Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)
Daniel J. Klionsky1, Amal Kamal Abdel-Aziz2, Sara Abdelfatah3, Mahmoud Abdellatif4 +2980 more•Institutions (777)
TL;DR: In this article, the authors present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes.
Abstract: In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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TL;DR: The results indicate that persistent autophagy after AKI induces pro-fibrotic cytokines in renal tubular cells, promoting renal fibrosis and chronic kidney disease.
Abstract: Autophagy, a highly conserved catabolic pathway in eukaryotic cells, contributes to the maintenance of the homeostasis and function of the kidney. Upon acute kidney injury (AKI), autophagy is activated in renal tubular cells to act as an intrinsic protective mechanism. However, the role of autophagy in the development of chronic kidney pathologies including renal fibrosis after AKI remains unclear. In this study, we detected a persistent autophagy activation in mouse kidneys after nephrotoxicity of repeated low dose cisplatin (RLDC) treatment. 3-methyladenine (3-MA) and chloroquine (CQ), respective inhibitors of autophagy at the initiation and degradation stages, blocked autophagic flux and improved kidney repair in post-RLDC mice, as indicated by kidney weight, renal function, and less interstitial fibrosis. In vitro, RLDC induced a pro-fibrotic phenotype in renal tubular cells, including the production and secretion of pro-fibrotic cytokines. Notably, autophagy inhibitors blocked RLDC-induced secretion of pro-fibrotic cytokines in these cells. Together, the results indicate that persistent autophagy after AKI induces pro-fibrotic cytokines in renal tubular cells, promoting renal fibrosis and chronic kidney disease.
10 citations
TL;DR: The inhibition of TGF-β signaling is recognized as a promising therapeutic strategy for advanced HCC, and the rationale for targeting this pathway for HCC treatment, alone or in combination with other agents is highlighted.
Abstract: Simple Summary Transforming growth factor β (TGF-β) signaling is a preeminent regulator of diverse cellular and physiological processes. Frequent dysregulation of TGF-β signaling has been implicated in cancer. In hepatocellular carcinoma (HCC), the most prevalent form of primary liver cancer, the autocrine and paracrine effects of TGF-β have paradoxical implications. While acting as a potent tumor suppressor pathway in the early stages of malignancy, TGF-β diverts to a promoter of tumor progression in the late stages, reflecting its bright and dark natures, respectively. Within this context, targeting TGF-β represents a promising therapeutic option for HCC treatment. We discuss here the molecular properties of TGF-β signaling in HCC, attempting to provide an overview of its effects on tumor cells and the stroma. We also seek to evaluate the dysregulation mechanisms that mediate the functional switch of TGF-β from a tumor suppressor to a pro-tumorigenic signal. Finally, we reconcile its biphasic nature with the therapeutic implications. Abstract Hepatocellular carcinoma (HCC) is associated with genetic and nongenetic aberrations that impact multiple genes and pathways, including the frequently dysregulated transforming growth factor β (TGF-β) signaling pathway. The regulatory cytokine TGF-β and its signaling effectors govern a broad spectrum of spatiotemporally regulated molecular and cellular responses, yet paradoxically have dual and opposing roles in HCC progression. In the early stages of tumorigenesis, TGF-β signaling enforces profound tumor-suppressive effects, primarily by inducing cell cycle arrest, cellular senescence, autophagy, and apoptosis. However, as the tumor advances in malignant progression, TGF-β functionally switches to a pro-tumorigenic signal, eliciting aggressive tumor traits, such as epithelial–mesenchymal transition, tumor microenvironment remodeling, and immune evasion of cancer cells. On this account, the inhibition of TGF-β signaling is recognized as a promising therapeutic strategy for advanced HCC. In this review, we evaluate the functions and mechanisms of TGF-β signaling and relate its complex and pleiotropic biology to HCC pathophysiology, attempting to provide a detailed perspective on the molecular determinants underlying its functional diversion. We also address the therapeutic implications of the dichotomous nature of TGF-β signaling and highlight the rationale for targeting this pathway for HCC treatment, alone or in combination with other agents.
10 citations
TL;DR: Applications of cryo-ET to cell biology research on plant and algal systems and the special opportunities they offer for understanding the organization of eukaryotic organelles with unprecedently resolution are discussed.
Abstract:
Recent developments in both instrumentation and image analysis algorithms have allowed three-dimensional electron microscopy (3D-EM) to increase automated image collections through large tissue volumes using serial block-face scanning EM (SEM) and to achieve near-atomic resolution of macromolecular complexes using cryo-electron tomography (cryo-ET) and sub-tomogram averaging. In this review, we discuss applications of cryo-ET to cell biology research on plant and algal systems and the special opportunities they offer for understanding the organization of eukaryotic organelles with unprecedently resolution. However, one of the most challenging aspects for cryo-ET is sample preparation, especially for multicellular organisms. We also discuss correlative light and electron microscopy (CLEM) approaches that have been developed for ET at both room and cryogenic temperatures.
10 citations
TL;DR: It is shown that ATG9A-positive vesicles are mobilized during chemotactic stimulation to participate in the extension and stabilization of leading-edge protrusions.
Abstract: Campisi et al. uncover a new function of the core autophagy protein ATG9A in cell migration by demonstrating that ATG9A-positive vesicles are mobilized during chemotactic stimulation to participate in the extension and stabilization of leading-edge protrusions.
9 citations
TL;DR: In this paper, the effects of FSH on dairy goat Sertoli cell autophagy and the role of autophagia in protein clearance were investigated, and it was found that FSH treatment of primary SERToli cells was found to enhance the expression level of LC3-II, reduce p62 degradation and the number of lysosomes, and downregulate the levels of LAMP2 protein and lysomal gene mRNAs.
9 citations
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TL;DR: A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original.
Abstract: The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.
70,111 citations
TL;DR: This paper identified the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes.
7,192 citations
TL;DR: It is demonstrated that the rat microtubule‐associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing.
Abstract: Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3-II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II. The amount of LC3-II is correlated with the extent of autophagosome formation. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes.
6,244 citations
Clotilde Théry1, Kenneth W. Witwer2, Elena Aikawa3, María José Alcaraz4 +414 more•Institutions (209)
TL;DR: The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities, and a checklist is provided with summaries of key points.
Abstract: The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
5,988 citations
TL;DR: A molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1, is demonstrated and a signalling mechanism for UlK1 regulation and autophagic induction in response to nutrient signalling is revealed.
Abstract: Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.
5,314 citations