Methods in Mammalian Autophagy Research

doi:10.1016/J.CELL.2010.01.028

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Methods in Mammalian Autophagy Research

Journal Article•DOI•

Methods in Mammalian Autophagy Research

Noboru Mizushima¹, Tamotsu Yoshimori², Beth Levine³•Institutions (3)

Tokyo Medical and Dental University¹, Osaka University², University of Texas Southwestern Medical Center³

05 Feb 2010-Cell (Cell Press)-Vol. 140, Iss: 3, pp 313-326

TL;DR: Methods to monitor autophagy and to modulate autophagic activity are discussed, with a primary focus on mammalian macroautophagy.

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About: This article is published in Cell.The article was published on 2010-02-05 and is currently open access. It has received 3998 citations till now. The article focuses on the topics: Autophagy database & MAP1LC3B.

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Journal Article•DOI•

Guidelines for the use and interpretation of assays for monitoring autophagy

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Daniel J. Klionsky¹, Fábio Camargo Abdalla², Hagai Abeliovich³, Robert T. Abraham⁴ +1284 more•Institutions (463)

01 Apr 2012-Autophagy

TL;DR: These guidelines are presented 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.

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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.

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4,316 citations

Journal Article•DOI•

Autophagy and the Integrated Stress Response

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Guido Kroemer¹, Guillermo Mariño¹, Guillermo Mariño², Beth Levine³•Institutions (3)

French Institute of Health and Medical Research¹, Institut Gustave Roussy², University of Texas Southwestern Medical Center³

22 Oct 2010-Molecular Cell

TL;DR: Autophagy is a cell biological process that is a central component of the integrated stress response and can be integrated with other cellular stress responses through parallel stimulation of autophagy and other stress responses by specific stress stimuli.

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3,002 citations

Cites background from "Methods in Mammalian Autophagy Rese..."

...The lipidated form of LC3 is stably associated with the autophagosome membrane, and its biochemical and microscopic detection is widely used to measure cellular autophagy (Mizushima et al., 2010) (Figure 1C)....
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Journal Article•DOI•

Autophagy in immunity and inflammation

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Beth Levine¹, Noboru Mizushima², Herbert W. Virgin³•Institutions (3)

University of Texas Southwestern Medical Center¹, Tokyo Medical and Dental University², Washington University in St. Louis³

20 Jan 2011-Nature

TL;DR: A crucial role is revealed for the autophagy pathway and proteins in immunity and inflammation, and they balance the beneficial and detrimental effects of immunity andinflammation, and thereby may protect against infectious, autoimmune and inflammatory diseases.

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Abstract: Autophagy is an essential, homeostatic process by which cells break down their own components. Perhaps the most primordial function of this lysosomal degradation pathway is adaptation to nutrient deprivation. However, in complex multicellular organisms, the core molecular machinery of autophagy - the 'autophagy proteins' - orchestrates diverse aspects of cellular and organismal responses to other dangerous stimuli such as infection. Recent developments reveal a crucial role for the autophagy pathway and proteins in immunity and inflammation. They balance the beneficial and detrimental effects of immunity and inflammation, and thereby may protect against infectious, autoimmune and inflammatory diseases.

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2,757 citations

Cites background from "Methods in Mammalian Autophagy Rese..."

...Autophagy is a general term for pathways by which cytoplasmic material, including soluble macromolecules and organelles, is delivered to lysosomes for degradation 6 ....
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Journal Article•DOI•

The Role of Atg Proteins in Autophagosome Formation

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Noboru Mizushima¹, Tamotsu Yoshimori², Yoshinori Ohsumi³•Institutions (3)

Tokyo Medical and Dental University¹, Osaka University², Tokyo Institute of Technology³

10 Oct 2011-Annual Review of Cell and Developmental Biology

TL;DR: The molecular mechanism of autophagosome formation is described with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagous membrane membrane.

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Abstract: Macroautophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of cytoplasm for delivery to the lysosome. Autophagosome formation is dynamically regulated by starvation and other stresses and involves complicated membrane reorganization. Since the discovery of yeast Atg-related proteins, autophagosome formation has been dissected at the molecular level. In this review we describe the molecular mechanism of autophagosome formation with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagosome membrane.

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2,522 citations

Journal Article•DOI•

TFEB links autophagy to lysosomal biogenesis.

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Carmine Settembre, Chiara Di Malta, Vinicia Assunta Polito¹, Vinicia Assunta Polito², Moises Garcia Arencibia³, Francesco Vetrini¹, Serkan Erdin¹, Serkan Erdin², Serpil Uckac Erdin², Serpil Uckac Erdin¹, Tuong Huynh¹, Tuong Huynh², Diego L. Medina, Pasqualina Colella, Marco Sardiello¹, Marco Sardiello², David C. Rubinsztein³, Andrea Ballabio - Show less +14 more•Institutions (3)

Baylor College of Medicine¹, Boston Children's Hospital², University of Cambridge³

17 Jun 2011-Science

TL;DR: A mitogen-activated protein kinase–dependent mechanism regulates autophagy by controlling the biogenesis and partnership of two distinct cellular organelles during starvation.

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Abstract: Autophagy is a cellular catabolic process that relies on the cooperation of autophagosomes and lysosomes. During starvation, the cell expands both compartments to enhance degradation processes. We found that starvation activates a transcriptional program that controls major steps of the autophagic pathway, including autophagosome formation, autophagosome-lysosome fusion, and substrate degradation. The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinated this program by driving expression of autophagy and lysosomal genes. Nuclear localization and activity of TFEB were regulated by serine phosphorylation mediated by the extracellular signal-regulated kinase 2, whose activity was tuned by the levels of extracellular nutrients. Thus, a mitogen-activated protein kinase-dependent mechanism regulates autophagy by controlling the biogenesis and partnership of two distinct cellular organelles.

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2,409 citations

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References

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Journal Article•DOI•

Autophagy in the Pathogenesis of Disease

[...]

Beth Levine¹, Guido Kroemer², Guido Kroemer³, Guido Kroemer⁴•Institutions (4)

University of Texas Southwestern Medical Center¹, University of Paris-Sud², Institut Gustave Roussy³, French Institute of Health and Medical Research⁴

11 Jan 2008-Cell

TL;DR: This Review summarizes recent advances in understanding the physiological functions of autophagy and its possible roles in the causation and prevention of human diseases.

...read moreread less

6,301 citations

"Methods in Mammalian Autophagy Rese..." refers background in this paper

...Under physiological conditions, autophagy has a number of vital roles such as maintenance of the amino acid pool during starvation, preimplantation development, prevention of neurodegeneration, antiaging, tumor suppression, clearance of intracellular microbes, and regulation of innate and adaptive immunity (Cecconi and Levine, 2008; Deretic and Levine, 2009; Levine and Kroemer, 2008; Mizushima et al., 2008; Rubinsztein, 2006)....
[...]
...in part, from increased autophagy (based on microscopic visualization of increased numbers of early intermediates in the pathway) when, in reality, the accumulation of early intermediates in such diseases likely represents a block in later stages of the autophagy pathway (Levine and Kroemer, 2008; Mizushima et al., 2008; Rubinsztein, 2006)....
[...]
...…microscopic visualization of increased numbers of early intermediates in the pathway) when, in reality, the accumulation of early intermediates in such diseases likely represents a block in later stages of the autophagy pathway (Levine and Kroemer, 2008; Mizushima et al., 2008; Rubinsztein, 2006)....
[...]
...…preimplantation development, prevention of neurodegeneration, antiaging, tumor suppression, clearance of intracellular microbes, and regulation of innate and adaptive immunity (Cecconi and Levine, 2008; Deretic and Levine, 2009; Levine and Kroemer, 2008; Mizushima et al., 2008; Rubinsztein, 2006)....
[...]
...Moreover, increasing evidence suggests that the deregulation of autophagy may contribute to a broad spectrum of mammalian diseases (Levine and Kroemer, 2008; Mizushima et al., 2008)....
[...]

Journal Article•DOI•

LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing

[...]

Yukiko Kabeya¹, Noboru Mizushima¹, Noboru Mizushima², Takashi Ueno³, Akitsugu Yamamoto⁴, Takayoshi Kirisako¹, Takayoshi Kirisako⁵, Takeshi Noda¹, Takeshi Noda⁵, Eiki Kominami³, Yoshinori Ohsumi⁵, Yoshinori Ohsumi¹, Tamotsu Yoshimori¹, Tamotsu Yoshimori⁵ - Show less +10 more•Institutions (5)

National Institute for Basic Biology, Japan¹, National Presto Industries², Juntendo University³, Kansai Medical University⁴, Graduate University for Advanced Studies⁵

01 Nov 2000-The EMBO Journal

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.

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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.

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6,244 citations

"Methods in Mammalian Autophagy Rese..." refers background in this paper

...To date, only microtubule-associated protein light chain 3 (LC3), a mammalian homolog of yeast Atg8, is known to exist on autophagosomes, and therefore, this protein serves as a widely used marker for autophagosomes (Figures 1 and 4) (Kabeya et al., 2000; Mizushima et al., 2004)....
[...]
...However, the low pH inside the lysosome quenches the fluorescent signal of GFP, which makes it difficult to trace the delivery of GFP-LC3 to lysosomes; indeed, most GFP-LC3 punctate signals do not colocalize with lysosomes (Bampton et al., 2005; Kabeya et al., 2000)....
[...]
...The amount of LC-II usually correlates well with the number of autophagosomes (or more precisely, in theory, the amount of autophagic membrane labeled with LC3-II) (Kabeya et al., 2000)....
[...]
...ery of GFP-LC3 to lysosomes; indeed, most GFP-LC3 punctate signals do not colocalize with lysosomes (Bampton et al., 2005; Kabeya et al., 2000)....
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Journal Article•DOI•

Autophagy fights disease through cellular self-digestion

[...]

Noboru Mizushima¹, Beth Levine², Ana Maria Cuervo³, Daniel J. Klionsky⁴•Institutions (4)

Tokyo Medical and Dental University¹, University of Texas at Dallas², Yeshiva University³, University of Michigan⁴

28 Feb 2008-Nature

TL;DR: Understanding autophagy may ultimately allow scientists and clinicians to harness this process for the purpose of improving human health, and to play a role in cell death.

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Abstract: 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.

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5,831 citations

"Methods in Mammalian Autophagy Rese..." refers background in this paper

...Under physiological conditions, autophagy has a number of vital roles such as maintenance of the amino acid pool during starvation, preimplantation development, prevention of neurodegeneration, antiaging, tumor suppression, clearance of intracellular microbes, and regulation of innate and adaptive immunity (Cecconi and Levine, 2008; Deretic and Levine, 2009; Levine and Kroemer, 2008; Mizushima et al., 2008; Rubinsztein, 2006)....
[...]
...in part, from increased autophagy (based on microscopic visualization of increased numbers of early intermediates in the pathway) when, in reality, the accumulation of early intermediates in such diseases likely represents a block in later stages of the autophagy pathway (Levine and Kroemer, 2008; Mizushima et al., 2008; Rubinsztein, 2006)....
[...]
...…microscopic visualization of increased numbers of early intermediates in the pathway) when, in reality, the accumulation of early intermediates in such diseases likely represents a block in later stages of the autophagy pathway (Levine and Kroemer, 2008; Mizushima et al., 2008; Rubinsztein, 2006)....
[...]
...…preimplantation development, prevention of neurodegeneration, antiaging, tumor suppression, clearance of intracellular microbes, and regulation of innate and adaptive immunity (Cecconi and Levine, 2008; Deretic and Levine, 2009; Levine and Kroemer, 2008; Mizushima et al., 2008; Rubinsztein, 2006)....
[...]
...Moreover, increasing evidence suggests that the deregulation of autophagy may contribute to a broad spectrum of mammalian diseases (Levine and Kroemer, 2008; Mizushima et al., 2008)....
[...]

Journal Article•DOI•

Regulation Mechanisms and Signaling Pathways of Autophagy

[...]

Congcong He¹, Daniel J. Klionsky¹•Institutions (1)

University of Michigan¹

17 Nov 2009-Annual Review of Genetics

TL;DR: The 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 autophagic for cell survival and homeostasis are presented.

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Abstract: 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.

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3,249 citations

"Methods in Mammalian Autophagy Rese..." refers background in this paper

...Details of the molecular regulation and machinery of autophagy have been reviewed elsewhere (He and Klionsky, 2009; Longatti and Tooze, 2009)....
[...]
...Moreover, p62, as well as LC3, can be transcriptionally regulated during autophagy (He and Klionsky, 2009; Nakaso et al., 2004), which may confound the interpretation of p62 and LC3 levels as indicators of autophagic flux....
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Journal Article•DOI•

The role of autophagy during the early neonatal starvation period

[...]

Akiko Kuma¹, Masahiko Hatano², Makoto Matsui³, Makoto Matsui⁴, Akitsugu Yamamoto⁵, Haruaki Nakaya², Tamotsu Yoshimori⁶, Yoshinori Ohsumi⁴, Takeshi Tokuhisa², Noboru Mizushima¹, Noboru Mizushima⁴, Noboru Mizushima³ - Show less +8 more•Institutions (6)

National Presto Industries¹, Chiba University², Institute of Medical Science³, Graduate University for Advanced Studies⁴, Nagahama Institute of Bio-Science and Technology⁵, National Institute of Genetics⁶

23 Dec 2004-Nature

TL;DR: The results suggest that the production of amino acids by autophagic degradation of ‘self’ proteins, which allows for the maintenance of energy homeostasis, is important for survival during neonatal starvation.

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Abstract: At birth the trans-placental nutrient supply is suddenly interrupted, and neonates face severe starvation until supply can be restored through milk nutrients. Here, we show that neonates adapt to this adverse circumstance by inducing autophagy. Autophagy is the primary means for the degradation of cytoplasmic constituents within lysosomes. The level of autophagy in mice remains low during embryogenesis; however, autophagy is immediately upregulated in various tissues after birth and is maintained at high levels for 3-12 h before returning to basal levels within 1-2 days. Mice deficient for Atg5, which is essential for autophagosome formation, appear almost normal at birth but die within 1 day of delivery. The survival time of starved Atg5-deficient neonates (approximately 12 h) is much shorter than that of wild-type mice (approximately 21 h) but can be prolonged by forced milk feeding. Atg5-deficient neonates exhibit reduced amino acid concentrations in plasma and tissues, and display signs of energy depletion. These results suggest that the production of amino acids by autophagic degradation of 'self' proteins, which allows for the maintenance of energy homeostasis, is important for survival during neonatal starvation.

...read moreread less

2,775 citations

"Methods in Mammalian Autophagy Rese..." refers background in this paper

...Another consideration with RNAi-mediated approaches to autophagy inhibition is that certain Atg proteins (e.g., Atg5; Hosokawa et al., 2006) still function normally in autophagy when present at very low levels; in such cases, RNAi-mediated silencing will require nearly complete suppression of protein expression to observe effective autophagy inhibition....
[...]
...However, abnormally elongated membranes are observed in Atg3 knockout cells (Sou et al., 2008), Atg5 knockout cells (Mizushima et al., 2001; Nishiyama et al., 2007), and Atg4BC74A-expressing cells (Fujita et al., 2008a), suggesting that these factors, which belong to the Atg12 and Atg8/LC3 conjugation systems, may also be important for the complete closure of autophagosomes (Figure 1)....
[...]
...The phenotypes of targeted mutant mice deficient in Atg3 (Sou et al., 2008), Atg5 (Kuma et al., 2004), Atg7 (Komatsu et al., 2005), Atg9a (Saitoh et al., 2009), and Atg16L1 (Saitoh et al., 2008) are essentially the same (neonatal lethality), whereas embryonic lethality is observed in mice deficient in Beclin 1 (Qu et al., 2003; Yue et al., 2003), FIP200 (Gan et al., 2006), and Ambra1 (Fimia et al., 2007)....
[...]
...The phenotypes of targeted mutant mice deficient in Atg3 (Sou et al., 2008), Atg5 (Kuma et al., 2004), Atg7 (Komatsu et al., 2005), Atg9a (Saitoh et al., 2009), and Atg16L1 (Saitoh et al., 2008) are essentially the same (neonatal lethality), whereas embryonic lethality is observed in mice deficient…...
[...]
...To date, autophagy deficiency/reduction has been confirmed in cells lacking Atg3 (Sou et al., 2008), Atg5 (Mizushima et al., 2001), Beclin 1 (Qu et al., 2003; Yue et al., 2003), Atg7 (Komatsu et al., 2005), Atg9a (Saitoh et al., 2009), Atg16L1 (Cadwell et al., 2008; Saitoh et al., 2008), FIP200 (Hara et al., 2008) and Ambra1 (Fimia et al., 2007)....
[...]