BCL-2 family.Apoptosis is finely regulated by intracellular events, which at its onset, appear to be under the control of members of the BCL-2 family (1). The main site of action of these proteins appears to be the mitochondrion, particularly through the control of the release of apoptogenic factors from this organelle (2, 3). Members of the BCL-2 family can be anti-apoptotic or proapoptotic and totally or partially mitochondrial-bound or cytosolic (4). In most cells, one of the crucial and most regulated steps in the implementation of apoptosis is the control of the translocation of Bax ␣ from the cytosol to the mitochondria (4). Anti-apoptotic proteins such as Bcl-2 or Bcl-XL act as inhibitors of Bax function, whereas proapoptotic BH3 only members can either hinder this inhibition (e. g. Bad) or directly activate Bax ␣ (e. g. Bid), which in both cases would promote the association of Bax ␣ with the mitochondrial outer membrane (MOM) 1 (5, 6). Bcl-2 is anchored into MOM bilayer by a short hydrophobic domain close to the C terminus (CT) with its N terminus (NT) exposed toward the cytosol (7-9). Bcl-2 belongs to a class of membrane proteins called tail-anchored (TA) proteins, which are associated with different intracellular membranes, usually MOM and the endoplasmic reticulum (10). Based on the homology with Bcl-2, it has been proposed that Bax became inserted to mitochondria by its hydrophobic ␣9 helix located at its Cterminal end (for example, see Ref. 11). Several studies have shown that the Bax ␣ CT is not required for its interaction with mitochondria, whereas others have found it mandatory for Bax ␣ function (12-16). We have observed that a chimeric Bcl-XL construct in which the natural Bcl-XL CT was substituted by t...