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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Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin-like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP-L1 to damaged mitochondria through its amino-terminal LC3-interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.

The regulated clearance of mitochondria is a well recognized but poorly understood aspect of cellular homeostasis, and defects in this process have been linked to aging, degenerative diseases, and cancer. Mitochondria are recycled through an autophagy-related process, and reticulocytes, which completely eliminate their mitochondria during maturation, provide a physiological model to study this phenomenon. Here, we show that mitochondrial clearance in reticulocytes requires the BCL2-related protein NIX (BNIP3L). Mitochondrial clearance does not require BAX, BAK, BCL-X L, BIM, or PUMA, indicating that NIX does not function through established proapoptotic pathways. Similarly, NIX is not required for the induction of autophagy during terminal erythroid differentiation. NIX is required for the selective elimination of mitochondria, however, because mitochondrial clearance, in the absence of NIX, is arrested at the stage of mitochondrial incorporation into autophagosomes and autophagosome maturation. These results yield insight into the mechanism of mitochondrial clearance in higher eukaryotes. Furthermore, they show a BAX-and BAK-independent role for a BCL2-related protein in development.autophagy ͉ mitochondria ͉ BCL2 family B CL2-related proteins play essential roles in the regulation of programmed cell death. Members of the BCL2 family are divided into subgroups based on the presence of one or more BCL2 homology domains (BH1-BH4) (1). BCL2-related proteins possessing a single BH3 domain (BH3-only proteins) are activated by diverse death-inducing stimuli including DNA damage, glucocorticoids, and growth factor deprivation, and their signals are integrated at the mitochondria by the multidomain proapoptotic proteins BAX and BAK (2). BH3-only proteins activate BAX and BAK either directly or indirectly, through binding to and inhibiting the function of antiapoptotic BCL2-related proteins (3-5). BAX or BAK activation in turn causes cytochrome c release, caspase activation, and apoptosis (6, 7). BNIP3 and NIX (also known as BNIP3L) are related proteins with limited homology to BH3-only proteins in a BH3-like domain (8-10). BNIP3 and NIX have uncertain biological function. BNIP3 and NIX localize to the mitochondria when overexpressed, induce cytochrome c release, and cause apoptosis (11-13), however, BNIP3 also causes necrosis-like cell death (14). Hypoxia induces and retinoblastoma protein represses BNIP3 expression through HIF-1␣ and E2F binding sites in the BNIP3 promoter, respectively (15-17). In contrast, NIX is induced by G q -coupled hypertrophic agonists in neonatal rat cardiomyocytes, by p53 in U2OS osteosarcoma cells, and by differentiation of human erythroid cells (18)(19)(20). Accordingly, NIX functions as an effector of G q -dependent cardiomyopathy and negatively regulates tumor growth in nude mice injected with U2OS osteosarcoma cells (19,21). NIX has a role in erythroid development, because Nix Ϫ/Ϫ mice exhibit anemia and erythroid hyperplasia (22). Results and DiscussionDefective Erythropoiesis in Nix ؊...