Open Access
Guidelines for the use and interpretatoin of assays for monitoring autophagy
D. J. Klionsky,F. C. Abdalla,H. Abeliovich,R. T. Abraham,A. Acevedo-Arozena,K. Adeli,L. Agholme,M. Agnello,A. M. Engelbrecht,B. Loos +9 more
TLDR
In this paper, the authors present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macro-autophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes.Abstract:
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.read more
Citations
More filters
Induction of autophagy by spermidine promotes longevity
Tobias Eisenberg,Heide Knauer,Alexandra Schauer,Sabrina Büttner,Christoph Ruckenstuhl,Didac Carmona-Gutierrez,Julia Ring,Sabrina Schroeder,Christoph Magnes,Lucia Antonacci,Heike Fussi,Luiza Deszcz,Luiza Deszcz,Regina Hartl,Regina Hartl,Elisabeth Schraml,Alfredo Criollo,Evgenia Megalou,Daniela Weiskopf,Peter Laun,Gino Heeren,Michael Breitenbach,Beatrix Grubeck-Loebenstein,Eva Herker,Birthe Fahrenkrog,Kai-Uwe Fröhlich,Frank Sinner,Nektarios Tavernarakis,Nadège Minois,Nadège Minois,Nadège Minois,Guido Kroemer,Frank Madeo +32 more
TL;DR: Administration of spermidine markedly extended the lifespan of yeast, flies and worms, and human immune cells and inhibited oxidative stress in ageing mice, and found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.
Suppression of autophagy precipitates neuronal cell death following low doses of methamphetamine
TL;DR: It is shown that autophagy is rapidly up‐regulated in response to methamphetamine, and the signaling pathway and the significance (protective vs. toxic) of autophile activation and the convergence of the autophagic and the ubiquitin‐proteasome pathways at the level of the same intracellular bodies in a simple cell model of methamphetamine toxicity are investigated.
References
More filters
Journal ArticleDOI
LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing
Yukiko Kabeya,Noboru Mizushima,Noboru Mizushima,Takashi Ueno,Akitsugu Yamamoto,Takayoshi Kirisako,Takayoshi Kirisako,Takeshi Noda,Takeshi Noda,Eiki Kominami,Yoshinori Ohsumi,Yoshinori Ohsumi,Tamotsu Yoshimori,Tamotsu Yoshimori +13 more
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.
Journal ArticleDOI
AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1
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.
Journal ArticleDOI
Role of AMP-activated protein kinase in mechanism of metformin action
Gaochao Zhou,Robert W. Myers,Ying Li,Yuli Chen,Xiaolan Shen,Judy Fenyk-Melody,Margaret Wu,John Ventre,Thomas W. Doebber,Nobuharu Fujii,Nicolas Musi,Michael F. Hirshman,Laurie J. Goodyear,David E. Moller +13 more
TL;DR: It is reported that metformin activates AMPK in hepatocytes; as a result, acetyl-CoA carboxylase (ACC) activity is reduced, fatty acid oxidation is induced, and expression of lipogenic enzymes is suppressed.
Journal ArticleDOI
Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.
Nathan C. Shaner,Robert E. Campbell,Robert E. Campbell,Paul Steinbach,Ben N G Giepmans,Amy E. Palmer,Roger Y. Tsien +6 more
TL;DR: The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1, and three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
Journal ArticleDOI
Methods in Mammalian Autophagy Research
TL;DR: Methods to monitor autophagy and to modulate autophagic activity are discussed, with a primary focus on mammalian macroautophagy.