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
Autophagy, or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation, with an astonishing number of connections to human disease and physiology. For example, autophagic dysfunction is associated with cancer, neurodegeneration, microbial infection and ageing. Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. Understanding autophagy may ultimately allow scientists and clinicians to harness this process for the purpose of improving human health.At first glance, it may seem perplexing that a process of cellular self-eating could be beneficial. In its simplest form, however, autophagy probably represents a single cell's adaptation to starvation-if there is no food available in the surroundings, a cell is forced to break down part of its own reserves to stay alive until the situation improves. In single-cell organisms such as yeasts, this starvation response is one of the primary functions of autophagy, but in fact this role extends up through to humans. For example, even on a day-to-day basis, autophagy is activated between meals in organs such as the liver to maintain its metabolic functions, supplying amino acids and energy through catabolism 1,2 .There are various types of autophagy, including micro-and macroautophagy, as well as chaperone-mediated autophagy (CMA), and they differ in their mechanisms and functions ( Fig. 1) 3,4 . Both micro-and macroautophagy have the capacity to engulf large structures through both selective and non-selective mechanisms, whereas CMA degrades only soluble proteins, albeit in a selective manner. The capacity for large-scale degradation is important in autophagic function, but it carries a certain risk, because unregulated degradation of the cytoplasm is likely to be lethal. On the other hand, basal levels of autophagy are important for maintaining normal cellular homeostasis. Thus, it is important that autophagy be tightly regulated (Fig. 2) so that it is induced when needed, but otherwise maintained at a basal level. Although a complete picture of autophagy regulation is not available, many aspects have been covered in recent reviews 5-8 .Both the non-selective and selective nature of autophagy, as well as basal and induced levels, are important in regard to the role of this process in human health and disease. Perhaps the most fundamental point is that either too little or too much autophagy can be deleterious, a Autophagy in cell survival and cell deathThe pro-survival function of autophagy has been demonstrated at the cellular and organismal level in different contexts, including during nutrient and growth factor deprivation, endoplasmic reticulum stress, development, microbial infection, and diseases characterized by the accumulation of protein aggregates 8-11 . This pro-survival function is generally believed to be adaptive, but, in the context of cancer, is potentially maladaptive 12 . Metabolic stress is a common feature of the tumour microenvironment and m...
Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.
Autophagy is a process of self-degradation of cellular components in which double-membrane autophagosomes sequester organelles or portions of cytosol and fuse with lysosomes or vacuoles for breakdown by resident hydrolases. Autophagy is upregulated in response to extra-or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection. Defective autophagy plays a significant role in human pathologies, including cancer, neurodegeneration, and infectious diseases. We present our current knowledge on the key genes composing the autophagy machinery in eukaryotes from yeast to mammalian cells and the signaling pathways that sense the status of different types of stress and induce autophagy for cell survival and homeostasis. We also review the recent advances on the molecular mechanisms that regulate the autophagy machinery at various levels, from transcriptional activation to post-translational protein modification.
Macroautophagy is a dynamic process involving the rearrangement of subcellular membranes to sequester cytoplasm and organelles for delivery to the lysosome or vacuole where the sequestered cargo is degraded and recycled. This process takes place in all eukaryotic cells. It is highly regulated through the action of various kinases, phosphatases, and guanosine triphosphatases (GTPases). The core protein machinery that is necessary to drive formation and consumption of intermediates in the macroautophagy pathway includes a ubiquitin-like protein conjugation system and a protein complex that directs membrane docking and fusion at the lysosome or vacuole. Macroautophagy plays an important role in developmental processes, human disease, and cellular response to nutrient deprivation.
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.
Autophagy, the process by which cells recycle cytoplasm and dispose of excess or defective organelles, has entered the research spotlight largely owing to the discovery of the protein components that drive this process. Identifying the autophagy genes in yeast and finding orthologs in other organisms reveals the conservation of the mechanism of autophagy in eukaryotes and allows the use of molecular genetics and biology in different model systems to study this process. By mostly morphological studies, autophagy has been linked to disease processes. Whether autophagy protects from or causes disease is unclear. Here, we summarize current knowledge about the role of autophagy in disease and health.Cellular homeostasis requires a constant balance between biosynthetic and catabolic processes. Eukaryotic cells primarily use two distinct mechanisms for large-scale degradation, the proteasome and autophagy; but only autophagy has the capacity to degrade entire organelles. The three types of autophagy are macroautophagy, microautophagy, and chaperone-mediated autophagy (1). Here, we will focus on macroautophagy, hereafter called autophagy, which plays an important physiological role in human health. In autophagy, a double-or multimembrane-bound structure, called the autophagosome or autophagic vacuole, is formed de novo to sequester cytoplasm. Then, the vacuole membrane fuses with the lysosome to deliver the contents into the organelle lumen, where they are degraded and the resulting macromolecules recycled (Fig. 1).Autophagy occurs at basal levels in most tissues and contributes to the routine turnover of cytoplasmic components. However, autophagy can be induced by a change of environmental conditions such as nutrient depletion. In addition to turnover of cellular components, autophagy is involved in development, differentiation, and tissue remodeling in various organisms (2). Autophagy is also implicated in certain human diseases. Paradoxically, autophagy can serve to protect cells but may also contribute to cell damage (Table 1). Here, we will summarize the current connections between autophagy and human disease and aging. Programmed Cell DeathAutophagy is involved in programmed cell death (PCD). Type I PCD, apoptosis, is characterized by condensation of cytoplasm and chromatin, DNA fragmentation, and cell fragmentation into apoptotic bodies, followed by removal and degradation of the dying cells by phagocytosis. Type II PCD (autophagic) is characterized by the accumulation of autophagic vesicles (autophagosomes and autophagolysosomes) and is often observed when massive cell elimination is demanded or when phagocytes do not have easy access to the dying cells. One feature that distinguishes apoptosis from autophagic cell death is the source of the lysosomal enzymes used for most of the dying cells' degradation. Apoptotic cells use phagocytic cell lysosomes for this process, whereas cells with autophagic morphology use the dying cells'
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