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

Lipins are evolutionarily conserved proteins found from yeasts to humans. Mammalian and yeast lipin proteins have been shown to control gene expression and to enzymatically convert phosphatidate to diacylglycerol, an essential precursor in triacylglcerol (TAG) and phospholipid synthesis. Loss of lipin 1 in the mouse, but not in humans, leads to lipodystrophy and fatty liver disease. Here we show that the single lipin orthologue of Drosophila melanogaster (dLipin) is essential for normal adipose tissue (fat body) development and TAG storage. dLipin mutants are characterized by reductions in larval fat body mass, whole-animal TAG content, and lipid droplet size. Individual cells of the underdeveloped fat body are characterized by increased size and ultrastructural defects affecting cell nuclei, mitochondria, and autophagosomes. Under starvation conditions, dLipin is transcriptionally upregulated and functions to promote survival. Together, these data show that dLipin is a central player in lipid and energy metabolism, and they establish Drosophila as a genetic model for further studies of conserved functions of the lipin family of metabolic regulators.Neutral lipids, or triacylglycerols (TAG), are principal energy stores of the eukaryotic cell. Metazoans have evolved specialized tissues that store TAG and make free fatty acids or other derivatives of TAG available to other tissues. Besides having storage functions, these specialized adipose tissues participate in the control of energy homeostasis by producing and releasing hormones and other signaling molecules (21, 26).Severe underdevelopment of the adipose tissue is observed in mice carrying the fatty liver dystrophy (fld) mutation. Lack of fat tissue is associated with transient postnatal accumulation of TAG in the liver, defects in the peripheral nervous system, and insulin resistance (15,16,29). Cloning of the fld gene (renamed lipin 1 [22]) revealed that it encodes a member of an evolutionarily old family of proteins found in a wide variety of eukaryotic organisms, including fungi, plants, and protozoans (22). Both yeast and mammalian lipin proteins act as type 1 phosphatidate phosphatases (PAP1), converting phosphatidate to diacylglycerol (DAG), and as transcriptional coregulators (4, 5, 11, 32). The protein domains responsible for these activities are conserved in lipin proteins of other species, indicating that this functional dichotomy is both evolutionarily old and central to lipin function. DAG produced by mammalian lipin 1 serves as a direct biosynthetic precursor of TAG and phospholipids, whereas the transcriptional coregulator function contributes to the control of genes involved in hepatic ␤-oxidation of fatty acids, the tricarboxylic acid (TCA) cycle, and mitochondrial oxidative phosphorylation (5). While these processes appear to be upregulated by lipin 1, enzymes involved in fatty acid and TAG synthesis are downregulated. In yeast, lipin suppresses genes involved in phospholipid synthesis (32). Loss of lipin in yeast leads to the overgrowth of i...

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Pipsqueak (Psq) belongs to a family of proteins defined by a phylogenetically old protein-protein interaction motif. Like the GAGA factor and other members of this family, Psq is an important developmental regulator in Drosophila, having pleiotropic functions during oogenesis, embryonic pattern formation, and adult development. The GAGA factor controls the transcriptional activation of homeotic genes and other genes by binding to control elements containing the GAGAG consensus motif. Binding is associated with formation of an open chromatin structure that makes the control regions accessible to transcriptional activators. We show here that Psq contains a novel DNA-binding domain, which binds, like the GAGA factor zinc finger DNA-binding domain, to target sites containing the GAGAG consensus motif. Binding is suppressed, as in the GAGA factor and other proteins of the family, by the associated protein-protein interaction motif. The DNA-binding domain, which we call the Psq domain, is identical with a previously identified region consisting of four tandem repeats of a conserved 50-amino acid sequence, the Psq motif. The Psq domain seems to be structurally related to known DNA-binding domains, both in its repetitive character and in the putative three-␣-helix structure of the Psq motif, but it lacks the conserved sequence signatures of the classical eukaryotic DNA-binding motifs. Psq may thus represent the prototype of a new family of DNA-binding proteins. Members of the BTB1 /POZ protein family play important roles in development and reproduction of Drosophila melanogaster. These proteins contain a protein-protein interaction motif that was first identified in zinc finger proteins encoded by the Drosophila Broad-Complex and tramtrack genes (1, 2), and later also in the bric à brac gene product (3). The domain, which was thereupon designated as BTB (Broad-Complex, Tramtrack, Bric à brac) domain (3), has since been found in proteins of a variety of species, as diverse as slime molds (4) and humans (for a review, see Ref. 5). Many of these proteins are DNA-binding C 2 H 2 zinc finger proteins, but the presence of the domain in a family of pox virus proteins (6) soon indicated that coupling to a DNA-binding domain is not mandatory. The domain is therefore also referred to as the POZ (pox virus, zinc finger) domain (7). In BTB/POZ proteins that contain a zinc finger DNA-binding motif, DNA binding is strongly inhibited by the BTB/POZ domain. This inhibitory effect on DNA binding is also observed in chimeric proteins in which the BTB/POZ domain is associated with a heterologous DNA-binding domain, for instance a POU domain (7). Inhibition of DNA binding appears to be the result of oligomerization through proteinprotein interactions mediated by the BTB/POZ domain.The tendency of BTB/POZ proteins to oligomerize in solution and their localization in distinct nuclear substructures (7-10) suggests that they might act by modifying chromatin structure (5). In fact, such a mode of action is supported by different lines of eviden...

Many prokaryotic and eukaryotic DNA-binding proteins use a helix-turn-helix (HTH) structure for DNA recognition. Here we describe a new family of eukaryotic HTH proteins, the Pipsqueak (Psq) family, which includes proteins from fungi, sea urchins, nematodes, insects, and vertebrates. Three subgroups of the Psq family can be distinguished. Like the HTH proteins of the prokaryotic resolvase family, members of the CENP-B/transposase subgroup catalyze site-specific recombination reactions. This functional conservation, together with a primary sequence similarity between the resolvase and Psq DNA-binding domains, suggests that the resolvase and Psq families are evolutionarily linked. More than half of the newly identified Drosophila Psq proteins contain a BTB protein-protein interaction domain. All proteins of this BTB subgroup belong to the conserved Tramtrack group of BTB-domain proteins. About half of the members of the Tramtrack group contain a Psq domain, while the other half is made up of proteins that contain a zinc finger domain. Thus, nearly all members of this group appear to be DNA-binding proteins. Among other developmental regulators, the Drosophila cell death protein E93 was found to contain a Psq motif and to define a third subgroup of Psq domain proteins. The high sequence conservation of the E93 Psq motif allowed the identification of E93 orthologs in humans and lower metazoans.