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There are 113 results for Autophagy (displaying 11 to 20).

Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19

Autophagy is the major pathway for the delivery of cytoplasmic material to the vacuole or lysosome. Selective autophagy is mediated by cargo receptors, which link the cargo to the scaffold protein Atg11 and to Atg8 family proteins on the forming autophagosomal membrane. We show that the essential kinase Hrr25 activates the cargo receptor Atg19 by phosphorylation, which is required to link cargo to the Atg11 scaffold, allowing selective autophagy to proceed. We also find that the Atg34 cargo receptor is regulated in a similar manner, suggesting a conserved mechanism.

GFP‐Atg11 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM copper sulfate for 3 h, and autophagy was induced by treating cells for 1 h with rapamycin. Scale bar, 5 μm. Ape1‐mRuby atg19Δ cells containing GFP‐Atg19 wild‐type or GFP‐Atg19‐3A were analyzed in log phase. Scale bar, 5 μm. GFP‐Atg8 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were analyzed as in (A). Scale bar, 5 μm.

… Macroautophagy (hereafter autophagy) is an important mechanism for the bulk degradation of cellular material. Autophagy is a sequential process beginning with the formation of a double‐membrane sheet termed phagophore at the pre‐autophagosomal structure (PAS). The phagophore expands to enwrap the cargo in the autophagosome, which subsequently fuses with the lysosome or vacuole where the cargo is degraded by resident hydrolases . This evolutionarily conserved process is integral for cellular …

Thaddaeus Pfaffenwimmer et al. EMBO Reports July 2014

The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy

… and the Unc‐51‐like kinase 1 ( ULK 1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a‐dependent trafficking of the ULK 1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62‐positive puncta reminiscent of the p62 pathology observed in C9 ALS / FTD patients. Finally, basal levels of autophagy were markedly …

HEK293 cells treated with non‐targeting (Ctrl) or FIP200 siRNA were co‐transfected with EGFP‐LC3 and either empty vector control (EV), Myc‐C9orf72S, or Myc‐C9orf72L. 24 h post‐transfection cells were treated with vehicle or 100 nM BafA1 for 6 h. Samples were lysed and subjected to SDS–PAGE and immunoblot. Autophagy levels were determined by immunoblot for EGFP‐LC3‐I and II. Expression of Myc‐C9orf72 was confirmed using anti‐Myc (* indicates a nonspecific band). FIP200 knockdown was confirmed …

HEK293 cells were transfected with non‐targeting (Ctrl) or C9orf72 siRNA. Cells were treated with rapamycin for 6 h to induce autophagy. Activation of ULK1 was determined on immunoblots using phospho‐ULK1 (Ser757), total ULK1, and GAPDH Abs (loading control). HeLa cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were transfected with mCherry‐FIP200. Twenty‐four hours post‐transfection, cells were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 …

… Macroautophagy (hereafter termed autophagy) is a conserved lysosomal degradation pathway that is an essential process to maintain cellular homeostasis. A double lipid membrane, the phagophore, engulfs cellular components targeted for degradation including proteins and organelles. The resulting autophagosome fuses with lysosomes, and its content is degraded to release cell components that can be reused. Autophagy is induced in response to starvation to boost nutrient availability, but is also …

Christopher P Webster et al. The EMBO Journal August 2016

Autophagy‐based unconventional secretory pathway for extracellular delivery of IL‐1β

Autophagy controls the quality and quantity of the eukaryotic cytoplasm while performing two evolutionarily highly conserved functions: cell‐autonomous provision of energy and nutrients by cytosol autodigestion during starvation, and removal of defunct organelles and large aggregates exceeding the capacity of other cellular degradative systems. In contrast to these autodigestive processes, autophagy in yeast has additional, biogenesis functions. However, no equivalent biosynthetic roles have …

GRASP55 controls autophagy initiation. (A, B) Effect of GRASP55 on autophagy induction by measuring LC3‐II. BMM cells were transfected with GRASP55 siRNAs or scramble (Scr) control. At 72 h post transfection, cells were induced for autophagy, treated or not with Bafilomycin A1 (Baf) to inhibit autophagic degradation and LC3‐II/actin ratios determined by immunoblotting (A) followed by densitometry (B). Data represent mean values±s.d. (n⩾3); *P<0.05. (C, D) RAW 264.7 was transfected with GRASP55 …

HMGB1 is an autophagy‐based alternative secretion substrate. (A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs, pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with 20 μM nigericin (Nig; inflammasome agonist) while incubated in EBSS for induction of autophagy by starvation. Cell culture supernatants were assayed for murine HMGB1 by ELISA. Data (normalized to sample with maximum HMGB1 secretion in each experimental repeat; Cre−and Nig) represent mean values±s.d. (n⩾3); *P<0.05. (B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of HMGB1 was determined by immunoblotting. Figure source data can be found in Supplementary data.

Rab8a is required for autophagy‐activated IL‐1β secretion. (A) Colocalization of Rab8a with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), Rab8a (blue, Alexa633). BMMs from GFP–LC3 knock‐in mice were pretreated with LPS and analysed by immunofluorescence microscopy. Arrows indicate triple colocalization. (B) Line tracing analysis of fluorescence signal intensity. (C) Pearson's colocalization coefficient for IL‐1β and Rab8a …

Autophagy is a fundamental biological process in eukaryotic cells, where it plays a number of roles associated with quality and quantity control of the cytoplasm ( ; ). For the most part, autophagy enables cellular viability ( ; ) by removing damaged organelles, for example, depolarized or leaky mitochondria ( ), digesting potentially toxic protein aggregates ( ), killing intracellular microbes ( ), and supplying energy and nutrients to the cell ( ) and the whole body ( ) by bulk autodigestion …

Nicolas Dupont et al. The EMBO Journal November 2011

Autolysosomal β‐catenin degradation regulates Wnt‐autophagy‐p62 crosstalk

The Wnt/β‐catenin signalling and autophagy pathways each play important roles during development, adult tissue homeostasis and tumorigenesis. Here we identify the Wnt/β‐catenin signalling pathway as a negative regulator of both basal and stress‐induced autophagy. Manipulation of β‐catenin expression levels in vitro and in vivo revealed that β‐catenin suppresses autophagosome formation and directly represses p62/SQSTM1 (encoding the autophagy adaptor p62) via TCF4. Furthermore, we show …

Working model summarising the crosstalk between Wnt/β‐catenin signalling and autophagy described in this study. β‐Catenin is a cellular integration point coordinating proliferative signalling with autophagy. Under normal physiological conditions (nutrient rich) when autophagy is required at basal levels only, β‐catenin limits autophagy and functions as a transcriptional co‐repressor of p62. During nutrient deprivation (starvation), the inhibitory function of β‐catenin on autophagy is reduced, p62 becomes derepressed, and β‐catenin is targeted for autophagic degradation.

Autophagy induction reduces Wnt/β‐catenin signalling. (A) Western blotting showing that autophagy was induced after 2 h starvation, as evidenced by decreased p62 protein and increased LC3‐II protein expression. β‐Catenin protein expression decreased over a 24‐h period of starvation in HT29 cells. (B) Autophagy decreased following Atg7 knockdown as shown by increased p62 and decreased LC3‐II protein expression. β‐Catenin protein expression increased following Atg7 siRNA. Reduction of LC3 puncta …

β‐Catenin directly interacts with the autophagy protein LC3. (A) Co‐immunoprecipitation of YFP–LC3 or negative control YFP in HT29 cells. Binding of endogenous β‐catenin to YFP–LC3 was detected after 8 h of autophagy induction by starvation and starvation in the presence of lysosomal autophagy flux inhibitor chloroquine (10 μM). Input and immunodepleted lysates are shown. (B) Immunoprecipitation of endogenous LC3 in HT29 cells. IgG was used as a negative control. (C) β‐Catenin contains a W/YXXI …

… conditions, including hypoxia ( ) and oxidative stress ( ). This involves diverting β‐catenin away from TCF to favour quiescence and adaptation‐promoting transcription, mitigating the deleterious consequences that could occur if proliferation were allowed to continue unabated during stress. Another key adaptive response to microenvironmental stress is macroautophagy (herein referred to as autophagy), a vital mechanism for recycling cellular components to sustain viability when cells have outstripped …

Katy J Petherick et al. The EMBO Journal July 2013

MCL‐1 is a stress sensor that regulates autophagy in a developmentally regulated manner

Apoptosis has an important role during development to regulate cell number. In differentiated cells, however, activation of autophagy has a critical role by enabling cells to remain functional following stress. In this study, we show that the antiapoptotic BCL‐2 homologue MCL‐1 has a key role in controlling both processes in a developmentally regulated manner. Specifically, MCL‐1 degradation is an early event not only following induction of apoptosis, but also under nutrient deprivation …

Inhibition of autophagy in MCL‐1‐deficient neurons activates apoptosis. (A) Brain morphology of P14 MCL‐1Δ/Δ Beclin‐1+/− mice. Brain sections were stained with cresyl violet and imaged using a × 1 objective. (B–D) Active BAX (6A7; B) and active caspase‐3 (C) were quantified by staining the cortex of 14‐day‐old MCL‐1Δ/Δ and MCL‐1Δ/Δ Beclin‐1+/− mice. The number of BAX‐positive LC3‐negative cells was also quantified. (D) Representative images from at least three sections from each animal were …

Apoptosis and autophagy are activated in a developmentally regulated manner in MCL‐1‐null animals. (A) Brains from E12.5 Foxg1 Cre MCL‐1 animals were analysed by western blot for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate p130CAS and the autophagic markers LC3 and p62. (B) Autophagy in Foxg1 Cre MCL‐1Δ/Δ mice. Brain sections from E15.5 animals with the indicated genotypes were stained for LC3 (green). Confocal images were taken using × 20 (bottom panel) or × 63 (top …

MCL‐1 degradation is an early event following starvation‐induced autophagy. (A) H1299 cells stably expressing GFP–LC3 were incubated for the indicated times in EBSS in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence for cyt c or active BAX (6A7 epitope). As a positive control, cells were infected with 5 p.f.u. per cell Ad HA‐tBID for 7 h. Data are expressed as the average of three experiments±s.d. (B) H1299 cells stably expressing GFP–LC3 were incubated for 4 h in either EBSS …

… Macroautophagy (hereafter referred to as autophagy) is a catabolic process, in which portions of cytoplasm or organelles are delivered to lysosomes for bulk degradation ( ; ). Autophagy is activated under starvation conditions in order to recycle the nutrients necessary for survival. A basal level of autophagy also exists in nutrient‐replete cells to allow the turnover of damaged organelles and long‐lived proteins ( ; ). This basal autophagy is of particular importance in terminally …

Marc Germain et al. The EMBO Journal January 2011

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L ‐Leucyl‐L‐leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which …

Decrease in the number of GFP‐Gal3 puncta is dependent on time and autophagy. (A, B) NIH3T3 cells stably expressing GFP‐Gal3 and either empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP‐Gal3 or Lamp1 puncta per cell was quantified using G‐Count (see also Supplementary Figure S4A and B). Then, the percent of GFP‐Gal3‐positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.

tfGal3 GFP signal in puncta attenuates in an autophagy‐dependent manner. (A) Diagram of the primary structure of tandem fluorescence‐tagged Galectin‐3 (tfGal3). (B) Schematic diagram of the fate of tfGal3 recruited to damaged lysosomes. (C–E) HeLa cells transfected with tfGal3 and either One‐STrEP‐FLAG‐tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell …

Autophagy‐deficient renal tubules exhibit severe injury and dysfunction under hyperuricaemia. (A) Plasma creatinine and urea nitrogen in Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA. The values displayed represent means±s.e. Statistically significant differences (*P<0.05) are indicated. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. (B, C) PAS‐stained renal cortical region of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (n=4–7). Bars: 40 μm (B). Kidney injury score …

… impair lysosomal membranes in vivo . These substances can cause pathologies, including neurodegenerative disorders such as Parkinson's disease, inflammation, and the development of hyperuricemic nephropathy ( ; ; ; ; ). Autophagy is an intracellular bulk degradation system that is drastically induced under several cellular stress conditions such as nutrient starvation, and plays diverse physiological and pathological roles as a prosurvival mechanism of cells through maintaining cellular …

Ikuko Maejima et al. The EMBO Journal August 2013

ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase

It has been proposed that cell growth and autophagy are coordinated in response to cellular nutrient status, but the relationship between them is not fully understood. Here, we have characterized the fly mutants of Autophagy‐specific gene 1 ( ATG1 ), an autophagy‐regulating kinase, and found that ATG1 is a negative regulator of the target of rapamycin (TOR)/S6 kinase (S6K) pathway. Our Drosophila studies have shown that ATG1 inhibits TOR/S6K‐dependent cell growth and development by interfering …

Functional interaction between Drosophila Autophagy‐specific gene 1 and Drosophila S6 kinase. (A) Quantification of adults that survived after eclosion for denoted genotypes; n=3. Bars indicate mean±s.d. Fifteen larvae of each genotype were examined in each experiment. (B,C) Activation of dS6K in DmATG1 mutants. Phosphorylation of dS6K was examined by immunoblot analyses using phosphospecific dS6K T398 antibody in the third instar larvae or pupae of denoted genotypes. Tubulin immunoblot …

Inhibition of S6 kinase Thr 389 phosphorylation by Autophagy‐specific gene 1α. (A,B) HEK 293T cells were transfected with HA–Akt (A) or Myc–RSK (B), and/or Flag–ATG1α WT or KI plasmids. The immunoprecipitated Akt and RSK proteins were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel). (C) HEK 293T cells were transfected with HA–S6K Thr 389 Glu mutant and/or Flag–ATG1α WT or KI …

Involvement of Drosophila Autophagy‐specific gene 1 in Drosophila Target of rapamycin‐dependent cell growth and development. (A) Genomic structure of CG10967. The P‐element insertion site of EP3348 (DmATG11) is denoted. (B) The transcriptional levels of DmATG1 in the third instar larvae were analysed by qRT–PCR. Ribosomal protein 49 (rp49) was used as an internal control; n=3. Bars indicate mean±s.d. (C) Images of the larvae of denoted genotypes at 3 days (top) and 6 days (middle) after egg …

… with the wild‐type ( w1118 ) fly ( ; ; ). TOR is also involved in the regulation of autophagy. Autophagy is a process conserved among all eukaryotic cells and is required for rapid degradation of large portions of the cytoplasm and organelles in the lysosomal lumen, occurring as a result of nutrient deprivation or normal developmental processes ( ). Under nutrient‐rich conditions, TOR blocks the initiation step of autophagy by facilitating dissociation of Autophagy‐specific gene (Atg) 13 from …

Sung Bae Lee et al. EMBO Reports March 2007

Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway

Autophagy is an intracellular bulk degradation system that is ubiquitous for eukaryotic cells. In this process, cytoplasmic components are enclosed in autophagosomes and delivered to lysosomes/vacuoles. We recently found that a protein conjugation system, in which Apg12p is covalently attached to Apg5p, is indispensable for autophagy in yeast. Here, we describe a novel coiled‐coil protein, Apg16p, essential for autophagy. Apg16p interacts with Apg12p‐conjugated Apg5p and less preferentially …

Phenotype of the apg16Δ strain. (A) Disruption constructs of YMR159c (APG16). Disruption of YMR159c was achieved by insertion of the LEU2 marker gene at the AccI site or by replacing an AccI–PvuII fragment with the LEU2 gene. (B) Autophagy‐negative phenotype of the apg16Δ strain. The autophagic ability of wild‐type (TN125), apg12Δ (YNM107) and apg16Δ cells (YNM114) was measured by the alkaline phosphatase assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars …

Autophagy is an intracellular degradation system in which cytoplasmic components are sequestered to the lysosome/vacuole by a membrane‐mediated process ( ; ). There are two major classes of autophagy: microautophagy and macroautophagy. Microautophagy is a process of incorporation of cytoplasmic components by invagination of the lysosomal/vacuolar membrane. In macroautophagy, cytoplasmic components are first enclosed in double membrane structures termed autophagosomes, and then delivered …

Noboru Mizushima et al. The EMBO Journal July 1999

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy

… ) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of non‐fusing mitochondria that were found to have reduced Δψ m and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1 K38A or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired …

Consequences of fusion–fission events. (A) Schematic illustrations of mitochondrial tracking in four different experiments (i–iv) and corresponding Δψm traces (iv). Colored arrows indicate generation of hyperpolarizing (red) and depolarizing (green) daughters during fission. Note that after a fission event, fusion (black arrows) preferably occurred in the hyperpolarized daughter mitochondrion. (B) Depolarized mitochondria are selectively targeted for autophagy after a multi‐hour time lag. APs …

Inhibition of fission or autophagy results in increased protein oxidative damage. (A) Level of oxidized mitochondrial protein in mitochondria purified from FIS1 RNAi (n=3), DRP1‐DN (n=4) and their respective control cells. The blot detects carbonyl groups on amino‐acid side chains (Oxiblot analysis, see Materials and methods). Loading control CIII, mitochondrial ComplexIII Core I (*P<0.03, **P<0.04). (B) COS7 cells coexpressing DRP1‐DN and mtDsRed (red) were fixed, permeabilized and stained …

A model of the mitochondrion's life cycle that integrates mitochondrial dynamics and turnover. The mitochondrion cyclically shifts between a post fusion state (network) and a post fission state (solitary). Fusion is brief and triggers fission. Following a fission event, the daughter mitochondrion may either maintain intact membrane potential (red line) or depolarize (green line). If it depolarizes, it is unlikely to proceed to a subsequent fusion, unless it re‐polarizes. After being depolarized and solitary for a few hours, the mitochondrion is removed by autophagy.

… to be determined if networks are capable of giving rise to detached mitochondria that are functionally dissimilar from their source network. Addressing this question requires characterization of the consequences of the individual fission event. While fusion may recruit dysfunctional mitochondria into the active pool, autophagy targets depolarized mitochondria for digestion and elimination ( ; ); the mechanism that sorts mitochondria between the two fates is still unclear ( ). If fusion and autophagy

Gilad Twig et al. The EMBO Journal January 2008

TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic

Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain‐containing protein, TBC 1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB 11‐positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi‐subunit tethering complex and GEF for RAB 1, as an interactor of TBC 1D14. TBC 1D14 …

… cells. Western blot indicates ULK1 knockdown. Scale bars, 20 μm. Model for the role of TBC1D14 and the TRAPP complex in mammalian autophagy and ATG9 traffic. Recycling endosomes harbour a population of TBC1D14 bound to RAB11, and a population of ATG9 molecules which may traffic to and from the ATG9 compartment. (i) Upon amino acid starvation, TBC1D14 and RAB11 induce a vesicle trafficking step from RAB11‐ to RAB1‐positive membranes at a tubulo‐vesicular transport intermediate (ii). This results …

… To remain healthy, cells must clear their cytosol of misfolded proteins, dysfunctional organelles and intracellular pathogens. To this end, eukaryotes employ the evolutionarily conserved autophagy pathways (Mizushima et al , ). Macroautophagy (here referred to as autophagy) is a highly dynamic process involving the formation of a double‐membrane cisterna (the phagophore or isolation membrane), which expands to engulf portions of the cytosol, closing to form an autophagosome (Lamb et al …

Christopher A Lamb et al. The EMBO Journal February 2016
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