Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Autophagy, Journal Year: 2016, Volume and Issue: 12(1), P. 1 - 222

Published: Jan. 2, 2016

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, on this topic has continued to accelerate, and many new scientists have entered field. Our knowledge base relevant technologies also been expanding. Accordingly, it is important update these monitoring autophagy different organisms. Various reviews described range assays that used purpose. Nevertheless, there continues be confusion regarding acceptable methods measure autophagy, especially multicellular eukaryotes. For example, a key point needs emphasized difference between measurements monitor numbers or volume autophagic elements (e.g., autophagosomes autolysosomes) at any stage process versus those flux through pathway (i.e., complete including amount rate cargo sequestered degraded). particular, block macroautophagy results autophagosome accumulation must differentiated from stimuli increase activity, defined as increased induction coupled with delivery to, degradation within, lysosomes (in most higher eukaryotes some protists such Dictyostelium) vacuole plants fungi). other words, investigators field understand appearance more does not necessarily equate fact, cases, accumulate because trafficking without concomitant change biogenesis, whereas an autolysosomes may reflect reduction degradative activity. It worth emphasizing here lysosomal digestion evaluating its competence crucial part evaluation flux, autophagy. Here, present selection interpretation use by who aim examine related processes, well reviewers need provide realistic reasonable critiques papers are focused processes. These meant formulaic rules, appropriate depend question being asked system used. addition, emphasize no individual assay guaranteed one every situation, strongly recommend multiple Along lines, potential pleiotropic effects due blocking genetic manipulation, imperative target gene knockout RNA interference than autophagy-related protein. Atg proteins, groups involved cellular pathways implying all proteins can specific marker process. guidelines, consider various assessing what information can, cannot, obtained them. Finally, discussing merits limits particular assays, hope encourage technical innovation

Language: Английский

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Anti-Vascular Endothelial Growth Factors Protect Retinal Pigment Epithelium Cells Against Oxidation by Modulating Nitric Oxide Release and Autophagy

Cellular Physiology and Biochemistry, Journal Year: 2017, Volume and Issue: 42(5), P. 1725 - 1738

Published: Jan. 1, 2017

To investigate whether oxidative stress modulates vascular endothelial growth factor (VEGF)-A and VEGF-C expression polarized secretion in a human retinal pigment epithelium cell line (ARPE-19).Long-term culture of ARPE-19 cells Dulbecco's modified Eagle medium (DMEM)/F12 containing 1% fetal bovine serum (FBS) on transwell filters (12 mm or 6 mm, pore size 0.4 microm) was performed to produce (RPE) monolayers. The integrity monolayer established by measurement transepithelial resistance (TER) presence tight junctions assessed zonula occludens (ZO-1) occludin apical Na/K ATPase localization. Paracellular permeability studied using radiolabeled mannitol. Confluent were treated with tertiary butyl hydrogen peroxide (tBH) for varying durations (0-5 h) doses (50-200 microM). VEGF-A -C evaluated western blot quantitative RT-PCR, while the basolateral surfaces quantitated ELISA.Polarity verified localization junction proteins, ZO-1 its binding partner confocal microscopy as well Na,K-ATPase at surface. TER confluent averaged 48.7+/-2.1 Omega. cm(2) tBH treatment did not alter significantly (46.9+/-1.9 cm(2); p>0.05 versus controls) expression. Whole mRNA nonpolarized increased 5 h both increase significant (p<0.05 vs controls). A similar, maximal also observed cellular protein levels. ARPE showed an exposure. levels higher monolayers stimulated domains. 150 microM function time (1-5 increases from 410 2080 pg/10(6) 290 1680 pattern similar. dose-dependent range 50-200 tended be than secretion.Our data show that RPE upregulates C. side Given role VEGF choroidal neovascularization, these may value understanding pathogenic mechanisms designing antiangiogenic therapies.

Language: Английский

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Guidelines for the use and interpretation of assays for monitoring autophagy

Autophagy, Journal Year: 2012, Volume and Issue: 8(4), P. 445 - 544

Published: April 1, 2012

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, on this topic has continued to accelerate, and many new scientists have entered field. Our knowledge base relevant technologies also been expanding. Accordingly, it is important update these monitoring autophagy different organisms. Various reviews described range assays that used purpose. Nevertheless, there continues be confusion regarding acceptable methods measure autophagy, especially multicellular eukaryotes. A key point needs emphasized a difference between measurements monitor numbers or volume autophagic elements (e.g., autophagosomes autolysosomes) at any stage process vs. those flux through pathway (i.e., complete process); thus, block macroautophagy results autophagosome accumulation differentiated from stimuli result increased activity, defined as induction coupled with delivery to, degradation within, lysosomes (in most higher eukaryotes some protists such Dictyostelium) vacuole plants fungi). other words, investigators field understand appearance more does not necessarily equate fact, cases, accumulate because trafficking without concomitant change biogenesis, whereas an increase autolysosomes may reflect reduction degradative activity. Here, present selection interpretation use by who aim examine related processes, well reviewers need provide realistic reasonable critiques papers are focused processes. These meant formulaic rules, appropriate depend part question being asked system used. addition, emphasize no individual assay guaranteed one every situation, strongly recommend multiple guidelines, consider various assessing what information can, cannot, obtained them. Finally, discussing merits limits particular assays, hope encourage technical innovation

Language: Английский

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Antimicrobial Autophagy: A Conserved Innate Immune Response in Drosophila

Journal of Innate Immunity, Journal Year: 2013, Volume and Issue: 5(5), P. 444 - 455

Published: Jan. 1, 2013

Autophagy is a major route by which cytoplasmic contents are delivered to the lysosome for degradation. Many autophagy-related (ATG) genes have been identified in yeast. Although most of them conserved human, molecular composition Atg1 complex appears differ between yeast and mammals. In yeast, forms with Atg11, Atg13, Atg17, Atg29 Atg31, whereas mammalian (ULK1/2) interacts Atg13 FIP200. Here, we identify novel binding protein, named Atg101. Atg101 shows no homology other Atg proteins, various eukaryotes, but not Saccharomyces cerevisiae. associates ULK-Atg13-FIP200 complex, likely through direct interaction Atg13. siRNA-treated cells, present solely as monomer. Interaction stable, regulated nutrient conditions. GFP-Atg101 localizes isolation membrane/phagophore. GFP-LC3 dot formation suppressed endogenous LC3-I accumulates suggesting that critical factor autophagy. Furthermore, important stability basal phosphorylation ULK1. These data suggest protein functions together ULK,

Language: Английский

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Regulation Mechanisms and Signaling Pathways of Autophagy

Annual Review of Genetics, Journal Year: 2009, Volume and Issue: 43(1), P. 67 - 93

Published: Aug. 4, 2009

Autophagy is a process of self-degradation cellular components in which double-membrane autophagosomes sequester organelles or portions cytosol and fuse with lysosomes vacuoles for breakdown by resident hydrolases. upregulated response to extra- intracellular stress signals such as starvation, growth factor deprivation, ER stress, pathogen infection. Defective autophagy plays significant role human pathologies, including cancer, neurodegeneration, infectious diseases. We present our current knowledge on the key genes composing machinery eukaryotes from yeast mammalian cells signaling pathways that sense status different types induce cell survival homeostasis. also review recent advances molecular mechanisms regulate at various levels, transcriptional activation post-translational protein modification.

Language: Английский

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Autophagy: cellular and molecular mechanisms

The Journal of Pathology, Journal Year: 2010, Volume and Issue: 221(1), P. 3 - 12

Published: Feb. 3, 2010

Abstract Autophagy is a self‐degradative process that important for balancing sources of energy at critical times in development and response to nutrient stress. also plays housekeeping role removing misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria, endoplasmic reticulum peroxisomes, well eliminating intracellular pathogens. Thus, autophagy generally thought survival mechanism, although its deregulation has been linked non‐apoptotic cell death. can be either non‐selective selective the removal specific ribosomes protein aggregates, mechanisms regulating aspects are not fully worked out. In addition elimination aggregates promotes cellular senescence surface antigen presentation, protects against genome instability prevents necrosis, giving it key preventing diseases cancer, neurodegeneration, cardiomyopathy, diabetes, liver disease, autoimmune infections. This review summarizes most up‐to‐date findings on how executed regulated molecular level disruption lead disease. Copyright © 2010 Pathological Society Great Britain Ireland. Published by John Wiley & Sons, Ltd.

Language: Английский

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Autophagy and the Integrated Stress Response

Molecular Cell, Journal Year: 2010, Volume and Issue: 40(2), P. 280 - 293

Published: Oct. 1, 2010

Language: Английский

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TFEB Links Autophagy to Lysosomal Biogenesis

Science, Journal Year: 2011, Volume and Issue: 332(6036), P. 1429 - 1433

Published: May 27, 2011

Starvation activates a transcriptional program controlling autophagosome formation, lysosome fusion, and substrate degradation.

Language: Английский

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Fiber Types in Mammalian Skeletal Muscles

Physiological Reviews, Journal Year: 2011, Volume and Issue: 91(4), P. 1447 - 1531

Published: Oct. 1, 2011

Mammalian skeletal muscle comprises different fiber types, whose identity is first established during embryonic development by intrinsic myogenic control mechanisms and later modulated neural hormonal factors. The relative proportion of the types varies strikingly between species, in humans shows significant variability individuals. Myosin heavy chain isoforms, complete inventory expression pattern are now available, provide a useful marker for both four major forms present trunk limb muscles minor head neck muscles. However, diversity involves all functional cell compartments, including membrane excitation, excitation-contraction coupling, contractile machinery, cytoskeleton scaffold, energy supply systems. Variations within each compartment limited need matching type properties compartments. Nerve activity mechanism profile, multiple signaling pathways implicated activity-dependent changes fibers. characterization these raising increasing interest clinical medicine, given potentially beneficial effects switching prevention treatment metabolic diseases.

Language: Английский

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Mechanisms of Diabetic Complications

Physiological Reviews, Journal Year: 2013, Volume and Issue: 93(1), P. 137 - 188

Published: Jan. 1, 2013

It is increasingly apparent that not only a cure for the current worldwide diabetes epidemic required, but also its major complications, affecting both small and large blood vessels. These complications occur in majority of individuals with type 1 2 diabetes. Among most prevalent microvascular are kidney disease, blindness, amputations, therapies slowing disease progression. Impaired function, exhibited as reduced glomerular filtration rate, risk factor macrovascular such heart attacks strokes. There have been number new tested clinical trials diabetic with, general, rather disappointing results. Indeed, it remains to be fully defined which pathways essentially protective than pathological, terms their effects on underlying process. Furthermore, seemingly independent showing significant interactions each other exacerbate pathology. Interestingly, some these may play key roles development per se. This review aims comprehensively discuss well validated, putative mechanisms involved complications. In addition, fields research, warrant further investigation potential therapeutic targets future, will highlighted.

Language: Английский

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