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

Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 prevents cells from undergoing apoptosis in response to a variety of stimuli. Cytosolic cytochrome c is necessary for the initiation of the apoptotic program, suggesting a possible connection between Bcl-2 and cytochrome c, which is normally located in the mitochondrial intermembrane space. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria. Overexpression of Bcl-2 prevented the efflux of cytochrome c from the mitochondria and the initiation of apoptosis. Thus, one possible role of Bcl-2 in prevention of apoptosis is to block cytochrome c release from mitochondria.

A sudden increase in permeability of the inner mitochondrial membrane, the so-called mitochondrial permeability transition, is a common feature of apoptosis and is mediated by the mitochondrial permeability transition pore (mtPTP). It is thought that the mtPTP is a protein complex formed by the voltage-dependent anion channel, members of the pro-and anti-apoptotic BAX-BCL2 protein family, cyclophilin D, and the adenine nucleotide (ADP/ATP) translocators (ANTs) 1,2 . The latter exchange mitochondrial ATP for cytosolic ADP and have been implicated in cell death. To investigate the role of the ANTs in the mtPTP, we genetically inactivated the two isoforms of ANT [3][4][5] in mouse liver and analysed mtPTP activation in isolated mitochondria and the induction of cell death in hepatocytes. Mitochondria lacking ANT could still be induced to undergo permeability transition, resulting in release of cytochrome c. However, more Ca 2+ than usual was required to activate the mtPTP, and the pore could no longer be regulated by ANT ligands. Moreover, hepatocytes without ANT remained competent to respond to various initiators of cell death. Therefore, ANTs are non-essential structural components of the mtPTP, although they do contribute to its regulation.To investigate the role of ANTs in the mtPTP, we inactivated both the heart-muscle (Ant1) and the systemic (Ant2) ANT isoform genes in the mouse liver. Humans have three ANT genes 6,7 , whereas results of complementary DNA library screening 5 and northern and western analyses 3 have suggested that mouse has only two Ant genes. To verify this, we screened the Celera and Ensembl mouse genome assemblies, as well as the respective EST 1a). Targeted embryonic stem cells (Fig. 1b) were used to introduce the conditional floxed allele into the mouse germ line. Liver-specific inactivation of the Ant2 fl allele was achieved by breeding Ant2 fl mice with an Alb-Cre line of transgenic mice that expresses CRE under control of the liver-specific albumin promoter, which results in the excision of exons 3 and 4 of Ant2 (Fig. 1a) Endogenous respiration rates of ANT1/ANT2-deficient mitochondria were almost twice that of control mitochondria (34.58 ± 1.6 versus 18.12 ± 1.1 nmol O min -1 per mg protein) (Fig. 2b) and the mitochondrial membrane potential (ΔP = ΔΨ + ΔpH) of the ANT-deficient mitochondria was higher than that of controls (191.7 ± 4.9 versus 172.9 ± 3.5 mV). Analysis of the specific activity of OXPHOS enzyme complexes in ANT-deficient mitochondria revealed that complex IV (cytochrome c oxidase, COX) was increased more than twofold compared with controls (P < 0.01) (Fig. 2c). This was confirmed by western blot analysis, which revealed that the mitochondrial COX subunit I (COI) and cytochrome c proteins were more abundant in the ANT-deficient mitochondria (Fig. 2d). Hence, the increased respiration rate is likely to be the result of the specific upregulation in COX activity, suggesting that COX activity may modulate respiration rate. Because ΔP is the product of proton pumpin...

Mitochondrial cytochrome c (cyt c) has been found to have dual functions in controlling both cellular energetic metabolism and apoptosis. Through interaction with apoptotic protease activating factors (Apaf), cyt c can initiate the activation cascade of caspases once it is released into the cytosol. The loss of a component of the mitochondrial electron transport chain also triggers the generation of superoxide. Although cyt c can be released independent of the mitochondrial permeability transition (MPT), the accompanying cellular redox change can trigger the MPT. Since another apoptotic protease, AIF, is released by MPT, the two separate pathways provide redundancy that ensures effective execution of the cell death program. Anti-apoptotic Bcl-2 family proteins function as gatekeepers to prevent the release of both cyt c and AIF. In spite of their stabilization effect on the mitochondrial outer membrane, Bcl-2 proteins may also be involved in the direct binding of Apaf molecules as regulatory elements further downstream from the mitochondrial apoptotic signals.

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