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

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

Fru-2,6-P2 (fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure-function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2. The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.

Protozoan Kinetoplastida, including the pathogenic trypanosomatids of the genera Trypanosoma and Leishmania, compartmentalize several important metabolic systems in their peroxisomes which are designated glycosomes. The enzymatic content of these organelles may vary considerably during the life-cycle of most trypanosomatid parasites which often are transmitted between their mammalian hosts by insects. The glycosomes of the Trypanosoma brucei form living in the mammalian bloodstream display the highest level of specialization; 90% of their protein content is made up of glycolytic enzymes. The compartmentation of glycolysis in these organelles appears essential for the regulation of this process and enables the cells to overcome short periods of anaerobiosis. Glycosomes of all other trypanosomatid forms studied contain an extended glycolytic pathway catalyzing the aerobic fermentation of glucose to succinate. In addition, these organelles contain enzymes for several other processes such as the pentose-phosphate pathway, beta-oxidation of fatty acids, purine salvage, and biosynthetic pathways for pyrimidines, ether-lipids and squalenes. The enzymatic content of glycosomes is rapidly changed during differentiation of mammalian bloodstream-form trypanosomes to the forms living in the insect midgut. Autophagy appears to play an important role in trypanosomatid differentiation, and several lines of evidence indicate that it is then also involved in the degradation of old glycosomes, while a population of new organelles containing different enzymes is synthesized. The compartmentation of environment-sensitive parts of the metabolic network within glycosomes would, through this way of organelle renewal, enable the parasites to adapt rapidly and efficiently to the new conditions.

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Phosphoglycerate kinase (PGK), a key enzyme in glycolysis, catalyses the transfer of a phosphoryl-group from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP. Despite extensive kinetic and structural investigations over more than two decades, the conformation assumed by this enzyme during catalysis remained unknown. Here we present the 2.8 A crystal structure of a ternary complex of PGK from Trypanosoma brucei, the causative agent of sleeping sickness. This structure determination relied on a procedure in which fragments containing less than 10% of the scattering mass were successively positioned in the unit cell to obtain phases. The PGK ternary complex exhibits a dramatic closing of the large cleft between the two domains seen in all previous studies, thereby bringing the two ligands, 3-phosphoglycerate and ADP into close proximity. Our results demonstrate that PGK is a hinge-bending enzyme, reveal a novel mechanism in which substrate-induced effects combine synergistically to induce major conformational changes and, to our knowledge, afford the first observation of the PGK active site in a catalytic conformation.

In trypanosomes the first part of glycolysis takes place in specialized microbodies, the glycosomes. Most glycolytic enzymes of Trypanosoma brucei have been purified and characterized kinetically. In this paper a mathematical model of glycolysis in the bloodstream form of this organism is developed on the basis of all available kinetic data. The fluxes and the cytosolic metabolite concentrations as predicted by the model were in accordance with available data as measured in non-growing trypanosomes, both under aerobic and under anaerobic conditions. The model also reproduced the inhibition of anaerobic glycolysis by glycerol, although the amount of glycerol needed to inhibit glycolysis completely was lower than experimentally determined. At low extracellular glucose concentrations the intracellular glucose concentration remained very low, and only at 5 mM of extracellular glucose, free glucose started to accumulate intracellularly, in close agreement with experimental observations. This biphasic relation could be related to the large difference between the affinities of the glucose transporter and hexokinase for intracellular glucose. The calculated intraglycosomal metabolite concentrations demonstrated that enzymes that have been shown to be near-equilibrium in the cytosol must work far from equilibrium in the glycosome in order to maintain the high glycolytic flux in the latter.

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