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

In metazoans, Bcl-2 family proteins are major regulators of mitochondrially mediated apoptosis; however, their evolution remains poorly understood. Here, we describe the molecular characterization of the four members of the Bcl-2 family in the most primitive metazoan, Trichoplax adhaerens. All four trBcl-2 homologs are multimotif Bcl-2 group, with trBcl-2L1 and trBcl-2L2 being highly divergent antiapoptotic Bcl-2 members, whereas trBcl-2L3 and trBcl-2L4 are homologs of proapoptotic Bax and Bak, respectively. trBax expression permeabilizes the mitochondrial outer membrane, while trBak operates as a BH3-only sensitizer repressing antiapoptotic activities of trBcl-2L1 and trBcl-2L2. The crystal structure of a trBcl-2L2:trBak BH3 complex reveals that trBcl-2L2 uses the canonical Bcl-2 ligand binding groove to sequester trBak BH3, indicating that the structural basis for apoptosis control is conserved from T. adhaerens to mammals. Finally, we demonstrate that both trBax and trBak BH3 peptides bind selectively to human Bcl-2 homologs to sensitize cancer cells to chemotherapy treatment.

In this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2–TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER–mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2–TOM20 interaction is thus an additional player in the control of apoptosis.