A comprehensive glossary of autophagy-related molecules and processes

Klionsky, Daniel J.; Codogno, Patrice; Cuervo, Ana María; Deretic, Vojo; Elazar, Zvulun; Fueyo, Juan; Gewirtz, David A.; Kroemer, Guido; Levine, Beth; Mizushima, Noboru; Rubinsztein, David C.; Thumm, Michael; Tooze, Sharon A.

doi:10.4161/auto.6.4.12244

Cited by 199 publications

(178 citation statements)

References 175 publications

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“…The following chemicals and reagents were used in the preparation of this manuscript: Phosphate buffered saline (PBS; Invitrogen, 1160381), Dulbecco's modified Eagle's medium/F-12 mixture (DMEM/F12; Invitrogen, 11330057), DMEM custom medium (made by Life Technologies and is a standard DMEM medium depleted of all amino acids, glucose and pyruvate), minimal essential medium (Invitrogen, 22571020), fetal bovine serum (FBS; Life Technologies, 16000-044), gentamycin (Invitrogen, 15750), ascorbic acid (Sigma-Aldrich, A5960), b-glycerophosphate (bGP; Sigma-Aldrich, G9422), doxycycline (dox; Sigma-Aldrich, D3447), bafilomycin A 1 (LC laboratories, B-1080), concanamycin A (Sigma-Aldrich, 27689), chloroquine (Sigma-Aldrich, C6628), dexamethasone (Sigma-Aldrich, D1756), Torin1 (Tocris Biosciences, 4247), hyaluronidase (Sigma-Aldrich, H3506), leupeptin (VWR, a2183.0025), 33 ECL western blotting substrate (Perkin Elmer, NEL104001EA), 2x Laemmli buffer (Sigma-Aldrich, S3401), alcian blue (SigmaAldrich, A5268), digoxigenin (Roche Inc., 11277073910), bovine serum albumin (Sigma-Aldrich, A8806), IGF1 (SigmaAldrich, I3769), Magic Cathepsin B kit (ImmunoChemistry Technologies, 937), rat-tail collagen (Invitrogen, A1048301), microscope cover glasses (VWR International, ECN 631-1578), 3-methyladenine (Sigma-Aldrich, M9281), 7 Vectastatin avidinbiotin complex (ABC) kit (Vector laboratories, PK-6100), 3, 3 0 -diaminobenzidine kit (DAKO, K3468), and normal horse serum Cell culture The RCJ3.1C5.18 nontransformed mesenchymal rat chondrogenic cell-line (C5.18) was obtained from Dr. Anna Spagnoli (Rush University Medical Center, Illinois, USA). 18 C5.18 cells were maintained in minimal essential medium containing 15% FBS with 10 ¡7 M dexamethasone (maintenance medium).…”

Section: Methodsmentioning

confidence: 99%

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

et al. 2015

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These authors contributed equally to this publication.Keywords: autophagy, bafilomycin, bone, chondrocyte, lysosome, MTOR, MTORC1Abbreviations: 3-MA, 3-methyladenine; ACAN, aggrecan; ACP5/TRAP, acid phosphatase 5, tartrate-resistant; ACTB, actin, b; ALPL, alkaline phosphatase, liver/bone/kindey; ATG, autophagy related; ATG5cKO, ATG5 conditional knockout in chondrocytes; BC, avidin-biotin complex; Baf, bafilomycin A 1 ; bGP, b-glycerophosphate; BrdU, bromodeoxyuridine; C5.18 cells, RCJ 3.1C5.18 rat mesenchymal cell line; COL2A1, collagen, type II, a 1; COL10A1, collagen, type X, a 1; CQ, chloroquine; DAPI, 4 0 , 6-diamidino-2-phenylindole, dihydrochloride; Dox, doxycycline; EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1; FBS, fetal bovine serum; GAG, glycosaminoglycan; IGF1, insulin-like growth factor 1 (somatomedin C); LAMP2, lysosomalassociated membrane protein 2; MEFs, mouse embryonic fibroblasts; MAP1LC3A, microtubule-associated protein 1 light chain 3 a;MTOR, mechanistic target of rapamycin (serine/threonine kinase); MTORC, MTOR complex; p-, phosphorylated-; PFA, paraformaldehyde; RPS6, ribosomal protein S6; RPS6KB1/p70(S6K)-a/PS6K/S6K1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; RPTOR, regulatory associated protein of MTOR, complex 1; SGK1, serum/glucocorticoid regulated kinase 1; TSC, tuberous sclerosis complex; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; v-ATPase, vacuolar-type H C -ATPase.Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H C -ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A 1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.

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Section: Methodsmentioning

confidence: 99%

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

et al. 2015

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“…Autophagy is a cellular process in which cytoplasmic contents are degraded within the lysosome/vacuole, and the resulting macromolecular constituents are recycled [1]. Macroautophagy is one type of autophagic process in which the substrates are sequestered within cytosolic double-membrane vesicles termed autophagosomes.…”

Section: The Definition Of Autophagymentioning

confidence: 99%

The machinery of macroautophagy

et al. 2013

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Autophagy is a primarily degradative pathway that takes place in all eukaryotic cells. It is used for recycling cytoplasm to generate macromolecular building blocks and energy under stress conditions, to remove superfluous and damaged organelles to adapt to changing nutrient conditions and to maintain cellular homeostasis. In addition, autophagy plays a critical role in cytoprotection by preventing the accumulation of toxic proteins and through its action in various aspects of immunity including the elimination of invasive microbes and its participation in antigen presentation. The most prevalent form of autophagy is macroautophagy, and during this process, the cell forms a double-membrane sequestering compartment termed the phagophore, which matures into an autophagosome. Following delivery to the vacuole or lysosome, the cargo is degraded and the resulting macromolecules are released back into the cytosol for reuse. The past two decades have resulted in a tremendous increase with regard to the molecular studies of autophagy being carried out in yeast and other eukaryotes. Part of the surge in interest in this topic is due to the connection of autophagy with a wide range of human pathophysiologies including cancer, myopathies, diabetes and neurodegenerative disease. However, there are still many aspects of autophagy that remain unclear, including the process of phagophore formation, the regulatory mechanisms that control its induction and the function of most of the autophagy-related proteins. In this review, we focus on macroautophagy, briefly describing the discovery of this process in mammalian cells, discussing the current views concerning the donor membrane that forms the phagophore, and characterizing the autophagy machinery including the available structural information.

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“…S2B). Furthermore, we examined autophagic flux by exposing the cells to NH 4 Cl, which raises the lysosomal pH and prevents the turnover of LC3 (similar, in effect, to deleting the PEP4 gene in yeast). The presence of NH 4 Cl resulted in elevated levels of LC3-II, indicating that the knockdown of SIN3A/B caused an increase in basal autophagy, and not just an increase in the level of LC3.…”

Section: Sin3a and Sin3b Play Redundant Roles In Regulating Lc3 Exprementioning

confidence: 99%

“…Furthermore, we examined autophagic flux by exposing the cells to NH 4 Cl, which raises the lysosomal pH and prevents the turnover of LC3 (similar, in effect, to deleting the PEP4 gene in yeast). The presence of NH 4 Cl resulted in elevated levels of LC3-II, indicating that the knockdown of SIN3A/B caused an increase in basal autophagy, and not just an increase in the level of LC3. Note that in human fibroblasts, the lack of a clear difference in LC3 levels in the absence, but not the presence, of NH 4 Cl indicates a rapid rate of lysosomal turnover of this protein (21).…”

Section: Sin3a and Sin3b Play Redundant Roles In Regulating Lc3 Exprementioning

confidence: 99%

Ume6 transcription factor is part of a signaling cascade that regulates autophagy

Bartholomew

Suzuki

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Autophagy has been implicated in a number of physiological processes important for human heath and disease. Autophagy involves the formation of a double-membrane cytosolic vesicle, an autophagosome. Central to the formation of the autophagosome is the ubiquitin-like protein autophagy-related (Atg)8 (microtubuleassociated protein 1 light chain 3/LC3 in mammalian cells). Following autophagy induction, Atg8 shows the greatest change in expression of any of the proteins required for autophagy. The magnitude of autophagy is, in part, controlled by the amount of Atg8; thus, controlling Atg8 protein levels is one potential mechanism for modulating autophagy activity. We have identified a negative regulator of ATG8 transcription, Ume6, which acts along with a histone deacetylase complex including Sin3 and Rpd3 to regulate Atg8 levels; deletion of any of these components leads to an increase in Atg8 and a concomitant increase in autophagic activity. A similar regulatory mechanism is present in mammalian cells, indicating that this process is highly conserved.lysosome | membrane biogenesis | phagophore | stress | vacuole M acroautophagy, hereafter referred to as autophagy, is an evolutionarily conserved process used by eukaryotic cells for the bulk degradation of intracellular proteins and organelles (1). Autophagy is not only vital for cell survival in nutrient-poor conditions (2) but is also linked to various physiological processes, including immune defense, tumor suppression, and prevention of neurodegeneration (3). Whereas autophagy plays a primarily protective role, it can also contribute to cell death; thus, the magnitude of autophagy must be carefully regulated.Central to autophagy is the formation of autophagosomes (4), double-membrane-bound structures that engulf and deliver cytoplasmic materials to the vacuole/lysosome for degradation. During autophagosome formation, the autophagy-related ubiquitin-like protein Atg8/microtubule-associated protein 1 light chain 3 (LC3) covalently modifies phosphatidylethanolamine (PE). Almost one-fourth of the characterized autophagy-related (Atg) proteins in yeast are involved in the formation or stability of Atg8-PE, which plays a critical role in controlling expansion of the phagophore (the initial sequestering membrane), and in determining autophagosome size, thereby regulating autophagy activity (5, 6). Upon starvation, the level of ATG8 mRNA sharply increases, leading to a subsequent induction of the Atg8 protein level (7,8). The increase in the amount of Atg8 during autophagy is critical for supplying a sufficient amount of this protein to maintain normal levels of autophagy; yeast strains deficient in Atg8 induction generate abnormally small autophagosomes (5). Thus, characterization of how Atg8 protein levels are modulated is of tremendous importance both in understanding the regulation of autophagy and for the elucidation of potential therapeutic targets. However, little is known about the mechanisms regulating ATG8 transcription. ResultsUme6-Sin3-Rpd3 Complex Represse...

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A comprehensive glossary of autophagy-related molecules and processes

Cited by 199 publications

References 175 publications

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

The machinery of macroautophagy

Ume6 transcription factor is part of a signaling cascade that regulates autophagy

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