Per O. Seglen

Methods for monitoring autophagy from yeast to human

The increasing interest in autophagy in a wide range of organisms, accompanied by an ever-growing influx of researchers into this field, necessitates a good understanding of the methodologies available to monitor this process. In this review we discuss current approaches that can be used to follow the overall process of autophagy, as well as individual steps, from yeast to human. The majority of the review considers methods that apply to macroautophagy; however, we also consider alternative types of degradation including chaperone-mediated autophagy and microautophagy. This information is meant to provide a resource for newcomers as well as a stimulus for experienced researchers who may be prompted to develop additional assays to examine autophagy-related pathways.

Does bafilomycin A1 block the fusion of autophagosomes with lysosomes

Bafilomycin A1 is a specific inhibitor of the vacuolar type H+-ATPase (V-ATPase) in cells, and inhibits the acidification of organelles containing this enzyme, such as lysosomes and endosomes. Recently, while editing and reviewing chapters on autophagy for Methods in Enzymology, we noticed repeated references to the effect of bafilomycin A1 in blocking the fusion of autophagosomes with lysosomes. Of course we have seen this in various research papers as well, but reading this routinely in chapters written by various people over a short period of time really caused this to stand out. Every one of these chapters referred to the paper by Yamamoto et al. In that paper, treatment with 100 nM bafilomycin A1 for 1 h blocks the fusion of autophagosomes with lysosomes in the rat hepatoma H-4-II-E cell line, based on data from electron microscopy. However, data from one of our labs noted an apparently different result in a relatively recent manuscript. Therefore, we decided to look into this more carefully.

Isolation and Characterization of Rat Liver Amphisomes: EVIDENCE FOR FUSION OF AUTOPHAGOSOMES WITH BOTH EARLY AND LATE ENDOSOMES

Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.

Prelysosomal convergence of autophagic and endocytic pathways.

[14C]Lactose, electroinjected into the cytosol of isolated rat hepatocytes, was sequestered by autophagy, transferred to lysosomes and eventually hydrolysed. Asparagine prevented the fusion between prelysosomal autophagic vacuoles and lysosomes, and caused lactose to accumulate in the former. However, if the hepatocytes were simultaneously allowed to endocytose added beta-galactosidase, no lactose accumulation occurred. These results suggest that autophagically sequestered lactose and endocytosed beta-galactosidase were delivered to the same prelysosomal vacuole, where the lactose was hydrolysed by the enzyme. The name amphisome is suggested for this new functional compartment, common to the autophagic and endocytic pathways.

Seeing is believing: The impact of electron microscopy on autophagy research

Autophagy was first discovered by transmission electron microscopy more than 50 years ago. For decades, electron microscopy was the only way to reliably detect autophagic compartments in cells because no specific protein markers were known. In the 1970s, however, the introduction of biochemical methods enabled quantitative studies of autophagic-lysosomal degradation, and in the 1980s specific biochemical assays for autophagic sequestration became available. Since the identification of autophagy-related genes in the 1990s, combined fluorescence microscopy, biochemical and genetic methods have taken the leading role in autophagy research. However, electron microscopy is still needed to confirm and verify results obtained by other methods, and also to produce novel knowledge that would not be achievable by any other experimental approach. Confocal microscopy, with its ever-improving resolution, is probably the best-suited morphological approach to investigate the dynamic aspects of autophagy. However, for analyzing the ultrastructural details of the many novel organelles and mechanisms involved in specific subtypes of autophagy, the electron microscope is still indispensable. This review will summarize the impact that electron microscopy has had on autophagy research since the discovery of this self-degradation process in the mid-1950s. Astonishingly, some of the “novel” concepts and principles of autophagy, presented in the recent studies, were already proposed several decades ago by the pioneering, accurate and passionate work of virtuoso electron microscopists.

Amino acid inhibition of the autophagic/lysosomal pathway of protein degradation in isolated rat hepatocytes

Protein degradation in isolated rat hepatocytes, as measured by the release of [14C]valine from pre-labelled protein, is partly inhibited by a physiologically balanced mixture of amino acids. The inhibition is largely due to the seven amino acids leucine, phenylalanine, tyrosine, tryptophan, histidine, asparagine and glutamine. When the amino acids are tested individually at different concentrations, asparagine and glutamine are the strongest inhibitors. However, when various combinations are tested, a mixture of the first five amino acids as well as a combination of leucine and asparagine inhibit protein degradation particularly strongly. The inhibition brought about by asparagine plus leucine is not additive to the inhibition by propylamine, a lysosomotropic inhibitor; thus indicating that the amino acids act exclusively upon the lysosomal pathway of protein degradation. Following a lag of about 15 min the effect of asparagine plus leucine is maximal and equal to the effect of propylamine, suggesting that their inhibition of the lysosomal pathway is complete as well as specific. Degradation of endocytosed 125I-labelled asialofetuin is not affected by asparagine plus leucine, indicating that the amino acids do not affect lysosomes directly, but rather inhibit autophagy at a step prior to the fusion of autophagic vacuoles with lysosomes. The aminotransferase inhibitor, aminooxyacetate, does not prevent the inhibitory effect of any of the amino acids, i.e. amino acid metabolites are apparently not involved.

Autoregulation of glycolysis, respiration, gluconeogenesis and glycogen synthesis in isolated parenchymal rat liver cells under aerobic and anaerobic conditions

Abstract 1. 1. Parenchymal cells isolated from the livers of 16-h-fasted rats have a very low ability to utilize glocuse. At 20 mM glucose, there is no net glucose consumption, no stimulation of respiration, and very low rates of glycolysis and glycogen synthesis. Cells from fed rats consume glucose more readily. 2. 2. Fructose is utilized very effectively by isolated liver cells from both fed and fasted rats. At 20 mM fructose, respiration is approximately doubled (i.e. there is no Crabtree effect), the rate of gluconeogenesis is high, glycogen accumulates and the glycolytic rate is 10 – 30 times higher than with glucose under aerobic as well as anaerobic conditions. 3. 3. Gluconeogenesis (formation of glucose + glycogen) from lactate, pyruvate or fructose is not subject to end-product inhibition by glucose at physiological concentrations (5 – 20 mM) in cells from fasted rats. However, glocose has a stimulatory and probably direct effect on glycogen synthesis, thereby directing a greater fraction of the gluconeogenic precursors into glycogen. Liver cells from 40-h-starved rats have largely lost the ability to synthetize glycogen. 4. 4. Glycogins (from fructose) is strongly inhibited by the end-product lactate under aerobic conditions. 5. 5. Lactate, but bit pyruvate, also slightly inhibits glycogen synthesis from high concentrations of fructose. Quinolinate, which blocks gluconeogenesis from lactate (but not from fructose) does not prevent lactate inhibition of glycogen synthesis, but since quinolinate alone stimulates glycogen synthesis the specificity of this inhibitor is doubtful. 6. 6. Under anaerobic conditions, glycolysins from fructose is strongly stimulated (classical Pasteur effect) whereas gluconeogenesis and glycogen synthesis are blocked. The end-product inhibition of glycolysis by lactate also largely disappears during anaerobis. Since previous experiments have shown that anoxia furthermore stimulates glycogenolysis and abolishes the glycogenolytic control function of glucose, the concept of “generalized Pasteur effect” introduced in order to emphasize the multiplicity of oxygen effects on liver cabohydrate metabolism.

Incorporation of radioactive amino acids into protein in isolated rat hepatocytes

The incorporation of radioactivity from a 14C-labelled amino acid mixture (algal protein hydrolysate) into protein in isolated rat hepatocytes has been studied. The incorporation rate declined with increasing cell concentration, an effect which could be explained partly by isotope consumption, partly (and largely) by isotope dilution due to the formation of non-labelled amino acids by the cells. At a high extracellular amino acid concentration, the rate of incorporation into protein became independent of cell concentration, because the isotope dilution effect was now quantitatively insignificant. The time course of protein labelling at various cells concentrations correlated better with the intracellular than with the extracellular amino acid specific activity, suggesting that amino acids for protein synthesis were taken from an intracellular pool. With increasing extracellular amino acid concentrations both the intracellular amino acid concentration, the intracellular radioactivity and the rate of incorporation into protein increased. Protein labelling exhibited a distinct time lag at high amino acid concentrations, presumably reflecting the time-dependent expansion of the intracellular amino acid pool. The gradual increase in the rate of protein labelling could be due either to an increased intracellular specific activity, or to a real stimulation of protein synthesis by amino acids, depending on whether the total intracellular amino acid pool or just the expandable compartment is the precursor pool for protein synthesis.

How shall i eat thee

If you work in the field of autophagy we do not really need to tell you that this research area has grown tremendously. Along with that growth has developed a need for some unification of the nomenclature. In 2003, researchers working with the yeast model system proposed the use of the acronym ATG to denote AuTophaGy-related genes,1 and this designation has also been adopted for most of the genes involved in autophagy in higher eukaryotes. Similarly, a common nomenclature for isoforms of lysosome associated protein type 2 (LAMP-2) was recently proposed, hopefully reducing some of the confusion resulting from the use of multiple names.2 At this time we thought it worthwhile to consider the terms being used to describe different types of lysosomal or vacuolar degradative pathways. Many names are being introduced, and this is reasonable to the extent that these various processes have distinct features; each unique process needs a specific name to avoid confusion, and to eliminate the need for a lengthy description. It would be helpful, however, if the community agreed on their use. Finally, the addition or use of a name that implies a unique process must be backed up by data that justify the nomenclature. Thus, researchers should verify that a process is specific before using a name that implies specificity. For example, to demonstrate selectivity in organelle degradation it is incumbent upon the researcher to show that the organelle in question, and not other organelles, is sequestered and/or degraded with kinetics that distinguish it from a bulk, nonspecific process. There are many types of autophagy. To our knowledge, the term “autophagy” (from the Greek “auto” for “self ” and “phagein” meaning “to eat”) was first used in a 1963 review article by Christian de Duve.3 The first reference we have found in a research paper is in regard to a possible role of autophagy in lung cancer;4 however, as this work was published in an Italian journal, we are not able to comment on this in any authoritative manner. The following year, de Duve published a highly referenced review,5 and by this time the authors unquestionably refer to the process of macroautophagy, although the actual term was introduced later.6 Perhaps the most distinguishing feature of macroautophagy for the purposes of this discussion is that it involves the generally nonspecific (see ref. 7 for an exception) sequestration of cytoplasm within a non-lysosomal/vacuolar compartment, usually delimited by a double or multiple membrane; this compartment is typically referred to as an autophagosome. Another long-standing term that has not seen tremendous usage of late, but that is experiencing renewed interest, is “crinophagy” that is derived from the Greek “crin” meaning, “to secrete”. As far as we can tell this name also derives from de Duve.8 “Crinophagy” was originally used to describe the direct fusion of secretory vesicles with lysosomes (e.g., see refs. 9–11), resulting in the formation of a “crinosome.”12 This topic has attracted recent attention because of possible connections with diabetes, as crinophagy appears to be used for the regulated degradation of vesicle-stored insulin.13 It is not known whether insulin degradation incorporates any aspects of macroautophagy, but that possibility has not been ruled out. We suggest that we retain the use of the term “crinophagy” as it was originally described; if it turns out that the degradation of insulin does involve a macroautophagic process, we think we will need to introduce another name. We note that “insulinophagy” should be avoided because the target of degradation would presumably be the vesicles that contain insulin rather than the hormone itself. One possibility would be “secrephagy” to note that the target is secretory vesicles, or alternatively “macrocrinophagy.” There is probably no controversy about the use or meaning of the name “chaperonemediated autophagy” (CMA),14 which is a process involving the direct translocation of

Inhibition of hepatocytic autophagy by adenosine, aminoimidazole-4- carboxamide riboside, and N6-mercaptopurine riboside. Evidence for involvement of AMP-activated protein kinase

To examine the role of AMP-activated protein kinase (AMPK; EC 2.7.1.109) in the regulation of autophagy, rat hepatocytes were incubated with the AMPK proactivators, adenosine, 5-amino-4-imidazole carboxamide riboside (AICAR), orN 6-mercaptopurine riboside. Autophagic activity was inhibited by all three nucleosides, AICAR andN 6-mercaptopurine riboside being more potent (IC50 = 0.3 mm) than adenosine (IC50 = 1 mm). 2′-Deoxycoformycin, an adenosine deaminase (EC 3.5.4.4) inhibitor, increased the potency of adenosine 5-fold, suggesting that the effectiveness of adenosine as an autophagy inhibitor was curtailed by its intracellular deamination. 5-Iodotubercidin, an adenosine kinase (EC 2.7.1.20) inhibitor, abolished the effects of all three nucleosides, indicating that they needed to be phosphorylated to inhibit autophagy. A 5-iodotubercidin-suppressible phosphorylation of AICAR to 5-aminoimidazole-4-carboxamide riboside monophosphate was confirmed by chromatographic analysis. AICAR, up to 0.4 mm, had no significant effect on intracellular ATP concentrations. Because activated AMPK phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (EC 1.1.1.88), the rate-limiting enzyme in cholesterol synthesis, the strong inhibition of hepatocytic cholesterol synthesis by all three nucleosides confirmed their ability to activate AMPK under the conditions used. Lovastatin and simvastatin, inhibitors of HMG-CoA reductase, strongly suppressed cholesterol synthesis while having no effect on autophagic activity, suggesting that AMPK inhibits autophagy independently of its effects on HMG-CoA reductase and cholesterol metabolism.